重组4-α-糖基转移酶转化淀粉制备大元环糊精的条件优化
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摘要
4-α-糖基转移酶能够以淀粉为底物通过分子内转糖基作用制备大元环糊精。本研究成功构建了产超嗜热菌Aquifex aeolicus来源的4-α-糖基转移酶重组菌的E.coli BL21 (DE3)/pET-24a(+)-AaAM。对重组菌进行摇瓶发酵,培养27h破壁上清酶活达到0.99 U/mL。以马铃薯淀粉为底物,对酶转化生成大元环糊精进行了条件优化。结果表明,在底物质量浓度为1 g/dL时,经异淀粉酶脱支预处理后,调节初始pH为7.0,反应温度为75℃,加酶量为20 U/g,在10 h时大元环糊精的转化率达到最高24.8%,为大元环糊精的工业生产奠定了基础。
4-α-glucanotransferase can catalyze intramolecular glucan transfer reactions of amylose to produce cycloamylose. In this study,a recombinant E. coli BL21(DE3)/pET-24 a(+)-AaAM derived from Aquifex aeolicus was constructed for intracellular production of 4-α-glucanotransferase. The Aa AM produced in culture medium activity was 0.99 U/mL after 27 h in shake flask. Furthermore,the condition for cycloamylose preparation with potato starch by this enzyme was optimized. The optimized conditions for the conversion as follows: substrate concentration was 1 g/dL debranched potato starch, the initial pH was 7.0, the reaction temperature was 75 ℃, 4-α-glucanotransferase concentration was 20 U/g starch. Under this conditions, the cycloamylose yield is 24.8% for 10 h, which paved the way for indnstrial production of cycloamylose.
4-α-glucanotransferase can catalyze intramolecular glucan transfer reactions of amylose to produce cycloamylose. In this study,a recombinant E. coli BL21(DE3)/pET-24 a(+)-AaAM derived from Aquifex aeolicus was constructed for intracellular production of 4-α-glucanotransferase. The Aa AM produced in culture medium activity was 0.99 U/mL after 27 h in shake flask. Furthermore,the condition for cycloamylose preparation with potato starch by this enzyme was optimized. The optimized conditions for the conversion as follows: substrate concentration was 1 g/dL debranched potato starch, the initial pH was 7.0, the reaction temperature was 75 ℃, 4-α-glucanotransferase concentration was 20 U/g starch. Under this conditions, the cycloamylose yield is 24.8% for 10 h, which paved the way for indnstrial production of cycloamylose.
引文
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[13] WANG Shuixing,FJ Jinheng,GONG Yan. MalQgene cloning from Streptomyces ST66 and the original nuclear fusion expression[J]. Journal of Agricultural Biotechnology,2007,15(6):1024-1028.(in Chinese)
[14] XU Y,ZHOU X,BAI Y X. Cycloamylose production from amylomaize by isoamylase and Thermus aquaticus 4-α-glucanotransferase[J]. Carbohydrate Polymers,2014,102:66-73.
[15] MINSU K,JUNHYUCK J,YOUNGWAN K. Overproduction of a thermostable 4-α-glucanotransferase by codon optimization at N-terminus region[J]. Journal of the Science of Food and Agriculture,2013,93:2683-2690.
[16] SHAKHAWAT H B,MOTOMITSU K. A cycloamylose-forming hyperthermostable 4-α-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli[J]. Journal of Molecular Catalysis B:Enzymatic,2003,22:45-53.
[17] WANG Ning,WU Dan,CHEN Sheng,et al. Preparation ofα-cyclodextrin by mutation Y89D of cyclodextrin glycosyltransferase from Paenibacillus macerans[J]. Food Science,2011(3):165-170.(in Chinese)
[18]王磊. Bacillus clarkii 7364γ-环糊精葡萄糖基转移酶的重组表达及其应用[D].无锡:江南大学,2010.
[19] TAKESHI T,MICHIYO Y. Cyclic glucans produced by the intramolecular transglycosylation activity of potato D-enzyme on amylopectin[J]. Biochemical and Biophysical Research Communicatons,1998,247:493-497.
