嗜热真菌糖苷酶的基因克隆、表达与分子改造
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摘要
纤维素是地球上最丰富的碳水化合物,可被纤维素酶降解转化成为葡萄糖,对于人类社会解决能源危机、食物短缺和环境污染具有重大的现实意义。传统上采用化学方法控制纤维素降解难度大、成本高,对环境污染严重,而纤维素的生物降解有效克服了这些问题。纤维素酶是一类能够水解纤维素β-D-糖苷键生成葡萄糖的多组分酶的总称,纤维素的彻底降解需要内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶三种组分的协同作用,β-葡萄糖苷酶是纤维素酶解的瓶颈。在纤维素水解时,增加β-葡萄糖苷酶活性,会有效提高纤维素酶解效率。近些年来,许多专家学者通过分子生物学等手段不断提高酶活力和获得新性能的重组纤维素酶。
糖化酶是一种具有外切活性的酶,它能从非还原性末端水解淀粉、糊精、糖原等的α-1, 4-葡萄糖苷键,得到终产物β-D-葡萄糖,是淀粉转化为葡萄糖过程中的主要酶类之一。糖化酶已在酿造、食品、医学等工业和农业领域中得到了广泛的应用,具有重要商业价值。但市场应用的糖化酶主要来源于常温菌,因其酶活力和产率低,热稳定性差导致产品成本高,储存期短,而制约糖化酶在农业、工业上的应用。由此可见,热稳定性糖化酶的研究和开发具有重要意义。
疏绵状嗜热丝孢菌(Thermomyces lanuginosus)和嗜热毛壳菌(Chaetomium thermophilum CT2)是广泛分布的,生长上限温度较高的嗜热真菌,从这两种真菌中已分离了多种嗜热酶,具有极大的研究和应用价值。本研究通过RT-PCR和RACE-PCR从嗜热毛壳菌中首次分离到了一个新的β-葡萄糖苷酶基因,GenBank登录号为EF648280,序列分析表明该基因全长核酸序列为3213 bp,包含一个2604 bp的开放阅读框ORF,编码867个氨基酸的蛋白质,酵母表达后酶蛋白表达量为1.644 U/mg,经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为119 kDa,比推测的蛋白分子量稍大,可能与该蛋白的糖基化有关。重组酶的最适反应温度和最适pH值分别为60℃和5.0,该酶具有较高的热稳定性,70℃保温10 min后剩余酶活为29.7 %。
本研究还从疏绵状嗜热丝孢菌中克隆到一种糖化酶基因(GenBank登录号为EF545003)并将其在毕赤酵母中进行了高效表达,经甲醇诱导表达后酶活最高可达11.6 U/mL,经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为67 kDa,该重组酶的最适反应温度和最适pH值分别为60℃和5.0,在pH 4.0-9.0的范围内是很稳定的。
对来自嗜热毛壳菌的β-葡萄糖苷酶基因的非保守氨基酸和保守氨基酸进行定点突变。三个非保守氨基酸的突变为K275R、N276S和279D,表达筛选后发现与原始菌GS-BGL相比,三个突变子的酶活都有不同程度的降低,突变子的酶活分别是原始菌酶活的81.3 %、63 %和65 %,但最适反应温度均提高了5℃;突变子N276S和G279D的最适pH由5变为4,而突变子K275R最适pH值仍保持在5;在热稳定性方面,三个突变子表现不同,60℃处理60 min,突变子G279D稳定性提高最多,仍有80.6 %的酶活,突变子N276S稳定性次之,酶活剩余72.1 %,原始酶仅剩69.55 %的活性,而突变子K275R酶活剩余59.5 %,与原始酶相比稳定性下降。而当保守氨基酸第287位的天冬氨酸突变为非亲核的甘氨酸后,突变子失去了糖苷水解酶的活性,证明了该氨基酸为维持水解酶活性的必需氨基酸,但目前还没有检测到糖苷合成酶的活性。
由于定点突变主要是针对天然酶蛋白中少数的氨基酸残基进行替换,蛋白的高级结构基本保持不变,而且本研究中的β-葡萄糖苷酶的结构迄今为止还没有报道,因而对酶蛋白的改造有限。为了提高β-葡萄糖苷酶的酶活力和稳定性,采用定向进化的方法对β-葡萄糖苷酶的基因进行改造。本研究以β-葡萄糖苷酶bgl基因为出发基因,采用易错PCR的方法建立突变体文库,以高活力为筛选压力,筛选方法采用平板荧光初筛和小量发酵法相结合。经过突变和筛选,获得了一个酶活力提高0.74倍的突变体,序列测定表明:突变基因与出发基因比较,突变子BE857有6个氨基酸发生了变化。通过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析等步骤纯化了该蛋白,酶学性质与出发酶进行了比较,该突变酶的最适温度为65℃,比出发酶提高了5℃,最适pH与出发酶一致均为5.0,在稳定性方面,突变子株BE857有所提高,60℃处理1 h后酶活仍剩余75.58 %,而出发菌株剩余酶活为69.55 %。
Cellulose is the most abundant carbohydrate available on earth, it can be converted into glucose by cellulases. It has great practical significance to solve the energy crisis, food shortage and environmental pollution. Traditionally, the chemical method to break down cellulose uncontrollable, costly and environmental pollution, but the biological degradation of cellulose effectively overcome these problems. Cellulase is a general term for multi-component enzyme that can hydrolyze cellulose byβ-D-glucoside bond. The completely degradation of cellulose requires synergy of three components: endoglucanase, exo-glucanase andβ-glucosidase, theβ-glucosidase is the bottleneck of cellulose hydrolysis. Increased mount or enhencedβ-glucosidase activity, will effectively improve the efficiency of cellulose hydrolysis. In recent years, many experts and scholars continue to improve the performance of activity and access to new recombinant cellulase by means of molecular biology.
Glucoamylase is an exo-acting enzyme that removes the glucose unites from the non-reducing ends of amylose, amylopectin and glycogen by hydrolyzingα-1, 4 linkages in a consecutive manner, producingβ-D-glucose as the sole product. Glucoamylase was widely used in dextrose production, baking industry, brewing of low calorie beer and whole grain hydrolysis for the alcohol industry. In recent years glucoamylases from thermophilic fungi, which are expected to be thermostable, have aroused increasing attention among researchers. The glucoamylase used in the market mainly from room temperature microorganisms. Due to high production costs, poor thermal stability, low activity and yield, the application of glucoamylase is restricted in agriculture and industrial applications. Thus, the research and development of thermorphilic glucoamylase have great importance.
T.lanuginosus and C.thermophilum are widely distributed soil-inhibiting fungi of considerable interest producers of thermostable enzymes, which have great value in research and application. In this study, aβ-glucosidase gene was isolated firstly from C.thermophilum CT2 by RT-PCR and RACE-PCR. The GenBank accession number was EF648280, sequence analysis showed that the full nucleotide sequence of the gene was 3213 bp, contained a 2604bp open reading frame, encoding 867 amino acids. The amount of enzyme protein was 1.644 U/mg after yeast expression. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. The SDS-PAGE showed that the recombinant protein was about 119 kDa, slightly larger than the deduced molecular weight, which may be related to the glycosylation. The optimal temperature and optimal pH were 60℃and 5.0 separately, the enzyme was relatively stable, after incubated at 70℃for 10 min still remained 29.7 % activity.
In this study, a glucoamylase gene from T.lanuginosus was also cloned (GenBank accession number is EF545003) and highly expressed in P.pastoris, the enzyme activity was 11.6 U/mL. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. SDS-PAGE showed that the recombinant protein was about 67 kDa, the optimal temperature and the optimum pH were 60℃and 5.0, separately. The enzyme was stable in the range pH 4.0-9.0.
The site-directed mutagenesis was acted toβ-glucosidase from C.thermophilum CT2. Three non-conservative amino acid mutation K275R, N276S, and 279D were expressed in p.pastoris, three mutants activities were 81.3 %, 63 % and 65 % respectively compared to GS-BGL, but the optimum temperature of three mutants were increased 5℃; mutants N276S and G279D optimum pH changed from 5 into 4 and mutant K275R optimum pH remained at 5. The performance of the three mutants were different in thermal stability, after incubated at 60℃for 60 min, G279D still have 80.6 % of the activity, N276S have 72.1 % of the activity, the activity of K275R was 59.5 %, while the original enzyme activity remaining 69.55 %. The conservative amino acid Asp287(nucleophile) mutated to Gly(non-nucleophile), After expressed in p.pastoris, found the mutant lost the glycoside hydrolase activity, but not yet detected glycoside synthase activity.
Since site-directed mutagenesis is mainly directed against a small number of amino acid residues, the high-level protein structure remained unchanged. The structure ofβ-glucosidase in this study enzyme was not reported so far, in order to improve theβ-glucosidase activity and stability, the directed evolution was used to alert theβ-glucosidase gene. In this study, the mutant library was constructed by error-prone PCR, fluorescence screening and small amount ferment were used to screen mutants with higher activity. A mutant BE857 with 0.74-fold increased enzyme activity was acquired. The sequencing showed the mutant have 6 amino acids changed. The optimum temperature of BE857 was 65℃, 5℃higher than the starting enzyme, the optimum pH kept invariant; the stability also increased, after treatment at 60℃for 1 h, the mutant still remaining 75.58 % activity, while the remaining activity of wild enzyme was 69.55 %.
糖化酶是一种具有外切活性的酶,它能从非还原性末端水解淀粉、糊精、糖原等的α-1, 4-葡萄糖苷键,得到终产物β-D-葡萄糖,是淀粉转化为葡萄糖过程中的主要酶类之一。糖化酶已在酿造、食品、医学等工业和农业领域中得到了广泛的应用,具有重要商业价值。但市场应用的糖化酶主要来源于常温菌,因其酶活力和产率低,热稳定性差导致产品成本高,储存期短,而制约糖化酶在农业、工业上的应用。由此可见,热稳定性糖化酶的研究和开发具有重要意义。
疏绵状嗜热丝孢菌(Thermomyces lanuginosus)和嗜热毛壳菌(Chaetomium thermophilum CT2)是广泛分布的,生长上限温度较高的嗜热真菌,从这两种真菌中已分离了多种嗜热酶,具有极大的研究和应用价值。本研究通过RT-PCR和RACE-PCR从嗜热毛壳菌中首次分离到了一个新的β-葡萄糖苷酶基因,GenBank登录号为EF648280,序列分析表明该基因全长核酸序列为3213 bp,包含一个2604 bp的开放阅读框ORF,编码867个氨基酸的蛋白质,酵母表达后酶蛋白表达量为1.644 U/mg,经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为119 kDa,比推测的蛋白分子量稍大,可能与该蛋白的糖基化有关。重组酶的最适反应温度和最适pH值分别为60℃和5.0,该酶具有较高的热稳定性,70℃保温10 min后剩余酶活为29.7 %。
本研究还从疏绵状嗜热丝孢菌中克隆到一种糖化酶基因(GenBank登录号为EF545003)并将其在毕赤酵母中进行了高效表达,经甲醇诱导表达后酶活最高可达11.6 U/mL,经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为67 kDa,该重组酶的最适反应温度和最适pH值分别为60℃和5.0,在pH 4.0-9.0的范围内是很稳定的。
对来自嗜热毛壳菌的β-葡萄糖苷酶基因的非保守氨基酸和保守氨基酸进行定点突变。三个非保守氨基酸的突变为K275R、N276S和279D,表达筛选后发现与原始菌GS-BGL相比,三个突变子的酶活都有不同程度的降低,突变子的酶活分别是原始菌酶活的81.3 %、63 %和65 %,但最适反应温度均提高了5℃;突变子N276S和G279D的最适pH由5变为4,而突变子K275R最适pH值仍保持在5;在热稳定性方面,三个突变子表现不同,60℃处理60 min,突变子G279D稳定性提高最多,仍有80.6 %的酶活,突变子N276S稳定性次之,酶活剩余72.1 %,原始酶仅剩69.55 %的活性,而突变子K275R酶活剩余59.5 %,与原始酶相比稳定性下降。而当保守氨基酸第287位的天冬氨酸突变为非亲核的甘氨酸后,突变子失去了糖苷水解酶的活性,证明了该氨基酸为维持水解酶活性的必需氨基酸,但目前还没有检测到糖苷合成酶的活性。
由于定点突变主要是针对天然酶蛋白中少数的氨基酸残基进行替换,蛋白的高级结构基本保持不变,而且本研究中的β-葡萄糖苷酶的结构迄今为止还没有报道,因而对酶蛋白的改造有限。为了提高β-葡萄糖苷酶的酶活力和稳定性,采用定向进化的方法对β-葡萄糖苷酶的基因进行改造。本研究以β-葡萄糖苷酶bgl基因为出发基因,采用易错PCR的方法建立突变体文库,以高活力为筛选压力,筛选方法采用平板荧光初筛和小量发酵法相结合。经过突变和筛选,获得了一个酶活力提高0.74倍的突变体,序列测定表明:突变基因与出发基因比较,突变子BE857有6个氨基酸发生了变化。通过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析等步骤纯化了该蛋白,酶学性质与出发酶进行了比较,该突变酶的最适温度为65℃,比出发酶提高了5℃,最适pH与出发酶一致均为5.0,在稳定性方面,突变子株BE857有所提高,60℃处理1 h后酶活仍剩余75.58 %,而出发菌株剩余酶活为69.55 %。
Cellulose is the most abundant carbohydrate available on earth, it can be converted into glucose by cellulases. It has great practical significance to solve the energy crisis, food shortage and environmental pollution. Traditionally, the chemical method to break down cellulose uncontrollable, costly and environmental pollution, but the biological degradation of cellulose effectively overcome these problems. Cellulase is a general term for multi-component enzyme that can hydrolyze cellulose byβ-D-glucoside bond. The completely degradation of cellulose requires synergy of three components: endoglucanase, exo-glucanase andβ-glucosidase, theβ-glucosidase is the bottleneck of cellulose hydrolysis. Increased mount or enhencedβ-glucosidase activity, will effectively improve the efficiency of cellulose hydrolysis. In recent years, many experts and scholars continue to improve the performance of activity and access to new recombinant cellulase by means of molecular biology.
Glucoamylase is an exo-acting enzyme that removes the glucose unites from the non-reducing ends of amylose, amylopectin and glycogen by hydrolyzingα-1, 4 linkages in a consecutive manner, producingβ-D-glucose as the sole product. Glucoamylase was widely used in dextrose production, baking industry, brewing of low calorie beer and whole grain hydrolysis for the alcohol industry. In recent years glucoamylases from thermophilic fungi, which are expected to be thermostable, have aroused increasing attention among researchers. The glucoamylase used in the market mainly from room temperature microorganisms. Due to high production costs, poor thermal stability, low activity and yield, the application of glucoamylase is restricted in agriculture and industrial applications. Thus, the research and development of thermorphilic glucoamylase have great importance.
T.lanuginosus and C.thermophilum are widely distributed soil-inhibiting fungi of considerable interest producers of thermostable enzymes, which have great value in research and application. In this study, aβ-glucosidase gene was isolated firstly from C.thermophilum CT2 by RT-PCR and RACE-PCR. The GenBank accession number was EF648280, sequence analysis showed that the full nucleotide sequence of the gene was 3213 bp, contained a 2604bp open reading frame, encoding 867 amino acids. The amount of enzyme protein was 1.644 U/mg after yeast expression. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. The SDS-PAGE showed that the recombinant protein was about 119 kDa, slightly larger than the deduced molecular weight, which may be related to the glycosylation. The optimal temperature and optimal pH were 60℃and 5.0 separately, the enzyme was relatively stable, after incubated at 70℃for 10 min still remained 29.7 % activity.
In this study, a glucoamylase gene from T.lanuginosus was also cloned (GenBank accession number is EF545003) and highly expressed in P.pastoris, the enzyme activity was 11.6 U/mL. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. SDS-PAGE showed that the recombinant protein was about 67 kDa, the optimal temperature and the optimum pH were 60℃and 5.0, separately. The enzyme was stable in the range pH 4.0-9.0.
