黑曲霉内切β-1，4-葡聚糖酶基因的克隆，优化及在毕赤酵母中的表达研究
|
摘要
内切β-1,4-葡聚糖酶(EC3.2.1.4)是一种重要的工业用酶,广泛应用于饲料工业,酿造业等领域。随着生产的发展,β-1,4-葡聚糖酶需求量日益增加。本实验的主要目的就是,从分泌热稳定性的β-1,4-葡聚糖酶的黑曲霉高产菌株中,克隆该基因,利用基因工程手段实现高效表达,满足工业生产的需求。
实验首先是利用RT-PCR技术,提取黑曲霉(Aspergillus niger)L3的总RNA进行反转录,并通过PCR扩增得到去除天然信号肽的β-1,4-葡聚糖酶基因egⅠ,将其插入到毕赤酵母表达载体pPIC9k上,使之位于α-因子信号肽下游,并与之同框,构建重组表达质粒pPIC9k-egⅠ,电击转化毕赤酵母GS115,经MM、MD、CMC-Na和G418平板筛选,得到重组毕赤酵母工程菌1-1#和1-5#,在摇瓶发酵水平上,以0.5%的β-葡聚糖为底物检测,酶活分别达到1 456 U/mL和1 928 U/mL。重组酶最适pH为5.0,最适反应温度为70℃,在70℃时保温30 min后仍然具有90%以上的活力。
为了进一步提高表达量,根据毕赤酵母偏爱的密码子优化来源于A.niger L3的内切β-1,4-葡聚糖酶的DNA序列,共改变193个碱基,G+C%由54%下降到44.22%。设计了14对寡聚核苷酸引物,采用重叠延伸PCR三步法获得全长基因序列共改变。将其插入到毕赤酵母表达载体pPIC9K上,构建重组表达质粒pPIC9k-syn-egⅠ,电击转化毕赤酵母GS115,经MM、MD、CMC-Na和G418平板以及摇瓶筛选,得到重组毕赤酵母工程菌2-7#。摇瓶发酵条件下,以0.5%的葡聚糖和1%的CMC-Na为底物检测,酶活分别达到3 658 U/mL和591.9 U/mL;采用50 L发酵罐进行发酵,酶活则分别达到65 649.6 U/mL和39 728.32 U/mL。
Endo-β-1,4-glucanase(EC3.2.1.4),as an important industrial enzyme,has been widely used in the many fields such as brewing and animal feed industry.With the developing of production,the need for it is increasing day and day.The purpose of this research is to clone thermostable endo-β-1,4-glucanase from a strain of Aspergillus niger, which effectively secreted EGⅠ,to improve the expression level with the methods of molecular biology and gene engineering and meet the need in industrial production.
The gene encoding endo-β-1,4-glucanase from Aspergillus niger L3 was isolated with RT-PCR method.egⅠwas eliminated its native signal peptide by PCR and inserted into the pPIC9K vector of Pichia pastoris in reading frame withα-factor secreting signal peptide sequence to construct the recombinant plasmid pPIC9K-egⅠ.The recombinant plasmid pPIC9K-egⅠwas transformed into P.pastoris GS115 with electroporation.Two of recombinant P.pastoris stains 1-1# and 1-5# were obtained by screening with MM,MD, CMC-Na and G418 plates and the activity of recombinant EGⅠcould reach to 1456 U/mL and 1928U/mL respectively.The optimal temperature for the recombinant EGⅠwas 70℃and the optimal pH was 5.0.
To improve expression efficiency in recombinantβ-1,4-glucanase in P.pastoris,the egⅠgene from A.niger L3 was modified with codon optimization.A total of 193 nucleotides are changed,the G+C ratio was decreased from 54%to 44.22%.14 pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egⅠgene was synthesized using PCR-basic three-step DNA synthesis method.The modified syn-egⅠgene was inserted into the pPIC9K vector to construct the recombinant plasmid pPIC9K-syn-egⅠ.The recombinant plasmid pPIC9K-syn-egⅠwas transformed into P. pastoris GS115 with electroporation.A recombinant P.pastoris stains 2-7# was obtained by screening with MM,MD,CMC-Na and G418 plates.The activity of recombinant EGⅠat shaking flask level could reach to 3658 U/mL and 591.9 U/mL with 0.5%barleyβ-glucan and 1%CMC-Na as substrate,respectively.At 50 L fermentor level,theβ-1, 4-glucanase activity was 65649.6 and 39728.32 U/mL with 0.5%barleyβ-glucan and 1% CMC-Na as substrate.
