黑曲霉木聚糖酶基因在毕赤酵母中的高效表达及其酶学性质研究
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摘要
木聚糖广泛存在于植物细胞壁中,是自然界中含量仅次于纤维素的可再生生物资源。木聚糖的结构复杂,降解木聚糖酶需要多种水解酶的共同参与,其中木聚糖酶是降解木聚糖最关键的酶类,它能够作用于木聚糖分子中的β-1,4糖苷键,产生不同长度的木寡糖和少量的木糖,从而生成可以再次利用的小分子物质。为了利用丰富的木聚糖资源,就必须获得性质优良的木聚糖酶。
本文以黑曲霉(Aspergillus niger)F19来源的野生型木聚糖酶基因xylB和突变的木聚糖酶基因ST7(Ser~(33)和Thr~(186)替换为为Cys,以此来引入1个二硫键,同时将酶表面的Ser~(39)、Ser~(54)、Thr~(101)、Thr~(105)和Thr~(151)替换为Arg)为研究对象,研究了xylB和ST7基因在毕赤酵母中的表达、重组木聚糖酶的纯化及其酶学性质,探索了突变体酶对热稳定性的影响,并对突变体木聚糖酶重组毕赤酵母的诱导条件进行了优化研究。主要结论如下:
1.野生型木聚糖酶基因xylB和突变体木聚糖酶基因ST7在毕赤酵母中的表达
将木聚糖酶基因xylB和ST7克隆至毕赤酵母表达载体pPIC9K,重组表达载体转化毕赤酵母GS115,经过甲醇诱导,木聚糖酶基因都获得特异性表达,而且木聚糖酶能够分泌到培养基中,筛选到两株高酶活性的重组毕赤酵母,野生型和突变体木聚糖酶重组毕赤酵母的酶活性分别达到82.9 U/ml和390.2 U/ml。
2.野生型木聚糖酶和突变体木聚糖酶的纯化
利用黑曲霉木聚糖酶可以与木聚糖进行特异性结合的特性,对木聚糖酶进行分离纯化,纯化的木聚糖酶在SDS-PAGE上均呈现出单一的蛋白条带,表明木聚糖酶得到纯化。
3.野生型木聚糖酶和突变体木聚糖酶的酶学性质分析
与野生型木聚糖酶相比,突变体木聚糖酶的最适反应温度从45℃提高到50℃,50℃时酶的半衰期从小于10 min提高到120 min,而且酶反应过程中生成物积累量的幅度从36.9%提高到276.3%,酶的热稳定性有了较大的提高。突变体木聚糖酶的比酶活性从2127.9±83.9 U/mg提高到3330.9±173.4 U/mg,K_m值从56.9 mg/ml降低到37.2 mg/ml,酶与底物的亲和性有较大提高。但V_(max)从82.9 mmol/ml.min.mg降低至27.0 mmol/ml.min.mg。
4.突变体木聚糖酶重组酵母诱导条件的优化
采用四因素三水平的正交设计试验研究了甲醇加入量、起始细胞浓度、pH和诱导时间四个因素对重组酵母产酶的影响,四个因素对产酶的影响大小为:甲醇加入量＞诱导时间＞pH＞起始细胞浓度。最佳的诱导条件为:BYPN培养基(pH 8.0),每隔12 h加入1%甲醇,起始细胞浓度OD_(600)=3,诱导培养7d,在此条件下对重组酵母进行诱导培养,产酶水平可达到1850.6±25.2 U/ml。
目前本实验室正在进行利用生物发酵罐对突变体木聚糖酶重组毕赤酵母进行诱导产酶的研究,以期提高木聚糖酶的产酶水平。
Xylan which is mainly found in plant cell wall is the most abundant regenerate resource secondary to cellulose. For complicated structure of xylan , the degradation of it needs many kinds of hydrolases. Xylanase is the key hydrolases for degradation of xylan. Xylanase can catalyze the hydrolysis ofβ-1,4 xylose linkage of xylan into xylooligosaccharides and xylose. With this catalyze procedure, micromolecul carbohydrate which can be reused is produced. For exploitation of xylan resource, we must get xylanase with good property.
