木聚糖酶和纤维素酶多酶嵌合体的构建
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摘要
为探究木聚糖酶和纤维素酶形成的多酶嵌合体降解纤维素的效果,此研究设计了2个含有不同来源锚定域的重组木聚糖酶基因和重组纤维素酶基因,并设计了含有2个不同来源的粘附域和纤维素结合域的脚手架蛋白基因,体外构建了大肠杆菌E.coil BL21(DE3)表达载体,表达纯化了脚手架蛋白、重组木聚糖酶和纤维素酶。同时,体外组装三种元件形成多酶嵌合体,以微晶粉末纤维素为底物检测其降解纤维素的效率。结果显示,纯化所得的脚手架蛋白Scaf、重组木聚糖酶Xyn10B和纤维素酶Cel48F可在体外组装形成多酶嵌合体,该多酶嵌合体酶活(0.107±0.013 U/mL)显著高于游离酶混合物的酶活(0.084±0.001 U/mL)(P<0.05)。试验表明木聚糖酶Xyn10B和纤维素酶Cel48F可与脚手架蛋白Scaf体外组装形成多酶嵌合体,并可实现对纤维素的高效降解。
The purpose of this experiment was to design an active cellulosome chimera formed by scaffoldin and two recombinase. A recombinant xylanase gene and a recombinant cellulase gene containing different dockerins were designed,and a scaffolding gene containing two different origins of cohesins and a carbohydrate-binding module(CBM),and E.coli BL21(DE3) expression vectors were constructed, the scaffolding, recombinant xylanase and cellulase were overexpressed and purified separately. The three elements were assembled in vitro to form a cellulosome chimeras and used avicel to detect the cellulose efficiency. The result showed that purified scaffolding, recombinant xylanases Xyn10B and cellulases Cel48F could be assembled with the scaffolding Scaf in vitro to form the cellulosome chimera. The enzyme activity(0.107±0.013 U/mL) of cellulosome chimera was significantly higher than that of the free enzymes mixture(0.084±0.001 U/mL). The experiment proved that the xylanase Xyn10B and cellulase Cel48 F could be assembled with the scaffolding Scaf in vitro to form the cellulosome chimera and achieve efficient degradation of cellulose.
The purpose of this experiment was to design an active cellulosome chimera formed by scaffoldin and two recombinase. A recombinant xylanase gene and a recombinant cellulase gene containing different dockerins were designed,and a scaffolding gene containing two different origins of cohesins and a carbohydrate-binding module(CBM),and E.coli BL21(DE3) expression vectors were constructed, the scaffolding, recombinant xylanase and cellulase were overexpressed and purified separately. The three elements were assembled in vitro to form a cellulosome chimeras and used avicel to detect the cellulose efficiency. The result showed that purified scaffolding, recombinant xylanases Xyn10B and cellulases Cel48F could be assembled with the scaffolding Scaf in vitro to form the cellulosome chimera. The enzyme activity(0.107±0.013 U/mL) of cellulosome chimera was significantly higher than that of the free enzymes mixture(0.084±0.001 U/mL). The experiment proved that the xylanase Xyn10B and cellulase Cel48 F could be assembled with the scaffolding Scaf in vitro to form the cellulosome chimera and achieve efficient degradation of cellulose.
引文
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[26] BAYER E A,MORAG E,LAMED R.The cellulosome:A treasure-trove for biotechnology[J].Trends in Biotechnology,1994,12(9):379-386.
[2] BAYER E A,LAMED R,WHITE B A,et al.From cellulosomes to cellulosomics[J].Chemical Record,2008,8(6):364-337.
[3] TSAI S L,DASILVA N A,CHEN W.Functional display of complex cellulosomes on the yeast surface via adaptive assembly[J].Acs Synthetic Biology,2013,2(1):14.
[4] FONTES C M G A,GILBERT H J.Cellulosomes:Highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates[J].Annual Review of Biochemistry,2010,79(1):655.
[5] LYTLE B,MYERS C,KRUUS K,et al.Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein,CipA[J].Journal of Bacteriology,1996,178(4):1 200-1 203.