The site-directed mutagenesis was acted toβ-glucosidase from C.thermophilum CT2. Three non-conservative amino acid mutation K275R, N276S, and 279D were expressed in p.pastoris, three mutants activities were 81.3 %, 63 % and 65 % respectively compared to GS-BGL, but the optimum temperature of three mutants were increased 5℃; mutants N276S and G279D optimum pH changed from 5 into 4 and mutant K275R optimum pH remained at 5. The performance of the three mutants were different in thermal stability, after incubated at 60℃for 60 min, G279D still have 80.6 % of the activity, N276S have 72.1 % of the activity, the activity of K275R was 59.5 %, while the original enzyme activity remaining 69.55 %. The conservative amino acid Asp287(nucleophile) mutated to Gly(non-nucleophile), After expressed in p.pastoris, found the mutant lost the glycoside hydrolase activity, but not yet detected glycoside synthase activity.
Since site-directed mutagenesis is mainly directed against a small number of amino acid residues, the high-level protein structure remained unchanged. The structure ofβ-glucosidase in this study enzyme was not reported so far, in order to improve theβ-glucosidase activity and stability, the directed evolution was used to alert theβ-glucosidase gene. In this study, the mutant library was constructed by error-prone PCR, fluorescence screening and small amount ferment were used to screen mutants with higher activity. A mutant BE857 with 0.74-fold increased enzyme activity was acquired. The sequencing showed the mutant have 6 amino acids changed. The optimum temperature of BE857 was 65℃, 5℃higher than the starting enzyme, the optimum pH kept invariant; the stability also increased, after treatment at 60℃for 1 h, the mutant still remaining 75.58 % activity, while the remaining activity of wild enzyme was 69.55 %.
引文
1.常理文.寡糖的毛细管电泳.分析化学, 1994, 22: 975-979
2.陈红歌.野油菜黄单胞菌α-淀粉酶性质及其体外定向进化研究.南京农业大学博士论文, 2005
3.陈惠黎,王克夷.糖复合物的结构和功能.上海医科大学出版社, 1997, 629-653
4.陈启和,何国庆.糖化酶及其基因研究进展.微生物学杂志, 2000, 20: 46-50
5.陈卫红,李多川.嗜热真菌热稳定性β-葡萄糖苷酶的克隆表达、纯化及酶学性质细胞生物学杂志. 2009, 31: 535-540
6.陈卫红,李多川.嗜热子囊菌光孢变种β-葡萄糖苷酶的基因克隆、表达及酶学性质分析应用与环境生物学报.2009, 15: 549-553
7.陈向东,藤尾雄策.日本根霉IF05318胞外β-葡萄糖苷酶的纯化及部分特性.微生物学报, 1997, 37: 368-373
8.楚春雪,李多川,郭润芳.一株嗜热毛壳菌β-葡萄糖苷酶的分离纯化及特性.菌物学报, 2004, 23: 397-402
9.方柏山,洪燕,夏启容.酶体外定向进化(Ⅱ)-文库筛选的方法及其应用.华侨大学学报(自然科学版), 2005, 26:113-116
10.方柏山,郑媛媛.酶体外定向进化(Ⅰ)-突变基因文库构建技术及其新进展.华侨大学学报(自然科学版), 2004, 25: 337-342
11.方芳,曹以诚,陈晓曦,曾炳佳. 49P(del)点突变提高中性纤维素内切酶EGV热稳定性的初步研究.现代生物医学进展, 2009, 9: 2634-2636
12.高伦江,董全,唐春红.纤维素酶的研究进展及前景展望.江苏食品与发酵, 2007, : 14-17
13.顾卫民.β-葡萄糖苷酶的特性及其在食品工业中的应用.江苏食品与发酵, 2003, 1: 5-7
14.关汉成,严自成,张树政.黑曲霉突变株葡萄糖淀粉酶的底物特异性.微生物学报, 1994, 31: 20-18
15.关铜,张世华.纤维素酶在饲料工业中的研究与应用.贵州畜牧兽医, 2003, 27: 7-9
16.韩如旸,陈美慈,赵宇华,闵航,马晓航.一种嗜热厌氧纤维素降解细菌的分离纯化方法.微生物学通报, 2000, 27: 363-366
17.韩笑,陈介南,王义强,何钢,刘海波,谭力.β-葡萄糖苷酶基因的克隆与表达研究进展.生物技术通报, 2008, 3: 8-12
18.何冰芳,欧阳平凯.极端微生物与工业生物催化剂开发.化工进展, 2006, 25: 1124-1127
19.侯温甫,杨文鸽.糖链及其蛋白质糖基化.生物技术通报, 2005, 3: 14-17
20.黄培堂,王嘉玺,朱厚础等译.分子克隆实验指南.北京:科学出版社, 2003. 1080-1087
21.黄瑛,蔡勇,杨江科,闫云君.基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化.生物工程学报, 2008, 3: 445-451
22.姜涌明,戴祝英,陈俊刚,胡健.分子酶学导论,中国农业大学出版社,中国,北京, 2000, 145-158
23.李洪钊,李亮助,孙强明,冮宏映.巴斯德毕赤酵母表达系统优化策略.微生物学报, 2003, 43: 288-292
24.刘玉芳,陈玉梅,张博润.糖化酶基因的克隆和在酿酒酵母中的表达.微生物学报, 1996, 36: 423-427
25.刘增然,何秀萍,龙章富,张博润,刘世贵.淀粉酶基因的克隆及其在工业啤酒酵母中的表达.食品科学, 2004, 25: 78-82
26.卢丽丽,肖敏,赵晗,王鹏,钱新民. 1-氟代糖在化学-酶法合成糖类中的应用.有机化学, 2006, 26:1631-1639
27.卢丽丽,肖敏,赵晗,王鹏,钱新民.糖苷合成酶-一类新型的寡糖高效合成工具.生物化学与生物物理学进展, 2006, 33: 310-320
28.马丽娜,陈喜文,甘睿,陈洁,陈德富.葡萄糖淀粉酶的结构和功能研究进展.生物技术通讯, 2005, 16: 677-680
29.马丽娜,陈喜文,陈德富,王绘砖,宋峥.曲霉属糖化酶基因的克隆及其在毕赤酵母中的表达.南开大学学报, 2007, 40: 85-90
30.马向东,马立新,薛征峰.一种鉴定淀粉酶活性及其产生菌的新方法.华中农业大学学报, 2000, 19: 95-99
31.马原松.寡糖及其开发利用.安微农业科学, 2006, 34: 3886-3888
32.孟雷,陈冠军,王怡,高培基.纤维素酶的多型性.纤维素科学与技术, 2002, 10: 47-55
33.邵金辉,赵云,毛爱军,朱雅新,董志扬.扣囊复膜孢酵母β-葡萄糖苷酶基因在毕赤酵母中的克隆与表达.微生物学报, 2005, 45: 792-794
34.宋志刚.低聚糖的开发利用.饲料博览, 2001, 1: 40-41
35.孙志浩,柳志强.酶的定向进化及其应用.生物加工过程, 2005, 3: 7-13
36.滕芳超,李多川,李亚玲,李浙江.嗜热毛壳菌一种β-葡萄糖苷酶的分离纯化及特性.菌物学报, 2006, 25: 481-487
37.王华夫,游小青.茶叶中β-葡萄糖苷酶活性的测定.中国茶叶, 1996, 3: 16-17
38.王元.现代酶工程技术的新进展.中国食品, 2000, 21: 22-23
39.文立民,于松涛,李浩,赵大健.糖化酵母糖化酶基因在酿酒醉母中的克隆和表达.食品与发醉工业, 1994, 118: 20-25
40.吴东儒.糖类的生物化学.北京:高等教育出版社, 1987
41.吴梧桐.蛋白质工程技术与新型生物催化剂设计.药物生物术, 2004, 11: 1-6
42.夏黎明,萧庆,余世袁.碳源对固定里氏木霉合成纤维素酶的影响.纤维素科学与技术, 1994, 2: 72-77
43.徐卉芳,张先恩,张治平,张用梅.大肠杆菌碱性磷酸酶的体外定向进化研究.生物化学与生物物理进展, 2003, 30: 89-94
44.徐威,朱春宝,朱宝泉.酶定向进化的研究策略.中国医药工业杂志, 2004, 35: 436-441
45.许钦坤.蛋白质定向进化的研究进展及其应用前景.生物技术通讯. 2005, 16: 191-193
46.阎伯旭,高培基.纤维素酶分子结构与功能研究进展.生命科学, 1995, 5: 22-25
47.阎伯旭,齐飞,张颖舒,高培基.纤维素酶分子结构和功能研究进展.生物化学与生物物理进展, 1999, 26: 233-237
48.杨清香,曹军卫,黄国锦.糖基转移酶和去糖基化酶.氨基酸和生物资源, 1997, 19: 38-43
49.杨雪鹏,杨寿钧,韩北忠,金城.非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究.生物工程学报, 2005, 21: 84-91
50.杨依军,李多川,沈崇尧.葡萄糖淀粉酶研究进展.微生物学通报, 1996, 23: 312-315
51.张浩,毛秉智.定点突变技术的研究进展.免疫学杂志, 2000, 16: 108-110
52.张今.进化生物技术-酶定向进化.北京:科学出版社, 2004
53.张龙翔,张庭芳,李令媛.生化实验方法和技术.高等教育出版社, 2005
54.张晓勇,陈秀霞,高向阳.纤维素酶的蛋白质工程.纤维素科学与技术, 2006,14 :55-58
55.张秀艳.β-葡聚糖酶的定向进化及热稳定性研究.浙江大学博士论文, 2006
56.赵永芳主编.生物化学技术原理及应用.科学出版社, 2002, 1-502
57.朱国萍,滕脉坤,伍传金,王玉珍. G138P定点突变对葡萄糖异构酶热稳定性的改善.生物化学与生物物理学报, 1998, 30: 607-610
58. Aharoni A, Griffiths A D, Tawfik D S. High-throughput screens and selections of enzyme encoding genes. CurrOp in Chem Biol, 2005, 9: 210-216
59. Aikawa J, Prak Y N. Replacements of amino acid properties of Rhizomu corpusillus pepsin, an aspartic proteinase from Rhizomu corpusillus. J Bio Chem(Tokyo), 2001, 129: 791
60. Ali S, Hossain Z, Mahmood S, Alam R. Purification of glucoamylase from Aspergillus terrus. World J Microbiol Biotechol, 1990, 6: 19-22
61. Alison M D, Beatrice A M, Sabine L F. Synthesis and modifications of carbohydrates, using biotransformations. Curr Opin In Chem Biol, 2004, 8:106-113
62. Allen S J, Holbrook J J. Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution. Protein engineering, 2000, 13: 5-7
63. Allison D S, Rey M W, Berka R M, Armstrong G, Dunn-Coleman N S. Transformation of the thermophilic fungus Humicola grisea var. thermoidea and overproduction of Humicola glucoamylase. Current Genetics, 1992, 21: 225-229
64. Alzari P M, Souchon H, Dominguez R. The crystal structure of endoglucansase celA, a family 8 glucosylhydroase from Clostridium thermocellum. Structure, 1996, 4: 265-275
65. Amidon W J, Pfeil J E, Gal S. Modification of luciferase to be a substrate for plant aspartic proteinase. Biochem J, 1999, 343: 425-433
66. Amirul A A, Khoo S L, Nazalan M N, Razip M S, Azizan M N. Purification and properties of two forms of glucoamylase from Aspergillus niger. Folia Microbiol, 1996, 41: 165-174
67. Amold F H. In IBC Directed Enzyme Evolution. San Diego, 1998
68. Aquino A C, Jorge J A, Terenzi H F, Polizeli M L. Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum. Folia Microbiol (Praha), 2001, 46: 11-16
69. Aro N, Ilmén M, Saloheimo A, Penttil? M. ACEI of Trichoderma reesei is a repressor of cellulose and xylanase expression. Applied and Environmental Microbiology, 2003, 69: 56-65
70. Arrizubieta M J, Polaina J. Increased thermal resistance and modification of the catalytic properties of a beta-glucosidase by random mutagenesis and in vitro recombination. Biological Chemistry, 2000, 275: 28843-28848
71. Bartoszewicz K. Glucoamylase of Aspergillus niger. Acta Biochim pol, 1986, 33: 17-29
72. Bauer M W, Kelly R M. The family 1β-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism. Biochemistry, 1998, 37: 17170-17178
73. Bauer S, Vasu P, Persson S, Mort A J, Somerville C R. Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc Natl Acad Sci U S A, 2006, 103:11417-11422
74. Bause E, Legler G. Isolation and amino acid sequence of a hexadecapeptide from the active site of beta-glucosidase A3 from Aspergillus wentii. Hoppe Seylers Z Physiol Chem, 1974 , 355: 438-442
75. Beguin P, Aubert J P. The biological degradation of cellulose. FEMS Microbiology Review, 1994, 13: 25-58
76. Benoliel B, Pocas F M J, Torres F A, Moraes L M. Expression of a Glucose-tolerant beta-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae. Appl Biochem Biotechnol, 2010, 160: 2036-2044
77. Berger R G. Application of genetic methods to the generation of volatile flavours. In: Hui H Y: Katchatourians G. (eds.): Food Biotechnology. VCH Weinheim. 1995, 291-295
78. Bhat M K, Bhat S. Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances, 1997, 15: 583-620
79. Bhatia Y, Mishra S, Bisaria V S. Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsiiβ-glucosidaseⅡwith high hydrolytic activity on sophorose. Appl Microbiol Biotechnol, 2005, 66: 527-535
80. Bhatti H N, Rashid M H , Asgher R N M, Perveen R, Jabbar A. Purification and characterization of a novel glucoamylase from Fusarium solani. Food Chemistry, 2007, 103: 338-343
81. Boel E, Hansen M T, Hjort I, Hoegh I, Fiil, N P. Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger. EMBO Journal, 1984, 3: 1581-1585
82. Boel E, Hjort I, Svensson B, Norris F, Norris K E, Fiil NP. Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs. EMBO Journal, 1984, 3: 1097-1102
83. Bornscheuer U T, Altenbuchner J, Meyer H H. Directed evolution of an esterase: Screening of enzyme libraries based on pH-indicators and a growth assay. Bioorganic and Medicinal Chemistry, 1999, 7: 2169-2173
84. Brunner F, Wirtz W, Rose J K, Jocelyn K. C. Roseb, 1, Alan G. Darvillb, Francine Goversc, Scheela D and Nürnberger T. A beta-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans. Phytochemistry, 2002, 59: 689-696
85. Brzobohaty B, Moore I, Kristoffersen P, Bako L, Campos N, Schell J, Palme K. Release of active cytokinin by aβ-glucosidase localized to the maize root meristem. Science, 1993, 262: 1051-1054
86. Buchholz F, Angrand P O, Stewart A F. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nature Biotechnol, 1998, 16: 657-662
87. Butler G, Rasmussen MD, Lin MF, Santos MA, Sakthikumar S, Munro CA, Rheinbay E, Grabherr M, Forche A, Reedy JL, Agrafioti I, Arnaud MB, Bates S, Brown AJ, Brunke S, Costanzo MC, Fitzpatrick DA, de Groot PW, Harris D, Hoyer LL, Hube B, Klis FM, Kodira C, Lennard N, Logue ME, Martin R, Neiman AM, Nikolaou E, Quail MA, Quinn J, Santos MC, Schmitzberger FF, Sherlock G, Shah P, Silverstein KA, Skrzypek MS, Soll D, Staggs R, Stansfield I, Stumpf MP, Sudbery PE, Srikantha T, Zeng Q, Berman J, Berriman M, Heitman J, Gow NA, Lorenz MC, Birren BW, Kellis M, Cuomo CA. Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature, 2009, 459:657-662
88. Cadwell R C, Joyce G F. Mutagenic PCR. PCR Methods Application, 1994, 6:136-140
89. Caldwell R C, Joyee G F. Randomization of genes by PCR mutagenesis. In PCR Methods Applic, 1992, 2: 28-33
90. Catcheside D E, Rasmussen J P, Yeadon P J. Diversification of exogenous genes in vivo in Neurospora. Appl Microbiol Biotechnol, 2003, 62: 544-549
91. Catherine M C, Patrick G M, Stuart D, John P M, Lucy B, Tuula T T, Maria G T. Molecular cloning and expression analysis of two distinctβ-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii. Mycological research, 2007, 111: 840-849
92. Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS micrrobiol, Rev., 2001, 24: 45-66
93. Chen J, Zhang Y Q, Zhao C Q, Li A N,.Zhou Q X, Li D C. Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris. Journal of Applied Microbiology, 2007, 103: 2277-2284
94. Chen K, Arnold F H. Enzyme engineering for nonaqueous solvents: Random mutagenesis to enhance activity of subtilisin E inpolar organic media. Bio/Technology, 1991, 9: 1073-1077
95. Chen K, Arnold F H. Tuning the activity of an enzyme for unusual environments: Sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc. Natl. Acad. Sci. USA, 1993, 90: 5618-5622
96. Cheong T K, Oriel P J. Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherrichia coli and marked enhancement of amidase expression using directed evolution. Enzyme and Microbial Technology, 2000, 26:152-158
97. Cherry J R, Lamsa M H, Schneider P, Vind J, Svendsen A, Jones A, Pedersen A H. Directed evolution of a fungal peroxidase. Nature Biotechnol, 1999, 17: 379-384
98. Clarke A J, Svensson B. Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from Aspergillus niger. Carlsberg Res Commun, 1984, 49: 559-566
99. Cole G E, Mccabe P L, Inlow D. Expression of Aspergillus anmanari glucoamylase in distiller's yeast. Bio/Technology, 1998, 6:417
100.Cortes M L, Felipe P, Martin V, Hughes MA, Izquierdo M. Successful use of a plant gene in the treatment of cancer in vivo. Gene Ther, 1998, 5: 1499-1507
101.Crameri A, Dawes G, Rodriguez E, Silver S, Stemmer W P C. Molecular evolution of an arsenate detoxify-catio pathway by DNA shuffling. Nature Biotechnol, 1997, 15: 436-438
102.Crameri A, Raillard S A, Bermudez E, Stemmer W P C. DNA Shuffling of a family of genes from diverse species accelerates evolution. Nature, 1998, 391: 288-291
103.Cregg J M, Cereghino J L, Shi J Y. Recombiant Protein Expression in Pichia pastoris. Molecular Biotechnology, 2000, 16: 23-52
104.Creighton T E, Proteins: Structures and Molecular Properties, second ed., Freeman&Company, New York, 1997
105.Damude H G, Withers S G, Kilburn D G, Miller R C, Warren R A. Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi. Biochemistry, 1995, 34: 2220-2224
106.Dan S, Marton I, Dekel M, Bravdo B A, He S, Withers S G, Shoseyov O. Cloning, Expression, Characterization, and Nucleophile Identification of Family 3, Aspergillus nigerβ-Glucosidase. Biological Chemistry, 2000, 275: 4973-4980