实验首先是利用RT-PCR技术,提取黑曲霉(Aspergillus niger)L3的总RNA进行反转录,并通过PCR扩增得到去除天然信号肽的β-1,4-葡聚糖酶基因egⅠ,将其插入到毕赤酵母表达载体pPIC9k上,使之位于α-因子信号肽下游,并与之同框,构建重组表达质粒pPIC9k-egⅠ,电击转化毕赤酵母GS115,经MM、MD、CMC-Na和G418平板筛选,得到重组毕赤酵母工程菌1-1#和1-5#,在摇瓶发酵水平上,以0.5%的β-葡聚糖为底物检测,酶活分别达到1 456 U/mL和1 928 U/mL。重组酶最适pH为5.0,最适反应温度为70℃,在70℃时保温30 min后仍然具有90%以上的活力。
为了进一步提高表达量,根据毕赤酵母偏爱的密码子优化来源于A.niger L3的内切β-1,4-葡聚糖酶的DNA序列,共改变193个碱基,G+C%由54%下降到44.22%。设计了14对寡聚核苷酸引物,采用重叠延伸PCR三步法获得全长基因序列共改变。将其插入到毕赤酵母表达载体pPIC9K上,构建重组表达质粒pPIC9k-syn-egⅠ,电击转化毕赤酵母GS115,经MM、MD、CMC-Na和G418平板以及摇瓶筛选,得到重组毕赤酵母工程菌2-7#。摇瓶发酵条件下,以0.5%的葡聚糖和1%的CMC-Na为底物检测,酶活分别达到3 658 U/mL和591.9 U/mL;采用50 L发酵罐进行发酵,酶活则分别达到65 649.6 U/mL和39 728.32 U/mL。
Endo-β-1,4-glucanase(EC3.2.1.4),as an important industrial enzyme,has been widely used in the many fields such as brewing and animal feed industry.With the developing of production,the need for it is increasing day and day.The purpose of this research is to clone thermostable endo-β-1,4-glucanase from a strain of Aspergillus niger, which effectively secreted EGⅠ,to improve the expression level with the methods of molecular biology and gene engineering and meet the need in industrial production.
The gene encoding endo-β-1,4-glucanase from Aspergillus niger L3 was isolated with RT-PCR method.egⅠwas eliminated its native signal peptide by PCR and inserted into the pPIC9K vector of Pichia pastoris in reading frame withα-factor secreting signal peptide sequence to construct the recombinant plasmid pPIC9K-egⅠ.The recombinant plasmid pPIC9K-egⅠwas transformed into P.pastoris GS115 with electroporation.Two of recombinant P.pastoris stains 1-1# and 1-5# were obtained by screening with MM,MD, CMC-Na and G418 plates and the activity of recombinant EGⅠcould reach to 1456 U/mL and 1928U/mL respectively.The optimal temperature for the recombinant EGⅠwas 70℃and the optimal pH was 5.0.
To improve expression efficiency in recombinantβ-1,4-glucanase in P.pastoris,the egⅠgene from A.niger L3 was modified with codon optimization.A total of 193 nucleotides are changed,the G+C ratio was decreased from 54%to 44.22%.14 pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egⅠgene was synthesized using PCR-basic three-step DNA synthesis method.The modified syn-egⅠgene was inserted into the pPIC9K vector to construct the recombinant plasmid pPIC9K-syn-egⅠ.The recombinant plasmid pPIC9K-syn-egⅠwas transformed into P. pastoris GS115 with electroporation.A recombinant P.pastoris stains 2-7# was obtained by screening with MM,MD,CMC-Na and G418 plates.The activity of recombinant EGⅠat shaking flask level could reach to 3658 U/mL and 591.9 U/mL with 0.5%barleyβ-glucan and 1%CMC-Na as substrate,respectively.At 50 L fermentor level,theβ-1, 4-glucanase activity was 65649.6 and 39728.32 U/mL with 0.5%barleyβ-glucan and 1% CMC-Na as substrate.