In this dissertation, native xylanase xylB and mutational xylanase ST7(Ser~(33) and Thr~(186) were substituted with Cys. In order to introduce a disulfide bridge into catalytic domain. Ser~(39)、Ser~(54)、Thr~(101)、Thr~(105) and Thr~(151) were substituted with Arg.) were our research objects. xylB and ST7 gene were expressed in Pichia pastoris GS115. Native xylanase and mutational xylanase were purified. Characterization and thermostability of recombinant xylanase were researched. Inducing condition of mutational xylanase recombined Pichia pastoris was optimized, and the results showed that:
1. Expression of native xylanase xylB and mutational xylanase ST7 in Pichia pastoris
xylB and ST7 were cloned into Pichia pastoris expression vector pPIC9K, then transferd them into Pichia pastoris GS115. Xylanase were successfully expressed after inducing with methanol. Xylanase were secreted into medium. We got two recombinants with high xylanase activity, xylanase activity can reach 82.9 U/ml and 390.2 U/ml.
2. Purification of native xylanase and mutational xylanase
With the knowledge that some xylanase can bind xylan specific, Aspergillus niger xylanase were purified with xylan. Single protein band were found in the SDS-PAGE , demonstrated that xylanase were purified.
3. Characterization of native xylanase and mutational xylanase
Compared with native xylanase, optimal reaction temperature of mutational xylanase increased from 45℃to 50℃. Half-life of the mutational xylanase increased from lower than 10 min to 120 min. Accumulation amplitude of enzyme react product increased from 36.9% to 276.3%. These dates confirmed that thermostability of mutational xylanase was greatly improved. Specific activity of xylanase increased from 2127.9±83.9 U/mg to 3330.9±173.4 U/mg. K_m of xylanase decreased from 56.9 mg/ml to 37.2 mg/ml, affinity of xylanase was greatly improved. V_(max) of xylanase decresed from 82.9 mmol/ml.min.mg to 27.0 mmol/ml.min.mg.
4. Optimization the induce condition of the mutational xylanase Pichia pastoris
Influence of methanol quantity、initial cell density、pH and inducing time to enzyme production were reseached with four factors and three levels Orthogonal design. The importance of these factors for enzyme production were : methanol quantity＞inducing time＞pH＞initial cell density. The best inducing condition were : BYPN medium(pH8.0), added 1% methanol every 12 h, initial cell density was OD_(600)=3, induced 7 d. Activity of mutational xylanase can reached 1850.6±25.2 U/ml under the best inducing condition.
With the purpose of improving the yield of xylanase, we are reseaching on the inducing condition of mutational xylanase production with bio-fermenter.
本文以黑曲霉(Aspergillus niger)F19来源的野生型木聚糖酶基因xylB和突变的木聚糖酶基因ST7(Ser~(33)和Thr~(186)替换为为Cys,以此来引入1个二硫键,同时将酶表面的Ser~(39)、Ser~(54)、Thr~(101)、Thr~(105)和Thr~(151)替换为Arg)为研究对象,研究了xylB和ST7基因在毕赤酵母中的表达、重组木聚糖酶的纯化及其酶学性质,探索了突变体酶对热稳定性的影响,并对突变体木聚糖酶重组毕赤酵母的诱导条件进行了优化研究。主要结论如下:
1.野生型木聚糖酶基因xylB和突变体木聚糖酶基因ST7在毕赤酵母中的表达
将木聚糖酶基因xylB和ST7克隆至毕赤酵母表达载体pPIC9K,重组表达载体转化毕赤酵母GS115,经过甲醇诱导,木聚糖酶基因都获得特异性表达,而且木聚糖酶能够分泌到培养基中,筛选到两株高酶活性的重组毕赤酵母,野生型和突变体木聚糖酶重组毕赤酵母的酶活性分别达到82.9 U/ml和390.2 U/ml。
2.野生型木聚糖酶和突变体木聚糖酶的纯化
利用黑曲霉木聚糖酶可以与木聚糖进行特异性结合的特性,对木聚糖酶进行分离纯化,纯化的木聚糖酶在SDS-PAGE上均呈现出单一的蛋白条带,表明木聚糖酶得到纯化。
3.野生型木聚糖酶和突变体木聚糖酶的酶学性质分析
与野生型木聚糖酶相比,突变体木聚糖酶的最适反应温度从45℃提高到50℃,50℃时酶的半衰期从小于10 min提高到120 min,而且酶反应过程中生成物积累量的幅度从36.9%提高到276.3%,酶的热稳定性有了较大的提高。突变体木聚糖酶的比酶活性从2127.9±83.9 U/mg提高到3330.9±173.4 U/mg,K_m值从56.9 mg/ml降低到37.2 mg/ml,酶与底物的亲和性有较大提高。但V_(max)从82.9 mmol/ml.min.mg降低至27.0 mmol/ml.min.mg。
4.突变体木聚糖酶重组酵母诱导条件的优化
采用四因素三水平的正交设计试验研究了甲醇加入量、起始细胞浓度、pH和诱导时间四个因素对重组酵母产酶的影响,四个因素对产酶的影响大小为:甲醇加入量＞诱导时间＞pH＞起始细胞浓度。最佳的诱导条件为:BYPN培养基(pH 8.0),每隔12 h加入1%甲醇,起始细胞浓度OD_(600)=3,诱导培养7d,在此条件下对重组酵母进行诱导培养,产酶水平可达到1850.6±25.2 U/ml。
目前本实验室正在进行利用生物发酵罐对突变体木聚糖酶重组毕赤酵母进行诱导产酶的研究,以期提高木聚糖酶的产酶水平。
Xylan which is mainly found in plant cell wall is the most abundant regenerate resource secondary to cellulose. For complicated structure of xylan , the degradation of it needs many kinds of hydrolases. Xylanase is the key hydrolases for degradation of xylan. Xylanase can catalyze the hydrolysis ofβ-1,4 xylose linkage of xylan into xylooligosaccharides and xylose. With this catalyze procedure, micromolecul carbohydrate which can be reused is produced. For exploitation of xylan resource, we must get xylanase with good property.
