米曲霉giF-10内切葡聚糖酶基因的克隆表达及酶学性质分析
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摘要
纤维素酶是一组复合酶的统称,主要包括内切葡聚糖酶,外切葡聚糖酶和β－葡萄糖苷酶,这三种酶协同作用将纤维素转化成葡萄糖。天然微生物产生的纤维素酶活力不够高,生产成本较高。通过基因工程手段,提高纤维素酶的活性是降低纤维素酶生产成本的一个有效途径。
根据国际基因库(GenBank Accession No.:D83732.1)中注册的米曲霉内切葡聚糖酶CelB基因序列设计引物,序列分析发现该基因无内含子,故直接以提取的米曲霉基因组DNA为模板,PCR高保真扩增得到一条1200bp左右的特异条带,连接到pMD19-T-simple上。测序表明,其长度为1251bp,OFR为1251bp,编码417个氨基酸,5端有一个编码17个氨基酸的信号肽序列,其全长理论分子量为44.46KDa,序列比对发现与参考的内切葡聚糖酶基因序列相似性高达100%。将内切葡聚糖酶基因eg在GenBank中注册,登录号为Accession No. HQ739052。
以克隆的内切葡聚糖酶基因eg为基础,将eg定向插入到大肠杆菌表达载体pET32a上,得到重组表达质粒pET32a-eg,在大肠杆菌BL21(DE3)中表达,水解圈筛选结果表明已成功表达。
以克隆的内切葡聚糖酶基因eg为基础,将eg定向插入到酵母表达载体pPICZaA上,将pPICZaA-eg线性化后电转化酵母宿主菌X33感受态细胞获得大量阳性转化子,水解圈筛选结果表明己成功表达。SDS-PAGE分析表明,表达产物分子量约为65KDa。在摇瓶培养条件下,对酵母表达工程菌X33-eg进行发酵条件的优化,结果显示最适甲醇诱导浓度为0.75%,用1L三角瓶诱导培养五天达到最高酶活120U/mL。
对原始酶与重组酶的酶学性质比较发现,原始酶的最高酶活可达250U/mL,重组酶的单位酶活力为120U/mL,原始酶的最适pH和温度分别为4.0和55℃,在45℃-65℃和pH3.4-4.4范围内可保持内切葡聚糖酶最高活力80%以上。重组酶的最适pH和温度分别为4.0和45℃,在35℃-45℃和pH3.4-4.4范围范围内可保持内切葡聚糖酶最高活力80%以上。
Endoglucanase is one of the most important cellulose enzymes, which cooperates with cellobihydrolase andβ-1,4-glucosidase to translate cellulose to glucose completely. Cellulase produced by natural microorganism has low activity which make the production cost very high. By means of genetic engineering to improve the activity of cellulase is an effective way to reduce the cost of cellulase production.
According to the sequence of endoglucanase CelB gene (GenBank Accession No.: D83732.1) from Aspergillus oryzae, a pair of primers was designed and synthesized, sequence analysis revealed that the gene has no intron, and a high-fidelity polymerase was used for amplifying endoglucanase eg gene by using Aspergillus oryzae giF-10 genomic DNA as template, PCR amplified a specific band about 1200bp, then connected it to pMD19-T simple vector.
Sequencing showed that the length of eg gene was 1251bp, OFR was 1251bp, encoding 417 amino acids,5'end had a signal peptide sequence coding 17 amino acid, The full gene had an approximate molecular mass of 44.46KDa. Sequence alignment of endoglucanase gene with GenBank database showed it shared a 100% sequence identity with the CelB gene from Aspergillus oryzae submitted in GenBank. Then submitted endoglucanase eg gene sequence in GenBank, accession number was HQ739052.
The eg gene was cloned into the E.coli expression vector pET32a, then obtained recombinant plasmid pET32a-eg, expressed in Escherichia coli BL21 (DE3). Hydrolysis cycle tests showed that the expression was successful.
The eg gene was cloned into the yeast expression vector pPICZaA,then the pPICZaA-eg plasmid was linearized and electroporated into yeast competent cells of X33 host strain and get a lot of positive transformants, hydrolysis cycle tests showed that the expression was successful. and the SDS-PAGE showed that the molecular weight was about 65 KDa In the condition of shake flask culture, fermentation conditions of yeast expression engineering strain were optimized, the results showed that the optimal concentration of methanol induction was 0.75%, five-day induction culture with 1L flask reached the highest activity 120 U/mL.