引文
1.贝锦龙,陈庄,杨林,廖玲,王章,蒋宗勇.人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕赤酵母系统中的高效表达.生物工程学报,2001,17(3):254-258
2.陈惠,赵海霞,王红宁,杨婉身,吴琦,倪燕.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量.中国生物化学与分子生物学报,2005,21(2):171-175
3.冯德江,刘翔,李旭刚.tRNA丰度与基因表达的关系.中国生物工程杂志.2002,(6):4-7
4.郭美锦,朱泰承,张明,储炬,庄英萍,张嗣良.重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响.中国生物工程杂志,2007,27(7):7-11
5.黄火清,罗会颖,柏映国,王亚茹,姚斌,孟昆,袁铁铮,杨培龙.来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达.微生物学报,2006,46(6):945-950
6.李相前,邵蔚蓝.海栖热袍菌内切葡聚糖酶Cell2B与木聚糖酶XynA CBD结构域融合基因的构建、表达及融合酶性质分析.微生物学报,2006,46(5):726-729
7.李寅,高海军,陈坚编著.高细胞密度发酵技术,北京:化学工业出版社,2006,271-279
8.马冬梅,白俊杰,简清,劳海华,叶星,罗建仁.中华鲟cystatin酵母工程菌的发酵条件和表达产物的纯化.华中农业大学学报,2003,22(6):584-587
9.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(2):47-55
10.彭日荷,熊爱生,李贤,范惠琴,姚泉洪,郭美锦,张嗣良。应用毕赤酵母高效表达高温植酸酶,生物化学与生物物理学报,2002,34(6):725-730
11.王清路,李俏俏,薛金艳,窦烨,王红艳,张玉军,张杰.巴斯德毕赤酵母表达系统的特点及应用.生物技术通讯,2006,17(4):640-643
12.韦石拓,甘风琼.巴斯德毕赤酵母新型分泌表达载体构建.广西农业生物科学,2003,22(1):37-40
13.许善峰.苜蓿根瘤菌内切β-1,4-葡聚糖酶EndS-1在Escherichia coli和Pichia pastoris中表达及定点突变的研究(硕士学位论文).扬州:扬州大学生技学院.2006
14.谢涛,王红宁.外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展.生物技术通讯,2006,17(3):415-416
15.熊爱生,彭日荷,李贤,范惠琴,姚泉洪,郭美锦,张嗣良.信号肽序列对毕赤酵母表达 外源蛋白质的影响.生物化学与生物物理学报.2003,35(2):154-160
16.熊涛,徐立荣,曾哲灵.β-葡聚糖酶的研究进展.四川食品与发酵,2006,6:1-4
17.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(3):233-237
18.殷幼平,曹月青,何正波,董亚敏。桑粒肩天牛3种纤维素消化酶的分布.林业科学,2000,40(6):103-106
19.于平.巴斯德毕赤酵母表达系统研究进展.工业微生物,2005,35(3):50-54
20.张朝春 梁伟锋 杨希才.神经营养素-3基因的人工合成及其在毕赤酵母中的分泌性表达.微生物学报,2004,44(2):210-214
21.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(2).58-58
22.Aho S.Arffman A,Korhola M.Saccharomgces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases.Appl.Microbiol.Biotechnol.1996,46(1):36-45
23.Akiba S,Kimura Y,Yamamoto K,Kumagai H.Purification and characterization of a protease-resistant cellulose from Aspergillus niger.Journal of Fermentation and Bioengineering.1995,79(2):125-130
24.Beer M U,Wood P J,Weisz j.Molecular weight distribution and(1,3)-(1,4)-β-D-glucan content of consecutive extracts of various oat and barley cultivars.Cereal Chemistry.1997,74:476-780
25.Beguin P,Aubert J P.The biological degradation of cellulose.FEMS Microbiology Reviews,1994,13(1):25-58
26.Brierley R A.Secretion of recombinant human insulin-like growth factor Ⅰ(IGF-1).Methods in Molecular Biology.1998,103:149-177
27.Cereghino J L,Cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiology Reviews,2000,24:45-66
28.Chung B H,Nam S W,Kim B M.Communication to the editor highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides.Biotechnology and Bioengineering,1996,49:473-479
29.David M H,Jacek L.DNAWorks:an automated method for designing oligonucleotides for PCR-based gene synthesis.Nucleic Acids Research,2002,30:e43
30.Dominguez R,Souchon H,Spinelli S,Dauter Z,.Wilson K S,Chauvaux S,Beguin P,Alzari P M.A common protein fold and similar active site in two distinct families of beta-glycanases. Nature Structural Biology , 1995 , 2(2): 569-576
31. Delroisse J M, Dannau M, Gilsoul J J. Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris. Protein Expression and Purification, 2005,42: 286-294
32. Gao X, Yo P, Keith A, RaganT J, Harris TK. Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for highfidelity assembly of longer gene sequences. Nucleic Acids Research, 2003, 31, e143