In this dissertation, native xylanase xylB and mutational xylanase ST7(Ser~(33) and Thr~(186) were substituted with Cys. In order to introduce a disulfide bridge into catalytic domain. Ser~(39)、Ser~(54)、Thr~(101)、Thr~(105) and Thr~(151) were substituted with Arg.) were our research objects. xylB and ST7 gene were expressed in Pichia pastoris GS115. Native xylanase and mutational xylanase were purified. Characterization and thermostability of recombinant xylanase were researched. Inducing condition of mutational xylanase recombined Pichia pastoris was optimized, and the results showed that:
1. Expression of native xylanase xylB and mutational xylanase ST7 in Pichia pastoris
xylB and ST7 were cloned into Pichia pastoris expression vector pPIC9K, then transferd them into Pichia pastoris GS115. Xylanase were successfully expressed after inducing with methanol. Xylanase were secreted into medium. We got two recombinants with high xylanase activity, xylanase activity can reach 82.9 U/ml and 390.2 U/ml.
2. Purification of native xylanase and mutational xylanase
With the knowledge that some xylanase can bind xylan specific, Aspergillus niger xylanase were purified with xylan. Single protein band were found in the SDS-PAGE , demonstrated that xylanase were purified.
3. Characterization of native xylanase and mutational xylanase
Compared with native xylanase, optimal reaction temperature of mutational xylanase increased from 45℃to 50℃. Half-life of the mutational xylanase increased from lower than 10 min to 120 min. Accumulation amplitude of enzyme react product increased from 36.9% to 276.3%. These dates confirmed that thermostability of mutational xylanase was greatly improved. Specific activity of xylanase increased from 2127.9±83.9 U/mg to 3330.9±173.4 U/mg. K_m of xylanase decreased from 56.9 mg/ml to 37.2 mg/ml, affinity of xylanase was greatly improved. V_(max) of xylanase decresed from 82.9 mmol/ml.min.mg to 27.0 mmol/ml.min.mg.
4. Optimization the induce condition of the mutational xylanase Pichia pastoris
Influence of methanol quantity、initial cell density、pH and inducing time to enzyme production were reseached with four factors and three levels Orthogonal design. The importance of these factors for enzyme production were : methanol quantity＞inducing time＞pH＞initial cell density. The best inducing condition were : BYPN medium(pH8.0), added 1% methanol every 12 h, initial cell density was OD_(600)=3, induced 7 d. Activity of mutational xylanase can reached 1850.6±25.2 U/ml under the best inducing condition.
With the purpose of improving the yield of xylanase, we are reseaching on the inducing condition of mutational xylanase production with bio-fermenter.