Comparing original enzyme with recombinant of the enzymatic properties found that the original enzyme activity was 250 U/mL, the recombinant enzyme unit activity of was 120 U/mL, the original enzyme's optimum pH and temperature were 4.0 and 55℃, respectively.In the range of 45℃-65℃and pH3.4-4.4 it could maintain above 80% of the highest endoglucanase activity. Recombinant enzyme optimal pH and temperature were 4.0 and 45℃,respectively. In the range of 35℃-45℃and pH3.4-4.4 it could maintain above 80% of the highest endoglucanase activity.
根据国际基因库(GenBank Accession No.:D83732.1)中注册的米曲霉内切葡聚糖酶CelB基因序列设计引物,序列分析发现该基因无内含子,故直接以提取的米曲霉基因组DNA为模板,PCR高保真扩增得到一条1200bp左右的特异条带,连接到pMD19-T-simple上。测序表明,其长度为1251bp,OFR为1251bp,编码417个氨基酸,5端有一个编码17个氨基酸的信号肽序列,其全长理论分子量为44.46KDa,序列比对发现与参考的内切葡聚糖酶基因序列相似性高达100%。将内切葡聚糖酶基因eg在GenBank中注册,登录号为Accession No. HQ739052。
以克隆的内切葡聚糖酶基因eg为基础,将eg定向插入到大肠杆菌表达载体pET32a上,得到重组表达质粒pET32a-eg,在大肠杆菌BL21(DE3)中表达,水解圈筛选结果表明已成功表达。
以克隆的内切葡聚糖酶基因eg为基础,将eg定向插入到酵母表达载体pPICZaA上,将pPICZaA-eg线性化后电转化酵母宿主菌X33感受态细胞获得大量阳性转化子,水解圈筛选结果表明己成功表达。SDS-PAGE分析表明,表达产物分子量约为65KDa。在摇瓶培养条件下,对酵母表达工程菌X33-eg进行发酵条件的优化,结果显示最适甲醇诱导浓度为0.75%,用1L三角瓶诱导培养五天达到最高酶活120U/mL。
对原始酶与重组酶的酶学性质比较发现,原始酶的最高酶活可达250U/mL,重组酶的单位酶活力为120U/mL,原始酶的最适pH和温度分别为4.0和55℃,在45℃-65℃和pH3.4-4.4范围内可保持内切葡聚糖酶最高活力80%以上。重组酶的最适pH和温度分别为4.0和45℃,在35℃-45℃和pH3.4-4.4范围范围内可保持内切葡聚糖酶最高活力80%以上。
Endoglucanase is one of the most important cellulose enzymes, which cooperates with cellobihydrolase andβ-1,4-glucosidase to translate cellulose to glucose completely. Cellulase produced by natural microorganism has low activity which make the production cost very high. By means of genetic engineering to improve the activity of cellulase is an effective way to reduce the cost of cellulase production.
According to the sequence of endoglucanase CelB gene (GenBank Accession No.: D83732.1) from Aspergillus oryzae, a pair of primers was designed and synthesized, sequence analysis revealed that the gene has no intron, and a high-fidelity polymerase was used for amplifying endoglucanase eg gene by using Aspergillus oryzae giF-10 genomic DNA as template, PCR amplified a specific band about 1200bp, then connected it to pMD19-T simple vector.
Sequencing showed that the length of eg gene was 1251bp, OFR was 1251bp, encoding 417 amino acids,5'end had a signal peptide sequence coding 17 amino acid, The full gene had an approximate molecular mass of 44.46KDa. Sequence alignment of endoglucanase gene with GenBank database showed it shared a 100% sequence identity with the CelB gene from Aspergillus oryzae submitted in GenBank. Then submitted endoglucanase eg gene sequence in GenBank, accession number was HQ739052.
The eg gene was cloned into the E.coli expression vector pET32a, then obtained recombinant plasmid pET32a-eg, expressed in Escherichia coli BL21 (DE3). Hydrolysis cycle tests showed that the expression was successful.