[6] YARON S,MORAG E,BAYER E A,et al.Expression,purification and subunit-binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome[J].Febs Letters,1995,360(2):121-124.
[7] LYND L R,WEIMER P J,VAN ZYL W H,et al.Microbial cellulose utilization:fundamentals and biotechnology[J].Microbiology & Molecular Biology Reviews,2002,66(3):506.
[8] FIEROBE H P,PAGèS S,BéLA?CH A,et al.Cellulosome from Clostridium cellulolyticum:molecular study of the dockerin/cohesin interaction[J].Biochemistry,1999,38(39):12 822-12 832.
[9] CASPI J,IRWIN D,LAMED R,et al.Thermobifida fusca family-6 cellulases as potential designer cellulosome components[J].Biocatalysis,2006,24(1/2):3-12.
[10] BACHMANN S L,MCCARTHY A J.Purification and cooperative activity of enzymes constituting the xylan-degrading system of Thermomonospora fusca[J].Applied & Environmental Microbiology,1991,57(8):2 121-2 130.
[11] WILSON D B.Studies of Thermobifida fusca plant cell wall degrading enzymes[J].Chemical Record,2004,4(2):72-82.
[12] MORA?S S,BARAK Y,CASPI J,et al.Cellulase-xylanase synergy in designer cellulosomes for enhanced degradation of a complex cellulosic substrate[J].Microbiology,2010,1(5):e00285-10.
[13] SARAH M,ELY M,YOAV B,et al.Deconstruction of lignocellulose into soluble sugars by native and designer cellulosomes[J].Microbiology,2012,3(6):214 104-214 104.
[14] JOHANNA S,SARAH M,RAPHAEL L,et al.Adaptor scaffoldins:An original strategy for extended designer cellulosomes,inspired from nature[J].Microbiology,2016,7(2):e00083.
[15] 萨姆布鲁克,拉塞尔.分子克隆实验指南[M].3版.黄培堂,译.北京:科学出版社,2008.
[16] FIEROBE H P,MECHALY A,TARDIF C,et al.Design and production of active cellulosome chimeras:Selective incorporation of dockerin-containing enzymes into defined functional complexes[J].Journal of Biological Chemistry,2001,276(24):21 257-21 261.
[17] PAGèS S,BéLA?CH A,BéLA?CH J P,et al.Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum:Prediction of specificity determinants of the dockerin domain[J].Proteins-structure Function & Bioinformatics,1997,29(4):517-527.
[18] FIEROBE H P,MINGARDON F,MECHALY A,et al.Action of designer cellulosomes on homogeneous versus complex substrates:controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin[J].Journal of Biological Chemistry,2005,280(16):16 325-16 334.
[19] FIEROBE H P,BAYER E A,TARDIF C,et al.Degradation of cellulose substrates by cellulosome chimeras[J].Journal of Biological Chemistry,2002,277(51):49 621-49 630.
[20] VAZANA Y,MORA?S S,BARAK Y,et al.Designer cellulosomes for enhanced hydrolysis of cellulosic substrates[J].Methods in Enzymology,2012,510:429-452.
[21] KIM J H,IRWIN D,WILSON D B.Purification and characterization of Thermobifida fusca xylanase 10B[J].Canadian Journal of Microbiology,2004,50(10):835-843.
[22] DAVIDI L,MORA?S S,ARTZI L,et al.Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(39):10 854.
[23] 陈富荣,朱雅新,东秀珠,等.瘤胃木质纤维素降解菌及降解酶基因的研究进展[J].微生物学报,2010,50(8):981-987.
[24] SAJJAD M,KHAN M I M,AKBAR N S,et al.Enhanced expression and activity yields of Clostridium thermocellum xylanases without non-catalytic domains[J].Journal of Biotechnology,2010,145(1):38-42.
[25] REVERBELLEROY C,BELAICH A,BERNADAC A,et al.Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli.[J].Microbiology,1996,142 (4):1 013-1 023.
[26] BAYER E A,MORAG E,LAMED R.The cellulosome:A treasure-trove for biotechnology[J].Trends in Biotechnology,1994,12(9):379-386.