一株γ-多聚谷氨酸生产菌的分离筛选与鉴定  

Isolation and identification of Bacillus strain producing γ-polyglutamic acid

作　　者：何剑[1] 雍晓雨[1,2] 周俊[1,2] 王舒雅[1,2] 陈怡露[1] 郑涛[1,2] 

机构地区：[1]南京工业大学生物与制药工程学院,南京211800 [2]南京工业大学生物能源研究所,南京211800

出　　处：《生物加工过程》2014年第4期87-93,共7页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础研究发展计划(973计划)(2013CB733904;2013CB7335002;2011CBA00807);国家高技术研究发展计划(863计划)(2012AA023406);江苏省自然科学基金(BK20130932);江苏省高校自然科学基金(13KJB530009)

摘　　要：从菜园土壤中取样,在含有谷氨酸的筛选培养基上采用梯度稀释涂布、平板划线的方法,以菌落/菌液黏稠度为指示,分离筛选生产γ-多聚谷氨酸的菌株。利用氨基酸分析仪测定提取纯化后的γ-多聚谷氨酸的产量,并通过形态学、生理生化特征以及16S rDNA基因序列分析鉴定该菌株,并对其合成γ-多聚谷氨酸的功能基因进行PCR扩增。结果表明:筛选到1株产γ-多聚谷氨酸的细菌C1,其液体摇瓶发酵产量为18.4 g/L,相对分子质量为1.8×106;该菌株为革兰氏阳性,菌落黏稠、菌体呈杆状、产芽胞、且形成荚膜;主要生理生化特点为能利用葡萄糖和蔗糖发酵,水解淀粉,H2O2酶阳性,产吲哚等;经16S rDNA鉴定与Bacillus amyloliquefaciens ATCC23350同源性为100%,故命名为Bacillus amyloliquefaciens C1,且拥有γ-多聚谷氨酸合成的相关基因pgsA、pgsB和pgsC。Soil sample was collected from farmland. The strains were screened by the methods of gradient dilution and plate streaking on the selected medium containing glutamic acid and under the indication of the viscosity of their fermentation broth. The γ-polyglutamic acid production of the strains was obtained by analyzing the glutamic acid content of the hydrolysate of γ-polyglutamic acid using amino acid analyser. Then, the isolated strain was identified according to the morphological features, physiological and biochemical characteristics, as well as phylogenetic analysis of 16S rDNA sequences. Finally, the pgsA, pgsB, and pgsC gene, related to the γ-polyglutamic acid synthesis were amplified by PCR. A Bacillus, called the C1, was isolated with aγ-polyglutamic acid production of 18？4 g/L in flask fermentation culture. The molecular mass ofγ-polyglutamic acid produced by strain C1 was 1？8＆#215;106 detected by GPC. C1 was a gram-positive bacilli, which fermented both glucose and sucrose, hydrolysed starch, produced indole, and shared 100% sequence identity of its 16S rDNA with Bacillus amyloliquefaciens ATCC 23350, thus it was called the Bacillus amyloliquefaciens C1. It was testified to harboring pgsA, pgsB, and pgsC genes.

关 键 词：解淀粉芽胞杆菌 鉴定 分离筛选 γ 多聚谷氨酸 

分 类 号：TQ922[轻工技术与工程—发酵工程]