The eg gene was cloned into the yeast expression vector pPICZaA,then the pPICZaA-eg plasmid was linearized and electroporated into yeast competent cells of X33 host strain and get a lot of positive transformants, hydrolysis cycle tests showed that the expression was successful. and the SDS-PAGE showed that the molecular weight was about 65 KDa In the condition of shake flask culture, fermentation conditions of yeast expression engineering strain were optimized, the results showed that the optimal concentration of methanol induction was 0.75%, five-day induction culture with 1L flask reached the highest activity 120 U/mL.
Comparing original enzyme with recombinant of the enzymatic properties found that the original enzyme activity was 250 U/mL, the recombinant enzyme unit activity of was 120 U/mL, the original enzyme's optimum pH and temperature were 4.0 and 55℃, respectively.In the range of 45℃-65℃and pH3.4-4.4 it could maintain above 80% of the highest endoglucanase activity. Recombinant enzyme optimal pH and temperature were 4.0 and 45℃,respectively. In the range of 35℃-45℃and pH3.4-4.4 it could maintain above 80% of the highest endoglucanase activity.
引文
[1]ChtistianP.The trichoderma cellulose regulartory puzzle[J].enzyme, Microb. Technol.1993,5(2):90-99
[2]M.K. Bhat, S. Bhat. Cellulose degrading enzymes and their potentialindustrial applications[J]. Biotechnol. Adv.1997,15(3-4):583-620
[3]M.E. Himmel, M.F. Ruth, C.E. Wyman. Cellulase for commodityproducts from cellulosic biomass[J]. Curr. Opin. Biotechnol.1999,10(4):358-64
[4]R.A. Laymon, W.S. Adney, A. Mohagheghi, et.al. Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli[J]. Appl. Biochem.Biotechnol.1996,57-58:389-97
[5]S. Takashima, A. Nakamura, H. Masaki, et.al. Cloning,sequencing, and expression of a thermostable cellulase gene of Humicola grisea[J].Biosci. Biotechnol. Biochem. 1997,61(2):245-50
[6]J. Hong, H. Tamaki, S. Akiba, K. Yamamoto, H. Kumagai. Cloningof a gene encoding a highly stable endo-B-1,4-Glucanase from Aspergillus niger and its expression in yeast[J] J. Biosci. Bioeng.2001,92(5):434-41
[7]高培基,曲音波,汪天虹.微生物降解纤维素机制的分子生物学研究进展[J].纤维素科学与技术.1995,3(2)：16-19
[8]阎伯旭,齐飞,张颖舒.纤维素酶分子结构和功能研究进展[J].生物化学与生物物理进,1999,26(3)：235-237
[9]谢占玲,吴润.纤维素酶的研究进展.2004,21(4)72-78
[10]钟发刚,王新华.饲用纤维素酶研究进展[J].中国微生态学杂志.2002,14(5)：308-309
[11]王娟.长梗木霉外切纤维素酶基因的克隆及其表达[D].福建：生命科学学院,2010
[12]张梁.里氏木霉BGLl基因的克隆及其在酿酒酵母中的表达[J].酿酒科技.2006,9：17-20
[13]Xiao Z,Wang T.Cloning and expression of Trichoderma reesei endoglucanase III(EGIII)gene in Saccharomyces cerevasiae[J].Wei Sheng Wu Xue Bao,2001,41(4):391-396
[14]乔宇.早氏木霉内切葡聚糖酶基因在毕赤酵母中的表达及酶学性质研究[J].菌物学报.2004,23(3)：388-396
[15]Takashima S,Nakamura A.Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei[J].Biochem(Tokyo).1999,125(4):728-736
[16]田生礼等.碱性纤维素酶基因在巴氏毕赤酵母中的表达及重组酵母菌发酵工艺的优化[J].中国生物制品学杂志.2009,22(5)：425-430
[17]蔡勇等.芽孢杆菌CY1-3株碱性纤维素酶基因celC的克隆及其在大肠杆菌中的表达[J].中国兽医科学.2006,36(12)：961-966