107.Davies G J, Ducros V, Lewis R J. Oligosaccharide specifity of a family 7 endoglucanase insertion of potential sugar-binding subsites. Biotechnology, 1997, 57: 91-100
108.Davies G J, Dauter M, Brzozowski A M, Bj?rnvad M E, Andersen K V, Schülein M. Structure of the Bacillus agaradherans family 5 endoglucanase at 1.6 ? and its cellobiose complex at 2.0 ? resolution. Biochemistry, 1998, 37: 1926-1932
109.Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure, 1995, 3: 853-859
110.De Groeve R M, De Baere M, Hoflack L, Lieve Hoflack, Tom Desmet, Erick J V and Soetaert W. Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase. Protein Engineering, Design & Selection, 2009, 22: 393-399
111.Denisov V P. Orientational disorder and entropy of water in protein cavities. J. Phy. Chem, 1997, 101: 380-389
112.Dharmawardhana D P, Ellis B E, Carlson J E. Aβ-glucosidase from lodgpole pine xylem specific for the lignin precursor coniferin. Plant Physiol., 1995, 107: 331-339
113.Dill K A. Dominant forces in protein folding. Biochemistry, 1990, 29: 7133-7155
114.Ding S, Ge W, Buswell J A. Molecular cloning and transcriptional expression analysis of an intracellular beta-glucosidase, a family 3 glycosyl hydrolase, from the edible straw mushroom, Volvariella volvacea. FEMS Microbiol Lett, 2007, 267: 221-229
115.Doi R H, Park J S, Liu C C, Malburg L M, Tamaru Y, Ichiishi A, Ibrahim A. Cellulosome and noncellulosomal cellulase of Clostridium cellulovorans. Extremophiles, 1998, 2: 53-60
116.Dominguez R, Souchon H, Lascombe M B. A common protein fold and similar active in two distinct families of beta-glycanases. Nature Structural & Molecular Biology, 1995, 2: 569-576
117.Driskill L E, Bauer M W, Kelly R M. Synergistic interactions amongβ-laminarinase,β-1,4-glucanase, andβ-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus during hydrolysis ofβ-1,4-,β-1,3-, and mixed-linked polysaccharides. Biotechnology and Bioengineering, 1999, 66: 51-60
118.Dujon B, Sherman D, Fischer G, Gilles Fischer1, Pascal Durrens6,7, Serge Casaregola8, Ingrid Lafontaine1, Genome evolution in yeasts. Nature, 2004, 430: 35-44
119.Edward J, Bylina E J, Coleman W J, Dilworth M R, Robles S J, Tanner M A, Yang M M, Youvan D C. Solid-phase enzyme screening. ASM News, 2000, 66: 211-217
120.Fairweather J K, Faijes M, Driguez H, Planas A. Specificity studies of Bacillus 1,3-1,4-β-glucanases and application to glycosynthase-catalyzed transglycosylation.Chem Biochem, 2002, 3: 866-873
121.Fairweather J K, Hrmova M, Rutten S J, Fincher G B, Driguez H. Synthesis of complex oligosaccharides by using a mutated (1, 3)-β-D-glucan endohydrolase from barley. Chemistry, 2003, 9: 2603-2610
122.Faure D, Henrissat B, Ptacek D, Bekri M A, Vanderleyden J. The celA gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in Azospirillum irakense, is required for optimal growth on cellobiosides. Appl Environ Microbiol, 2001, 67: 2380-2383
123.Fort S, Boyer V, Greffe L, Gideon J. Davies, Moroz O, Christiansen L, Schülein M, Cottaz S, Driguez H. Highly efficient synthesis ofβ-(1→4)-oligo-and-polysaccharides using a mutant cellulase. J Am Chem Soc, 2000, 122: 5429-5437
124.Freeman A, Cohen-Hadar N, Abramov S, Modai-Hod R, Dror Y, Georgiou G. Screening of large protein libraries by the‘cell immobilized on adsorbed bead’approach. Biotechnology and Bioengineering, 2004, 86: 196-200
125.Fromant M, Blanquet S, Plateau P. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction . Anal Biochem, 1995, 224: 347-353
126.Gibbons I. Microfluidic assays for high-throughput submicroliter assays using capillary electrophoresis. Drug Discov. Today, 2000, 5: 33-36
127.Gomes I, Gomes J, Gomes D J, Steiner W. Simultaneous production of high activities of thermostable endoglucanase andβ-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Appl Microbiol Biotechnol, 2000, 53: 461-468
128.Gonzalez B G,Sanz A J,Gonzalez B, Hermoso J A, Polaina J. Directed evolution of beta-glucosidase A from Paenibacillus polymyxa to thermal resistance. J Biol Chem,2000,275: 13708-13712
129.Goto M, Semimaru T, Furukawa K and Hayashida S. Analysis of the raw starch-binding domain by mutation of a glucoamylase from Aspergillus awamori var. kawachi expressed in Saccharomyces cerevisiae. Applied and Environment Microbiology, 1994, 60: 3926-3930
130.Graham L D, Haggett K D, Jennings P A, Le Brocque D S, Whittaker R G, Schober P A. Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes weith increased activities and altered primary specificities. Biochemistry, 1993, 32: 6250-6258
131.Hagglund P, Bunkenborg J, Elortza F, Jensen O N, Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J Proteome Res,2004, 3: 556-566
132.Hansson L O, Widersten M, Mannervik B. An approach to optimizing the active site in a glutathione transferase by evolution in Vitro. Biochem, 1999, 344: 93-100
133.Harhangi H R, Steenbakkers P J, Akhmanova A, Jetten M S, van der Drift C, Opden Camp H J. A highly expressed family 1 beta-glucosidase with transglycosylation capacity from the anaerobic fungus Piromyces sp. E2. Biochim Biophys Acta, 2002, 12: 293-303
134.Harnpicharnchai P, Champreda V, Sornlake W, Eurwilaichitr L. (2009) A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars. Protein Expr Purif, 2009, 67: 61-69
135.Hayashi S, Sako S, Yokoi H, Takasaki Y, Imada K. Purification and characterization of the intracellularβ-glucosidase from Aureobasidium sp ATCC 20524. Journal of Industrial Microbiology & Biotechnology,1999, 22: 160-163
136.Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science, 2001, 291: 2364-2369
137.Henrissat B. A classification of glycosyl-hydrolases based on aminoacid sequence similarities. Biochem. J, 1991, 293: 781-788
138.Hilge M, Gloor S M, Rypniewski W, Sauer O, Heightman TD, Zimmermann W, Winterhalter K, Piontek K. High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca substrate specificity in glycosylhydrolase family 5. Structure, 1998, 6: 1433-1444
139.Hong J, Tamaki H, Kumagai H. Unusual hydrophobic linker region of beta-glucosidase (BGLII) from Thermoascus aurantiacus is required for hyper-activation by organic solvents. Appl Microbiol Biotechnol, 2006, 73: 80-88
140.Hong J, Tamaki H, Yamamoto K,Kumagai H. Cloning and functional expression of thermostableβ-glucosidase gene from Thermoascus aurantiacus. Appl Microbiol Biotechnol, 2007, 73: 1331-1339
141.Howard S,Withers S G. Bromoketone C-glycosides, a new class of beta-glucanase inactivators. J Am Chem Soc, 1998, 120: 10326-10331
142.Hrmova M, Imai T, Rutten S J, Fairweather J K, Pelosi L, Bulone V, Driguez H, Fincher G B. Mutated barley (1,3)-β-D-glucan endohydrolases synthesize crystalline (1,3)-β-D-glucans. J Biol Chem, 2002, 277: 30102-30111
143.Huang C Y, Chang A K. Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobiles pyruvate decarboxylase: effect on activity and pH dependence. Eur J Biochem, 2001, 268: 3558-3565
144.Iffland A, Tafelmeyer P, Saudan C, Johnsson K. Directed molecular evolution of cytochrome c peroxidase. Biochemistry, 2000, 39: 10790-10798
145.Jahn M, Chen H, Mullegger J, Warren R A, Withers S G. Thioglycosynthases: double mutant glycosidases that serve as scaffolds for thioglycoside synthesis. Chem Commun, 2004, 7: 274-275
146.Jahn M, Marles J, Warren R A J, Withers S G. Thioglycoligases: mutant glycosidases for thioglycoside synthesis. Angew Chem Int Ed, 2003, 42: 352-354
147.Jahn M, Stoll D, Warren R A, SzabóL, Singh P, Gilbert H J, Ducros V M, Davies G J, Withers S G. Expansion of the glycosynthase repertoire to produce defined manno-oligosaccharides. Chem Commun, 2003, 12: 1327-1329
148.Jakeman D L, Withers S G. On expanding the repertoire of glycosynthases: mutantβ-galactosidases formingβ-(1,6)-linkages. Can J Chem, 2002, 80: 866-870
149.James J A, Lee B H. Glucoamylases: microbial sources, industrial applications and molecular biology– a review. Journal of Food Biochemistry, 1997, 21: 1-52
150.Jeffries T W, Grigoriev I V, Grimwood J, Laplaza J M, Aerts A, Salamov A, Schmutz J, Lindquist E, Dehal P, Shapiro H, Jin Y S, Passoth V, Richardson P M. Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis. Nat Biotechnol, 2007, 25: 319-326
151.Jeya M, Joo A R, Lee K M, Tiwari M K, Lee K M, Kim S H, Lee J K. Characterization of beta-glucosidase from a strain of Penicillium purpurogenum KJS506. Appl Microbiol Biotechnol, 2010, 86:1473-1484
152.Johnson P E, Brun E, MacKenzie L F, Withers S G, McIntosh L P. The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations. Journal of molecular biology, 1999, 287: 609-625
153.Joo H, Lin Z, Arnold F H. Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature, 1999, 399: 670-673
154.Kadam K L, Wollweber K L, Grosch J C. Investigations on plasmid instability in amylase producing Bacillus subtilis using continuous culture. Biotechnol Bioeng, 1987, 29: 859-872
155.Kaper T, Brouns S J, Geerling A C, De Vos WM, Van der Oost J. DNA Family shuffling of hyperthermostable beta-glycosidases. Biochem. J., 2002, 368: 461-470
156.Kawai R, Yoshida M, Tani T, Igarashi K, Ohira T, Nagasawa H, Samejima M. Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris. Biosci Biotechnol Biochem, 2003, 67: 1-7
157.Kempton J B. Withers S G. Mechanism of Agrobacteriumβ-glucosidase: Kinetic studies. Biochemistry, 1992, 31: 9961-9969
158.Kikuchi M, Ohnishi K, Harayama S. An effective family shuffling method using single-stranded DNA. Gene, 2000, 243: 133-137
159.Kim B, Hathaway T R, Loeb L A. Human immunodeficiency virus revers traverse transcriptase Functional mutants obtained by random mutagenesis coupled with gentic selection in Escherichia coli. J Biol. Chem., 1996, 271: 4872-4878
160.Kim Y S, Jung H C, Pan J G. Bacterial cell surface display of an enzyme library for selective screening of improved cellulose variants. Applied Environmental Microbiology, 2000, 66: 788-793
161.Kim Y W, Lee S S, Warren R A, Withers S G. Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J. Bio. Chem, 2004, 279: 42787-42793
162.Kirschner A, Bornscheuer U T. Directed evolution of a Baeyer-Villiger monooxygenase to enhance enantioselectivity. Appl. Microbiol. Biotechnol., 2008, 81: 465-472
163.Kleyweg G J, Zou J Y, Divne C. The crystal structure of the catlytic domian of endoglucanaseⅠfrom Trichoderma reesei. Molecular Biology, 1997, 272: 383-397
164.Knaust R K C, Nordlund P. Screening for soluble expression of recombinant proteins in a 96-well format. Analytical Biochemistry, 2001, 297: 79-85
165.Knecht W, Munch-Petersen B, Piskur J. Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster. Molecular Biology, 2000, 30: 827-837
166.Kobata A. Structures and function of sugar chains of glycoproteins. Eur J Biochem, 1992, 209: 483-501
167.Kolkman J A, Stemmer W P C. Directed evolution of proteins byexon shuffling. Nature Biotechnology, 2001, 19: 423-428
168.Komeda H, Ishikawa N, Asano Y. Enhancement of the thermostability and catalytic activity of D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3 by directed evolution. J . Mol. Catal. B : Enz. , 2003 , 21: 283-290
169.Kong X, Liu Y, Gou X, Zhu S, Zhang H, Wang X, Zhang J. Directed evolution ofαaspartyl dipeptidase from Salmonella typhimurium. Biochem Biophys Res Commun, 2001, 289: 137-142
170.Koshland D E J. Steriochemistry and the mechanism of enzymatic reactions. Biol Rev,1953, 28: 416-436
171.Krogh K B, Harris P V, Olsen C L, Johansen K S, Hojer-Pedersen J, Borjesson J, Olsson L. Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum. Appl Microbiol Biotechnol, 2010, 86:143-154
172.Kumar S, Satyanarayana T. Purification and kinetics of a raw starch-hydrolyzing, thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae. Biotechnol Prog, 2003, 19: 936-944
173.Ladbury J E, Wynn R, Thomson J A, Sturtevant J M. Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin result in a change in heat capacity of the native protein. Biochemistry, 1995, 34: 2148-2152
174.Lanio T, Jeltsch A, Pingoud A. Towards the design of rare cutting restriction endonucleases: using directed wvolution to generate variants of EcoRⅤdiffering in their substrate specificity by two orders of magnitude. Mol Biol, 1998, 283: 59-69
175.Leah R, Kigel J, Svendsen I, Mundy J. Biochemical and molecular characterization of barley seedβ-glucosidase. J Biol Chem., 1995, 270: 15789-15797
176.Lebbink J H, Kaper T, Bron P, van der Oost J, de Vos W M. Improving lowtemperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution. Biochemistry, 2000, 39: 3656-3665
177.Leung D W, Chen E, Goeddel D V. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1989, 1: 11-15
178.Li D C, Yang Y J, Peng Y L, Shen C Y. Purification and characterization of extracellular glucoamylase from the thermophilic Thermomyces lanuginosus. Mycol Res. 1998, 102: 568-572
179.Li X L, Ljungdahl L G, Ximenes E A, Chen H, Felix C R, Cotta M A, Dien B S. Properties of a recombinant beta-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis. Appl Biochem Biotechnol, 2004, 113-116:233-50
180.Li Y K, Chir J, Chen F Y. Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247. Biochem J. 2001, 355: 835-840
181.Lima A O, Davis D F, Swiatek G, McCarthy J K, Yernool D, Pizzirani-Kleiner A A, Eveleigh D E. Evaluation of GFP tag as a screening reporter in directed evolution of a hyperthermophilicβ-Glucosidase. Mol Biotechnol, 2009, 42: 205-215
182.Lin H, Tao H, Cornish V W. Directed evolution of a glycosynthase via chemicalcomplementation. Am. Chem. Soc., 2004, 126: 15051-15059
