一株热反硝化地芽孢杆菌Y62产耐高温α-淀粉酶的异源表达  

Heterologous Expression of a Thermostableα-Amylase Produced by Geobacillus thermodenitrificans Y62

作　　者：李佳欣 顾燕 汤伟 张军 鞠晓月 何增国 LI Jia-xin;GU Yan;TANG Wei;ZHANG Jun;JU Xiao-yue;HE Zeng-guo(School of Medicine and Pharmacy,Ocean University of China,Qingdao 266000,Shandong,China;Marine Biomedical Research Institute of Qingdao,Qingdao 266000,Shandong,China;Qingdao Bioantan Biotechnology Co.,Ltd.,Qingdao 266000,Shandong,China)

机构地区：[1]中国海洋大学医药学院,山东青岛266000 [2]青岛海洋生物医药研究院,山东青岛266000 [3]青岛百奥安泰生物科技有限公司,山东青岛266000

出　　处：《食品研究与开发》2023年第10期194-200,207,共8页Food Research and Development

基　　金：广东省重点领域研发计划项目(2020B0202010001);泰山产业领军人才工程专项(tscy202006017)。

摘　　要：为获得耐高温α-淀粉酶的菌株,该研究从常年覆盖热油的土壤中分离筛选产耐高温淀粉酶的细菌,通过形态学、生理生化分析和16S rRNA鉴定菌株种属,对筛选出的菌株进行中试工程化发酵产酶,下游酶分离中利用简便的硫酸铵沉淀法分离粗酶,并且对分离纯化的耐高温淀粉酶提取物进行热稳定评价。克隆菌株耐高温淀粉酶的基因序列并用于构建表达载体,在大肠杆菌BL21中成功进行异源表达,并对所产淀粉酶进行生物信息学分析。结果表明:通过筛选获得一株产淀粉酶的热反硝化地芽孢杆菌(Geobacillus thermodenitrificans)Y62,中试放大后在48 h取得112.7 U/mL酶活,其粗酶回收率可达90.2%。该淀粉酶具有良好的耐高温特性,在100℃下仍能保持37.8%的活性。异源表达产物以包涵体的形式存在,改变诱导条件后溶出可溶性蛋白,纯化产物经过质谱鉴定后确定该淀粉酶为α-淀粉酶。生物信息学分析及预测表明该耐高温淀粉酶编码511个氨基酸,分子量为59.86 kDa,含2个跨膜区域、3个催化位点和2个Ca2+结合位点,与已报道的α-淀粉酶GTA相似。To obtain strains producing thermostableα-amylase,this study isolated bacteria with thermostableα-amylase producing potency from the samples derived from heat oil immersed soil further identified the strains by morphological,physiological and biochemical analysis as well as 16S rRNA sequencing.A set of techniques were established at pilot-scale for upstream fermentation and downstream enzyme isolation by ammonium sul-fate precipitation.The thermal stability of the purified thermostable amylase extract was evaluated.Moreover,the gene encoding the thermostable enzyme was cloned and successfully expressed in Escherichia coli BL21 by het-erologous expression,while the amylase was analyzed by bioinformatics methods.The results showed that a strain of amylase-producing Geobacillus thermodenitrificans Y62 was finally obtained after screening.The en-zymatic activity was 112.7 U/mL after 48 h of pilot amplification,while the crude enzyme recovery rate was 90.2%.This amylase had excellent thermostability,as it could maintain 37.8%of the activity at a high tempera-ture up to 100℃.The enzyme was detected in the form of inclusion bodies in heterologous expression and fur-ther solubilized by adjusting the induction conditions.The purified product was identified asα-amylase by mass spectrometry.Bioinformatics analysis showed that the thermostableα-amylase was composed of 511 amino acids with a molecular weight of 59.86 kDa.It contained two transmembrane regions,three catalytic sites and two Ca2+-binding sites,which were similar to the previously reportedα-amylase GTA.

关 键 词：热反硝化地芽孢杆菌 分离鉴定 耐高温Α-淀粉酶 异源表达 生物信息学分析 

分 类 号：TQ925.1[轻工技术与工程—发酵工程]