细菌纤维素外切酶基因的筛选及耐热纤维素酶基因在枯草杆菌系统里的表达
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摘要
筛选性能优良的纤维素酶组分和建立高效分泌纤维素酶的表达系统对于木质纤维素的利用和生物炼制行业成本的降低非常重要。构建分泌纤维素酶的重组菌有很多的优势,比如产酶营养需求低,便于在分子水平上对产酶基因进行表达调控等。
本研究首先借助Escherichia coli BL21 (DE3)/pET-28a (+)表达系统,筛选了来自热纤梭菌(Clostridium thermocellum),解纤维梭菌(Clostridium cellulolyticum)和野油菜黄单孢菌(Xanthomonas campestris pv. campestris)的近20种细菌纤维素外切酶基因,比较了其外切酶的酶活。同时为了实现纤维素酶的胞外分泌表达,建立了枯草杆菌表达系统Bacillus subtilis WB600 (WB800)/pP43JM2,随后将来源于热纤梭菌的外切葡聚糖酶CbhA, CelK, Cel48S, Cel48Y, CelO和内切葡聚糖CelA以及欧文氏菌(Erwinia carotovora subsp.)的内切葡聚糖酶CelV,嗜热放线菌(Thermomonospora fusca)的内切酶CelE在枯草杆菌表达系统里进行了表达。实验结果表明,除了外切酶CelO外,其它四种纤维素外切酶和三种不同来源的纤维素内切酶在枯草杆菌WB600和WB800中都实现了成功的分泌型表达,以磷酸处理的微晶纤维素为底物,外切酶在枯草杆菌W13800表达系统中的酶活要普遍高于相应的WB600的酶活,外切酶CbhA释放的还原糖的量可以达到300mg/L,而WB600分泌的内切酶CelA释放的还原糖可以高达1250 mg/L。对于微晶纤维素和稀酸处理的秸秆等结晶度较高的底物,外切酶CbhA和内切酶CelA呈现良好的协同作用。在枯草杆菌里表达纤维素酶为实现纤维素酶多组分的人工组装以及枯草杆菌整合生物工艺菌种的构建奠定了基础。
The screening for the powerful cellulase components remains to be a challenging work for the biorefinery research and development. The engineered bacterial strains for production of cellulase would provide many advantages, such as the low nutrients requirement, easy manipulation of genetic engineering, etc.
In this study, about 20 cellobiohydrolase genes sourced from Clostridium thermocellum DSM 1237, Clostridium cellulolyticum DSM 5812 and Xanthomonas campestris pv. campestris strain 8004, were tested to be expressed in Escherichia coli BL21 (DE3)/pET-28a(+) system for activity evaluation.In order to realize the secretion of cellulases extracellularly, Bacillus subtilis WB600 (WB800)/pP43JM2 expression system was constructed. When the targeting genes including cellobiohydrolase genes from Clostridium thermocellum, endoglucanse genes from Clostridium thermocellum, Erwinia carotovora subsp. and Thermomonospora fusca.were cloned into pP43JM2 and expressed in B. subtilis WB600 and WB800, all the cellulases, except for celO, were expressed and secreted into the broth and showed the activities with phosphoric acid swollen cellulose as the substrate. For cellobiohydrolases, the enzyme activities from WB800 were much higher than that from WB600, CbhA can produce 300 mg/L reducing sugars. While for endoglucanase CelA, the reducing sugars reached 1250 mg/L. Through the combination of CbhA and CelA, it showed a clear synergistic effect towards Avicel and dilute-acid pretreated corn stover.
The established B. subtilis expression system was proven to be a useful platform for secretive expression and would be applied for multiple cellulase production in the future research. Efficient secretion of active cellulase is the first step in the development of B. subtilis consolidated bioprocessing cells that can produce cellulase, hydrolyze cellulose, and ferment the soluble sugars to products.