引文
1.崔罗生.黑曲霉木聚糖酶基因克隆、表达与热稳定性改造研究.[硕士学位论文].武汉:华中农业大学图书馆,2008
2.邓萍,曹云鹤,陆文清,郭小华.黑曲霉木聚糖酶基因xynB在大肠杆菌中的表达及重组木聚糖酶的特性.农业生物技术学报,2006,14(5):774-778
3.符丹丹,李爱江,谢慧,邬敏辰.宇佐美曲霉木聚糖酶的纯化及其性质.食品科学,2006,27(2):103-108
4.高艳华,袁建军,段峰.食品级木聚糖酶在面食制品改良中的应用试验.中国科技论文在线
5.怀文辉,何秀萍,郭文杰.微生物木聚糖酶降解酶研究进展及应用前景.微生物学通报,2000,27(2):137-139
6.江正兵,宋慧婷,马立新.短小芽孢杆菌木聚糖酶基因在毕赤酵母中的分泌表达及酶学性质研究.生物工程学报,2003,19(1):50-55
7.江正强.海栖热袍菌xynB基因的克隆和表达、重组木聚糖酶的提纯及其酶学性质.[博士学位论文].北京:中国农业大学,2001
8.刘成,孙中涛,田林茂,杜金华.黑曲霉产木聚糖酶固态发酵条件及部分酶学性质的研究.生物技术,2007,17(4):82-85
9.李春华,李翔,马立新.酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达.微生物学通报,2005,32(6):89-95
10.李海燕,祝令香,毛爱军,董志扬.黑曲霉木聚糖酶在工业酒精酵母中的分泌表达.微生物学报,2005,45(1):135-138
11.李洪钊,李亮助,孙强明.巴斯德毕赤酵母表达系统优化策略.微生物学报,2003,43(2):288-292
12.陆健,曹钰,陈坚.运用定点突变提高重组木聚糖酶在毕赤氏酵母中的表达.微生物学报,2002,42(4):425-430
13.刘伟丰,毛爱军,祝令香,赵云,董志扬.耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究.微生物学报,2004,44(4):487-490
14.刘寅.黑曲霉木聚糖酶基因克隆及在酿酒酵母中的表达.[硕士学位论文].河南: 河南农业大学,2006
15.毛连山,余世袁.木聚糖酶在纸浆漂白中应用的研究现状.中国造纸学报,2006,21(3):93-98
16.王丽萍,彭德奎,陈守文,喻子牛,孙明.枯草芽胞杆菌重组木聚糖酶基因高效表达系统的构建.农业生物技术学报,2007,15(1):133-137
17.薛业敏,毛忠贵,邵蔚蓝.极端耐热木聚糖酶基因在大肠杆菌中的高效表达.食品与发酵工业,2003,29(11):20-25
18.殷尔康.疏棉状嗜热丝饱菌木聚糖酶在大肠杆菌中的高效表达.[硕士学位论文].南京:南京师范大学,2007
19.杨浩萌,柏映国,李江,罗会颖,王亚茹,伍宁丰,范云六,姚斌.木聚糖酶xynB的N46D突变、表达及酶学性质变化.中国生物化学与分子生物学报.2006b,22(3):204-210
20.杨浩萌,孟昆,罗会颖,王亚茹,袁铁铮,柏映国,姚斌,范云六.通过N端替换提高木聚糖酶的热稳定性.生物工程学报,2006a,22(1):26-32
21.杨浩萌,王亚茹,伍宁,姚斌。N13D、S40E定点突变提高木聚糖酶XYNB的稳定性.微生物学通报,2007,34(3):533-536
22.杨梦华,李颖,关国华,江正强.极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达.微生物学报,2005,45(2):236-240
23.杨培龙,姚斌,王亚茹,罗会颖,袁铁铮,柏映国,范云六.在烟草中表达的高比活性木聚糖酶xynB.作物学报,2006,32(2):176-181
24.岳晓禹.木聚糖酶的研究进展.酿酒科技,2007,4:113-120
25.周晨妍,白剑宇,邬敏辰,王武.宇佐美曲霉木聚糖酶(xynⅡ)基因在大肠杆菌中的表达.农业生物技术学报,2007,15(3):514-518
26.张红莲,姚斌,王亚茹,袁铁峥,张王照,伍宁丰,范云六.链霉菌Streptomyces olivaceoviridis A1木聚糖酶基因xynA在大肠杆菌及毕赤酵母中的高效表达.生物工程学报,2003b,19(1):41-45
27.张红莲,姚斌,王亚茄,张王照,袁铁铮.具有高比活性的新木聚糖酶xynB的酶学性质研究及其编码基因的克隆和表达.科学通报,2003a,48(4):364-368
28.张晶,王战勇,苏婷婷.产木聚糖酶黑曲霉LN0601的液体发酵.辽宁石油化工 大学学报,2007,27(2):10-12
29. 张晓晖,郭春华,江晓霞,项碧飞,张兴友.饲用木聚糖酶生产菌株的筛选及部分酶学性质的研究.饲料工业,2007,28(6):23-25
30. Autio K. Effects of cell wall components on the functionality of wheat gluten.Biotechnology Advances, 2006, 24: 633-635
31. Bailey M J, Biely P, Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. Journal Biotechnol, 1992, 23: 257-270
32. Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem,1976,72:248-254
33. Chakrit T, Khin L K, Khanok R. Purification of xylanase from alkaliphilic Bacillus sp.K-8 by using corn husk column. Process Biochemistry, 2006, 41: 2441-2445
34. Chen Y Z, Jian Y B, Shan S D, Jin W, Jie Z, Min C Wu, Wu W. Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli.Bioresource Technology, 2008, 99: 831-838
