低温、嗜盐α-淀粉酶Amy3的克隆、表达及重组酶性质

摘要

[目的]从分离自北极海底沉积物Pseudoalteromonas sp.K8菌株克隆、重组表达α-淀粉酶Amy3,并研究其酶学性质.[方法]基于Pseudoalteromonas haloplanktis TAC 125基因组分析,从亲缘关系较近的Pseudoalteromonas sp.K8克隆获得α-淀粉酶基因amy3,以大肠杆菌为宿主进行重组表达,经Ni-NTA亲和层析柱纯化获得重组蛋白Amy3.以可溶性淀粉等为底物,研究Amy3的酶学性质.[结果]Amy3最适催化pH为8.5,在pH 6.5-10.0范围内酶活力维持在40％以上;其在pH 7.5-8.5范围的稳定性较好,pH 8.0条件下的半衰期可达4h.Amy3在低温下较稳定,25℃半衰期为5h;最适反应温度为25℃,并且在0℃可以保持50％以上酶活力,显示良好的低温催化特性.NaCl能够有效提升Amy3的酶活力及稳定性;荧光光谱分析表明,NaC1并未引起Amy3酶蛋白三级结构的改变.动力学分析显示,NaC1影响了酶催化的Km及Kcat,进而提升了酶的催化效率.底物特异性分析表明,Amy3对支链淀粉的水解能力优于直链淀粉,并能够有效地水解小麦淀粉、玉米淀粉和木薯淀粉.[结论]来源于Pseudoalteromonassp.K8菌株的α-淀粉酶Amy3具有良好的低温催化及嗜盐性,在洗涤、食品、污水处理等行业中有潜在的应用前景.%[Objective] The present study was to clone a novel alpha-amylase from marine bacterium Pseudoalteromonas sp.K8 isolated from the sediment of Kongsfjorden and characterize the enzyme.[Methods] We cloned a cold-active and salt-tolerant α-amylase Amy3,from Pseudoalteromonas sp.K8.The protein was expressed in Escherichia coli,purified by Ni-NTA and characterized.[Results] Using soluble starch as substrate,the optimum pH of Amy3 was about pH 8.5,and more than 40％ of the maximal activity maintained in the pH range of 6.5 to 10.Amy3 showed the maximum activity at 25 ℃,and retained more than 50％ activity at 0 ℃.It exhibited improved catalytic activity and thermostability in NaC1 solution,with maximal activity occurring at 800 mmol/L NaC1/KC1,and more than 50％ of maximal activity retained after incubation in 2 mol/L NaC1 for 80 h at 25 ℃.NaC1 did not cause significant change of the global tertiary structure of Amy3,whereas influenced the catalysis efficiency.Amy3 hydrolyzed amylopectin preferentially,and could also hydrolyze wheat starch,corn starch and tapioca starch.[Conclusion] The results indicated that the alpha-amylase Amy3 is a cold-active and salt-tolerant α-amylase with potential use in basic research and industry.