来源于Laceyella sp.的α-淀粉酶基因克隆、重组表达及酶学性质研究  

Cloning,Recombinant Expression and Enzymatic Properties ofα-Amylase Gene from Laceyella sp.

作　　者：赵海燕[1] 宋晨斌 刘正亚 马兴荣 尚会会 李安华[1] 关现军[1] 王建设[1] ZHAO Hai-yan;SONG Chen-bin;LIU Zheng-ya;MA Xing-rong;SHANG Hui-hui;LI An-hua;GUAN Xian-jun;WANG Jian-she(School of Biotechnology and Food Science,Anyang Institute of Technology,Anyang Key Laboratory of Microbial Engineering,Anyang 455000;School of Bioengineering and Biotechnology,Tianshui Normal University,Tianshui 741001)

机构地区：[1]安阳工学院生物与食品工程学院安阳市微生物工程重点实验室,安阳455000 [2]天水师范学院生物工程与技术学院,天水741001

出　　处：《生物技术通报》2020年第8期23-33,共11页Biotechnology Bulletin

基　　金：农业部饲料生物技术重点实验室开放课题;河南省高等学校重点科研项目(15B230001);安阳市科技攻关计划项目;安阳工学院校科研基金项目(YJJ2017002)。

摘　　要：从土壤中筛选产淀粉酶菌株,对其淀粉酶基因进行克隆及异源表达,分析其酶学性质。采用固体淀粉筛选培养基,从安阳面粉厂附近土壤里分离得到一株产淀粉酶的菌株Laceyella sp.,编号为MF-8-1。然后利用同源克隆的方法得到淀粉酶编码基因AmyL。将AmyL与表达载体pET-22b(+)连接构建pET-22b-AmyL重组质粒,并转化至E.coli BL21(DE3)中进行表达。最后对重组酶进行分离纯化以及酶学性质测定。AmyL在E.coli中成功表达,重组酶AmyL分子量为55 kD,最适反应温度为45℃,最适pH值为6.0。在温度低于60℃时,有较好的温度稳定性;在pH 6.0-10.0内较稳定。酶的动力学研究表明,该酶的比活、Km和Vmax分别为185.39 U/mg、0.95 mg/mL、343.12μmol/(min·mg)。结果表明,AmyL在中温碱性条件下具有高活性且保持稳定,在洗涤剂、纺织、造纸等行业具有潜在的应用价值。Cloning and heterologous expression of amylase gene in the amylase-producing strain screened from soil were conducted and their enzymatic properties were analyzed.An amylase-producing strain was isolated and numbered MF-8-1,which was from the soil sample near the Anyang Flour Mill by using the solid starch screening medium.Then the amylase gene AmyL was cloned by corresponding primers with homology cloning.Further the recombinant plasmid of pET-22b-AmyL was constructed by ligating Amy and expression vector pET-22b(+),and transformed into expression host strain Escherichia coli BL21(DE3).Finally,the recombinant enzyme was isolated and purified and its enzymatic properties were determined.As results,AmyL was expressed successfully in E.coli BL21(DE3),and the molecular weight of recombinant enzyme AmyL was 55 kD.Recombinant enzyme presented maximum activity at pH 6.0 and 45℃.It had better temperature stability when the temperature was lower than 60℃.The enzyme was highly stable at pH 6.0-10.0.The kinetics studies of recombinant enzyme showed that the specific activity,Km and Vmax of the enzyme were 185.39 U/mg,0.95 mg/mL and 343.12μmol/(min•mg),respectively.AmyL is characterized by stable enzyme activity under medium temperature and alkalinity,thus it has potential application value in detergent,textile,paper making and other industries.

关 键 词：Laceyella sp. Α-淀粉酶 基因克隆 异源表达 酶学性质 

分 类 号：Q55[生物学—生物化学]