35. Cosson T, Vendrell A M P, Teresa B G. Enzymatic assays for xylanase and β-glucanase feed enzymes. Animal Feed Science and Technology, 1999, 77: 345-353
36. Dawn E S, Karl R, Kugen P. Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. Journal of Biotechnology, 2007, 127: 348-354
37. Duriya C, Kusol P, Verawat C, Pattanop K, Lily E. Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris. Protein Expression and Purification, 2006, 46: 143-149
38. Eun J S, Beauchemin K A, Hong S H. Exogenous enzymes added to untreated or ammoniated rice straw : Effects on in vit ro fermentation characteristics and degradability. Animal Feed Science and Technology, 2006, 131: 86-101
39. Ferreira V, Nolasco P C, Castro A M, Silva J N, Santos A S, Damaso M C, Pereira J N. Evaluation of cell recycle on Thermomyces lanuginosus Xylanase A production by Pichia pastoris GS115. Appl Biochem Biotechnol. 2006, 129: 226-33
40. Fred F, Matti L, Janne J. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-β-xylanase Ⅱ. Journal of Biotechnology, 2004, 108: 137-143
41. Gabriel P, Michael J O. Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus. Journal of Biotechnology, 2006, 125: 338-350
42. Hairong X, Fred F, Marti L. Engineering the thermostability of Trichoderma reesei endo-l,4-p-xylanase II by combination of disulphide bridges. Extremophiles, 2004, 8: 393-400
43. Hidenort T, Tomoko O, Satoshi M. Acidophilic Xylanase from Aureobasidium pullulans:Efficient Expression and Secretion in Pichia pastoris and Mutational Analysis. Journal of Bioscience and Bioengineering, 2004, 98(5): 338-343
44. Hirohito. Contribution of salt bridges to alkaliphily of Bacillus alkaline xylanase. Nucleic Acids Symposium Series, 2007, (51): 461-462
45. Jacques G, Frederic D L, Josette L B. An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: Structural basis and molecular study. Protein Science, 2000, 9: 466-475
46. Jean G B, Gary W, Antoine P. High-Level Production of Recombinant Fungal Endo-P-l,4-xylanase in the Methylotrophic Yeast Pichia pastoris. Protein Expression and Purification, 2000, 19: 179-187
47. Jian Y S, Ming Q L, Ying L X, Zi R X, Lin P, Hui G. Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement. Protein Expression and Purification, 2005, 42: 122-130
48. Ken K Y W, Larry U L T, John N S. Multiplicity of P-l,4-xylanase in microorganisms: Functions and Applications. Microbiological Reviews, 1988, 52: 305-317
49. Kentaro M, Misa Takenouchi, Hidemasa K. Thermal Stabilization of Bacillus subtilis Family-11 Xylanase by Directed Evolution. The Journal of Biological chemistry, 2006,281(15): 10236-10242.