[18]Kim JO, Park SR, Lim WJ, et.al. Cloning and Characterization of Thermostable Endoglucanase (Ce18Y) from the Hyperthermophilic Aquifex aeolicus VF5[J].Biochemical and Biophysical Research Communications,2000,279(2):420-6
[19]Hakamada Y, Endo K, Takizawa S,et.al. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans[J]. Biochimicaet Biophysica Acta (BBA)-General Subjects,2002,1570(3):174-180
[20]Lee SJ, Lee KS, Kim SR, et.al. A novel cellulase gene from the mulberry longicorn beetle, Apriona germari:gene structure, expression, and enzymatic activity[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2005,140(4):551-60
[21]Yuguo Wang, Xiaonan Wang, Rentao Tang,et.al. A novel thermostable cellulase from Fervidobacterium nodosum[J]. Journal of Molecular Catalysis B:Enzymatic, 2010,66(4):294-301
[22]Li J, Tang C, Shi H, Wu M,et.al. Cloning and optimized expression of a neutral endoglucanase gene (nce15A) from Volvariella volvacea WX32 in Pichia pastoris[J] Journal of Bioscience and Bioengineering,2011,111(5):537-40
[23]WatanabeH,TokudaG. Animalce Hulases. [J]CellMolLife Sci,2001,58(9):1167-1178
[24]Watanabe H,Noda H,Tokuda G,et al.Acellulase gene of termite origin[J].Nature,1998,394(6691): 330-331
[25]Smant G,Stokkemans JP Yan YT,etal.Endogenous cellulases in animals:isolation of beta-14-endoglucanase genes from two species of plant-par-asitic cyst[J].Acad Sci USA, 1998,95(9):4906-4911
[24]崔宗均,李美丹,朴哲,等.一组高效稳定纤维素分解菌复合系MC1的筛选及功能[J].环境科学,2002,23(3)：36-39
[27]Bhat M K. Cellulases and related enzymes in biotechnology[J].Biotechnol Adv,2000,18(5):355-383
[28]Nuc P, Nuc K.Recombinant protein production in Escherichia coli[J].Postepy Biochem,2006,52(4):448-456
[29]Dong X,Tang B, Li J,et al. Expression and Purification of Intact and Functional Soybean(Glycine max)Seed Ferrition Complex in E sch eichia coli[J].J Microbiol Biotechnol,2008,18(2):299-307
[30]解庭波.大肠杆菌表达系统的研究进展[J].长江大学学报.2008,5(3)：77-82
[31]罗会颖,姚斌,袁铁铮等.来源于Escherichia coli的高比活植酸酶基因的高效表达[J].生物工程学报,2004,20(1)：78-84
[32]Li-Kou Zou,Hong-Ning WANG,Xin PAN,et al. Design and Expression of a Synthetic phyC Gene Encoding the Neutral Phytase in pichia pastoris[J].Acta Biochimica et Biophysica 2006,38(11):803-811
[33]Siyi Hu,Liangwei Li,Jingjuan Qiao,et al.Codon optimization, expression and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris[J].Protein Expression and Purification,2006,47(1):249-257
[34]余祖华,王红宁.利用巴斯德毕赤酵母表达外源蛋白的研究进展[J].生物技术通讯,2004,15(6)：614-616
[35]Clare JJ,Rayment FB,Ballantine SP,et al.Hight-level expression Of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integration of the gene [J].Nature Biotechnology,1991,9(5):455-60
[36]Hasslacher M,Schall M,Hayn M,et al. High-level in treacllular expression of hydroxynituile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J].Protein Expre Purif,1997,11(1):61-71
[37]蔡尽忠.影响外源基因在巴斯德毕赤酵母表达的因素[J].海峡药业.2006,18(6)：7-9
[38]Cregg J M, Cereghino J L, Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. Mol Biotechnol,2000,16(1):23-52
[39]李洪钊,李亮助.孙强明.巴斯德毕赤酵母表达系统的优化策略[J].微生物学报,2003,43(2)：288-292
