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

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

Key words:  pullulanase  Klebsiella pneumoniae  isolation  identification  enzamatic characterization  gene cloning

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、16S rDNA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3291 bp,编码1096个氨基酸。

Abstract:

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 pneumoniae based on morphological, physiological characterization 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 Ca²⁺, Na⁺ and Li⁺, but inhibited by Cu²⁺, Zn²⁺, Co²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Ba²⁺ 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.