50. Khanok R, Khin L K, Morakot T. Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1. Applied and Environmental Microbiology, 1999, 65(2): 694-697
51. Kim J C, Simmins P H, Mullan B P. The digestible energy value of wheat for pigs, with special reference to the post-weaned animal. Animal Feed Science and Technology, 2005, 122: 257-287
52. Ming Q L, Guang F L. Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Protein Expression and Purification, 2008, 57: 101-107
53. Ming Q L, Xiao Y W, Jian Y S. Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan. Protein Expression and Purification2006, 48: 292-299
54. Mi Y J, Sanguk K,Cheol W Y. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No.236. Journal of Biotechnology, 2007, 127: 300-309
55. Mo^nica C D, Marcius S A, Eleonora K. Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris. Applied and Environmental Microbiology, 20003, 69(10): 6064-6072
56. Neeta K, Abhay S, Maraa R. Molecular and biotechnological aspects ofxylanase. FEMS Microbiology Reviews, 1999, 23: 411-456
57. Ossi T, Kirsikka E, Fred F. A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism. Journal of Biotechnology, 2001, 88: 37-46
58. Ossi T, Mika V, Fred F. Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Engineering, 2002, 15(2): 141-145
59. Ping D, Defa L, Yunhe C. Cloning of a gene encoding an acidophilic endo-β-1,4 xylanase obtained from Aspergillus niger CGMCC1067 and constitutive expression in Pichia pastoris. Enzyme and Microbial Technology, 2006, 39: 1096-1102
60. Qin W, Tao X. Enhancement of the activity and alkaline pH stability of Thermobifida fusca xylanase A by directed evolution. Biotechnol Lett, 2008, 30: 937-944
61. Rutchadaporn S, Krisana A, Jarupan G, Sutipa T, Verawat C, Lily E. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. Journal of Biotechnology, 2006, 126: 454-462
62. Santosh O R, Bruno M, Olle H. The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus. FEMS Yeast Research, 2005, 5: 839-850
63. Selinheimo E, Kruus K, Buchert J. Effects of laccase, xylanase and their combination on the rheological properties of wheat doughs. Journal of Cereal Science , 2006, 43: 152-159
64. Uysal H, Bilgicli N, Elgun A. Effect of dietary fibre and xylanase enzyme addition on the selected properties of wire-cut cookies. Journal of Food Engineering, 2007, 78: 1074-1078
65. Yang H M, Yao B, Meng K. Introduction of a disulWde bridge enhances the thermostability of a Streptomyces olivaceoviridis xylanase mutant. J Ind Microbiol Biotechnol, 2007, 34: 213-218
66. Yi F C, Chao H Y, Wen H L. Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme and Microbial Technology, 2005, 37: 541-546
67. Yun H C, Jia Y Q, Yi H L, Wen Q L. De novo synthesis, constitutive expression of Aspergillus sulphureus β-xylanase gene in Pichia pastoris and partial enzymic characterization. Appl Microbiol Biotechnol, 2007, 76: 579-585
2.邓萍,曹云鹤,陆文清,郭小华.黑曲霉木聚糖酶基因xynB在大肠杆菌中的表达及重组木聚糖酶的特性.农业生物技术学报,2006,14(5):774-778
3.符丹丹,李爱江,谢慧,邬敏辰.宇佐美曲霉木聚糖酶的纯化及其性质.食品科学,2006,27(2):103-108
4.高艳华,袁建军,段峰.食品级木聚糖酶在面食制品改良中的应用试验.中国科技论文在线
5.怀文辉,何秀萍,郭文杰.微生物木聚糖酶降解酶研究进展及应用前景.微生物学通报,2000,27(2):137-139
6.江正兵,宋慧婷,马立新.短小芽孢杆菌木聚糖酶基因在毕赤酵母中的分泌表达及酶学性质研究.生物工程学报,2003,19(1):50-55
7.江正强.海栖热袍菌xynB基因的克隆和表达、重组木聚糖酶的提纯及其酶学性质.[博士学位论文].北京:中国农业大学,2001
8.刘成,孙中涛,田林茂,杜金华.黑曲霉产木聚糖酶固态发酵条件及部分酶学性质的研究.生物技术,2007,17(4):82-85
9.李春华,李翔,马立新.酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达.微生物学通报,2005,32(6):89-95
10.李海燕,祝令香,毛爱军,董志扬.黑曲霉木聚糖酶在工业酒精酵母中的分泌表达.微生物学报,2005,45(1):135-138
11.李洪钊,李亮助,孙强明.巴斯德毕赤酵母表达系统优化策略.微生物学报,2003,43(2):288-292
12.陆健,曹钰,陈坚.运用定点突变提高重组木聚糖酶在毕赤氏酵母中的表达.微生物学报,2002,42(4):425-430
13.刘伟丰,毛爱军,祝令香,赵云,董志扬.耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究.微生物学报,2004,44(4):487-490
