菊欧氏杆菌β-1,4-内切葡聚糖酶celY基因在大肠杆菌中的克隆、表达及活性分析

以菊欧氏杆菌(Erwinia chrysanthemi)基因组DNA为模板,通过PCR方法扩增了该菌的β-1,4-内切葡聚糖酶celY基因及其调控元件并克隆至pUC19载体。测序结果经分析,该基因编码的蛋白序列与菊欧氏杆菌Ech586的β-1,4-内切葡聚糖酶同源性达到98%。将celY的开放阅读框基因插入到表达载体pET-28a中,转化大肠杆菌E.coli BL21(DE3),采用LB-CMC平板刚果红染色筛选含celY基因的转化子。12%SDS-PAGE显示,重组菌经IPTG诱导后表达了目的蛋白,其相对分子质量与预期结果相符。CMCase活力测定显示重组pET-28a-celY阳性菌分泌到培养基中的CelY酶活力为84.4 U/L,周质中的酶活力为12.7 U/L。β-1,4-内切葡聚糖酶CelY工程菌的获得,为研究菊欧氏杆菌纤维素酶基因在菊欧氏杆菌同源转化中的表达及工程菌的酶学特性提供了重要的资料。

Cloning and Expression of Endo-1,4-β-D-glucanase celY Gene from Erwinia chrysanthemi in Escherichia coli and Its Activity Analysis

The celY gene and its regulatory elements encoding endo-1,4-β-D-glucanase celY from a strain of Erwinia chrysanthemi was amplified by PCR and cloned into plasmid pUC19.Sequencing analysis showed that the sequence of endo-1,4-β-D-gluc-anase exhibited 98% homology with E.chrysanthemi 586’s.Then the ORF of celY gene was inserted into expression plasmid pET-28a and the recombinant plasmid was transformed into Escherichia coli BL21(DE3) and induced to express.Clones were screened for endoglucanase production using the Congo red method.From the results of the 12% SDS-PAGE,the recombinant plasmid could express the target protein by IPTG induction and its relative molecular mass met the expectation.The CMCase activity measurement showed that the enzyme activity in supernatant was 84.4 U/L,and 12.7 U/L in periplasm.The endo-1,4-β-D-glucanase engineering bacteria which were obtained provides the important material to study the expression of autogenic transformation for the Erwinia chrysanthemi cellulose genes and to analyze the characterstics of recombinant.