南极菌Pseudoalteromonas sp. A211-5产碱性α-淀粉酶Amy172的克隆表达及酶学性质研究

Clone and Expression of Alkaline α-Amylase Amy172 from Antarctic Bacterium Pseudoalteromonas sp. A211-5 and Its Enzymatic Property

对具有淀粉降解活性的南极菌Pseudoalteromonas sp.A211-5进行测序分析,根据基因注释结果,获得了一条基因序列为2 139 bp的淀粉酶基因,命名为amy172;通过特异性引物克隆获得Amy172的基因序列并进行异源表达;采用Ni-NTA层析柱对重组α-淀粉酶Amy172进行纯化,采用DNS法测定其酶学性质,采用薄层层析(TLC)技术对其酶解产物进行分析。结果表明:SDS-PAGE显示在分子量为79 kDa左右有一条明显的目的蛋白条带;重组α-淀粉酶Amy172最适温度为50℃,最适pH值为10.0,且在pH值5.0～10.0的范围内仍能保持80%以上的酶活,金属离子Fe^(3+)、Mn^(2+)、Mg^(2+)、Fe^(2+)、Ca^(2+)、K^+、Sr^(2+)、Ni^(2+)和Ba^(2+)对酶活性具有抑制作用;TLC分析Amy172的酶解产物为葡萄糖、麦芽糖、麦芽三糖和麦芽四糖。

We analyzed the complete genome of a starch degradation bacterium Pseudoalteromonas sp.A211-5 from Antarctic sediment sample by bioinformatics methods.Firstly,according to the results of gene annotation,we obtained amylase gene with the gene sequence of 2 139 bp,and named it amy172.Then,we obtained the gene sequence of Amy172 by cloning of specific primer,and heterologously expressed it.Finally,we purified the recombinant α-amylase Amy172 by Ni-NTA chromatography,determined its enzymatic property by the DNS method,and analyzed the enzymolysis products by thin layer chromatography(TLC).The SDS-PAGE indicates that there is a single band at a molecular weight of about 79 kDa.The optimum temperature of the recombinant α-amylase Amy172 is 50 ℃,the optimum pH value is 10.0,and it maintains more than 80% of the highest activity in the range of pH value of 5.0-10.0.Metal ions,such as Fe^3+,Mn^2+,Mg^2+,Fe^2+,Ca^2+,K^+,Sr^2+,Ni^2+,and Ba^2+,have inhibitory effects on the activity of the recombinant α-amylase Amy172.Moreover,the TLC analysis reveals that the enzymolysis products of Amy172 are glucose,maltose,maltotriose,and maltotetraose.