[40]邬小兵,乐国伟.影响毕赤酵母高效表达外源蛋白的因素[J].生物技术,2002,12(4)：44-46
[41]李欣,郭树华.影响外源基因在巴氏毕赤酵母中表达的因素[J].生物技术通讯,2000.11(2)：132-134
[42]Sreekrishna K, Brankamp R G, Kropp K E. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene, 1997,190:55-62
[43]郑淑霞,沈志扬,刘树滔等.黑曲霉发酵粉中一种β-葡萄糖苷酶的分离纯化与表征[J].福州大学学报(自然科学版),2004,32(1)：101-105
[44]J.萨姆布鲁克,K.W.拉塞尔著.黄培堂译.分子克隆试验指南[M].第三版,北 京,科学出版社,2002
[45]王晓丽,苟琳.生物化学实验教程[M].四川,四川科学技术出版社,2005
[46]Chou C H, Aristidow A A, Meng S Y,et al. Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in E.coli[J].Biotechnol Bioeng,1997,190(1):55-62
[47]廖美德,谢秋玲,林剑等.外源基因在大肠杆菌中的高效表达[J].生命科学,2002,14(5)：283-287
[48]Kwon I,Ekino K,Goto M,et al.Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75[J].Bioscience Biotechnology and Biochemistry,1999,63 (10):1714-1720
[49]Wilkinson DL,Harrison RG. Predicting the solubility of recombinant proteins in Escherichia coli[J]. Biotechnology,1991,9(5):443-448
[50]Cavaco.paulo A,Morgado J,Andreaus J,et al.Interactions of cotton with CBD peptides [J].Enzyme and Microbial Technology,1999,25:639-643
[51]常巧玲.热纤梭菌β-1,4-内切葡聚糖酶基因celD在大肠杆菌和毕赤酵母中的表达.浙江,动物科学学院,2006
[52]肖志壮,王婷.瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达[J].微生物学报,2001,41(4)：391-396
[53]Cheng C,Nofihiro T,Shigezo U.Nucleotide sequence of the cellobiohydrase gene from Trichoderma viride [J].Nucleic Acids Res,1990,18(18):55-59
[54]陈新爱,夏黎明.里氏木霉纤维二糖酶bgl2基因的cDNA克隆及其在大肠杆菌中的表达[J].菌物系统,2002,21 f21：223-227
[55]Rashid MH, Javed MR, Kawaguchi T, Sumitani J, Arai M.Improvement of Aspergillus oryzaefor hyperproduction of endoglucanase:expression cloning of cmc-1 gene of Aspergillus aculeatus[J].Biotechnol Lett.2008,30(12):2165-2172
[56]Yu L X Gray B N.Rutzke C J,et al.Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco[J] Journal of Biotechnology,2007,131(3):362-369
[57]陈惠,胥兵,廖俊华等.内切葡聚糖酶基因在巨大芽胞杆菌中的表达及其酶学性质研究[J].遗传,2008,30(5)：649-654
[58]聂志强.产纤维素酶真菌的筛选及其酶学性质的研究[J].中国酿造,2009,3：81-83
[59]张苏龙,吕淑霞,林英等.一株产纤维素酶真菌的筛选、鉴定及酶学性质初步研究[J].微生物学杂志,2009,29(3)：72-76
[60]K.B.R.M. Krogh, H. Kastberg, C.I. Jφrgensen, et al.Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins [J].Enzyme and Microbial Technology,2009,44:359-367
[2]M.K. Bhat, S. Bhat. Cellulose degrading enzymes and their potentialindustrial applications[J]. Biotechnol. Adv.1997,15(3-4):583-620
[3]M.E. Himmel, M.F. Ruth, C.E. Wyman. Cellulase for commodityproducts from cellulosic biomass[J]. Curr. Opin. Biotechnol.1999,10(4):358-64
[4]R.A. Laymon, W.S. Adney, A. Mohagheghi, et.al. Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli[J]. Appl. Biochem.Biotechnol.1996,57-58:389-97
[5]S. Takashima, A. Nakamura, H. Masaki, et.al. Cloning,sequencing, and expression of a thermostable cellulase gene of Humicola grisea[J].Biosci. Biotechnol. Biochem. 1997,61(2):245-50
[6]J. Hong, H. Tamaki, S. Akiba, K. Yamamoto, H. Kumagai. Cloningof a gene encoding a highly stable endo-B-1,4-Glucanase from Aspergillus niger and its expression in yeast[J] J. Biosci. Bioeng.2001,92(5):434-41