183.Lin L L, Ma Y J, Chien H R, Hsu W H. Construction of an amylolytic yeast by multiple integrate of the A .awamori GA gene into a S. cerevisiae chromosome. Enzyme and Microbiol Tech, 1998, 23: 360-366
184.Lin L L, Rumbak E, Zappe H, Thomson J A, Woods D R. Cloning, sequencing and analysis of expression of a Butyrivibrio jibrisofvens gene encoding aβ-glucosidase. Journal of General Microbiology,1990, 136:1567-1576
185.Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, Li C, Dong J, Xiao L, Liu Z. Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution. Appl Microbiol Biotechnol, 2009, 82: 671-679
186.Liu D R, Schultz P G. Progress toward the evolution of an organism with an expanded genetic code. Proc Natl Acad Sci USA,1999,96: 780-4785
187.Lorimer I A L, Pastan I. Random recombination of antibody single chain Fv sequence after fragmentation with DNase I in the presence of Mn2+. Nucleic Acids Res, 1995, 23: 3067-3068
188.Ma Y F, Evans D E. Mutations of barley beta-amylase that improve substrate-binding affinity and thermo stability. Mol Genet Genomics, 2001, 266: 345-352
189.Mackenzie L F, Wang Q, Warren R A J, Withers S G. Glycosynthases: mutant glycosidases for oligosaccharide synthesis. Am Chem Soc, 1998, 120: 5583-5584
190.Maheshwari R, Bharadwaj G, Bhat M K. Thermophilic fungi: Their physiology and enzymes. Microbiology and Molecular Biology Reviews, 2000, 64: 461-488
191.Malet C, Planas A. Fromβ-glucanase toβ-glucansynthase glycosyltransfer toα-glycosylfluorides catalyzed by a mutant endoglucanase lacking its catalytic nueleophile. FEBS. Lett., 1998, 440: 208-212
192.Manger J F, Muller T, Janssen M, Hofer M and Holker U. Isolation and sequencing of a new glucoamylase gene from an Aspergillus niger aggregate atrain (DSM 823) molecularly classified as Aspergillus tubingensis. Antonie Van Leeuwenhoek, 2005, 88: 267-275
193.Manjula Pandey, Saroj Mishra. Expression and characterization of Pichia etchellsiiβ-glucosidase Escherichia coli. Gene, 1997, 190: 45-51
194.Martel M B, Herve P C, Letoublon R, Fevre M. Purification and characterization of a glucoamylase secreted by the plant pathogen Scierotinia sclerotiorum. Can J Microbiol, 2002, 48: 212-218
195.Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M, Kubicek CP, Ferreira P, Ruiz-Duenas FJ, Martinez AT, Kersten P, Hammel KE, Vanden Wymelenberg A, Gaskell J, Lindquist E, Sabat G, Bondurant SS, Larrondo LF, Canessa P, Vicuna R, Yadav J, Doddapaneni H, Subramanian V, Pisabarro AG, Lavín JL, Oguiza JA, Master E, Henrissat B, Coutinho PM, Harris P, Magnuson JK, Baker SE, Bruno K, Kenealy W, Hoegger PJ, Kües U, Ramaiya P, Lucas S, Salamov A, Shapiro H, Tu H, Chee CL, Misra M, Xie G, Teter S, Yaver D, James T, Mokrejs M, Pospisek M, Grigoriev IV, Brettin T, Rokhsar D, Berka R, Cullen D. Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc Natl Acad Sci U S A. 2009, 106: 1954-1959
196.Matsuura T, Yomo T, Trakulnaleamsai S, Ohashi Y, Yamamoto K, Urabe I. Nonadditivity of mutational effects on the properties of catalase and its application to efficient directed evolution. Protein Engineering, 1998, 11: 789-795
197.May O, Nguyen P T, Arnold F H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nature Biotechnology, 2000, 18: 317-320
198.Mayer C, Zechel D L, Reid S P, Warren R A, Withers S G. The E358S mutant of Agrobacterium sp.β-glucosidase is a greatly improved glycosynthase. FEBS Lett, 2000, 466: 40-44
199.McBeath G, Kast P, Hilvert D. Redesigning enzyme topology by directed evolution. Science, 1998, 279: 958-961
200.McCarter J D, Withers S G. Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol, 1994, 4: 885-892
201.McCarthy J K, Uzelac A, Davis D F, Eveleigh D E. Improved Catalytic Efficiency and Active Site Modification of 1,4-β-D-Glucan Glucohydrolase A from Thermotoga neapolitana by Directed Evolution. Biological Chemistry, 2004, 279: 11495-11502
202.Michael W B, Robert M k, Biochemistry, 1992, 31: 9961-9969
203.Miller G L. Use of dinitrosalicyclic acid regent for determination of reducing sugar. Anal. Chem, 1951, 31: 426-428
204.Miyazaki K, Takenouchi M, Kondo H, Noro N, Suzuki M, Tsuda S. Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution. J Biol Chem, 2006, 281: 10236-10242
205.Moore J C, Jin H M, Kuchner O, Arnold F H. Strategies for the in vitro evolution ofprotein function: enzyme evolution by random recombination of improved sequences. Mol Biol, 1997, 272: 336-347
206.Moracci M, Trincone A, Perugino G, Ciaramella M, Rossi M. Restoration of the activity of active-site mutants of the hyperthermophilicβ-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles. Biochemistry, 1998, 37: 17262-17270
207.Moracci M, Trincone A. Mose rossi glycosynthases: new enzymes for oligosaccharide synthesis. Journal of Molecular Catalysis B: Enzymatic, 2001, 11:155-163
208.Morawski B, Lin Z, Cirino P, Joo H, Bandara G, Arnold F H. Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Picha pastoris. Protein Engineering, 2000, 13: 377-384
209.Mullegger J, Chen H, Warren R A J, Withers S.G. Glycosylation of a neoglycoprotein by using glycosynthase and thioglycoligase approaches: the generation of a thioglycoprotein. Angew Chem Int Ed Engl, 2006, 45: 2585- 2588
210.Murashima K, Kosugi A, Doi R H. Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with noncelluloso-mal endoglucanase EngD. Mol. Microbiol, 2002, 45: 617-626
211.Nagendra H G, Sukumar N, Vijayan M. Role of water in plasticity, stability, and action of proteins: the crystal structures of lysozyme at very low levels of hydration. Proteins, 1998, 32: 229-240
212.Nakamura A, Fukunmori F, Horinouchi. Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase. Biol. Chem, 1991, 266: 1579-1583
213.Nakazawa H, Okada K, Onodera T, Ogasawara W, Okada H, Morikawa Y. Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei. Appl Microbiol Biotechnol, 2009, 83: 649-657
214.Nakkharat P, Haltrich D. Purification and characterisation of an intracellular enzymeβ-glucosidase andβ-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58. Journal of Biotechnology, 2006, 123: 304-313
215.Nashiru O, Zechel D L, Stoll D, Mohammadzadeh T, Warren R A, Withers S G.β-Mannosynthase:synthesis ofβ-mannosides with a mutantβ-mannosidase. Angew Chem Int Ed, 2001, 40: 417-420
216.Ness J E, Welch M, Giver L, Bueno M, Cherry J R, Borchert T V, Stemmer W P, Minshull J. DNA shuffing of subgenomic sequences of subtilisin. Nature Biotechnol,1999, 17: 893-896
217.Nguyen Q D, Rezessy-SzabóJ M, Claeyssens M, Stals I, Hoschkeá. Purification and characterisation of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626. Enzyme and Microbial Technology, 2002, 31: 345-352
218.Nielsen B R, Lehmbeck J, Frandsen T P. Cloning, heterologous expression, and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii. Protein expression and purification, 2002, 26: 1-8
219.Niemeyer H M. Hydroxamic acid (4-hydroxy-1,4-benzoxazin-3-ones), defence chemicals in the gramineae. Phytochemistry, 1988, 27: 3349-3358
220.Norouzian D, Rostami K, Nouri I D. Subsite mapping of purified glucoamylases I, II, III produced by Arthrobotrys amerospora ATCC 34468. Microbiol Biotechnol, 2000, 8: 55-61
221.Ohnishi M, Higushi A, Todoriki S, Hiromi K, Oghushi M, Wada A. Characterazation on the conformation of glucoamylase from Rhizopus niveus and Rhizopus delemar. Starch/ Starke, 1990, 42: 273-276
222.Okuyama M, Mori H, Watanabe K, Kimura A, Chiba S.α-Glucosidase mutant catalyzes "α-glycosynthase"-type reaction. Biosci Biotechnol Biochem, 2002, 66: 928-933
223.Oren A. A thermophilic amyloglucosidase from Halobacterium sodomense, a halophilic bacterium from the Dead Sea. Curr Microbiol, 1983, 8: 225-230
224.Ozaki K, Sumitomo N, Hayashi Y. Site-directed mutagenesis of the putative active site of endoglucanase K from Bacillus sp. KAM-330. Biochim Biophys. Acta., 1994, 1207: 159-164
225.Palackal N, Brennan Y, Callen W N, Dupree P, Frey G, Goubet F, Hazlewood G P, Healey S, Kang Y E, Kretz K A, Lee E, Tan X, Tomlinson G L, Verruto J, Wong V W, Mathur E J, Short J M, Robertson D E, Steer B A. An evolutionary route to xylanase process fitness. Protein Sci, 2004, 13: 494-503
226.Palcic M M. Biocatalytic synthesis of oligosaccharides. Curr Opin Biotechnol, 1999, 10: 616-624
227.Pazur J H, Knull H R, Cepure A. Glucoamylase: Structure and properties of the two forms of glucoamylase from Aspergillus niger. Carbohyd Res, 1971, 20: 83-96
228.Pellerin P, Brillouet J M. Purification and properties of an exo-(1→3)-β-d-galactanase from Aspergillus niger. Carbohydrate Research, 1994, 264: 281-291
229.Perugino G, Trincone A, Giordano A, van der Oost J, Kaper T, Rossi M, Moracci M. Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH.Biochemistry, 2003, 42: 8484-8493
230.Perugino G, Trincone A, Rossi M, Moracci M. Oligosaccharide synthesis by glycosynthases. Trends Biotechnol, 2004, 22:31-37
231.PouLton J E. Cyanogenesis in Plants. Plant Physiol., 1990, 94: 401-405
232.Pretorius I S, Laing E, Pretorius G H, Marmur J. Expression of Bacillus amylase gene in yeast. Current Genetics, 1988, 14: 1-8
233.Ramasesh N, Srekantiah K R, Murthy V S. Strudies on the two forms of amyloglucosidase of Aspergillus niger van Teighem. Starch /St?rke, 1982, 34: 346-351
234.Reetz M T, Wang L W. Directed evolution of enantioselective hybrid catalysts: a novel concept in asymmetric catalysis, Tetrahedron, 2007, 63: 6404-6414
235.Rey M W, Brown K M, Golightly E J, Fuglasang C C, Nielsen B R, Hendriksen H V, Butterworth A, Xu. Cloning, Heterologous Expressionand Characterization of Thielavia terrestris Glucoamylase. Applied Biochemistry and Biotechnology, 2003, 3: 153-166
236.Riedel K, Bronnenmeier K. Active-site mutations which change the substrate specificity of the Clostridium ercorarium stcellulase CelZ implications for synergism. European Journal of Endocrinology, 1999, 262: 218-223
237.Rothstain S J, Larners K N, Lazarus C M. Expression and secretion of wheal amylase in Saccharomyoes cereviaiae. Gene, 1987, 55:353
238.Rouvinen J, Bergfors T, Teeri T. Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science, 1990, 249: 380-386
239.Rudd P M, Elliott T, Cresswell P, Wilson I A, Dwek R A. Glycosylation and the immune system. Science, 2001, 291: 2370-2376
240.Rudick M J, Elbein A D. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of a second,β-glucosidase. J Bacteriol, 1975, 124:534-541
241.Rye C S, Withers S G. Glycosidase mechanisms. Curr Opin Chem Biol, 2000, 4: 573-580
242.Sakai K, Fukui S, Yabuuchi S, Aoyagi S, Tsumura Y. Expression of the Saccharomyces diastaticus STA1 gene in brewing yeasts. J Am Soc Brew Chem, 1989, 47: 87-91
243.Sakon J, Adney W S, Himmel M E. Crystal structure of thermostable family 5 endocellu- lase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry, 1996, 35: 10648-10660
244.Sakon J, Irwin D, Wilson D B. Structure and mechanism of endo/exocellulase E4 from Thermobifida fusca. Nature Structural & Molecular Biology, 1997, 4: 810-818
245.Saloheimo M, Kuja-Panula J, Yl?sm?ki E, Ward M, Penttil? M. Enzymatic propertiesand intracellular localization of the novel Trichoderma reeseiβ-Glucosidase BGLII (Cel1A). Applied Environment Microbiology, 2002, 68: 4546-4553
246.Shao Z, Zhao H, Giver L, Arnold F H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucl Acids Res, 1998, 26: 681-683
247.Siddiqui K S, Rashid M H, Ghauri T M, Durrani I S, Rajoka M I. Short Communication: Purification and characterization of an intracellularβ-glucosidase from Cellulomonas biazotea. World Journal of Microbiology & Biotechnology, 1997, 13: 245-247
248.Sinnott M L. Catalytic mechanism of enzymic glycosyl transfer. Chem Rev, 1990, 90: 1171-1202
249.Smith M E B, Hibbert E G, Jones A B, Dalby P A, Hailes H C. Enhancing and reversing the stereoselectivity of escherichia coli transketolase via single-point mutations. Adv. Synth. Cat., 2008, 350: 2631-2638
250.Spee J H, Vos W M, Kuipes O P. Efficient random mutagenesis method with adjustable mutation by use of PCR and dITP. Nucl Acids Res, 1993, 21: 777-778
251.Spinelli B B L, Lourdes M, Polizeli T M. Biochemical characterization of glucoamylase from the hyper producer exo-1 mutant strain of Neurospora crassa. FEMS Microbiol Lett, 1996, 13: 3-7
252.Sreekrishna K, Brankamp R G, Kropp K E, Blankenship D T, Tsay J T, Smith P L, Wierschke J D, Subramaniam A, Birkenberger L A. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene, 1997, 190: 55-62
253.Stahlberg J, Divne C, Koivula A. Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolaseⅠfrom Trichoderma reesei. Journal of Molecular Biology, 1996, 264: 337-349
254.Steenbakkers P J, Harhangi H R, Bosscher M W, van der Hooft M M, Keltjens J T, van der Drift C, Vogels G D, op den Camp H J. beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase. Biochem J, 2003, 370: 963-970
255.Stemmer W P C. DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci, 1994, 91: 10747-10751
256.Stemmer W P C. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994, 370: 389-391
257.Stoffer B, Frandsen T P, Busk P K, Schneider P, Svendsen I, Svensson B. Production,purification and characterization of the catalytic domain of glucoamylase from Aspergillus niger. Biochem, 1993, 292: 197-202
258.Swift R J, Wiebe M G, Robson G D, Trinci A P. Recombinant glucoamylase production by Aspergillus niger B1 in chemostat and pH auxostat cultures. Fungal Genetics and Biology, 1998, 25:100-109