本研究首先借助Escherichia coli BL21 (DE3)/pET-28a (+)表达系统,筛选了来自热纤梭菌(Clostridium thermocellum),解纤维梭菌(Clostridium cellulolyticum)和野油菜黄单孢菌(Xanthomonas campestris pv. campestris)的近20种细菌纤维素外切酶基因,比较了其外切酶的酶活。同时为了实现纤维素酶的胞外分泌表达,建立了枯草杆菌表达系统Bacillus subtilis WB600 (WB800)/pP43JM2,随后将来源于热纤梭菌的外切葡聚糖酶CbhA, CelK, Cel48S, Cel48Y, CelO和内切葡聚糖CelA以及欧文氏菌(Erwinia carotovora subsp.)的内切葡聚糖酶CelV,嗜热放线菌(Thermomonospora fusca)的内切酶CelE在枯草杆菌表达系统里进行了表达。实验结果表明,除了外切酶CelO外,其它四种纤维素外切酶和三种不同来源的纤维素内切酶在枯草杆菌WB600和WB800中都实现了成功的分泌型表达,以磷酸处理的微晶纤维素为底物,外切酶在枯草杆菌W13800表达系统中的酶活要普遍高于相应的WB600的酶活,外切酶CbhA释放的还原糖的量可以达到300mg/L,而WB600分泌的内切酶CelA释放的还原糖可以高达1250 mg/L。对于微晶纤维素和稀酸处理的秸秆等结晶度较高的底物,外切酶CbhA和内切酶CelA呈现良好的协同作用。在枯草杆菌里表达纤维素酶为实现纤维素酶多组分的人工组装以及枯草杆菌整合生物工艺菌种的构建奠定了基础。
The screening for the powerful cellulase components remains to be a challenging work for the biorefinery research and development. The engineered bacterial strains for production of cellulase would provide many advantages, such as the low nutrients requirement, easy manipulation of genetic engineering, etc.
In this study, about 20 cellobiohydrolase genes sourced from Clostridium thermocellum DSM 1237, Clostridium cellulolyticum DSM 5812 and Xanthomonas campestris pv. campestris strain 8004, were tested to be expressed in Escherichia coli BL21 (DE3)/pET-28a(+) system for activity evaluation.In order to realize the secretion of cellulases extracellularly, Bacillus subtilis WB600 (WB800)/pP43JM2 expression system was constructed. When the targeting genes including cellobiohydrolase genes from Clostridium thermocellum, endoglucanse genes from Clostridium thermocellum, Erwinia carotovora subsp. and Thermomonospora fusca.were cloned into pP43JM2 and expressed in B. subtilis WB600 and WB800, all the cellulases, except for celO, were expressed and secreted into the broth and showed the activities with phosphoric acid swollen cellulose as the substrate. For cellobiohydrolases, the enzyme activities from WB800 were much higher than that from WB600, CbhA can produce 300 mg/L reducing sugars. While for endoglucanase CelA, the reducing sugars reached 1250 mg/L. Through the combination of CbhA and CelA, it showed a clear synergistic effect towards Avicel and dilute-acid pretreated corn stover.
The established B. subtilis expression system was proven to be a useful platform for secretive expression and would be applied for multiple cellulase production in the future research. Efficient secretion of active cellulase is the first step in the development of B. subtilis consolidated bioprocessing cells that can produce cellulase, hydrolyze cellulose, and ferment the soluble sugars to products.
引文
[1]So K, Brown R. Economic analysis of selected lignocellulose-to-ethanol conversion technologies[J]. Applied Biochemistry and Biotechnology,1999,79(1):633-640
[2]Pierre, Beguim. Molecuar biology of cellulose degradation[J]. Ann. Rev. Microbiol, 1990,44:219-248
[3]Tor-Magnus, Enari. Microbial cellulase. In:Willian M. F(ed.). Microbial Enzymes and Biotechndogy[M]. London and New York:Applied Science Publishers,1983,183-223
[4]贾丙志,范运梁,程文静.纤维素降解菌筛选的研究进展[J].现代农业科技,2008,21：315-317.
[5]Sarma, N.T.C.Bhalla. Bacteria from froest litter[J]. India J. Microbiol,1992,32:213-215
[6]Rohrmann,S. Molitoris, HP. Screening for wood-degrading enzymes in marine fungi[J]. Can.Bot./Rev.Can.Bot,1992,70(10):2116-2123
[7]Sharrock K:Cellulase assay methods:a review[J]. Journal of Biochemical and Biophysical Methods,1988,17(2):81-105.
[8]Nidetzky B, Hayn M, Macarron R, Steiner W:Synergism of Trichoderma reesei cellulases while degrading different celluloses [J].Biotechnology letters,1993,15(1):71-76.
[9]Henrissat B. A classification of glycosyl hydrolases based on amino-acid sequence similarities[J]. Biochem.J,1991,280:309-316
[10]侯配斌.粘细菌纤维堆囊菌降解纤维素多酶复合体的确证和性质分析[D].山东大学,2006
[11]Doi R, Kosugi A, Murashima K, Tamaru Y, Han S. Cellulosomes from mesophilic Bacteria[J]. Journal of Bacteriology,2003,18:5907-5914
[12]阎伯旭,曲音波,高培基.真菌和细菌纤维素酶的差别及内外切葡聚糖苷酶的底物专一性[J].1999,11：61-64
[13]Ghose T. Measurement of cellulase activities [J]. Pure Appl Chem,1987,59(2):257-268.
