β-甘露聚糖酶基因在枯草芽孢杆菌中的克隆及表达

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.图9表3参19

Cloning and Expression of β-mannanase Gene in Bacillus subtilis

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