摘要
本研究从广西防城港市北仑河口红树林土壤沉积物中筛选到1株产普鲁兰酶细菌GXM-1,利用形态学和16S rRNA序列进行分析鉴定,结果表明该菌株为Bacillus licheniforms;采用同源克隆策略获得了Bacillus licheniforms GXM-1普鲁兰酶基因(pulM),在Escherichia coli BL21中实现了pulM的可溶性表达。纯化普鲁兰酶的比活力为72.6 U/mg,最适反应温度为40℃,最适p H为6.5,K_(m)=(6.442±0.4668)mg/mL,V_(max)=(89.84±2.795)μmol·min^(-1)·mg^(-1),40℃保温4 h相对残余酶活力为40%,在pH 7.0~8.0缓冲液中保存12 h,相对残余酶活力保持在80%以上,金属离子Ca^(2+)、Na^(+)、Li^(+)和Ni^(+)对PulM的酶活力有明显的激活作用。研究结果为普鲁兰酶在工业中的应用研究提供了新的思路。
In this study,a pullulanase-producing strain GXM-1 was screened from the mangrove soil sediments in the Beilun Estuary of Fangchenggang,Guangxi.Based on the morphological observation and 16S rRNA sequences analysis,the strain was identified as Bacillus licheniformis GXM-1.The gene encoding pullulanase from Bacillus licheniforms GXM-1 was obtained by homologous cloning strategy,and the soluble expression of pulM was achieved in Escherichia coli BL21.After fermentation and purification,the specific activity of pullulanase was 72.6 U/mg,the optimum reaction temperature was 40℃,the optimum pH was 6.5,K_(m)=(6.442±0.4668)mg/mL,V_(max)=(89.84±2.795)μmol·min^(-1)·mg^(-1).The enzyme activity was kept at 40%at 40℃for 4 h.The enzyme activity was kept above 80%when it stored in pH 7.0~8.0 buffer for 12 h.The metal ions Ca^(2+),Na^(+),Li^(+)and Ni^(+)significantly activated the enzyme activity of PulM.It provides a new idea for the application research of pullulanase in industry.
作者
郭晓敏
李宁
唐玉
路卫卫
李伟亮
董宇辰
陆坚
黄日波
Guo Xiaomin;Li Ning;Tang Yu;Lu Weiwei;Li Weiliang;Dong Yuchen;Lu Jian;Huang Ribo(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,College of Life Science and Technology,Guangxi University,Nanning,530005;College of Animal Science and Technology,Guangxi University,Nanning,530005)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第9期3035-3043,共9页
Genomics and Applied Biology
基金
广西大学博士启动基金项目(XBZ160253)
广西重大科技创新基地建设项目(2018-15-Z03)
广西自然科学基金青年基金项目(2018GXNSFBA281017)共同资助
关键词
普鲁兰酶
筛选鉴定
基因克隆
重组表达
酶学性质
Pullulanase
Screeningand identification
Gene cloning
Recombinant expression
Enzymatic properties