[2] WANG Shuixing,GUO Yong,XU Yang. Cycloamylose and its application prospective in food industry[J]. Food and Machinery,2006,22(2):113-116.(in Chinese)
[3] WANG Shuixing,GUO Yong,LI Yanping. Novel annular starch——the latest research progress of cycloamylose[J]. Cereal and Feed Industry,2006,9(3):23-26.(in Chinese)
[4] YOSHINOBU T,KAZUTOSHI F,TAKESHI T. Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization:production of cycloamylose[J]. Applied and Environmental Microbiology,1999,65(3):910-915.
[5] MICHIYO Y,HIROKI T,AKESHI T. Cyclization reaction catalyzed by glycogen debranching enzyme(EC 2.4.1.25/EC3.2.1.33)and its potential for cycloamylose production[J]. Applied and Environmental Microbiology,2002,68(9):4233-4239.
[6] YOSHINOBU T,HARUYO S,TAKESHI T. Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases[J]. Applied and Environmental Microbiology,2001,67(4):1453-1460.
[7] TAKESHI T,MICHIYO Y,HIROKI T. Potato D-enzyme catalyzes the cyclization of amylose to produce cycloamylose,a novel cyclic glucan[J]. The Journal of Biological Chemistry,1996,271(6):2902-2908.
[8] INGO P,KOJI T,YOSHINOBU T. Crystal structure of amylomaltase from Thermus aquaticus,a glycosyltransferase catalysing the production of large cyclic glucans[J]. Journal of Molecular Biology,2000,296:873-886.
[9] HIROMI I,SHINYA F. Crystal structures of 4-α-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor[J]. The Journal of Biological Chemistry,2003,278(21):19378-19386.
[10] WIRAYA S,AREEYA P. A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products[J]. Journal of Inclusion Phenomena and Macrocyclic Chemistry,2011,70:369-375.
[11] KAZUTOSHI F,HIROTAKA M,YOSHINOBU T. Protein engineering of amylomaltase from Thermus aquaticus with random and saturation mutageneses[J]. Biologia Bratislava,2005,16:97-102.
[12] KAZUTOSHI F,HIROTAKA M,YOSHINOBU T. Use of random and saturation mutageneses To Improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses[J]. Applied and Environmental Microbiology,2005,71(10):5823-5827.
[13] WANG Shuixing,FJ Jinheng,GONG Yan. MalQgene cloning from Streptomyces ST66 and the original nuclear fusion expression[J]. Journal of Agricultural Biotechnology,2007,15(6):1024-1028.(in Chinese)
[14] XU Y,ZHOU X,BAI Y X. Cycloamylose production from amylomaize by isoamylase and Thermus aquaticus 4-α-glucanotransferase[J]. Carbohydrate Polymers,2014,102:66-73.
[15] MINSU K,JUNHYUCK J,YOUNGWAN K. Overproduction of a thermostable 4-α-glucanotransferase by codon optimization at N-terminus region[J]. Journal of the Science of Food and Agriculture,2013,93:2683-2690.
[16] SHAKHAWAT H B,MOTOMITSU K. A cycloamylose-forming hyperthermostable 4-α-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli[J]. Journal of Molecular Catalysis B:Enzymatic,2003,22:45-53.
[17] WANG Ning,WU Dan,CHEN Sheng,et al. Preparation ofα-cyclodextrin by mutation Y89D of cyclodextrin glycosyltransferase from Paenibacillus macerans[J]. Food Science,2011(3):165-170.(in Chinese)
[18]王磊. Bacillus clarkii 7364γ-环糊精葡萄糖基转移酶的重组表达及其应用[D].无锡:江南大学,2010.
[19] TAKESHI T,MICHIYO Y. Cyclic glucans produced by the intramolecular transglycosylation activity of potato D-enzyme on amylopectin[J]. Biochemical and Biophysical Research Communicatons,1998,247:493-497.