昆明假单胞菌HL22-2海藻糖合酶基因的克隆、表达及酶学性质研究

Cloning, expression and enzymatic characteristics of trehalose synthase gene from Pseudomonas kunmingensis HL22-2

采用热不对称交错聚合酶链式反应(Tail-PCR)克隆云南磷矿来源昆明假单胞菌(Pseudomonas kunmingensis)HL22-2的海藻糖合酶(TreS)基因HL22-2TreS,将该基因与表达载体pETM3C连接后在大肠杆菌(Escherichia coli)BL21(DE3)pLysS中进行异源表达,通过Ni-NTA柱纯化重组酶HL22-2TreS,并对其酶学特性进行分析。结果表明,HL22-2TreS基因全长3336 bp,编码1111个氨基酸,氨基酸序列与Genbank数据库中相关的海藻糖合酶具有极高的相似性。重组酶HL22-2TreS的分子质量约126 kDa,该酶的最适反应温度和pH值分别为40℃和7.0,在温度20~50℃及pH值6.0~9.0条件下比较稳定,Cu2+、Hg2+、Ba2+及Al3+对海藻糖合酶的活力有强烈的抑制效果。HL22-2TreS基因对麦芽糖和海藻糖的米氏常数(Km)分别为20.6 mmol/L和87.5 mmol/L,对麦芽糖具有更高的亲和性,更容易将麦芽糖转化成海藻糖。

The trehalose synthase(TreS)gene HL22-2TreS of Pseudomonas kunmingensis HL22-2 from Yunnan phosphate rock was cloned by thermal asymmetric interlace polymerase chain reaction(Tail-PCR).This gene was ligated to the expression vector pETM3C and heterologously expressed in Escherichia coli BL21(DE3)pLysS.The recombinase HL22-2TreS was purified by Ni-NTA column and the enzymatic characteristics were analyzed.The results showed that the HL22-2TreS gene was 3336 bp in length and encoded 1111 amino acids.The amino acid sequence was highly similar to the trehalose synthase associated with the Genbank database.The molecular mass of the recombinase HL22-2TreS was about 126 kDa.The optimal reaction temperature and pH of the recombinase was 40℃and 7.0,respectively,which was stable at temperature of 20-50℃and pH of 6.0-9.0.The Cu2+,Hg2+,Ba2+and Al3+had a strong inhibitory effect on the activity of trehalose synthase.The Michaelis constant(Km)of HL22-2TreS for maltose and trehalose was 20.6 mmol/L and 87.5 mmol/L,respectively.It had higher affinity for maltose and was easier to convert maltose into trehalose.