β-甘露聚糖酶基因在枯草芽孢杆菌中的克隆及表达  

Cloning and Expression of β-mannanase Gene in Bacillus subtilis

作　　者：刘项羽[1] 徐美娟[1] 杨套伟[1] 张显[1] 饶志明[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室和江南大学应用微生物与代谢工程研究室,无锡214122

出　　处：《应用与环境生物学报》2012年第4期672-677,共6页Chinese Journal of Applied and Environmental Biology

基　　金：教育部新世纪优秀人才计划(No.NCET-10-0459);国家重点基础研究发展计划(973计划)(No.2012CB725202);国家自然科学基金项目(No.30970056);国家高技术研究发展计划(863计划)(No.2011AA02A211);中央高校基本科研业务费专项资金(No.JUSRP31001);教育部"111"引智计划(No.111-2-06);江苏省优势学科工程项目资助~~

摘　　要：从Bacillus subtilis JNA 3-10中克隆出β-甘露聚糖酶基因成熟肽链编码序列manA1和含信号肽的β-甘露聚糖酶基因manA2,在B.subtilis 168中克隆表达,分别筛选获得高效分泌表达β-甘露聚糖酶的重组菌株BPM1001(pMA5-manA1/B.subtilis 168)和BPM1002(pMA5-manA2/B.subtilis 168),结果表明菌株BPM1002总酶活力是菌株BPM1001的9.65倍,是原始菌株的13.1倍.在基因manA2下游引入His序列克隆出β-甘露聚糖酶基因manA3,获得枯草芽孢杆菌168重组菌株BPM1003.采用Ni-NTA柱纯化重组菌株BPM1003分泌表达的β-甘露聚糖酶,并研究其酶学性质,该酶促反应的最适pH为6.5,最适温度为65℃,在37℃条件下保存一个月酶活力依然保留有77.8%.5 L发酵罐放大实验结果表明魔芋粉对于产β-甘露聚糖酶具有明显的诱导作用,酶活力最高可达2 748.82 U/mL.The manA1 gene encoding mature β-mannanase and the manA2 gene contained a signal peptide from the Bacillus subtilis JNA 3-10 were amplified. The two genes were inserted into expression vector pMA5, and the plasmid pMA5- manA1 and pMA5-manA2 were constructed and transformed into B. subtilis 168 respectively. The recombinant strains BPM1001 （pMA5-manA1 / B. subtilis 168） and BPM1002 （pMA5-manA2/B. subtilis 168） were therefore obtained. SDS-PAGE showed that the genes manA1 and manA2 were expressed successfully in recombinant B. subtilis 168. After incubated in shake-flask, the enzyme activity of strain BPM1002 was 9.65-fold higher than that of BPM1001. Subsequently, the β-mannanase gene manA3 was cloned with His-Tag added downstream of the gene manA2. Then the enzyme was purified successively by Ni affinity chromatography. The analysis of enzymatic properties showed that the optimum activity of the β-mannanase was at pH 6.5 and 65 ℃, and the enzyme activity was maintained 77.8% of original activity after incubated at 37 ℃ for 30 days. The activity of the β-mannanase reached 2 748.82 U/mL in a 5 L fermentor. Fig 9, Tab 3, Ref 19

关 键 词：Β-甘露聚糖酶 枯草芽孢杆菌 信号肽 克隆 表达 重组菌株 酶学性质 

分 类 号：Q939.97[生物学—微生物学] Q78