一种中温淀粉酶在解淀粉芽孢杆菌LT的重组表达

Recombinant Expression of a Medium-temperature Amylase Production in Bacillus amyloliquefaciens LT

解淀粉芽孢杆菌可用作多种食品工业酶的生产菌株,但其极低的转化效率影响了这类工业菌株的重组改造及优化。通过对1398解淀粉芽孢杆菌和一种地衣芽孢杆菌Jbac基因组的数据分析,发现这些微生物存在IV型DNA限制修饰系统。对这些微生物转化经甲基化和未经甲基化的质粒,证实外源DNA甲基化修饰会降低DNA的转化效率;利用将外源质粒进行相同类型甲基化的这种新转化策略,表达质粒p MK4E-dx被转入解淀粉芽孢杆菌LT中,发酵结果显示,其中重组菌株LT-J1/pMK4-dx和LT-J2/pMK4-dx分泌的中温淀粉酶活达到145.9和153.6 U/m L,分别为原菌株发酵淀粉酶活的3.65和3.84倍。

Bacillus amyloliquefaciens is one of best host cells for production of a wide spectrum of enzymes applied in food industry.However,studies for strain optimization are always difficult due to the low efficiency in transforming foreign DNA into those industrial strains.A novel type IV DNA methylation/restriction system was identified in B.amyloliquefaciens and other close species that was not found in model bacillus strain through genomic data mining,such as B.subtilis 164or 168.It was confirmed that transformation efficiency of non-methylated DNA was much higher than DNAs purified from E.coli strain with methylation activity.With the optimized vector preparation and transformation protocols,two recombinant B.amyloliquefaciens LT transformants expressing its native medium-temperature amylase were obtained,namely LT-J1and LT-J2,they produced the enzyme up to 145.9U/mL and 153.6U/mL,respectively,more than two times higher than the original strain that does not harbor the expression vector pMK4E-dx.