33. Gellissen G. Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 2000, 54: 741-750
34. Graham S, Francis Y.M. C. Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification. 2002, 26(1): 96-105
35. Himmel M E, Ruth M F, Wyman C E. Cellulase for commodity products from cellulosic biomass. Curr Opin Biotechnol, 1999, 10(4): 358-364
36. Hong J, Hisanori T, Shunichi A , Yamamtot K, Kumagai H. Cloning of a gene encoding a highly Stable endo-P-l,4-glucanse from Aspergillus niger and its expression in yeast. Journal of Bioscience and Bioengineering, 2001, 92(5): 434-439
37. Hong J, Wang Y H, Hidehiko K, Tamaki H. Construction of thermotolerant yeast expressing thermostable cellulose genes. Journal of Biotechnology, 2007, 130(2): 114-123
38. Hu S Y, Li L W, Qiao J J, Guo Y J. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expression and Purification, 2006, xxx - xxx
39. Kjeldsen T, Pettenss A F, Hich M. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnology and Applied Biochemistry. 1999, 29(1): 79-86
40. Koti Sreekrishna, Robert G Brankamp, Kropp K E, Blankenship D T, Tsay J T, Smith P L, Wierschke J D. Strategies for optimal synthesis and secretion of heterologous proteins in the methylptrophic yaest Pichia pastoris, Gene, 1997, 190: 55-62
41. Lam K H, Chow K C, Wong W K. Construction of an efficient Bacillus subcillus system for extracellular production of heterologous proteins. Journal of Biotechnology. 1998,63(3): 167-177
42. Li Z J, Xiong F, Lin Q S, d'Anjou M, Daugulis A J, Yang D S C, Hew C L:
43. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expression and Purification, 2001, 21(3): 438-445
44. Mansur M, Cabello C, Hernandezd L. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnology Letters 2005, 27: 339-345
45. Nakamura A, Fukunmori F, Horinouchi. Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase. J. Biol. Chem, 1991, 266: 1579-1583
46. N. Kitamoto M, Go T. Shibayama T, Kimura Y, Kito K, Ohmiya N, Tsukagoshi. Molecular cloning, purification and characterization of two endo-1,4-b-glucanases from Aspergillus oryzae KBN616. Appl Microbiol Biotechnol. 1996,46: 538-544
47. Olse O,Thomsen K K, Weber J. Transplanting two unique beta-glucanase catalytic activ -ities into one multienzyme which forms glucose. Biotechnology, 1996, 14(1): 71-76
48. Orengo C. Protein superfamilies and domain superfolds.Nature,1994 ,367(6464): 631-634
49. Oriol Cos, Ramon Ramon, Jose Luis Montesinos, Francisco Valero. Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review, Microbial Cell Factories 2006, 5:17
50. Outchkourov N S, Stiekema W J, Jongsma M A. Optimization of the expression of equistatin in Pichia pastoris. Protein Expression and Purification, 2002, 24: 18-24
51. Palomer X, inguez-Puigjaner E D, Vendrell M. Imma Llop-Tous. Study of the strawberry Cell endo-β-(1, 4)-glucanase protein accumulation and characterization of its in vitro activity by heterologous expression in Pichia pastoris. Plant Science , 2004, 167:509-518
52. Paradis, F W, Warren, R A J, Kilburn D G, Miller J. The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum. Gene. 1987, 61: 199-206
53. Stemmer W P, Crameri A, Ha K D, Brennan T M, Heyneker H L. Single-step assembly of a gene and entire plasmid from large number oligonucleotides. Gene, 1995, 164,49-53.