14.刘寅.黑曲霉木聚糖酶基因克隆及在酿酒酵母中的表达.[硕士学位论文].河南: 河南农业大学,2006
15.毛连山,余世袁.木聚糖酶在纸浆漂白中应用的研究现状.中国造纸学报,2006,21(3):93-98
16.王丽萍,彭德奎,陈守文,喻子牛,孙明.枯草芽胞杆菌重组木聚糖酶基因高效表达系统的构建.农业生物技术学报,2007,15(1):133-137
17.薛业敏,毛忠贵,邵蔚蓝.极端耐热木聚糖酶基因在大肠杆菌中的高效表达.食品与发酵工业,2003,29(11):20-25
18.殷尔康.疏棉状嗜热丝饱菌木聚糖酶在大肠杆菌中的高效表达.[硕士学位论文].南京:南京师范大学,2007
19.杨浩萌,柏映国,李江,罗会颖,王亚茹,伍宁丰,范云六,姚斌.木聚糖酶xynB的N46D突变、表达及酶学性质变化.中国生物化学与分子生物学报.2006b,22(3):204-210
20.杨浩萌,孟昆,罗会颖,王亚茹,袁铁铮,柏映国,姚斌,范云六.通过N端替换提高木聚糖酶的热稳定性.生物工程学报,2006a,22(1):26-32
21.杨浩萌,王亚茹,伍宁,姚斌。N13D、S40E定点突变提高木聚糖酶XYNB的稳定性.微生物学通报,2007,34(3):533-536
22.杨梦华,李颖,关国华,江正强.极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达.微生物学报,2005,45(2):236-240
23.杨培龙,姚斌,王亚茹,罗会颖,袁铁铮,柏映国,范云六.在烟草中表达的高比活性木聚糖酶xynB.作物学报,2006,32(2):176-181
24.岳晓禹.木聚糖酶的研究进展.酿酒科技,2007,4:113-120
25.周晨妍,白剑宇,邬敏辰,王武.宇佐美曲霉木聚糖酶(xynⅡ)基因在大肠杆菌中的表达.农业生物技术学报,2007,15(3):514-518
26.张红莲,姚斌,王亚茹,袁铁峥,张王照,伍宁丰,范云六.链霉菌Streptomyces olivaceoviridis A1木聚糖酶基因xynA在大肠杆菌及毕赤酵母中的高效表达.生物工程学报,2003b,19(1):41-45
27.张红莲,姚斌,王亚茄,张王照,袁铁铮.具有高比活性的新木聚糖酶xynB的酶学性质研究及其编码基因的克隆和表达.科学通报,2003a,48(4):364-368
28.张晶,王战勇,苏婷婷.产木聚糖酶黑曲霉LN0601的液体发酵.辽宁石油化工 大学学报,2007,27(2):10-12
29. 张晓晖,郭春华,江晓霞,项碧飞,张兴友.饲用木聚糖酶生产菌株的筛选及部分酶学性质的研究.饲料工业,2007,28(6):23-25
30. Autio K. Effects of cell wall components on the functionality of wheat gluten.Biotechnology Advances, 2006, 24: 633-635
31. Bailey M J, Biely P, Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. Journal Biotechnol, 1992, 23: 257-270
32. Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem,1976,72:248-254
33. Chakrit T, Khin L K, Khanok R. Purification of xylanase from alkaliphilic Bacillus sp.K-8 by using corn husk column. Process Biochemistry, 2006, 41: 2441-2445
34. Chen Y Z, Jian Y B, Shan S D, Jin W, Jie Z, Min C Wu, Wu W. Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli.Bioresource Technology, 2008, 99: 831-838
35. Cosson T, Vendrell A M P, Teresa B G. Enzymatic assays for xylanase and β-glucanase feed enzymes. Animal Feed Science and Technology, 1999, 77: 345-353
36. Dawn E S, Karl R, Kugen P. Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. Journal of Biotechnology, 2007, 127: 348-354
37. Duriya C, Kusol P, Verawat C, Pattanop K, Lily E. Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris. Protein Expression and Purification, 2006, 46: 143-149
38. Eun J S, Beauchemin K A, Hong S H. Exogenous enzymes added to untreated or ammoniated rice straw : Effects on in vit ro fermentation characteristics and degradability. Animal Feed Science and Technology, 2006, 131: 86-101
39. Ferreira V, Nolasco P C, Castro A M, Silva J N, Santos A S, Damaso M C, Pereira J N. Evaluation of cell recycle on Thermomyces lanuginosus Xylanase A production by Pichia pastoris GS115. Appl Biochem Biotechnol. 2006, 129: 226-33
40. Fred F, Matti L, Janne J. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-β-xylanase Ⅱ. Journal of Biotechnology, 2004, 108: 137-143
41. Gabriel P, Michael J O. Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus. Journal of Biotechnology, 2006, 125: 338-350
42. Hairong X, Fred F, Marti L. Engineering the thermostability of Trichoderma reesei endo-l,4-p-xylanase II by combination of disulphide bridges. Extremophiles, 2004, 8: 393-400
43. Hidenort T, Tomoko O, Satoshi M. Acidophilic Xylanase from Aureobasidium pullulans:Efficient Expression and Secretion in Pichia pastoris and Mutational Analysis. Journal of Bioscience and Bioengineering, 2004, 98(5): 338-343
44. Hirohito. Contribution of salt bridges to alkaliphily of Bacillus alkaline xylanase. Nucleic Acids Symposium Series, 2007, (51): 461-462
45. Jacques G, Frederic D L, Josette L B. An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: Structural basis and molecular study. Protein Science, 2000, 9: 466-475
