产碱性淀粉酶重组枯草芽孢杆菌的构建与发酵优化

Construction and Fermentation Optimization of a Recombinant Bacillus subtilis Producing Alkaline Amylase

摘要:

碱性淀粉酶催化淀粉在碱性环境中高效降解，广泛应用于纺织退浆、洗涤、制药等领域。通过信号肽筛选，将来源于嗜碱单胞菌Alkalimonas amylolytica 的碱性淀粉酶基因于枯草芽孢杆菌Bacillus subtilis WB600实现分泌表达，并优化了重组菌产酶的发酵条件。在最佳信号肽ywbN介导下，重组B. subtilis发酵51 h，胞外AmyK酶活最高达146.86 U/mL，且能在发酵上清液中检测到与AmyK理论相对分子质量（60 kDa）一致的蛋白质条带。通过单因素实验优化了发酵培养基（糊精32 g/L，蛋白胨12 g/L，酵母粉24 g/L，KH2PO4 2.32 g/L，K2HPO4·3H2O 16.43 g/L），并在发酵24 h后添加0.2 g/dL Na2CO3调节发酵液pH，发酵72 h胞外AmyK酶活达640.33 U/mL，较优化前提高了336%，是目前已报道重组B. subtilis产碱性淀粉酶的最高水平。研究结果为AmyK发酵生产的工业化提供了数据支撑。

Abstract:

Alkaline amylase，which could efficiently hydrolyze starch under alkaline conditions，has been widely used in textile desizing field，detergent industry，and pharmaceutical production. In this study，alkaline amylase from Alkalimonas amylolytica （AmyK） was screened through signal peptide and expressed in Bacillus subtilis WB600. The fermentation condition of the recombinant strain was then optimized. The extracellular AmyK activity of the recombinant B. subtilis could reach a maximal level of 146.86 U/mL with 51 h fermentation mediated by ywbN signal peptide. There was a protein band observed in the culture supernatant which was consistent for the theoretical molecular weight of 60 kDa for AmyK. The fermentation medium was optimize via a single factor test and determined as dextrin of 32 g/L，tryptone of 12 g/L，yeast extract of 24 g/L，KH2PO4 of 2.32 g/L，and K2HPO4·3H2O of 16.43 g/L with pH regulated by 0.2% w/v Na2CO3 after 24 h fermentation. The AmyK activity reached to 640.33 U/mL after 72 h，increasing by 336% when compared with the initial medium，which was the highest yield of alkaline amylase for the reported recombinant B. subtilis strains. This study provided a experimental support for the commercial production of AmyK from fermentation.