绿色糖单孢菌麦芽糖α-淀粉酶基因在枯草芽孢杆菌中的高效分泌表达

High Level Secretion Expression of Maltogenic α-amylase from Saccharomonospora viridis in Bacillus subtilis

关键词:  麦芽糖α-淀粉酶  枯草芽孢杆菌  高效表达  优化

Key words:  maltose α-amylase  Bacillus subtilis  high efficient expression  optimization

摘要:

[目的]构建高产麦芽糖α-淀粉酶的工程菌株并实现高效分泌表达。[方法]PCR扩增麦芽糖α-淀粉酶基因sva,再与大肠杆菌-枯草芽孢杆菌麦芽糖诱导型穿梭载体pHCMCO4-Pglv连接,构建重组质粒pHCMCO4-Pglv-sva并转入枯草芽孢杆菌进行表达,对重组酶进行SDS-PAGE分析,然后对重组菌株的生长及发酵条件进行优化。[结果]成功构建重组质粒pHCMCO4-Pglv-sva并在枯草芽孢杆菌中实现分泌表达,SDS-PAGE分析发现,在55kDa处得到特异性蛋白条带。单因素试验结果显示,重组菌的最适诱导温度为35℃,最适接种量为4%(V/V),最适装液量为30mL。正交实验结果显示,重组菌的最佳发酵条件组合是诱导温度37℃、接种量5%(V/V)、装液量25mL,在此条件下发酵液粗酶活力达到257.3698U/mL。装液量对重组菌的产酶量影响显著。[结论]成功构建能高效分泌表达麦芽糖α-淀粉酶的枯草芽孢杆菌工程菌株,经发酵条件优化后,重组酶产量显著提高10倍左右。

Abstract:

[Objective] Engineering strains for high yield of maltose alpha amylase were constructed in order to implement efficient secrection expression.[Methods] The gene that had a length of 1 440 bp was amplified by PCR.The recombinant plasmid pHCMCO4-Pglv-sva was constructed and transformed into Bacillus subtilis.The recombinant protein was analyzed by SDSPAGE, and the growing conditions of recombinant strains were further optimized.[Results] Therecombinant strain pHCMCO4-Pglv-sva was successfully constructed and expressed in Bacillus subtilis.The molecular weight of the recombinant protein was approximately 55 kDa by SDS-PAGE analyis.Single factor optimization of fermentation conditions revealed that the recombinant strain had the optimal induction temperature at 35℃, the optimal inoculated quantity of 4% (V/V)and the optimal loading fluid amount of 30 mL.Orthogonal experiment resultsshowed that the optimum fermentation conditions were induction temperature at 37℃, quantity of 5% (V/V)and fluid volume 25 mL, under which the enzyme activity of recombinant maltose alpha amylase reached to 257.3698U/mL.Among them, liquid loading quantity had significant effects on enzyme activity of maltose alpha amylase.[Conclusion] The recombinant plasmid pHCMCO4-Pglv-sva was successfully built and maltose alpha amylase was highly increased to ten times after optimizing fermentation condition.