54. Sue Macauley-Patrick, Mariana L. Fazenda, Brian McNeil. Heterologous protein production using the Pichia pastoris expression system. Yeast, 2005, 22: 249-270
55. Sulzenbacher G. Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group. Biochemistry, 1996 , 35 (48): 15280-15287
56. Teather R M, Wood P J. Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen. Applied and Environmental Microbiology, 1982, 8: 777-780
57. Teng D, Fan Y, YangY L. Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Applied Microbiology and Biotechnology, 2007, 74(5): 1074-1083
58. Vassileva A, chugh D A, swaminatha S. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 2001, 21(5): 71-75
59. Wood B E, Beall D S, Ingram L O. Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives of Escherichia coil KO11. Biotechnol Bioeng, 1997, 55(3): 547-555
60. Wood T M. The cellulase of fusarium solani purification and specificity of the (1, 4) - glucosidase components. Biochem, 1971, 121: 35
61. Wu J M, Lin J C, Chieng L L, Lee C K, Hsu T A. Combined use of GAP and AOX1 promoter to enhance the expression of human granulocyte-macrophage colony-stimulating factor in Pichia pastoris. Enz Microb Technol, 2003, 33(4): 453-459
62. Xiong A S, Yao Q H, Peng R H. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Research, 2004, 32, (12): e98
63. Xiong A S, Yao Q H, Peng R H. High level expression of a recombinant acid phytase gene in Pichia pastoris. Journal of Applied Microbiology 2005, 98, 418-428
64. Yadava A, Ockenhouse C F. Effect of Codon Optimization on Expression Levels of a Functionally Folded Malaria Vaccine Candidate in Prokaryotic and Eukaryotic Expression Systems. Infection and Immunity, 2003, 71(9): 4961-4969
2.陈惠,赵海霞,王红宁,杨婉身,吴琦,倪燕.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量.中国生物化学与分子生物学报,2005,21(2):171-175
3.冯德江,刘翔,李旭刚.tRNA丰度与基因表达的关系.中国生物工程杂志.2002,(6):4-7
4.郭美锦,朱泰承,张明,储炬,庄英萍,张嗣良.重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响.中国生物工程杂志,2007,27(7):7-11
5.黄火清,罗会颖,柏映国,王亚茹,姚斌,孟昆,袁铁铮,杨培龙.来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达.微生物学报,2006,46(6):945-950
6.李相前,邵蔚蓝.海栖热袍菌内切葡聚糖酶Cell2B与木聚糖酶XynA CBD结构域融合基因的构建、表达及融合酶性质分析.微生物学报,2006,46(5):726-729
7.李寅,高海军,陈坚编著.高细胞密度发酵技术,北京:化学工业出版社,2006,271-279
8.马冬梅,白俊杰,简清,劳海华,叶星,罗建仁.中华鲟cystatin酵母工程菌的发酵条件和表达产物的纯化.华中农业大学学报,2003,22(6):584-587
9.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(2):47-55
10.彭日荷,熊爱生,李贤,范惠琴,姚泉洪,郭美锦,张嗣良。应用毕赤酵母高效表达高温植酸酶,生物化学与生物物理学报,2002,34(6):725-730
11.王清路,李俏俏,薛金艳,窦烨,王红艳,张玉军,张杰.巴斯德毕赤酵母表达系统的特点及应用.生物技术通讯,2006,17(4):640-643
12.韦石拓,甘风琼.巴斯德毕赤酵母新型分泌表达载体构建.广西农业生物科学,2003,22(1):37-40
13.许善峰.苜蓿根瘤菌内切β-1,4-葡聚糖酶EndS-1在Escherichia coli和Pichia pastoris中表达及定点突变的研究(硕士学位论文).扬州:扬州大学生技学院.2006
14.谢涛,王红宁.外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展.生物技术通讯,2006,17(3):415-416
15.熊爱生,彭日荷,李贤,范惠琴,姚泉洪,郭美锦,张嗣良.信号肽序列对毕赤酵母表达 外源蛋白质的影响.生物化学与生物物理学报.2003,35(2):154-160
16.熊涛,徐立荣,曾哲灵.β-葡聚糖酶的研究进展.四川食品与发酵,2006,6:1-4
17.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(3):233-237
18.殷幼平,曹月青,何正波,董亚敏。桑粒肩天牛3种纤维素消化酶的分布.林业科学,2000,40(6):103-106
19.于平.巴斯德毕赤酵母表达系统研究进展.工业微生物,2005,35(3):50-54
20.张朝春 梁伟锋 杨希才.神经营养素-3基因的人工合成及其在毕赤酵母中的分泌性表达.微生物学报,2004,44(2):210-214
21.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(2).58-58
22.Aho S.Arffman A,Korhola M.Saccharomgces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases.Appl.Microbiol.Biotechnol.1996,46(1):36-45
