提高内切葡聚糖酶活力及其在毕赤酵母中的表达研究
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摘要
以中性内切葡聚糖酶基因EG和真菌Corticium rolfsii的碳水化合物结合模块(FCBM)为模块,构建融合基因重构体EG-FCBM和CD-FCBM,并利用高效表达载体在毕赤酵母中对其进行高效表达。酶活及性质分析显示,EG-FCBM和CD-FCBM在诱导表达84~96h后,其对微晶纤维素的活力分别为951和676 U·mL-1,较原始基因EG(526U·mL-1)提高81%、28%,而酶学性质两者间无较大差异。这一结果表明,FCBM在催化水解纤维素的过程中有着极为重要的作用,通过增加FCBM来提高纤维素酶活性方法可行。
Redesigned endoglucanases enhanced FCBM from the Corticium rolfsii,EG-FCBM and CD-FCBM respectively,were constructed in this study.The redesigned genes were expressed in P.pastoris,and their characters were also discussed.The enzymatic activities of EG,EG-FCBM and CD-FCBM in Pichia pastoris cultivation supernatant reached526,951 and 676 U·mL-1 respectively.EG-FCBM and CD-FCBM showed 81% and 28% enzymatic activity increase compared to EG.The optimal pH,temperature,pH stability and heat stability between EG-FCBM and CD-FCBM had little difference.The results indicated that the FCBM from the Corticium rolfsii can improve EG's catalytic power.
Redesigned endoglucanases enhanced FCBM from the Corticium rolfsii,EG-FCBM and CD-FCBM respectively,were constructed in this study.The redesigned genes were expressed in P.pastoris,and their characters were also discussed.The enzymatic activities of EG,EG-FCBM and CD-FCBM in Pichia pastoris cultivation supernatant reached526,951 and 676 U·mL-1 respectively.EG-FCBM and CD-FCBM showed 81% and 28% enzymatic activity increase compared to EG.The optimal pH,temperature,pH stability and heat stability between EG-FCBM and CD-FCBM had little difference.The results indicated that the FCBM from the Corticium rolfsii can improve EG's catalytic power.
引文
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[2]Bhat M K.Cellulases and related enzymes in biotechnology[J].Biotechnol Adv,2000,18(5):355-383.
[3]Galante Y M,De Conti.A,Monteverdi R.Application of Trichoderma enzymes in food and feed industries[C]//Harman G E,Kubicek C P.Trichoderma and Gliocladium-Enzymes,biological control and commercial applications.Boca Raton:CRC Press,1998:327-342.
[4]Ramadan M F,Thomas M J.Impact of enzymatic treatment on chemical composition,physicochemical properties and radical scavenging activity of goldenberry(Physalis peruviana L.)juice[J].J Sci Food Agric,2007,87(3/4):52-460.
[5]Buchert J,Koponen J M,Suutarinen M,et al.Effect of enzyme aided pressing on anthocyanin yield and profiles in bilberry and blackcurrant juices[J].J Sci Food Agric,2005,85(15):2548-2556.
[6]Kapasakalidis P G,Rastall R A,Gordon M.Effect of a cellulase treatment on extraction of antioxidant phenols from black currant(Ribes nigrum L.)pomace[J].J Agric Food Chem,2009,57(10):4342-4351.
[7]Davies G.Structures and mechamisms of glycosyl hydrolases[J].Structure,1995,9(3):853-859.
[8]Linder M.The roles and function of cellulose-binding domains[J].Journal of Biotechnology,1997,57(2):15-28.
[9]Black G W,Rixon J E,Clarke J H,et al.Cellulose binding domains and linker sequences potentiate the activity of hemicellulases against complex substrates[J].Journal of Biotechnology,1997,57(1/3):59-69.
[10]Bae H J,Turcotte G,Chamberland H,et al.A comparative study between an endoglucanaseⅣand its fused protein complex Cel5-CBM6[J].FEMS Microbio Lett,2003,227(2):175-181.
[11]Sunna A,Gibbs M D,Bergquist P L.The thermostabilizing domain,XynA,of Caldibacillus cellulovorans xylanase is a xylan binding domain[J].The J Biochem,2000,346(3):583-586.
[12]Meissner K,Wassenberg D,Liebl W.The thermostabilizing domain of the modular xylanase XynAof Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage beta-1,3/beta-1,4-glucan[J].Mol Microbio,2000,36(4):898-912.
[13]吴振芳,陈惠,曾民,等.内切葡聚糖酶基因在毕赤酵母中高效表达研究[J].农业生物技术学报,2009,17(3):67-72.
[14]Yasokawa D,Shimizu T,Nakagawa R,et al.Cloning,sequencing,and heterologous expression of a cellobiohydrolase cDNA from the basidiomycete Corticium rolfsii[J].Biosci Biotech Bioche,2003,67(6):1319-1326.
[15]谢占玲,吴润.纤维素酶的研究进展[J].草业科学,2004,21(4):45-51.
[16]Cai G H,LI Y Y,Zhang Z,et al.Fusion expression and analysis of the epitopes in tartary buckwheat allergen[J].Food Sci,2006,27(11):31-34.
[17]唐自钟.内切葡聚糖酶基因同源和异源结构域重构研究[D].雅安:四川农业大学,2011.
[18]Tolonen A C,Chilaka A C,Church G M.Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9hydrolase Cphy3367[J].Mol Microbiol,2009,74(6):1300-1313.
[19]Li X Q,Shao W L.The construction of Thermotoga maritima endoglucanase Cel12Bfused with CBD and the characterization of chimeric enzyme[J].Acta Microbiologica Sinica,2006,46(5):726-729.
[20]Lynd L R,Weimer P J,Van W H,et al.Microbial cellulose utilization:fundamentals and biotechnology[J].Microbiol Molecular Biol Rev,2002,66(3):506-577.
[21]Rubingh D N.Protein engineering from a bioindustrial point of view[J].Current Opinion in Biotech,1997,8(4):417-422.