产普鲁兰酶克雷伯氏菌的分离鉴定及酶学性质研究  

Isolation of Klebsiella pneumoniae Producing Pullulanase and Its Enzymatic Characterization

作　　者：谢能中[1] 王青艳[1] 米慧芝[1] 朱绮霞[1] 秦艳[1] 曹薇[1] 朱婧[1] 陆雁[1] 黄日波[1] 

机构地区：[1]广西科学院国家非粮生物质能源工程技术研究中心,非粮生物质酶解国家重点实验室,广西生物质产业化工程院,广西生物炼制重点实验室,广西南宁530007

出　　处：《广西科学》2013年第1期35-39,共5页Guangxi Sciences

基　　金：广西千亿元重大科技攻关工程项目(桂科攻11107008-4);广西自然科学基金项目(2012GXNSFBA053063);广西科学研究与技术开发计划项目(桂科重12118004-3;桂科重12118004-4);广西科学院基本科研业务费项目(12YJ25SW01;12YJ25SW02)资助

摘　　要：利用曲里苯蓝法从淀粉加工厂废水氧化池酸性污泥样品中筛选普鲁兰酶产生菌,对筛选到的GXAS-38菌株进行形态观察、生理生化特征分析、16SrDNA序列系统发育分析和普鲁兰酶酶学性质研究,并用PCR方法克隆GXAS-38菌株的普鲁兰酶基因。结果表明,GXAS-38菌株属于肺炎克雷伯氏菌(Klebsiella pneumoniae),其产普鲁兰酶的最适反应温度为60℃,在温度35～50℃时酶活较稳定;最适反应pH值5.5,在pH值5.0～7.5时酶活较稳定。Ca2+、Na+和Li+对GXAS-38菌株的普鲁兰酶活性有激活作用;Cu2+、Zn2+、Co2+、Mn2+、Fe2+、Fe3+和Ba2+对GXAS-38菌株的普鲁兰酶活性有抑制作用;螯合剂EDTA能够强烈地抑制GXAS-38菌株的普鲁兰酶活性,GXAS-38菌株的普鲁兰酶反应需要金属离子参与。GXAS-38菌株完整的普鲁兰酶编码基因全长3291bp,编码1096个氨基酸。A pullulanase producing bacterium, designated strain GXAS-38, was isolated from samples collected from the waste lagoon of a starch factor in Nanning,Guangxi. Strain GXAS- 38 was identified as Klebsiella pn eumon iae based on morphological, physiological characteriza- tion and 16S rDNA sequences analysis. The optimum temperature and pH for the enzyme were determined as 60℃ and 5.5 ,respectively. The stable temperature and pH range were 35-50℃ and 5.0-7.5, respectively. The enzyme could be stimulated by Ca^2＋ , Na^＋ and Li^＋ , but inhibited by Cu^2＋ , Zn^2＋ , Co^2＋ , Mn^2＋ , Fe^2＋ , Fe^3＋ , Ba^2＋ and EDTA. The pullulanase gene was amplified by PCR and sequenced. The full length of this gene was 3291 bp and presumably encoded 1096 amino acids.

关 键 词：普鲁兰酶 肺炎克雷伯氏菌 分离 鉴定 酶学性质 基因克隆 

分 类 号：Q814[生物学—生物工程]