淀粉液化芽孢杆菌b-1,3-1,4-葡聚糖酶基因的克隆及表达的优化

Optimization of cloning and expression of b-glucanase gene from Bacillus amyloliquefaciens BS5582

摘要:

为了比较不同的表达系统对b-1,3-1,4-葡聚糖酶基因(bgl)的效果, 本研究将高产b-1,3-1,4-葡聚糖酶的淀粉液化芽孢杆菌Bacillus amyloliquefaciens BS5582的bgl基因(GenBank Accession No. EU623974)克隆到3种不同的质粒载体中, 即构建pEGX-4T-1-bgl、pET20b(+)-bgl和pET28a(+)-bgl重组质粒。比较了pEGX-4T-1-bgl在不同Escherichia coli宿主中表达效果, 以及pET20b(+)-bgl和pET28a(+)-bgl 在E. coli BL21(DE3)中的表达效果。结果表明, E. coli BL21(DE3)-pET28a(+)-bgl能够表达最高的重组b-1,3-1,4-葡聚糖酶酶活, 其总酶活可达(322.0±8.8) U/mL, 是出发菌在最适摇瓶发酵条件下产酶活的40.1%。对该重组菌的产酶条件进行了分析, 结合IPTG和乳糖协同的诱导作用, 在基础产酶培养基中产最高总酶活为(1883.3±45.8) U/mL, 表明其具有良好的工业应用价值。

Abstract:

To compare of performance of β-1,3-1,4-glucanase gene (bgl) in different expression systems, the β-1,3-1,4-glucanase gene (GenBank Accession No. EU623974) was amplified from Bacillus amyloliquefaciens BS5582 by PCR and was cloned into three vectors pEGX-4T-1, pET20b(+) and pET28a(+) to construct pEGX-4T-1-bgl, pET20b(+)-bgl and pET28a(+)-bgl recombinant plasmids. The pEGX-4T-1-bgl was transformed into three different Escherichia coli host strains. The pET20b (+)-bgl and pET28a (+)-bgl were transformed into E. coli BL21 (DE3) respectively. Recombinant β-glucanase was expressed by IPTG inducement in these recombinants. E. coli BL21 (DE3)-pET28a (+)-bgl had the highest enzyme activity. In Luria-Bertani medium, the total enzyme activity was (322.0±8.8) U/mL, which was 40.1% of original strain in optimal shaking flask condition. This recombinant’s performance was studied in Terrific Broth medium under inducement of IPTG and lactose at the same time., and the highest total enzyme activity could reach (1883.3±45.8) U/mL (818.8% of the original), which indicate that the recombinant strain has a good value in industry application.