259.Takahashi T, Tsuchida Y, Irie M. Isolation of Two Inactive Fragments of a Rhizopus sp. Glucoamylase: Relationship among Three Forms of the Enzyme and the Isolated Fragments. Biochem, 1982, 92: 1623-1633
260.Takashi T, Inokushi N, Irie M. Purification and characterization of a glucoamylase from Aspergillus satoi. J Biochem, 1981, 89: 125-130
261.Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T. Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei. J Biochem, 1999, 125: 728-736
262.Tao J H, Xu J H. Biocatalysis in developmene of green pharmaceutical processes. Current Optinion in Chemical Biology, 2009, 13: 43-50
263.Taylor P M, Napier E J, Fleming I D. Some properties of a glucoamylase produced by the thermophilic fungus Humicola lanuginosa. Carbohyd Res, 1978, 16: 301-308
264.Teeri T T. Crystalline cellulose degradation: new insight into the function of cellobio- hydrolases. Trends Biotechnol, 1997, 15: 160-167
265.Thorsen T S, Johnsen A H, Josefsen K, Jensen B. Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus. Biochim Biophys Acta, 2006, 1764: 671-676
266.Tigue N J, Kay J. Active site mutants of human herpesvirus-6 proteinase. FEBS Lett , 1998, 441: 467-469
267.Tolborg J F, Petersen L, Jensen K J, Mayer C, Jakeman D L, Warren R A, Withers S G. Solid-phase oligosaccharide and glycopeptide synthesis using glycosynthases. J Org Chem, 2002, 67: 4143-4149
268.Trincone A, Giordano A, Perugino G, Rossi M, Moracci M. Glycosynthase-catalysed syntheses at pH below neutrality. Bioorg Med Chem Lett, 2003, 13: 4039-4042
269.Trincone A, Perugino G, Rossi M. A novel thermophilie glycosynthase that effects branching glycosylation. Bioorg. Med. Chem. Int. Ed., 2001, 40: 417-420
270.Trincone A, Perugino G, Rossi M, Moracci M. A novel thermophilic glycosynthase that effects branching glycosylation. Bioorg Med Chem Lett, 2000, 10: 365-368
271.Tsukada T, Igarashi K, Yoshida M, Samejima M. Molecular cloning and characterizationof two intracellular beta-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium. Appl Microbiol Biotechnol, 2006, 73: 807-814
272.Van den S P, Rudd P M, Dwek R A, Opdenakker G. Concepts and principles of O-linked glycosylation. Crit Rev Biochem Mol Biol, 1998, 33: 151-208
273.Van Kampen M D, Egmond M R. Directed evolution: From a staphylococcal lipase to phospholipase. Eur J Lipid Sci Technol, 2000, 102: 717-726
274.Van Kampen M D, Dekker N, Egmond M R, Verheij H M. Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as strdied by in vivo chimeragenesis. Biochemistry, 1998, 37: 3459-3466
275.Van Lieshout J, Faijes M, Nieto J, Van der O J, Planas A. Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the thermophile Pyrococcus furiosus. Archaea, 2004, 1: 285-292
276.Varrot A, Hastrup S, Davies G J. Crystal structure of the catalytic core domain of the family 6 cellobiohydrolaseⅡ. Biological Chemistry, 1999, 337: 297-304
277.Vaughan M D, Johnson K, DeFrees S, Tang X, Warren R A, Withers S G. Glycosynthase-mediated synthesis of glycosphingolipids. Am Chem Soc, 2006, 128: 6300- 6301
278.Wada M, Hsu C C, Franke D, Mitchell M, Heine A, Wilson I, Wong C H. Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions. Bioorg. Med. Chem., 2003, 11: 2091-2098
279.Wang Q, Tull D, Meinke A, Gilkes N R, Warren R A, Aebersold R, Withers S G. Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1, 4-glucanase. Journal of Biological Chemistry, 1993, 268: 14096-140102
280.Wang S X, Dunphy W G. Activation of Xenopus Chk1 by mutagenesis of threonine-377. FEBS Lett, 2000, 487: 277-281
281.Wang T, Liu X, Yu Q, Zhang X, Qu Y, Gao P, Wang T. Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei. Biomolecular Engineering, 2005, 22: 89-94
282.Washington S L, Yoon M S, Chagovetz A M, Li S X, Clairmont C A, Preston B D, Eckert K A, Sweasy J B. A genetic system to identify DNA polymerase beta mutator mutants. Proceedings of the National Academy of Sciences of the USA, 1997, 94: 1321-1326
283.Wintrode P L, Miyazaki K, Amold F H. Cold adaptation of a mesophilic subtilisin-likeprotease by laboratory evolution. Journal of Biological Chemistry, 2000, 275: 31635- 31640
284.Withers S G, Warren R A J, Street I P, Rupitz K, Kempton J B, Aebersold R. Unequivocal demonstration of the involvement of a glutamate residue as a nucleophile in the mechanism of a“retaining”glycosidase. J Am Chem Soc, 1990, 112: 5887-5889
285.Wolfgang D E, Wilson D B. Mechanistic studies of active site mutants of Thermomo- nospora fusca endocellulase E2. Biochemistry, 1999, 3: 9746-9751
286.Wood V, Gwilliam R, Rajandream M A. The genome sequence of Schizosaccharomyces pombe. Nature, 2002, 415: 871-880
287.Xu X, Huang J, Fang J, Lin C, Cheng J, Shen Z. Expression of a fungal glucoamylase in transgenic rice seeds. Protein Expr Purif, 2008, 61: 113-116
288.Yamashita I, Itoh T, Fukui S. Cloning and expression of the Saccharomycopsis fibuligera glucoamylase gene in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 1985, 23: 130-133
289.Yan T R, Liau J C. Synthesis of alkylβ-glucosides from cellobiose with Aspergillus nigerβ-glucosidase.ⅡBiotechnologyLetters, 1998, 20: 653-657
290.Yang L Q, Dai X J, Luo Y M, Ma C X, Hou J H, Wu Z Q, Wang C Y, Li M G. Cloning and expression of a new glucoamylase gene. Sheng Wu Gong Cheng Xue Bao, 2007, 23:477-482
291.Yang S H, Jia N B, Li M G, Wang J H. Heterologous expression and efficient ethanol production of a Rhizopus glucoamylase gene in Saccharomyces cerevisiae. Molecular Biology Reports, 2010,DOI 10.1007/s11033-010-0077-3
292.Yano T, Oue S, Kagamiyama H. Directed evolution of an aspartate aminotransferase with new substrate specificities. Proceedings of the National Academy of Sciences of the USA, 1998, 95: 5511-5515
293.Yoon J J, Igarashi K, Kajisa T. Purification, identification and molecular cloning of glycoside hydrolase family 15 glucoamylase from the brown-rot basidiomycete Fomitopsis palustris. FEMS Microbiology Letters, 2006, 259: 288-294
294.Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, Kitaoka M, Katayama T, Kumagai H. Purification, crystallization and preliminary X-ray analysis of beta-glucosidase from Kluyveromyces marxianus NBRC1777. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2009, 65:1190-1192
295.Zanoelo F F, Polizeli Mde L, Terenzi H F, Jorge J A. Beta-glucosidase activity from thethermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett, 2004, 240: 137-143
296.Zhang J H, Dawes G, Stemmer W P C. Directed evolution of a fucosidase from a galacto- sidase by DNA shuffling and screening. Proceedings of the National Academy of Sciences of the USA, 1997, 94: 4504-4509
297.Zhang Y H P, Himmel M E, Mielenz J R. Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, 2006, 24: 452-481
298.Zhao H, Arnold F H. Functional and non-functional mutations distinguished by random recombination of homologous genes. Proc Natl Acad Sci, 1997, 94: 7997-8000
299.Zhao H, Arnold F H. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res, 1997, 25: 1307-1308
300.Zhao H, Giver L, Shao Z, Affholter J A, Arnold F H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol, 1998, 16: 258-261
301.Zheng Y Y, Xue Y F, Zhang Y L, Zhou C, Schwaneberg U, Ma Y H. Cloning, expressionand characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4. Appl Microbiol Biotechnol, 2010, DOI 10.1007/s00253-010-2439-0
302.Zhou H, Wang H W, Zhu K, Sui S F, Xu P, Yang S F, Li N. The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding and beyond. Plant Physiol, 1999, 121: 913-919
303.Zopf D, Roth S. Oligosaccharide anti-infective agents. Lancet, 1996, 347: 1017-1021
2.陈红歌.野油菜黄单胞菌α-淀粉酶性质及其体外定向进化研究.南京农业大学博士论文, 2005
3.陈惠黎,王克夷.糖复合物的结构和功能.上海医科大学出版社, 1997, 629-653
4.陈启和,何国庆.糖化酶及其基因研究进展.微生物学杂志, 2000, 20: 46-50
5.陈卫红,李多川.嗜热真菌热稳定性β-葡萄糖苷酶的克隆表达、纯化及酶学性质细胞生物学杂志. 2009, 31: 535-540
6.陈卫红,李多川.嗜热子囊菌光孢变种β-葡萄糖苷酶的基因克隆、表达及酶学性质分析应用与环境生物学报.2009, 15: 549-553
7.陈向东,藤尾雄策.日本根霉IF05318胞外β-葡萄糖苷酶的纯化及部分特性.微生物学报, 1997, 37: 368-373
8.楚春雪,李多川,郭润芳.一株嗜热毛壳菌β-葡萄糖苷酶的分离纯化及特性.菌物学报, 2004, 23: 397-402
9.方柏山,洪燕,夏启容.酶体外定向进化(Ⅱ)-文库筛选的方法及其应用.华侨大学学报(自然科学版), 2005, 26:113-116
10.方柏山,郑媛媛.酶体外定向进化(Ⅰ)-突变基因文库构建技术及其新进展.华侨大学学报(自然科学版), 2004, 25: 337-342
11.方芳,曹以诚,陈晓曦,曾炳佳. 49P(del)点突变提高中性纤维素内切酶EGV热稳定性的初步研究.现代生物医学进展, 2009, 9: 2634-2636
12.高伦江,董全,唐春红.纤维素酶的研究进展及前景展望.江苏食品与发酵, 2007, : 14-17
13.顾卫民.β-葡萄糖苷酶的特性及其在食品工业中的应用.江苏食品与发酵, 2003, 1: 5-7
14.关汉成,严自成,张树政.黑曲霉突变株葡萄糖淀粉酶的底物特异性.微生物学报, 1994, 31: 20-18
15.关铜,张世华.纤维素酶在饲料工业中的研究与应用.贵州畜牧兽医, 2003, 27: 7-9
16.韩如旸,陈美慈,赵宇华,闵航,马晓航.一种嗜热厌氧纤维素降解细菌的分离纯化方法.微生物学通报, 2000, 27: 363-366
17.韩笑,陈介南,王义强,何钢,刘海波,谭力.β-葡萄糖苷酶基因的克隆与表达研究进展.生物技术通报, 2008, 3: 8-12
18.何冰芳,欧阳平凯.极端微生物与工业生物催化剂开发.化工进展, 2006, 25: 1124-1127
19.侯温甫,杨文鸽.糖链及其蛋白质糖基化.生物技术通报, 2005, 3: 14-17
20.黄培堂,王嘉玺,朱厚础等译.分子克隆实验指南.北京:科学出版社, 2003. 1080-1087
21.黄瑛,蔡勇,杨江科,闫云君.基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化.生物工程学报, 2008, 3: 445-451
22.姜涌明,戴祝英,陈俊刚,胡健.分子酶学导论,中国农业大学出版社,中国,北京, 2000, 145-158
23.李洪钊,李亮助,孙强明,冮宏映.巴斯德毕赤酵母表达系统优化策略.微生物学报, 2003, 43: 288-292
24.刘玉芳,陈玉梅,张博润.糖化酶基因的克隆和在酿酒酵母中的表达.微生物学报, 1996, 36: 423-427
25.刘增然,何秀萍,龙章富,张博润,刘世贵.淀粉酶基因的克隆及其在工业啤酒酵母中的表达.食品科学, 2004, 25: 78-82
26.卢丽丽,肖敏,赵晗,王鹏,钱新民. 1-氟代糖在化学-酶法合成糖类中的应用.有机化学, 2006, 26:1631-1639
27.卢丽丽,肖敏,赵晗,王鹏,钱新民.糖苷合成酶-一类新型的寡糖高效合成工具.生物化学与生物物理学进展, 2006, 33: 310-320
28.马丽娜,陈喜文,甘睿,陈洁,陈德富.葡萄糖淀粉酶的结构和功能研究进展.生物技术通讯, 2005, 16: 677-680
29.马丽娜,陈喜文,陈德富,王绘砖,宋峥.曲霉属糖化酶基因的克隆及其在毕赤酵母中的表达.南开大学学报, 2007, 40: 85-90
30.马向东,马立新,薛征峰.一种鉴定淀粉酶活性及其产生菌的新方法.华中农业大学学报, 2000, 19: 95-99
31.马原松.寡糖及其开发利用.安微农业科学, 2006, 34: 3886-3888
32.孟雷,陈冠军,王怡,高培基.纤维素酶的多型性.纤维素科学与技术, 2002, 10: 47-55
33.邵金辉,赵云,毛爱军,朱雅新,董志扬.扣囊复膜孢酵母β-葡萄糖苷酶基因在毕赤酵母中的克隆与表达.微生物学报, 2005, 45: 792-794
34.宋志刚.低聚糖的开发利用.饲料博览, 2001, 1: 40-41
35.孙志浩,柳志强.酶的定向进化及其应用.生物加工过程, 2005, 3: 7-13
36.滕芳超,李多川,李亚玲,李浙江.嗜热毛壳菌一种β-葡萄糖苷酶的分离纯化及特性.菌物学报, 2006, 25: 481-487
37.王华夫,游小青.茶叶中β-葡萄糖苷酶活性的测定.中国茶叶, 1996, 3: 16-17
38.王元.现代酶工程技术的新进展.中国食品, 2000, 21: 22-23
39.文立民,于松涛,李浩,赵大健.糖化酵母糖化酶基因在酿酒醉母中的克隆和表达.食品与发醉工业, 1994, 118: 20-25
40.吴东儒.糖类的生物化学.北京:高等教育出版社, 1987
41.吴梧桐.蛋白质工程技术与新型生物催化剂设计.药物生物术, 2004, 11: 1-6
42.夏黎明,萧庆,余世袁.碳源对固定里氏木霉合成纤维素酶的影响.纤维素科学与技术, 1994, 2: 72-77
43.徐卉芳,张先恩,张治平,张用梅.大肠杆菌碱性磷酸酶的体外定向进化研究.生物化学与生物物理进展, 2003, 30: 89-94
44.徐威,朱春宝,朱宝泉.酶定向进化的研究策略.中国医药工业杂志, 2004, 35: 436-441
45.许钦坤.蛋白质定向进化的研究进展及其应用前景.生物技术通讯. 2005, 16: 191-193
46.阎伯旭,高培基.纤维素酶分子结构与功能研究进展.生命科学, 1995, 5: 22-25
47.阎伯旭,齐飞,张颖舒,高培基.纤维素酶分子结构和功能研究进展.生物化学与生物物理进展, 1999, 26: 233-237
48.杨清香,曹军卫,黄国锦.糖基转移酶和去糖基化酶.氨基酸和生物资源, 1997, 19: 38-43
49.杨雪鹏,杨寿钧,韩北忠,金城.非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究.生物工程学报, 2005, 21: 84-91
50.杨依军,李多川,沈崇尧.葡萄糖淀粉酶研究进展.微生物学通报, 1996, 23: 312-315
51.张浩,毛秉智.定点突变技术的研究进展.免疫学杂志, 2000, 16: 108-110
52.张今.进化生物技术-酶定向进化.北京:科学出版社, 2004
53.张龙翔,张庭芳,李令媛.生化实验方法和技术.高等教育出版社, 2005
54.张晓勇,陈秀霞,高向阳.纤维素酶的蛋白质工程.纤维素科学与技术, 2006,14 :55-58
55.张秀艳.β-葡聚糖酶的定向进化及热稳定性研究.浙江大学博士论文, 2006
56.赵永芳主编.生物化学技术原理及应用.科学出版社, 2002, 1-502
57.朱国萍,滕脉坤,伍传金,王玉珍. G138P定点突变对葡萄糖异构酶热稳定性的改善.生物化学与生物物理学报, 1998, 30: 607-610
58. Aharoni A, Griffiths A D, Tawfik D S. High-throughput screens and selections of enzyme encoding genes. CurrOp in Chem Biol, 2005, 9: 210-216
59. Aikawa J, Prak Y N. Replacements of amino acid properties of Rhizomu corpusillus pepsin, an aspartic proteinase from Rhizomu corpusillus. J Bio Chem(Tokyo), 2001, 129: 791
60. Ali S, Hossain Z, Mahmood S, Alam R. Purification of glucoamylase from Aspergillus terrus. World J Microbiol Biotechol, 1990, 6: 19-22
61. Alison M D, Beatrice A M, Sabine L F. Synthesis and modifications of carbohydrates, using biotransformations. Curr Opin In Chem Biol, 2004, 8:106-113
62. Allen S J, Holbrook J J. Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution. Protein engineering, 2000, 13: 5-7
63. Allison D S, Rey M W, Berka R M, Armstrong G, Dunn-Coleman N S. Transformation of the thermophilic fungus Humicola grisea var. thermoidea and overproduction of Humicola glucoamylase. Current Genetics, 1992, 21: 225-229
64. Alzari P M, Souchon H, Dominguez R. The crystal structure of endoglucansase celA, a family 8 glucosylhydroase from Clostridium thermocellum. Structure, 1996, 4: 265-275
65. Amidon W J, Pfeil J E, Gal S. Modification of luciferase to be a substrate for plant aspartic proteinase. Biochem J, 1999, 343: 425-433
66. Amirul A A, Khoo S L, Nazalan M N, Razip M S, Azizan M N. Purification and properties of two forms of glucoamylase from Aspergillus niger. Folia Microbiol, 1996, 41: 165-174
67. Amold F H. In IBC Directed Enzyme Evolution. San Diego, 1998
68. Aquino A C, Jorge J A, Terenzi H F, Polizeli M L. Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum. Folia Microbiol (Praha), 2001, 46: 11-16
69. Aro N, Ilmén M, Saloheimo A, Penttil? M. ACEI of Trichoderma reesei is a repressor of cellulose and xylanase expression. Applied and Environmental Microbiology, 2003, 69: 56-65
70. Arrizubieta M J, Polaina J. Increased thermal resistance and modification of the catalytic properties of a beta-glucosidase by random mutagenesis and in vitro recombination. Biological Chemistry, 2000, 275: 28843-28848
71. Bartoszewicz K. Glucoamylase of Aspergillus niger. Acta Biochim pol, 1986, 33: 17-29
72. Bauer M W, Kelly R M. The family 1β-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism. Biochemistry, 1998, 37: 17170-17178
73. Bauer S, Vasu P, Persson S, Mort A J, Somerville C R. Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc Natl Acad Sci U S A, 2006, 103:11417-11422
74. Bause E, Legler G. Isolation and amino acid sequence of a hexadecapeptide from the active site of beta-glucosidase A3 from Aspergillus wentii. Hoppe Seylers Z Physiol Chem, 1974 , 355: 438-442