[14]汤憋站,童克中,陈慎等.枯草杆菌转化的研究Ⅰ.受体菌株的筛选[J].微生物学报,1964,10：189-194
[15]Kunst F, Ogasawara N, Moszer I, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis [J]. Nature,1997,390:249-256
[16]Simon M, Palva Ⅰ. Protein secretion in Bacillus species [J]. Microbiol Rev,1993,57: 109-137
[17]彭清忠,张惟材,朱厚础.枯草杆菌表达系统的研究进展[J].生物技术通讯,2001,12：220-225
[18]Tjalsma H, Antelmann H, Jongbloed JD, etal. Protemics of protein secretion by Bacillus subtilis [J]. Mol Gen Genet,1988,213:444-448
[19]von Heijne G, Abrahmsen L. Species-specific variation in signal peptide design, Implication for protein secretion in foreign hosts [J]. FEBS Lett,1989,244:439-446
[20]Bolhuis A, Tjalsma H, Smith H E. Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtiils [J]. Appl Environ Microbiol,1999,65:2934-2941
[21]Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The Carbohydrate-Active EnZymes database (CAZy):an expert resource for Glycogenomics. Nucleic Acids Research,2009,37(Database):D233-D238.
[22]Aminov RI, Golovchenko NP, Ohmiya K. Expression of a celE gene from Clostridium thermocellum in Bacillus[J]. Jounal of Fermentation and Bioengineering,1995,6: 530-537
[23]Zhang X, Zhang Z, Zhu Z, Sathitsuksanoh N, Yang Y, Zhang Y. The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg:heterologous expression, characterization, and processivity[J]. Applied Microbiology and Biotechnology,2010,86(2):525-533
[24]Petit MA, Joliff G, Mesas JM, etal. Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification[J]. Nature Biotechnology,1990,8:559-563
[25]Yao Q, Sun T, Chen G, Liu W. Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum[J]. Biotechnol Lett,2007,29: 1243-1247
[26]Morimoto T, Kadoya R, Endo K, etal. Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis[J]. DNA Res,2008,15:73-81
[27]Wu X, Lee W, Tran L, Wong S:Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases[J]. Journal of Bacteriology, 1991,173(16):4952.
[28]Cho H, Yukawa H, Inui M, Doi R, Wong S:Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800[J]. Applied and Environmental Microbiology,2004,70(9):5704.
[29]Arai T, Matsuoka S, Cho H, Yukawa H, Inui M, Wong S, Doi R:Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation[J]. Proceedings of the National Academy of Sciences,2007,104(5):1456.
[30]Den Haan R, Rose SH, Lynd LR, van Zyl WH. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae[J]. Metabb Eng,2007,9: 87-94
[31]Zhang XZ, Zhang YH. One-step production of biocommodities from lignocellulolytic biomass by recombinant cellulolytic Bacillus subtilis:Opportunities and challenges [J]. Eng Life Sci,2010,5:398-406
[32]Romero S, Merino E, Bolivar F, Gosset G, Martinez A. Metabolic engineering of Bacillus subtilis for ethanol production:lactate dehydrogenase plays a key role in fermentative metabolism[J]. Appl Environ Microbiol,2007,73:5190-5198
[33]Luria S, Delbrck M:Mutations of bacteria from virus sensitivity to virus Resistance[J]. Genetics,1943,28(6):491
[34]Johnson E, Madia A, Demain A:Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum[J]. Applied and Environmental Microbiology,1981,41(4):1060
[35]Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). Molecular cloning:a laboratory manual,2nd ed [M].1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[36]Spizizen J:Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J]. Proceedings of the National Academy of Sciences of the United States of America 1958,44(10):1072
[37]Matsuno Y, Ano T, Shoda M. High-efficiency transformation of Bacillus subtilis NB22, an antifungal antibiotic iturin producer by electroporation [J]. Jounal of Fermentation and Bioengineering,1992,4:261-264
[38]Zhang Y, Cui J, Lynd L, Kuangs L:A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid:evidence from enzymatic hydrolysis and supramolecular structure[J]. Biomacromolecules,2006,7(2):644-648
[39]Vazana Y, Morais S, Barak Y, Lamed R, Bayer EA:Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States[J]. Applied and Environmental Microbiology,2010, 76(10):3236-3243
[40]Zverlov V, Velikodvorskaya G, Schwarz W, Bronnenmeier K, Kellermann J, Staudenbauer W:Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA[J]. Journal of Bacteriology, 1998,180(12):3091.