[7]高培基,曲音波,汪天虹.微生物降解纤维素机制的分子生物学研究进展[J].纤维素科学与技术.1995,3(2)：16-19
[8]阎伯旭,齐飞,张颖舒.纤维素酶分子结构和功能研究进展[J].生物化学与生物物理进,1999,26(3)：235-237
[9]谢占玲,吴润.纤维素酶的研究进展.2004,21(4)72-78
[10]钟发刚,王新华.饲用纤维素酶研究进展[J].中国微生态学杂志.2002,14(5)：308-309
[11]王娟.长梗木霉外切纤维素酶基因的克隆及其表达[D].福建：生命科学学院,2010
[12]张梁.里氏木霉BGLl基因的克隆及其在酿酒酵母中的表达[J].酿酒科技.2006,9：17-20
[13]Xiao Z,Wang T.Cloning and expression of Trichoderma reesei endoglucanase III(EGIII)gene in Saccharomyces cerevasiae[J].Wei Sheng Wu Xue Bao,2001,41(4):391-396
[14]乔宇.早氏木霉内切葡聚糖酶基因在毕赤酵母中的表达及酶学性质研究[J].菌物学报.2004,23(3)：388-396
[15]Takashima S,Nakamura A.Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei[J].Biochem(Tokyo).1999,125(4):728-736
[16]田生礼等.碱性纤维素酶基因在巴氏毕赤酵母中的表达及重组酵母菌发酵工艺的优化[J].中国生物制品学杂志.2009,22(5)：425-430
[17]蔡勇等.芽孢杆菌CY1-3株碱性纤维素酶基因celC的克隆及其在大肠杆菌中的表达[J].中国兽医科学.2006,36(12)：961-966
[18]Kim JO, Park SR, Lim WJ, et.al. Cloning and Characterization of Thermostable Endoglucanase (Ce18Y) from the Hyperthermophilic Aquifex aeolicus VF5[J].Biochemical and Biophysical Research Communications,2000,279(2):420-6
[19]Hakamada Y, Endo K, Takizawa S,et.al. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans[J]. Biochimicaet Biophysica Acta (BBA)-General Subjects,2002,1570(3):174-180
[20]Lee SJ, Lee KS, Kim SR, et.al. A novel cellulase gene from the mulberry longicorn beetle, Apriona germari:gene structure, expression, and enzymatic activity[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2005,140(4):551-60
[21]Yuguo Wang, Xiaonan Wang, Rentao Tang,et.al. A novel thermostable cellulase from Fervidobacterium nodosum[J]. Journal of Molecular Catalysis B:Enzymatic, 2010,66(4):294-301
[22]Li J, Tang C, Shi H, Wu M,et.al. Cloning and optimized expression of a neutral endoglucanase gene (nce15A) from Volvariella volvacea WX32 in Pichia pastoris[J] Journal of Bioscience and Bioengineering,2011,111(5):537-40
[23]WatanabeH,TokudaG. Animalce Hulases. [J]CellMolLife Sci,2001,58(9):1167-1178
[24]Watanabe H,Noda H,Tokuda G,et al.Acellulase gene of termite origin[J].Nature,1998,394(6691): 330-331
[25]Smant G,Stokkemans JP Yan YT,etal.Endogenous cellulases in animals:isolation of beta-14-endoglucanase genes from two species of plant-par-asitic cyst[J].Acad Sci USA, 1998,95(9):4906-4911
[24]崔宗均,李美丹,朴哲,等.一组高效稳定纤维素分解菌复合系MC1的筛选及功能[J].环境科学,2002,23(3)：36-39
[27]Bhat M K. Cellulases and related enzymes in biotechnology[J].Biotechnol Adv,2000,18(5):355-383
[28]Nuc P, Nuc K.Recombinant protein production in Escherichia coli[J].Postepy Biochem,2006,52(4):448-456
[29]Dong X,Tang B, Li J,et al. Expression and Purification of Intact and Functional Soybean(Glycine max)Seed Ferrition Complex in E sch eichia coli[J].J Microbiol Biotechnol,2008,18(2):299-307
[30]解庭波.大肠杆菌表达系统的研究进展[J].长江大学学报.2008,5(3)：77-82
[31]罗会颖,姚斌,袁铁铮等.来源于Escherichia coli的高比活植酸酶基因的高效表达[J].生物工程学报,2004,20(1)：78-84
[32]Li-Kou Zou,Hong-Ning WANG,Xin PAN,et al. Design and Expression of a Synthetic phyC Gene Encoding the Neutral Phytase in pichia pastoris[J].Acta Biochimica et Biophysica 2006,38(11):803-811