75. Beguin P, Aubert J P. The biological degradation of cellulose. FEMS Microbiology Review, 1994, 13: 25-58
76. Benoliel B, Pocas F M J, Torres F A, Moraes L M. Expression of a Glucose-tolerant beta-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae. Appl Biochem Biotechnol, 2010, 160: 2036-2044
77. Berger R G. Application of genetic methods to the generation of volatile flavours. In: Hui H Y: Katchatourians G. (eds.): Food Biotechnology. VCH Weinheim. 1995, 291-295
78. Bhat M K, Bhat S. Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances, 1997, 15: 583-620
79. Bhatia Y, Mishra S, Bisaria V S. Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsiiβ-glucosidaseⅡwith high hydrolytic activity on sophorose. Appl Microbiol Biotechnol, 2005, 66: 527-535
80. Bhatti H N, Rashid M H , Asgher R N M, Perveen R, Jabbar A. Purification and characterization of a novel glucoamylase from Fusarium solani. Food Chemistry, 2007, 103: 338-343
81. Boel E, Hansen M T, Hjort I, Hoegh I, Fiil, N P. Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger. EMBO Journal, 1984, 3: 1581-1585
82. Boel E, Hjort I, Svensson B, Norris F, Norris K E, Fiil NP. Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs. EMBO Journal, 1984, 3: 1097-1102
83. Bornscheuer U T, Altenbuchner J, Meyer H H. Directed evolution of an esterase: Screening of enzyme libraries based on pH-indicators and a growth assay. Bioorganic and Medicinal Chemistry, 1999, 7: 2169-2173
84. Brunner F, Wirtz W, Rose J K, Jocelyn K. C. Roseb, 1, Alan G. Darvillb, Francine Goversc, Scheela D and Nürnberger T. A beta-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans. Phytochemistry, 2002, 59: 689-696
85. Brzobohaty B, Moore I, Kristoffersen P, Bako L, Campos N, Schell J, Palme K. Release of active cytokinin by aβ-glucosidase localized to the maize root meristem. Science, 1993, 262: 1051-1054
86. Buchholz F, Angrand P O, Stewart A F. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nature Biotechnol, 1998, 16: 657-662
87. Butler G, Rasmussen MD, Lin MF, Santos MA, Sakthikumar S, Munro CA, Rheinbay E, Grabherr M, Forche A, Reedy JL, Agrafioti I, Arnaud MB, Bates S, Brown AJ, Brunke S, Costanzo MC, Fitzpatrick DA, de Groot PW, Harris D, Hoyer LL, Hube B, Klis FM, Kodira C, Lennard N, Logue ME, Martin R, Neiman AM, Nikolaou E, Quail MA, Quinn J, Santos MC, Schmitzberger FF, Sherlock G, Shah P, Silverstein KA, Skrzypek MS, Soll D, Staggs R, Stansfield I, Stumpf MP, Sudbery PE, Srikantha T, Zeng Q, Berman J, Berriman M, Heitman J, Gow NA, Lorenz MC, Birren BW, Kellis M, Cuomo CA. Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature, 2009, 459:657-662
88. Cadwell R C, Joyce G F. Mutagenic PCR. PCR Methods Application, 1994, 6:136-140
89. Caldwell R C, Joyee G F. Randomization of genes by PCR mutagenesis. In PCR Methods Applic, 1992, 2: 28-33
90. Catcheside D E, Rasmussen J P, Yeadon P J. Diversification of exogenous genes in vivo in Neurospora. Appl Microbiol Biotechnol, 2003, 62: 544-549
91. Catherine M C, Patrick G M, Stuart D, John P M, Lucy B, Tuula T T, Maria G T. Molecular cloning and expression analysis of two distinctβ-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii. Mycological research, 2007, 111: 840-849
92. Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS micrrobiol, Rev., 2001, 24: 45-66
93. Chen J, Zhang Y Q, Zhao C Q, Li A N,.Zhou Q X, Li D C. Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris. Journal of Applied Microbiology, 2007, 103: 2277-2284
94. Chen K, Arnold F H. Enzyme engineering for nonaqueous solvents: Random mutagenesis to enhance activity of subtilisin E inpolar organic media. Bio/Technology, 1991, 9: 1073-1077
95. Chen K, Arnold F H. Tuning the activity of an enzyme for unusual environments: Sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc. Natl. Acad. Sci. USA, 1993, 90: 5618-5622
96. Cheong T K, Oriel P J. Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherrichia coli and marked enhancement of amidase expression using directed evolution. Enzyme and Microbial Technology, 2000, 26:152-158
97. Cherry J R, Lamsa M H, Schneider P, Vind J, Svendsen A, Jones A, Pedersen A H. Directed evolution of a fungal peroxidase. Nature Biotechnol, 1999, 17: 379-384
98. Clarke A J, Svensson B. Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from Aspergillus niger. Carlsberg Res Commun, 1984, 49: 559-566
99. Cole G E, Mccabe P L, Inlow D. Expression of Aspergillus anmanari glucoamylase in distiller's yeast. Bio/Technology, 1998, 6:417
100.Cortes M L, Felipe P, Martin V, Hughes MA, Izquierdo M. Successful use of a plant gene in the treatment of cancer in vivo. Gene Ther, 1998, 5: 1499-1507
101.Crameri A, Dawes G, Rodriguez E, Silver S, Stemmer W P C. Molecular evolution of an arsenate detoxify-catio pathway by DNA shuffling. Nature Biotechnol, 1997, 15: 436-438
102.Crameri A, Raillard S A, Bermudez E, Stemmer W P C. DNA Shuffling of a family of genes from diverse species accelerates evolution. Nature, 1998, 391: 288-291
103.Cregg J M, Cereghino J L, Shi J Y. Recombiant Protein Expression in Pichia pastoris. Molecular Biotechnology, 2000, 16: 23-52
104.Creighton T E, Proteins: Structures and Molecular Properties, second ed., Freeman&Company, New York, 1997
105.Damude H G, Withers S G, Kilburn D G, Miller R C, Warren R A. Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi. Biochemistry, 1995, 34: 2220-2224
106.Dan S, Marton I, Dekel M, Bravdo B A, He S, Withers S G, Shoseyov O. Cloning, Expression, Characterization, and Nucleophile Identification of Family 3, Aspergillus nigerβ-Glucosidase. Biological Chemistry, 2000, 275: 4973-4980
107.Davies G J, Ducros V, Lewis R J. Oligosaccharide specifity of a family 7 endoglucanase insertion of potential sugar-binding subsites. Biotechnology, 1997, 57: 91-100
108.Davies G J, Dauter M, Brzozowski A M, Bj?rnvad M E, Andersen K V, Schülein M. Structure of the Bacillus agaradherans family 5 endoglucanase at 1.6 ? and its cellobiose complex at 2.0 ? resolution. Biochemistry, 1998, 37: 1926-1932
109.Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure, 1995, 3: 853-859
110.De Groeve R M, De Baere M, Hoflack L, Lieve Hoflack, Tom Desmet, Erick J V and Soetaert W. Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase. Protein Engineering, Design & Selection, 2009, 22: 393-399
111.Denisov V P. Orientational disorder and entropy of water in protein cavities. J. Phy. Chem, 1997, 101: 380-389
112.Dharmawardhana D P, Ellis B E, Carlson J E. Aβ-glucosidase from lodgpole pine xylem specific for the lignin precursor coniferin. Plant Physiol., 1995, 107: 331-339
113.Dill K A. Dominant forces in protein folding. Biochemistry, 1990, 29: 7133-7155
114.Ding S, Ge W, Buswell J A. Molecular cloning and transcriptional expression analysis of an intracellular beta-glucosidase, a family 3 glycosyl hydrolase, from the edible straw mushroom, Volvariella volvacea. FEMS Microbiol Lett, 2007, 267: 221-229
115.Doi R H, Park J S, Liu C C, Malburg L M, Tamaru Y, Ichiishi A, Ibrahim A. Cellulosome and noncellulosomal cellulase of Clostridium cellulovorans. Extremophiles, 1998, 2: 53-60
116.Dominguez R, Souchon H, Lascombe M B. A common protein fold and similar active in two distinct families of beta-glycanases. Nature Structural & Molecular Biology, 1995, 2: 569-576
117.Driskill L E, Bauer M W, Kelly R M. Synergistic interactions amongβ-laminarinase,β-1,4-glucanase, andβ-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus during hydrolysis ofβ-1,4-,β-1,3-, and mixed-linked polysaccharides. Biotechnology and Bioengineering, 1999, 66: 51-60
118.Dujon B, Sherman D, Fischer G, Gilles Fischer1, Pascal Durrens6,7, Serge Casaregola8, Ingrid Lafontaine1, Genome evolution in yeasts. Nature, 2004, 430: 35-44
119.Edward J, Bylina E J, Coleman W J, Dilworth M R, Robles S J, Tanner M A, Yang M M, Youvan D C. Solid-phase enzyme screening. ASM News, 2000, 66: 211-217
120.Fairweather J K, Faijes M, Driguez H, Planas A. Specificity studies of Bacillus 1,3-1,4-β-glucanases and application to glycosynthase-catalyzed transglycosylation.Chem Biochem, 2002, 3: 866-873
121.Fairweather J K, Hrmova M, Rutten S J, Fincher G B, Driguez H. Synthesis of complex oligosaccharides by using a mutated (1, 3)-β-D-glucan endohydrolase from barley. Chemistry, 2003, 9: 2603-2610
122.Faure D, Henrissat B, Ptacek D, Bekri M A, Vanderleyden J. The celA gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in Azospirillum irakense, is required for optimal growth on cellobiosides. Appl Environ Microbiol, 2001, 67: 2380-2383
123.Fort S, Boyer V, Greffe L, Gideon J. Davies, Moroz O, Christiansen L, Schülein M, Cottaz S, Driguez H. Highly efficient synthesis ofβ-(1→4)-oligo-and-polysaccharides using a mutant cellulase. J Am Chem Soc, 2000, 122: 5429-5437
124.Freeman A, Cohen-Hadar N, Abramov S, Modai-Hod R, Dror Y, Georgiou G. Screening of large protein libraries by the‘cell immobilized on adsorbed bead’approach. Biotechnology and Bioengineering, 2004, 86: 196-200
125.Fromant M, Blanquet S, Plateau P. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction . Anal Biochem, 1995, 224: 347-353
126.Gibbons I. Microfluidic assays for high-throughput submicroliter assays using capillary electrophoresis. Drug Discov. Today, 2000, 5: 33-36
127.Gomes I, Gomes J, Gomes D J, Steiner W. Simultaneous production of high activities of thermostable endoglucanase andβ-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Appl Microbiol Biotechnol, 2000, 53: 461-468
128.Gonzalez B G,Sanz A J,Gonzalez B, Hermoso J A, Polaina J. Directed evolution of beta-glucosidase A from Paenibacillus polymyxa to thermal resistance. J Biol Chem,2000,275: 13708-13712
129.Goto M, Semimaru T, Furukawa K and Hayashida S. Analysis of the raw starch-binding domain by mutation of a glucoamylase from Aspergillus awamori var. kawachi expressed in Saccharomyces cerevisiae. Applied and Environment Microbiology, 1994, 60: 3926-3930
130.Graham L D, Haggett K D, Jennings P A, Le Brocque D S, Whittaker R G, Schober P A. Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes weith increased activities and altered primary specificities. Biochemistry, 1993, 32: 6250-6258
131.Hagglund P, Bunkenborg J, Elortza F, Jensen O N, Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J Proteome Res,2004, 3: 556-566
132.Hansson L O, Widersten M, Mannervik B. An approach to optimizing the active site in a glutathione transferase by evolution in Vitro. Biochem, 1999, 344: 93-100
133.Harhangi H R, Steenbakkers P J, Akhmanova A, Jetten M S, van der Drift C, Opden Camp H J. A highly expressed family 1 beta-glucosidase with transglycosylation capacity from the anaerobic fungus Piromyces sp. E2. Biochim Biophys Acta, 2002, 12: 293-303
134.Harnpicharnchai P, Champreda V, Sornlake W, Eurwilaichitr L. (2009) A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars. Protein Expr Purif, 2009, 67: 61-69
135.Hayashi S, Sako S, Yokoi H, Takasaki Y, Imada K. Purification and characterization of the intracellularβ-glucosidase from Aureobasidium sp ATCC 20524. Journal of Industrial Microbiology & Biotechnology,1999, 22: 160-163
136.Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science, 2001, 291: 2364-2369
137.Henrissat B. A classification of glycosyl-hydrolases based on aminoacid sequence similarities. Biochem. J, 1991, 293: 781-788
138.Hilge M, Gloor S M, Rypniewski W, Sauer O, Heightman TD, Zimmermann W, Winterhalter K, Piontek K. High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca substrate specificity in glycosylhydrolase family 5. Structure, 1998, 6: 1433-1444
139.Hong J, Tamaki H, Kumagai H. Unusual hydrophobic linker region of beta-glucosidase (BGLII) from Thermoascus aurantiacus is required for hyper-activation by organic solvents. Appl Microbiol Biotechnol, 2006, 73: 80-88
140.Hong J, Tamaki H, Yamamoto K,Kumagai H. Cloning and functional expression of thermostableβ-glucosidase gene from Thermoascus aurantiacus. Appl Microbiol Biotechnol, 2007, 73: 1331-1339
141.Howard S,Withers S G. Bromoketone C-glycosides, a new class of beta-glucanase inactivators. J Am Chem Soc, 1998, 120: 10326-10331
142.Hrmova M, Imai T, Rutten S J, Fairweather J K, Pelosi L, Bulone V, Driguez H, Fincher G B. Mutated barley (1,3)-β-D-glucan endohydrolases synthesize crystalline (1,3)-β-D-glucans. J Biol Chem, 2002, 277: 30102-30111
143.Huang C Y, Chang A K. Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobiles pyruvate decarboxylase: effect on activity and pH dependence. Eur J Biochem, 2001, 268: 3558-3565
144.Iffland A, Tafelmeyer P, Saudan C, Johnsson K. Directed molecular evolution of cytochrome c peroxidase. Biochemistry, 2000, 39: 10790-10798
145.Jahn M, Chen H, Mullegger J, Warren R A, Withers S G. Thioglycosynthases: double mutant glycosidases that serve as scaffolds for thioglycoside synthesis. Chem Commun, 2004, 7: 274-275
146.Jahn M, Marles J, Warren R A J, Withers S G. Thioglycoligases: mutant glycosidases for thioglycoside synthesis. Angew Chem Int Ed, 2003, 42: 352-354
147.Jahn M, Stoll D, Warren R A, SzabóL, Singh P, Gilbert H J, Ducros V M, Davies G J, Withers S G. Expansion of the glycosynthase repertoire to produce defined manno-oligosaccharides. Chem Commun, 2003, 12: 1327-1329