[41]Berger E, Zhang D, Zverlov V, Schwarz W:Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose Synergistically[J]. FEMS Microbiology Letters,2007,268(2):194-201
[42]Wang W, Kruus K, Wu J:Cloning and expression of the Clostridium thermocellum celS gene in Escherichia coli[J]. Applied Microbiology and Biotechnology,1994, 42(2):346-352
[43]Kataeva I, Li X, Chen H, Choi S, Ljungdahl L:Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum:evidence for gene duplication and recombination[J]. Journal of Bacteriology,1999,181(17):5288-5295
[44]Zverlov V, Velikodvorskaya G, Schwarz W:A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum:investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose[J]. Microbiology,2002,148(1):247
[45]Makrides S:Strategies for achieving high-level expression of genes in Escherichia coli[J]. Microbiology and Molecular Biology Reviews,1996,60(3):512
[46]Zhang X, Cui Z, Hong Q, Li S:High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800[J]. Applied and environmental microbiology,2005,71(7):4101
[47]Murashima K, Chen C, Kosugi A, Tamaru Y, Doi R, Wong S:Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes[J]. Journal of Bacteriology, 2002,184(1):76.
[2]Pierre, Beguim. Molecuar biology of cellulose degradation[J]. Ann. Rev. Microbiol, 1990,44:219-248
[3]Tor-Magnus, Enari. Microbial cellulase. In:Willian M. F(ed.). Microbial Enzymes and Biotechndogy[M]. London and New York:Applied Science Publishers,1983,183-223
[4]贾丙志,范运梁,程文静.纤维素降解菌筛选的研究进展[J].现代农业科技,2008,21：315-317.
[5]Sarma, N.T.C.Bhalla. Bacteria from froest litter[J]. India J. Microbiol,1992,32:213-215
[6]Rohrmann,S. Molitoris, HP. Screening for wood-degrading enzymes in marine fungi[J]. Can.Bot./Rev.Can.Bot,1992,70(10):2116-2123
[7]Sharrock K:Cellulase assay methods:a review[J]. Journal of Biochemical and Biophysical Methods,1988,17(2):81-105.
[8]Nidetzky B, Hayn M, Macarron R, Steiner W:Synergism of Trichoderma reesei cellulases while degrading different celluloses [J].Biotechnology letters,1993,15(1):71-76.
[9]Henrissat B. A classification of glycosyl hydrolases based on amino-acid sequence similarities[J]. Biochem.J,1991,280:309-316
[10]侯配斌.粘细菌纤维堆囊菌降解纤维素多酶复合体的确证和性质分析[D].山东大学,2006
[11]Doi R, Kosugi A, Murashima K, Tamaru Y, Han S. Cellulosomes from mesophilic Bacteria[J]. Journal of Bacteriology,2003,18:5907-5914
[12]阎伯旭,曲音波,高培基.真菌和细菌纤维素酶的差别及内外切葡聚糖苷酶的底物专一性[J].1999,11：61-64
[13]Ghose T. Measurement of cellulase activities [J]. Pure Appl Chem,1987,59(2):257-268.
[14]汤憋站,童克中,陈慎等.枯草杆菌转化的研究Ⅰ.受体菌株的筛选[J].微生物学报,1964,10：189-194
[15]Kunst F, Ogasawara N, Moszer I, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis [J]. Nature,1997,390:249-256
[16]Simon M, Palva Ⅰ. Protein secretion in Bacillus species [J]. Microbiol Rev,1993,57: 109-137
[17]彭清忠,张惟材,朱厚础.枯草杆菌表达系统的研究进展[J].生物技术通讯,2001,12：220-225
[18]Tjalsma H, Antelmann H, Jongbloed JD, etal. Protemics of protein secretion by Bacillus subtilis [J]. Mol Gen Genet,1988,213:444-448
[19]von Heijne G, Abrahmsen L. Species-specific variation in signal peptide design, Implication for protein secretion in foreign hosts [J]. FEBS Lett,1989,244:439-446
[20]Bolhuis A, Tjalsma H, Smith H E. Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtiils [J]. Appl Environ Microbiol,1999,65:2934-2941
[21]Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The Carbohydrate-Active EnZymes database (CAZy):an expert resource for Glycogenomics. Nucleic Acids Research,2009,37(Database):D233-D238.