23.Akiba S,Kimura Y,Yamamoto K,Kumagai H.Purification and characterization of a protease-resistant cellulose from Aspergillus niger.Journal of Fermentation and Bioengineering.1995,79(2):125-130
24.Beer M U,Wood P J,Weisz j.Molecular weight distribution and(1,3)-(1,4)-β-D-glucan content of consecutive extracts of various oat and barley cultivars.Cereal Chemistry.1997,74:476-780
25.Beguin P,Aubert J P.The biological degradation of cellulose.FEMS Microbiology Reviews,1994,13(1):25-58
26.Brierley R A.Secretion of recombinant human insulin-like growth factor Ⅰ(IGF-1).Methods in Molecular Biology.1998,103:149-177
27.Cereghino J L,Cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiology Reviews,2000,24:45-66
28.Chung B H,Nam S W,Kim B M.Communication to the editor highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides.Biotechnology and Bioengineering,1996,49:473-479
29.David M H,Jacek L.DNAWorks:an automated method for designing oligonucleotides for PCR-based gene synthesis.Nucleic Acids Research,2002,30:e43
30.Dominguez R,Souchon H,Spinelli S,Dauter Z,.Wilson K S,Chauvaux S,Beguin P,Alzari P M.A common protein fold and similar active site in two distinct families of beta-glycanases. Nature Structural Biology , 1995 , 2(2): 569-576
31. Delroisse J M, Dannau M, Gilsoul J J. Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris. Protein Expression and Purification, 2005,42: 286-294
32. Gao X, Yo P, Keith A, RaganT J, Harris TK. Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for highfidelity assembly of longer gene sequences. Nucleic Acids Research, 2003, 31, e143
33. Gellissen G. Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 2000, 54: 741-750
34. Graham S, Francis Y.M. C. Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification. 2002, 26(1): 96-105
35. Himmel M E, Ruth M F, Wyman C E. Cellulase for commodity products from cellulosic biomass. Curr Opin Biotechnol, 1999, 10(4): 358-364
36. Hong J, Hisanori T, Shunichi A , Yamamtot K, Kumagai H. Cloning of a gene encoding a highly Stable endo-P-l,4-glucanse from Aspergillus niger and its expression in yeast. Journal of Bioscience and Bioengineering, 2001, 92(5): 434-439
37. Hong J, Wang Y H, Hidehiko K, Tamaki H. Construction of thermotolerant yeast expressing thermostable cellulose genes. Journal of Biotechnology, 2007, 130(2): 114-123
38. Hu S Y, Li L W, Qiao J J, Guo Y J. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expression and Purification, 2006, xxx - xxx
39. Kjeldsen T, Pettenss A F, Hich M. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnology and Applied Biochemistry. 1999, 29(1): 79-86
40. Koti Sreekrishna, Robert G Brankamp, Kropp K E, Blankenship D T, Tsay J T, Smith P L, Wierschke J D. Strategies for optimal synthesis and secretion of heterologous proteins in the methylptrophic yaest Pichia pastoris, Gene, 1997, 190: 55-62
41. Lam K H, Chow K C, Wong W K. Construction of an efficient Bacillus subcillus system for extracellular production of heterologous proteins. Journal of Biotechnology. 1998,63(3): 167-177
42. Li Z J, Xiong F, Lin Q S, d'Anjou M, Daugulis A J, Yang D S C, Hew C L:
43. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expression and Purification, 2001, 21(3): 438-445
