微小毛霉凝乳酶蛋白质工程改造研究
|
摘要
微小毛霉凝乳酶(MPC)是微生物凝乳酶的主要来源之一。本研究将微小毛霉凝乳酶基因克隆到表达载体pET30a中并转化宿主BL21(DE3),使微小毛霉凝乳酶蛋白在大肠杆菌中表达。通过易错PCR技术对MPC进行随机突变建库,从中筛选出两株具有优良蛋白特性的突变体,为蛋白质结构与功能研究以及菌种改造奠定基础。
The rennet from mucor pusillus is one of the major sources of microbial rennet. In order to develop a rennet with suitable enzymatic properties,the gene of rennet was cloned from mucor pusillus,and the DNA fragment was inserted into the expression vector pET30a, forming pET30a-MPC. PET30a-MPC was further transformed into E. coli BL21(DE3). An error-prone PCR was performed with the pET30a-MPC as template.Expression of the enzyme was performed with IPTG induction. Two mutants with better enzymatic properties were confirmed by high-throughput screening,which will be helpful for the study of protein structure and function and the transformation of strain breeding.
The rennet from mucor pusillus is one of the major sources of microbial rennet. In order to develop a rennet with suitable enzymatic properties,the gene of rennet was cloned from mucor pusillus,and the DNA fragment was inserted into the expression vector pET30a, forming pET30a-MPC. PET30a-MPC was further transformed into E. coli BL21(DE3). An error-prone PCR was performed with the pET30a-MPC as template.Expression of the enzyme was performed with IPTG induction. Two mutants with better enzymatic properties were confirmed by high-throughput screening,which will be helpful for the study of protein structure and function and the transformation of strain breeding.
引文
[1]MANDY JACOBDORIS JAROS,HARAID ROHM.Recent advances in milk clotting enzymes[J].International journal of dairy technology,2011(1):14-33.
[2]SUPANNEE CHITPINITYOL,M JAMES C CRABBE.Chymosin and aspartic proteinases[J].Food chemistry,1998(4):395-418.
[3]宋曦,甘伯中,贺晓玲,等.天祝放牧牦牛生活环境土壤中一株产凝乳酶细菌的分离与鉴定[J].食品科学,2009(11):158-162.
[4]钟继才.凝乳酶在干酪生产中的应用[J].中国乳品工业,2006(1):54-56.
[5]杭锋,洪青,王钦博,等.凝乳酶的研究进展[J].食品科学,2016(3):273-279.
[6]李倬林,邵淑娟,李铁柱,等.响应面法优化微小毛霉固态发酵生产凝乳酶工艺研究[J].食品工业科技,2012(4):248-254.
[7]姜媛媛,王景会,李玉秋,等.微小毛霉凝乳酶基因的克隆与表达[J].中国乳品工业,2010(2):7-9.
[8]EIJSINK VG,GASEIDNES S,BORCHERT TV,et al.Directed evolution of enzyme stability[J].Biomolecular engineering,2005(1):21-30.
[9]TURNER NJ.Directed evolution of enzymes for applied biocatalysis[J].Trends biotechnol,2003(11):474-478.
[10]赵心清,姜如娇,白凤武.启动子和细胞全局转录机制的定向进化在微生物代谢工程中的应用[J].生物工程学报,2009(9):1312-1315.
[11]刘振民,骆承庠,酒药中凝乳酶特性及产酶条件的研究[J].食品科学,2000(2):3-6.
[2]SUPANNEE CHITPINITYOL,M JAMES C CRABBE.Chymosin and aspartic proteinases[J].Food chemistry,1998(4):395-418.
[3]宋曦,甘伯中,贺晓玲,等.天祝放牧牦牛生活环境土壤中一株产凝乳酶细菌的分离与鉴定[J].食品科学,2009(11):158-162.
[4]钟继才.凝乳酶在干酪生产中的应用[J].中国乳品工业,2006(1):54-56.
[5]杭锋,洪青,王钦博,等.凝乳酶的研究进展[J].食品科学,2016(3):273-279.
[6]李倬林,邵淑娟,李铁柱,等.响应面法优化微小毛霉固态发酵生产凝乳酶工艺研究[J].食品工业科技,2012(4):248-254.
[7]姜媛媛,王景会,李玉秋,等.微小毛霉凝乳酶基因的克隆与表达[J].中国乳品工业,2010(2):7-9.
[8]EIJSINK VG,GASEIDNES S,BORCHERT TV,et al.Directed evolution of enzyme stability[J].Biomolecular engineering,2005(1):21-30.
[9]TURNER NJ.Directed evolution of enzymes for applied biocatalysis[J].Trends biotechnol,2003(11):474-478.
[10]赵心清,姜如娇,白凤武.启动子和细胞全局转录机制的定向进化在微生物代谢工程中的应用[J].生物工程学报,2009(9):1312-1315.
[11]刘振民,骆承庠,酒药中凝乳酶特性及产酶条件的研究[J].食品科学,2000(2):3-6.