[22]Aminov RI, Golovchenko NP, Ohmiya K. Expression of a celE gene from Clostridium thermocellum in Bacillus[J]. Jounal of Fermentation and Bioengineering,1995,6: 530-537
[23]Zhang X, Zhang Z, Zhu Z, Sathitsuksanoh N, Yang Y, Zhang Y. The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg:heterologous expression, characterization, and processivity[J]. Applied Microbiology and Biotechnology,2010,86(2):525-533
[24]Petit MA, Joliff G, Mesas JM, etal. Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification[J]. Nature Biotechnology,1990,8:559-563
[25]Yao Q, Sun T, Chen G, Liu W. Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum[J]. Biotechnol Lett,2007,29: 1243-1247
[26]Morimoto T, Kadoya R, Endo K, etal. Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis[J]. DNA Res,2008,15:73-81
[27]Wu X, Lee W, Tran L, Wong S:Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases[J]. Journal of Bacteriology, 1991,173(16):4952.
[28]Cho H, Yukawa H, Inui M, Doi R, Wong S:Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800[J]. Applied and Environmental Microbiology,2004,70(9):5704.
[29]Arai T, Matsuoka S, Cho H, Yukawa H, Inui M, Wong S, Doi R:Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation[J]. Proceedings of the National Academy of Sciences,2007,104(5):1456.
[30]Den Haan R, Rose SH, Lynd LR, van Zyl WH. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae[J]. Metabb Eng,2007,9: 87-94
[31]Zhang XZ, Zhang YH. One-step production of biocommodities from lignocellulolytic biomass by recombinant cellulolytic Bacillus subtilis:Opportunities and challenges [J]. Eng Life Sci,2010,5:398-406
[32]Romero S, Merino E, Bolivar F, Gosset G, Martinez A. Metabolic engineering of Bacillus subtilis for ethanol production:lactate dehydrogenase plays a key role in fermentative metabolism[J]. Appl Environ Microbiol,2007,73:5190-5198
[33]Luria S, Delbrck M:Mutations of bacteria from virus sensitivity to virus Resistance[J]. Genetics,1943,28(6):491
[34]Johnson E, Madia A, Demain A:Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum[J]. Applied and Environmental Microbiology,1981,41(4):1060
[35]Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). Molecular cloning:a laboratory manual,2nd ed [M].1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[36]Spizizen J:Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J]. Proceedings of the National Academy of Sciences of the United States of America 1958,44(10):1072
[37]Matsuno Y, Ano T, Shoda M. High-efficiency transformation of Bacillus subtilis NB22, an antifungal antibiotic iturin producer by electroporation [J]. Jounal of Fermentation and Bioengineering,1992,4:261-264
[38]Zhang Y, Cui J, Lynd L, Kuangs L:A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid:evidence from enzymatic hydrolysis and supramolecular structure[J]. Biomacromolecules,2006,7(2):644-648
[39]Vazana Y, Morais S, Barak Y, Lamed R, Bayer EA:Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States[J]. Applied and Environmental Microbiology,2010, 76(10):3236-3243
[40]Zverlov V, Velikodvorskaya G, Schwarz W, Bronnenmeier K, Kellermann J, Staudenbauer W:Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA[J]. Journal of Bacteriology, 1998,180(12):3091.
[41]Berger E, Zhang D, Zverlov V, Schwarz W:Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose Synergistically[J]. FEMS Microbiology Letters,2007,268(2):194-201
[42]Wang W, Kruus K, Wu J:Cloning and expression of the Clostridium thermocellum celS gene in Escherichia coli[J]. Applied Microbiology and Biotechnology,1994, 42(2):346-352
[43]Kataeva I, Li X, Chen H, Choi S, Ljungdahl L:Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum:evidence for gene duplication and recombination[J]. Journal of Bacteriology,1999,181(17):5288-5295
[44]Zverlov V, Velikodvorskaya G, Schwarz W:A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum:investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose[J]. Microbiology,2002,148(1):247
[45]Makrides S:Strategies for achieving high-level expression of genes in Escherichia coli[J]. Microbiology and Molecular Biology Reviews,1996,60(3):512
[46]Zhang X, Cui Z, Hong Q, Li S:High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800[J]. Applied and environmental microbiology,2005,71(7):4101
[47]Murashima K, Chen C, Kosugi A, Tamaru Y, Doi R, Wong S:Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes[J]. Journal of Bacteriology, 2002,184(1):76.