44. Mansur M, Cabello C, Hernandezd L. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnology Letters 2005, 27: 339-345
45. Nakamura A, Fukunmori F, Horinouchi. Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase. J. Biol. Chem, 1991, 266: 1579-1583
46. N. Kitamoto M, Go T. Shibayama T, Kimura Y, Kito K, Ohmiya N, Tsukagoshi. Molecular cloning, purification and characterization of two endo-1,4-b-glucanases from Aspergillus oryzae KBN616. Appl Microbiol Biotechnol. 1996,46: 538-544
47. Olse O,Thomsen K K, Weber J. Transplanting two unique beta-glucanase catalytic activ -ities into one multienzyme which forms glucose. Biotechnology, 1996, 14(1): 71-76
48. Orengo C. Protein superfamilies and domain superfolds.Nature,1994 ,367(6464): 631-634
49. Oriol Cos, Ramon Ramon, Jose Luis Montesinos, Francisco Valero. Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review, Microbial Cell Factories 2006, 5:17
50. Outchkourov N S, Stiekema W J, Jongsma M A. Optimization of the expression of equistatin in Pichia pastoris. Protein Expression and Purification, 2002, 24: 18-24
51. Palomer X, inguez-Puigjaner E D, Vendrell M. Imma Llop-Tous. Study of the strawberry Cell endo-β-(1, 4)-glucanase protein accumulation and characterization of its in vitro activity by heterologous expression in Pichia pastoris. Plant Science , 2004, 167:509-518
52. Paradis, F W, Warren, R A J, Kilburn D G, Miller J. The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum. Gene. 1987, 61: 199-206
53. Stemmer W P, Crameri A, Ha K D, Brennan T M, Heyneker H L. Single-step assembly of a gene and entire plasmid from large number oligonucleotides. Gene, 1995, 164,49-53.
54. Sue Macauley-Patrick, Mariana L. Fazenda, Brian McNeil. Heterologous protein production using the Pichia pastoris expression system. Yeast, 2005, 22: 249-270
55. Sulzenbacher G. Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group. Biochemistry, 1996 , 35 (48): 15280-15287
56. Teather R M, Wood P J. Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen. Applied and Environmental Microbiology, 1982, 8: 777-780
57. Teng D, Fan Y, YangY L. Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Applied Microbiology and Biotechnology, 2007, 74(5): 1074-1083
58. Vassileva A, chugh D A, swaminatha S. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 2001, 21(5): 71-75
59. Wood B E, Beall D S, Ingram L O. Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives of Escherichia coil KO11. Biotechnol Bioeng, 1997, 55(3): 547-555
60. Wood T M. The cellulase of fusarium solani purification and specificity of the (1, 4) - glucosidase components. Biochem, 1971, 121: 35
61. Wu J M, Lin J C, Chieng L L, Lee C K, Hsu T A. Combined use of GAP and AOX1 promoter to enhance the expression of human granulocyte-macrophage colony-stimulating factor in Pichia pastoris. Enz Microb Technol, 2003, 33(4): 453-459
62. Xiong A S, Yao Q H, Peng R H. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Research, 2004, 32, (12): e98
63. Xiong A S, Yao Q H, Peng R H. High level expression of a recombinant acid phytase gene in Pichia pastoris. Journal of Applied Microbiology 2005, 98, 418-428
64. Yadava A, Ockenhouse C F. Effect of Codon Optimization on Expression Levels of a Functionally Folded Malaria Vaccine Candidate in Prokaryotic and Eukaryotic Expression Systems. Infection and Immunity, 2003, 71(9): 4961-4969