[33]Siyi Hu,Liangwei Li,Jingjuan Qiao,et al.Codon optimization, expression and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris[J].Protein Expression and Purification,2006,47(1):249-257
[34]余祖华,王红宁.利用巴斯德毕赤酵母表达外源蛋白的研究进展[J].生物技术通讯,2004,15(6)：614-616
[35]Clare JJ,Rayment FB,Ballantine SP,et al.Hight-level expression Of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integration of the gene [J].Nature Biotechnology,1991,9(5):455-60
[36]Hasslacher M,Schall M,Hayn M,et al. High-level in treacllular expression of hydroxynituile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J].Protein Expre Purif,1997,11(1):61-71
[37]蔡尽忠.影响外源基因在巴斯德毕赤酵母表达的因素[J].海峡药业.2006,18(6)：7-9
[38]Cregg J M, Cereghino J L, Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. Mol Biotechnol,2000,16(1):23-52
[39]李洪钊,李亮助.孙强明.巴斯德毕赤酵母表达系统的优化策略[J].微生物学报,2003,43(2)：288-292
[40]邬小兵,乐国伟.影响毕赤酵母高效表达外源蛋白的因素[J].生物技术,2002,12(4)：44-46
[41]李欣,郭树华.影响外源基因在巴氏毕赤酵母中表达的因素[J].生物技术通讯,2000.11(2)：132-134
[42]Sreekrishna K, Brankamp R G, Kropp K E. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene, 1997,190:55-62
[43]郑淑霞,沈志扬,刘树滔等.黑曲霉发酵粉中一种β-葡萄糖苷酶的分离纯化与表征[J].福州大学学报(自然科学版),2004,32(1)：101-105
[44]J.萨姆布鲁克,K.W.拉塞尔著.黄培堂译.分子克隆试验指南[M].第三版,北 京,科学出版社,2002
[45]王晓丽,苟琳.生物化学实验教程[M].四川,四川科学技术出版社,2005
[46]Chou C H, Aristidow A A, Meng S Y,et al. Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in E.coli[J].Biotechnol Bioeng,1997,190(1):55-62
[47]廖美德,谢秋玲,林剑等.外源基因在大肠杆菌中的高效表达[J].生命科学,2002,14(5)：283-287
[48]Kwon I,Ekino K,Goto M,et al.Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75[J].Bioscience Biotechnology and Biochemistry,1999,63 (10):1714-1720
[49]Wilkinson DL,Harrison RG. Predicting the solubility of recombinant proteins in Escherichia coli[J]. Biotechnology,1991,9(5):443-448
[50]Cavaco.paulo A,Morgado J,Andreaus J,et al.Interactions of cotton with CBD peptides [J].Enzyme and Microbial Technology,1999,25:639-643
[51]常巧玲.热纤梭菌β-1,4-内切葡聚糖酶基因celD在大肠杆菌和毕赤酵母中的表达.浙江,动物科学学院,2006
[52]肖志壮,王婷.瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达[J].微生物学报,2001,41(4)：391-396
[53]Cheng C,Nofihiro T,Shigezo U.Nucleotide sequence of the cellobiohydrase gene from Trichoderma viride [J].Nucleic Acids Res,1990,18(18):55-59
[54]陈新爱,夏黎明.里氏木霉纤维二糖酶bgl2基因的cDNA克隆及其在大肠杆菌中的表达[J].菌物系统,2002,21 f21：223-227
[55]Rashid MH, Javed MR, Kawaguchi T, Sumitani J, Arai M.Improvement of Aspergillus oryzaefor hyperproduction of endoglucanase:expression cloning of cmc-1 gene of Aspergillus aculeatus[J].Biotechnol Lett.2008,30(12):2165-2172
[56]Yu L X Gray B N.Rutzke C J,et al.Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco[J] Journal of Biotechnology,2007,131(3):362-369
[57]陈惠,胥兵,廖俊华等.内切葡聚糖酶基因在巨大芽胞杆菌中的表达及其酶学性质研究[J].遗传,2008,30(5)：649-654
[58]聂志强.产纤维素酶真菌的筛选及其酶学性质的研究[J].中国酿造,2009,3：81-83
[59]张苏龙,吕淑霞,林英等.一株产纤维素酶真菌的筛选、鉴定及酶学性质初步研究[J].微生物学杂志,2009,29(3)：72-76
[60]K.B.R.M. Krogh, H. Kastberg, C.I. Jφrgensen, et al.Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins [J].Enzyme and Microbial Technology,2009,44:359-367