46. Jean G B, Gary W, Antoine P. High-Level Production of Recombinant Fungal Endo-P-l,4-xylanase in the Methylotrophic Yeast Pichia pastoris. Protein Expression and Purification, 2000, 19: 179-187
47. Jian Y S, Ming Q L, Ying L X, Zi R X, Lin P, Hui G. Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement. Protein Expression and Purification, 2005, 42: 122-130
48. Ken K Y W, Larry U L T, John N S. Multiplicity of P-l,4-xylanase in microorganisms: Functions and Applications. Microbiological Reviews, 1988, 52: 305-317
49. Kentaro M, Misa Takenouchi, Hidemasa K. Thermal Stabilization of Bacillus subtilis Family-11 Xylanase by Directed Evolution. The Journal of Biological chemistry, 2006,281(15): 10236-10242.
50. Khanok R, Khin L K, Morakot T. Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1. Applied and Environmental Microbiology, 1999, 65(2): 694-697
51. Kim J C, Simmins P H, Mullan B P. The digestible energy value of wheat for pigs, with special reference to the post-weaned animal. Animal Feed Science and Technology, 2005, 122: 257-287
52. Ming Q L, Guang F L. Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Protein Expression and Purification, 2008, 57: 101-107
53. Ming Q L, Xiao Y W, Jian Y S. Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan. Protein Expression and Purification2006, 48: 292-299
54. Mi Y J, Sanguk K,Cheol W Y. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No.236. Journal of Biotechnology, 2007, 127: 300-309
55. Mo^nica C D, Marcius S A, Eleonora K. Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris. Applied and Environmental Microbiology, 20003, 69(10): 6064-6072
56. Neeta K, Abhay S, Maraa R. Molecular and biotechnological aspects ofxylanase. FEMS Microbiology Reviews, 1999, 23: 411-456
57. Ossi T, Kirsikka E, Fred F. A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism. Journal of Biotechnology, 2001, 88: 37-46
58. Ossi T, Mika V, Fred F. Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-β-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Engineering, 2002, 15(2): 141-145
59. Ping D, Defa L, Yunhe C. Cloning of a gene encoding an acidophilic endo-β-1,4 xylanase obtained from Aspergillus niger CGMCC1067 and constitutive expression in Pichia pastoris. Enzyme and Microbial Technology, 2006, 39: 1096-1102
60. Qin W, Tao X. Enhancement of the activity and alkaline pH stability of Thermobifida fusca xylanase A by directed evolution. Biotechnol Lett, 2008, 30: 937-944
61. Rutchadaporn S, Krisana A, Jarupan G, Sutipa T, Verawat C, Lily E. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. Journal of Biotechnology, 2006, 126: 454-462
62. Santosh O R, Bruno M, Olle H. The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus. FEMS Yeast Research, 2005, 5: 839-850
63. Selinheimo E, Kruus K, Buchert J. Effects of laccase, xylanase and their combination on the rheological properties of wheat doughs. Journal of Cereal Science , 2006, 43: 152-159
64. Uysal H, Bilgicli N, Elgun A. Effect of dietary fibre and xylanase enzyme addition on the selected properties of wire-cut cookies. Journal of Food Engineering, 2007, 78: 1074-1078
65. Yang H M, Yao B, Meng K. Introduction of a disulWde bridge enhances the thermostability of a Streptomyces olivaceoviridis xylanase mutant. J Ind Microbiol Biotechnol, 2007, 34: 213-218
66. Yi F C, Chao H Y, Wen H L. Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme and Microbial Technology, 2005, 37: 541-546
67. Yun H C, Jia Y Q, Yi H L, Wen Q L. De novo synthesis, constitutive expression of Aspergillus sulphureus β-xylanase gene in Pichia pastoris and partial enzymic characterization. Appl Microbiol Biotechnol, 2007, 76: 579-585