148.Jakeman D L, Withers S G. On expanding the repertoire of glycosynthases: mutantβ-galactosidases formingβ-(1,6)-linkages. Can J Chem, 2002, 80: 866-870
149.James J A, Lee B H. Glucoamylases: microbial sources, industrial applications and molecular biology– a review. Journal of Food Biochemistry, 1997, 21: 1-52
150.Jeffries T W, Grigoriev I V, Grimwood J, Laplaza J M, Aerts A, Salamov A, Schmutz J, Lindquist E, Dehal P, Shapiro H, Jin Y S, Passoth V, Richardson P M. Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis. Nat Biotechnol, 2007, 25: 319-326
151.Jeya M, Joo A R, Lee K M, Tiwari M K, Lee K M, Kim S H, Lee J K. Characterization of beta-glucosidase from a strain of Penicillium purpurogenum KJS506. Appl Microbiol Biotechnol, 2010, 86:1473-1484
152.Johnson P E, Brun E, MacKenzie L F, Withers S G, McIntosh L P. The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations. Journal of molecular biology, 1999, 287: 609-625
153.Joo H, Lin Z, Arnold F H. Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature, 1999, 399: 670-673
154.Kadam K L, Wollweber K L, Grosch J C. Investigations on plasmid instability in amylase producing Bacillus subtilis using continuous culture. Biotechnol Bioeng, 1987, 29: 859-872
155.Kaper T, Brouns S J, Geerling A C, De Vos WM, Van der Oost J. DNA Family shuffling of hyperthermostable beta-glycosidases. Biochem. J., 2002, 368: 461-470
156.Kawai R, Yoshida M, Tani T, Igarashi K, Ohira T, Nagasawa H, Samejima M. Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris. Biosci Biotechnol Biochem, 2003, 67: 1-7
157.Kempton J B. Withers S G. Mechanism of Agrobacteriumβ-glucosidase: Kinetic studies. Biochemistry, 1992, 31: 9961-9969
158.Kikuchi M, Ohnishi K, Harayama S. An effective family shuffling method using single-stranded DNA. Gene, 2000, 243: 133-137
159.Kim B, Hathaway T R, Loeb L A. Human immunodeficiency virus revers traverse transcriptase Functional mutants obtained by random mutagenesis coupled with gentic selection in Escherichia coli. J Biol. Chem., 1996, 271: 4872-4878
160.Kim Y S, Jung H C, Pan J G. Bacterial cell surface display of an enzyme library for selective screening of improved cellulose variants. Applied Environmental Microbiology, 2000, 66: 788-793
161.Kim Y W, Lee S S, Warren R A, Withers S G. Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J. Bio. Chem, 2004, 279: 42787-42793
162.Kirschner A, Bornscheuer U T. Directed evolution of a Baeyer-Villiger monooxygenase to enhance enantioselectivity. Appl. Microbiol. Biotechnol., 2008, 81: 465-472
163.Kleyweg G J, Zou J Y, Divne C. The crystal structure of the catlytic domian of endoglucanaseⅠfrom Trichoderma reesei. Molecular Biology, 1997, 272: 383-397
164.Knaust R K C, Nordlund P. Screening for soluble expression of recombinant proteins in a 96-well format. Analytical Biochemistry, 2001, 297: 79-85
165.Knecht W, Munch-Petersen B, Piskur J. Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster. Molecular Biology, 2000, 30: 827-837
166.Kobata A. Structures and function of sugar chains of glycoproteins. Eur J Biochem, 1992, 209: 483-501
167.Kolkman J A, Stemmer W P C. Directed evolution of proteins byexon shuffling. Nature Biotechnology, 2001, 19: 423-428
168.Komeda H, Ishikawa N, Asano Y. Enhancement of the thermostability and catalytic activity of D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3 by directed evolution. J . Mol. Catal. B : Enz. , 2003 , 21: 283-290
169.Kong X, Liu Y, Gou X, Zhu S, Zhang H, Wang X, Zhang J. Directed evolution ofαaspartyl dipeptidase from Salmonella typhimurium. Biochem Biophys Res Commun, 2001, 289: 137-142
170.Koshland D E J. Steriochemistry and the mechanism of enzymatic reactions. Biol Rev,1953, 28: 416-436
171.Krogh K B, Harris P V, Olsen C L, Johansen K S, Hojer-Pedersen J, Borjesson J, Olsson L. Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum. Appl Microbiol Biotechnol, 2010, 86:143-154
172.Kumar S, Satyanarayana T. Purification and kinetics of a raw starch-hydrolyzing, thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae. Biotechnol Prog, 2003, 19: 936-944
173.Ladbury J E, Wynn R, Thomson J A, Sturtevant J M. Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin result in a change in heat capacity of the native protein. Biochemistry, 1995, 34: 2148-2152
174.Lanio T, Jeltsch A, Pingoud A. Towards the design of rare cutting restriction endonucleases: using directed wvolution to generate variants of EcoRⅤdiffering in their substrate specificity by two orders of magnitude. Mol Biol, 1998, 283: 59-69
175.Leah R, Kigel J, Svendsen I, Mundy J. Biochemical and molecular characterization of barley seedβ-glucosidase. J Biol Chem., 1995, 270: 15789-15797
176.Lebbink J H, Kaper T, Bron P, van der Oost J, de Vos W M. Improving lowtemperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution. Biochemistry, 2000, 39: 3656-3665
177.Leung D W, Chen E, Goeddel D V. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1989, 1: 11-15
178.Li D C, Yang Y J, Peng Y L, Shen C Y. Purification and characterization of extracellular glucoamylase from the thermophilic Thermomyces lanuginosus. Mycol Res. 1998, 102: 568-572
179.Li X L, Ljungdahl L G, Ximenes E A, Chen H, Felix C R, Cotta M A, Dien B S. Properties of a recombinant beta-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis. Appl Biochem Biotechnol, 2004, 113-116:233-50
180.Li Y K, Chir J, Chen F Y. Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247. Biochem J. 2001, 355: 835-840
181.Lima A O, Davis D F, Swiatek G, McCarthy J K, Yernool D, Pizzirani-Kleiner A A, Eveleigh D E. Evaluation of GFP tag as a screening reporter in directed evolution of a hyperthermophilicβ-Glucosidase. Mol Biotechnol, 2009, 42: 205-215
182.Lin H, Tao H, Cornish V W. Directed evolution of a glycosynthase via chemicalcomplementation. Am. Chem. Soc., 2004, 126: 15051-15059
183.Lin L L, Ma Y J, Chien H R, Hsu W H. Construction of an amylolytic yeast by multiple integrate of the A .awamori GA gene into a S. cerevisiae chromosome. Enzyme and Microbiol Tech, 1998, 23: 360-366
184.Lin L L, Rumbak E, Zappe H, Thomson J A, Woods D R. Cloning, sequencing and analysis of expression of a Butyrivibrio jibrisofvens gene encoding aβ-glucosidase. Journal of General Microbiology,1990, 136:1567-1576
185.Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, Li C, Dong J, Xiao L, Liu Z. Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution. Appl Microbiol Biotechnol, 2009, 82: 671-679
186.Liu D R, Schultz P G. Progress toward the evolution of an organism with an expanded genetic code. Proc Natl Acad Sci USA,1999,96: 780-4785
187.Lorimer I A L, Pastan I. Random recombination of antibody single chain Fv sequence after fragmentation with DNase I in the presence of Mn2+. Nucleic Acids Res, 1995, 23: 3067-3068
188.Ma Y F, Evans D E. Mutations of barley beta-amylase that improve substrate-binding affinity and thermo stability. Mol Genet Genomics, 2001, 266: 345-352
189.Mackenzie L F, Wang Q, Warren R A J, Withers S G. Glycosynthases: mutant glycosidases for oligosaccharide synthesis. Am Chem Soc, 1998, 120: 5583-5584
190.Maheshwari R, Bharadwaj G, Bhat M K. Thermophilic fungi: Their physiology and enzymes. Microbiology and Molecular Biology Reviews, 2000, 64: 461-488
191.Malet C, Planas A. Fromβ-glucanase toβ-glucansynthase glycosyltransfer toα-glycosylfluorides catalyzed by a mutant endoglucanase lacking its catalytic nueleophile. FEBS. Lett., 1998, 440: 208-212
192.Manger J F, Muller T, Janssen M, Hofer M and Holker U. Isolation and sequencing of a new glucoamylase gene from an Aspergillus niger aggregate atrain (DSM 823) molecularly classified as Aspergillus tubingensis. Antonie Van Leeuwenhoek, 2005, 88: 267-275
193.Manjula Pandey, Saroj Mishra. Expression and characterization of Pichia etchellsiiβ-glucosidase Escherichia coli. Gene, 1997, 190: 45-51
194.Martel M B, Herve P C, Letoublon R, Fevre M. Purification and characterization of a glucoamylase secreted by the plant pathogen Scierotinia sclerotiorum. Can J Microbiol, 2002, 48: 212-218
195.Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M, Kubicek CP, Ferreira P, Ruiz-Duenas FJ, Martinez AT, Kersten P, Hammel KE, Vanden Wymelenberg A, Gaskell J, Lindquist E, Sabat G, Bondurant SS, Larrondo LF, Canessa P, Vicuna R, Yadav J, Doddapaneni H, Subramanian V, Pisabarro AG, Lavín JL, Oguiza JA, Master E, Henrissat B, Coutinho PM, Harris P, Magnuson JK, Baker SE, Bruno K, Kenealy W, Hoegger PJ, Kües U, Ramaiya P, Lucas S, Salamov A, Shapiro H, Tu H, Chee CL, Misra M, Xie G, Teter S, Yaver D, James T, Mokrejs M, Pospisek M, Grigoriev IV, Brettin T, Rokhsar D, Berka R, Cullen D. Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc Natl Acad Sci U S A. 2009, 106: 1954-1959
196.Matsuura T, Yomo T, Trakulnaleamsai S, Ohashi Y, Yamamoto K, Urabe I. Nonadditivity of mutational effects on the properties of catalase and its application to efficient directed evolution. Protein Engineering, 1998, 11: 789-795
197.May O, Nguyen P T, Arnold F H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nature Biotechnology, 2000, 18: 317-320
198.Mayer C, Zechel D L, Reid S P, Warren R A, Withers S G. The E358S mutant of Agrobacterium sp.β-glucosidase is a greatly improved glycosynthase. FEBS Lett, 2000, 466: 40-44
199.McBeath G, Kast P, Hilvert D. Redesigning enzyme topology by directed evolution. Science, 1998, 279: 958-961
200.McCarter J D, Withers S G. Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol, 1994, 4: 885-892
201.McCarthy J K, Uzelac A, Davis D F, Eveleigh D E. Improved Catalytic Efficiency and Active Site Modification of 1,4-β-D-Glucan Glucohydrolase A from Thermotoga neapolitana by Directed Evolution. Biological Chemistry, 2004, 279: 11495-11502
202.Michael W B, Robert M k, Biochemistry, 1992, 31: 9961-9969
203.Miller G L. Use of dinitrosalicyclic acid regent for determination of reducing sugar. Anal. Chem, 1951, 31: 426-428
204.Miyazaki K, Takenouchi M, Kondo H, Noro N, Suzuki M, Tsuda S. Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution. J Biol Chem, 2006, 281: 10236-10242
205.Moore J C, Jin H M, Kuchner O, Arnold F H. Strategies for the in vitro evolution ofprotein function: enzyme evolution by random recombination of improved sequences. Mol Biol, 1997, 272: 336-347
206.Moracci M, Trincone A, Perugino G, Ciaramella M, Rossi M. Restoration of the activity of active-site mutants of the hyperthermophilicβ-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles. Biochemistry, 1998, 37: 17262-17270
207.Moracci M, Trincone A. Mose rossi glycosynthases: new enzymes for oligosaccharide synthesis. Journal of Molecular Catalysis B: Enzymatic, 2001, 11:155-163
208.Morawski B, Lin Z, Cirino P, Joo H, Bandara G, Arnold F H. Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Picha pastoris. Protein Engineering, 2000, 13: 377-384
209.Mullegger J, Chen H, Warren R A J, Withers S.G. Glycosylation of a neoglycoprotein by using glycosynthase and thioglycoligase approaches: the generation of a thioglycoprotein. Angew Chem Int Ed Engl, 2006, 45: 2585- 2588
210.Murashima K, Kosugi A, Doi R H. Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with noncelluloso-mal endoglucanase EngD. Mol. Microbiol, 2002, 45: 617-626
211.Nagendra H G, Sukumar N, Vijayan M. Role of water in plasticity, stability, and action of proteins: the crystal structures of lysozyme at very low levels of hydration. Proteins, 1998, 32: 229-240
212.Nakamura A, Fukunmori F, Horinouchi. Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase. Biol. Chem, 1991, 266: 1579-1583
213.Nakazawa H, Okada K, Onodera T, Ogasawara W, Okada H, Morikawa Y. Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei. Appl Microbiol Biotechnol, 2009, 83: 649-657
214.Nakkharat P, Haltrich D. Purification and characterisation of an intracellular enzymeβ-glucosidase andβ-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58. Journal of Biotechnology, 2006, 123: 304-313
215.Nashiru O, Zechel D L, Stoll D, Mohammadzadeh T, Warren R A, Withers S G.β-Mannosynthase:synthesis ofβ-mannosides with a mutantβ-mannosidase. Angew Chem Int Ed, 2001, 40: 417-420
216.Ness J E, Welch M, Giver L, Bueno M, Cherry J R, Borchert T V, Stemmer W P, Minshull J. DNA shuffing of subgenomic sequences of subtilisin. Nature Biotechnol,1999, 17: 893-896
217.Nguyen Q D, Rezessy-SzabóJ M, Claeyssens M, Stals I, Hoschkeá. Purification and characterisation of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626. Enzyme and Microbial Technology, 2002, 31: 345-352
218.Nielsen B R, Lehmbeck J, Frandsen T P. Cloning, heterologous expression, and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii. Protein expression and purification, 2002, 26: 1-8
219.Niemeyer H M. Hydroxamic acid (4-hydroxy-1,4-benzoxazin-3-ones), defence chemicals in the gramineae. Phytochemistry, 1988, 27: 3349-3358
220.Norouzian D, Rostami K, Nouri I D. Subsite mapping of purified glucoamylases I, II, III produced by Arthrobotrys amerospora ATCC 34468. Microbiol Biotechnol, 2000, 8: 55-61
221.Ohnishi M, Higushi A, Todoriki S, Hiromi K, Oghushi M, Wada A. Characterazation on the conformation of glucoamylase from Rhizopus niveus and Rhizopus delemar. Starch/ Starke, 1990, 42: 273-276
222.Okuyama M, Mori H, Watanabe K, Kimura A, Chiba S.α-Glucosidase mutant catalyzes "α-glycosynthase"-type reaction. Biosci Biotechnol Biochem, 2002, 66: 928-933
