强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株。为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善。利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称的位置引入耶氏解脂酵母来源的脂肪酶基因lipY2。整合菌株在摇瓶发酵44h时,木聚糖酶、脂肪酶酶活力分别达(58±2.07)U/mL和(207±10.62)U/mL,其分泌表达促进了地衣芽孢杆菌对发酵培养基的分解与利用,提高了培养基中还原糖、上清总氮的含量和沉淀中含氮化合物的分解;细菌生物量较地衣芽孢杆菌原始菌株提高了11.76%,同时碱性蛋白酶的发酵周期较原始菌提前了4h,碱性蛋白酶产量提高了14.41%。地衣芽孢杆菌2709分泌酶系的丰富和发酵性能的改善为在饲料行业中作为微生物制剂的地衣芽孢杆菌提供了改造的方法。

Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in Bacillus licheniformis

Bacillus licheniformis 2709 is a strain of alkaline protease that has been put into industrial production due to its easy cultivation,GRAS status and perfect protein secretion ability.In order to improve the fermentation production performance of the strain,increase the utilization of culture medium components and the production of alkaline protease by the bacteria,the extracellular secretion enzyme system has been improved.With the help of homologous recombination mechanism,the xylanase gene(xynA)from Bacillus pumilus was introduced near the origin of replication and the lipase gene(lipY2)from Yarrowia lipolytica was introduced into the center of the origin of replication.The results showed that the activity of xylanase and lipase reached(58±2.07)U/mL and(207±10.62)U/mL,respectively when the strain was fermented in shake flask for 44 h.Their efficient secretion and expression ability promoted the utilization rate of the fermentation substrate by Bacillus licheniformis,thus increasing the content of reducing sugar and total nitrogen in the medium and decomposing nitrogen compounds in the precipitate.Compared with the original strain,the bacterial biomass of the mutant strain was increased by 11.76%,the fermentation cycle of alkaline protease was shortened by 4 h,and the yield of alkaline protease was increased by 14.41%.The enrichment of the secreted enzymes and the improvement of fermentation performance of Bacillus licheniformis 2709 provide a method for modification of Bacillus licheniformis as a microbial agent in the feed industry.