223.Oren A. A thermophilic amyloglucosidase from Halobacterium sodomense, a halophilic bacterium from the Dead Sea. Curr Microbiol, 1983, 8: 225-230
224.Ozaki K, Sumitomo N, Hayashi Y. Site-directed mutagenesis of the putative active site of endoglucanase K from Bacillus sp. KAM-330. Biochim Biophys. Acta., 1994, 1207: 159-164
225.Palackal N, Brennan Y, Callen W N, Dupree P, Frey G, Goubet F, Hazlewood G P, Healey S, Kang Y E, Kretz K A, Lee E, Tan X, Tomlinson G L, Verruto J, Wong V W, Mathur E J, Short J M, Robertson D E, Steer B A. An evolutionary route to xylanase process fitness. Protein Sci, 2004, 13: 494-503
226.Palcic M M. Biocatalytic synthesis of oligosaccharides. Curr Opin Biotechnol, 1999, 10: 616-624
227.Pazur J H, Knull H R, Cepure A. Glucoamylase: Structure and properties of the two forms of glucoamylase from Aspergillus niger. Carbohyd Res, 1971, 20: 83-96
228.Pellerin P, Brillouet J M. Purification and properties of an exo-(1→3)-β-d-galactanase from Aspergillus niger. Carbohydrate Research, 1994, 264: 281-291
229.Perugino G, Trincone A, Giordano A, van der Oost J, Kaper T, Rossi M, Moracci M. Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH.Biochemistry, 2003, 42: 8484-8493
230.Perugino G, Trincone A, Rossi M, Moracci M. Oligosaccharide synthesis by glycosynthases. Trends Biotechnol, 2004, 22:31-37
231.PouLton J E. Cyanogenesis in Plants. Plant Physiol., 1990, 94: 401-405
232.Pretorius I S, Laing E, Pretorius G H, Marmur J. Expression of Bacillus amylase gene in yeast. Current Genetics, 1988, 14: 1-8
233.Ramasesh N, Srekantiah K R, Murthy V S. Strudies on the two forms of amyloglucosidase of Aspergillus niger van Teighem. Starch /St?rke, 1982, 34: 346-351
234.Reetz M T, Wang L W. Directed evolution of enantioselective hybrid catalysts: a novel concept in asymmetric catalysis, Tetrahedron, 2007, 63: 6404-6414
235.Rey M W, Brown K M, Golightly E J, Fuglasang C C, Nielsen B R, Hendriksen H V, Butterworth A, Xu. Cloning, Heterologous Expressionand Characterization of Thielavia terrestris Glucoamylase. Applied Biochemistry and Biotechnology, 2003, 3: 153-166
236.Riedel K, Bronnenmeier K. Active-site mutations which change the substrate specificity of the Clostridium ercorarium stcellulase CelZ implications for synergism. European Journal of Endocrinology, 1999, 262: 218-223
237.Rothstain S J, Larners K N, Lazarus C M. Expression and secretion of wheal amylase in Saccharomyoes cereviaiae. Gene, 1987, 55:353
238.Rouvinen J, Bergfors T, Teeri T. Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science, 1990, 249: 380-386
239.Rudd P M, Elliott T, Cresswell P, Wilson I A, Dwek R A. Glycosylation and the immune system. Science, 2001, 291: 2370-2376
240.Rudick M J, Elbein A D. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of a second,β-glucosidase. J Bacteriol, 1975, 124:534-541
241.Rye C S, Withers S G. Glycosidase mechanisms. Curr Opin Chem Biol, 2000, 4: 573-580
242.Sakai K, Fukui S, Yabuuchi S, Aoyagi S, Tsumura Y. Expression of the Saccharomyces diastaticus STA1 gene in brewing yeasts. J Am Soc Brew Chem, 1989, 47: 87-91
243.Sakon J, Adney W S, Himmel M E. Crystal structure of thermostable family 5 endocellu- lase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry, 1996, 35: 10648-10660
244.Sakon J, Irwin D, Wilson D B. Structure and mechanism of endo/exocellulase E4 from Thermobifida fusca. Nature Structural & Molecular Biology, 1997, 4: 810-818
245.Saloheimo M, Kuja-Panula J, Yl?sm?ki E, Ward M, Penttil? M. Enzymatic propertiesand intracellular localization of the novel Trichoderma reeseiβ-Glucosidase BGLII (Cel1A). Applied Environment Microbiology, 2002, 68: 4546-4553
246.Shao Z, Zhao H, Giver L, Arnold F H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucl Acids Res, 1998, 26: 681-683
247.Siddiqui K S, Rashid M H, Ghauri T M, Durrani I S, Rajoka M I. Short Communication: Purification and characterization of an intracellularβ-glucosidase from Cellulomonas biazotea. World Journal of Microbiology & Biotechnology, 1997, 13: 245-247
248.Sinnott M L. Catalytic mechanism of enzymic glycosyl transfer. Chem Rev, 1990, 90: 1171-1202
249.Smith M E B, Hibbert E G, Jones A B, Dalby P A, Hailes H C. Enhancing and reversing the stereoselectivity of escherichia coli transketolase via single-point mutations. Adv. Synth. Cat., 2008, 350: 2631-2638
250.Spee J H, Vos W M, Kuipes O P. Efficient random mutagenesis method with adjustable mutation by use of PCR and dITP. Nucl Acids Res, 1993, 21: 777-778
251.Spinelli B B L, Lourdes M, Polizeli T M. Biochemical characterization of glucoamylase from the hyper producer exo-1 mutant strain of Neurospora crassa. FEMS Microbiol Lett, 1996, 13: 3-7
252.Sreekrishna K, Brankamp R G, Kropp K E, Blankenship D T, Tsay J T, Smith P L, Wierschke J D, Subramaniam A, Birkenberger L A. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene, 1997, 190: 55-62
253.Stahlberg J, Divne C, Koivula A. Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolaseⅠfrom Trichoderma reesei. Journal of Molecular Biology, 1996, 264: 337-349
254.Steenbakkers P J, Harhangi H R, Bosscher M W, van der Hooft M M, Keltjens J T, van der Drift C, Vogels G D, op den Camp H J. beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase. Biochem J, 2003, 370: 963-970
255.Stemmer W P C. DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci, 1994, 91: 10747-10751
256.Stemmer W P C. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994, 370: 389-391
257.Stoffer B, Frandsen T P, Busk P K, Schneider P, Svendsen I, Svensson B. Production,purification and characterization of the catalytic domain of glucoamylase from Aspergillus niger. Biochem, 1993, 292: 197-202
258.Swift R J, Wiebe M G, Robson G D, Trinci A P. Recombinant glucoamylase production by Aspergillus niger B1 in chemostat and pH auxostat cultures. Fungal Genetics and Biology, 1998, 25:100-109
259.Takahashi T, Tsuchida Y, Irie M. Isolation of Two Inactive Fragments of a Rhizopus sp. Glucoamylase: Relationship among Three Forms of the Enzyme and the Isolated Fragments. Biochem, 1982, 92: 1623-1633
260.Takashi T, Inokushi N, Irie M. Purification and characterization of a glucoamylase from Aspergillus satoi. J Biochem, 1981, 89: 125-130
261.Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T. Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei. J Biochem, 1999, 125: 728-736
262.Tao J H, Xu J H. Biocatalysis in developmene of green pharmaceutical processes. Current Optinion in Chemical Biology, 2009, 13: 43-50
263.Taylor P M, Napier E J, Fleming I D. Some properties of a glucoamylase produced by the thermophilic fungus Humicola lanuginosa. Carbohyd Res, 1978, 16: 301-308
264.Teeri T T. Crystalline cellulose degradation: new insight into the function of cellobio- hydrolases. Trends Biotechnol, 1997, 15: 160-167
265.Thorsen T S, Johnsen A H, Josefsen K, Jensen B. Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus. Biochim Biophys Acta, 2006, 1764: 671-676
266.Tigue N J, Kay J. Active site mutants of human herpesvirus-6 proteinase. FEBS Lett , 1998, 441: 467-469
267.Tolborg J F, Petersen L, Jensen K J, Mayer C, Jakeman D L, Warren R A, Withers S G. Solid-phase oligosaccharide and glycopeptide synthesis using glycosynthases. J Org Chem, 2002, 67: 4143-4149
268.Trincone A, Giordano A, Perugino G, Rossi M, Moracci M. Glycosynthase-catalysed syntheses at pH below neutrality. Bioorg Med Chem Lett, 2003, 13: 4039-4042
269.Trincone A, Perugino G, Rossi M. A novel thermophilie glycosynthase that effects branching glycosylation. Bioorg. Med. Chem. Int. Ed., 2001, 40: 417-420
270.Trincone A, Perugino G, Rossi M, Moracci M. A novel thermophilic glycosynthase that effects branching glycosylation. Bioorg Med Chem Lett, 2000, 10: 365-368
271.Tsukada T, Igarashi K, Yoshida M, Samejima M. Molecular cloning and characterizationof two intracellular beta-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium. Appl Microbiol Biotechnol, 2006, 73: 807-814
272.Van den S P, Rudd P M, Dwek R A, Opdenakker G. Concepts and principles of O-linked glycosylation. Crit Rev Biochem Mol Biol, 1998, 33: 151-208
273.Van Kampen M D, Egmond M R. Directed evolution: From a staphylococcal lipase to phospholipase. Eur J Lipid Sci Technol, 2000, 102: 717-726
274.Van Kampen M D, Dekker N, Egmond M R, Verheij H M. Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as strdied by in vivo chimeragenesis. Biochemistry, 1998, 37: 3459-3466
275.Van Lieshout J, Faijes M, Nieto J, Van der O J, Planas A. Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the thermophile Pyrococcus furiosus. Archaea, 2004, 1: 285-292
276.Varrot A, Hastrup S, Davies G J. Crystal structure of the catalytic core domain of the family 6 cellobiohydrolaseⅡ. Biological Chemistry, 1999, 337: 297-304
277.Vaughan M D, Johnson K, DeFrees S, Tang X, Warren R A, Withers S G. Glycosynthase-mediated synthesis of glycosphingolipids. Am Chem Soc, 2006, 128: 6300- 6301
278.Wada M, Hsu C C, Franke D, Mitchell M, Heine A, Wilson I, Wong C H. Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions. Bioorg. Med. Chem., 2003, 11: 2091-2098
279.Wang Q, Tull D, Meinke A, Gilkes N R, Warren R A, Aebersold R, Withers S G. Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1, 4-glucanase. Journal of Biological Chemistry, 1993, 268: 14096-140102
280.Wang S X, Dunphy W G. Activation of Xenopus Chk1 by mutagenesis of threonine-377. FEBS Lett, 2000, 487: 277-281
281.Wang T, Liu X, Yu Q, Zhang X, Qu Y, Gao P, Wang T. Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei. Biomolecular Engineering, 2005, 22: 89-94
282.Washington S L, Yoon M S, Chagovetz A M, Li S X, Clairmont C A, Preston B D, Eckert K A, Sweasy J B. A genetic system to identify DNA polymerase beta mutator mutants. Proceedings of the National Academy of Sciences of the USA, 1997, 94: 1321-1326
283.Wintrode P L, Miyazaki K, Amold F H. Cold adaptation of a mesophilic subtilisin-likeprotease by laboratory evolution. Journal of Biological Chemistry, 2000, 275: 31635- 31640
284.Withers S G, Warren R A J, Street I P, Rupitz K, Kempton J B, Aebersold R. Unequivocal demonstration of the involvement of a glutamate residue as a nucleophile in the mechanism of a“retaining”glycosidase. J Am Chem Soc, 1990, 112: 5887-5889
285.Wolfgang D E, Wilson D B. Mechanistic studies of active site mutants of Thermomo- nospora fusca endocellulase E2. Biochemistry, 1999, 3: 9746-9751
286.Wood V, Gwilliam R, Rajandream M A. The genome sequence of Schizosaccharomyces pombe. Nature, 2002, 415: 871-880
287.Xu X, Huang J, Fang J, Lin C, Cheng J, Shen Z. Expression of a fungal glucoamylase in transgenic rice seeds. Protein Expr Purif, 2008, 61: 113-116
288.Yamashita I, Itoh T, Fukui S. Cloning and expression of the Saccharomycopsis fibuligera glucoamylase gene in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 1985, 23: 130-133
289.Yan T R, Liau J C. Synthesis of alkylβ-glucosides from cellobiose with Aspergillus nigerβ-glucosidase.ⅡBiotechnologyLetters, 1998, 20: 653-657
290.Yang L Q, Dai X J, Luo Y M, Ma C X, Hou J H, Wu Z Q, Wang C Y, Li M G. Cloning and expression of a new glucoamylase gene. Sheng Wu Gong Cheng Xue Bao, 2007, 23:477-482
291.Yang S H, Jia N B, Li M G, Wang J H. Heterologous expression and efficient ethanol production of a Rhizopus glucoamylase gene in Saccharomyces cerevisiae. Molecular Biology Reports, 2010,DOI 10.1007/s11033-010-0077-3
292.Yano T, Oue S, Kagamiyama H. Directed evolution of an aspartate aminotransferase with new substrate specificities. Proceedings of the National Academy of Sciences of the USA, 1998, 95: 5511-5515
293.Yoon J J, Igarashi K, Kajisa T. Purification, identification and molecular cloning of glycoside hydrolase family 15 glucoamylase from the brown-rot basidiomycete Fomitopsis palustris. FEMS Microbiology Letters, 2006, 259: 288-294
294.Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, Kitaoka M, Katayama T, Kumagai H. Purification, crystallization and preliminary X-ray analysis of beta-glucosidase from Kluyveromyces marxianus NBRC1777. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2009, 65:1190-1192
295.Zanoelo F F, Polizeli Mde L, Terenzi H F, Jorge J A. Beta-glucosidase activity from thethermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett, 2004, 240: 137-143
296.Zhang J H, Dawes G, Stemmer W P C. Directed evolution of a fucosidase from a galacto- sidase by DNA shuffling and screening. Proceedings of the National Academy of Sciences of the USA, 1997, 94: 4504-4509
297.Zhang Y H P, Himmel M E, Mielenz J R. Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, 2006, 24: 452-481
298.Zhao H, Arnold F H. Functional and non-functional mutations distinguished by random recombination of homologous genes. Proc Natl Acad Sci, 1997, 94: 7997-8000
299.Zhao H, Arnold F H. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res, 1997, 25: 1307-1308
300.Zhao H, Giver L, Shao Z, Affholter J A, Arnold F H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol, 1998, 16: 258-261
301.Zheng Y Y, Xue Y F, Zhang Y L, Zhou C, Schwaneberg U, Ma Y H. Cloning, expressionand characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4. Appl Microbiol Biotechnol, 2010, DOI 10.1007/s00253-010-2439-0
302.Zhou H, Wang H W, Zhu K, Sui S F, Xu P, Yang S F, Li N. The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding and beyond. Plant Physiol, 1999, 121: 913-919
303.Zopf D, Roth S. Oligosaccharide anti-infective agents. Lancet, 1996, 347: 1017-1021