4种芽孢杆菌来源木聚糖酶基因在大肠杆菌中的表达及酶活分析

Expression and enzymatic properties analysis of four sources of bacillus xylanase in Escherichia coli

研究旨在比较4种不同来源木聚糖酶基因在大肠杆菌E.coli BL21中表达后的酶活等性质，为生产中筛选高表达量及耐极性木聚糖酶基因提供依据、根据已发表的木聚糖酶基因序列设计引物，分别扩增出浸麻芽孢杆菌（B．nlaeerans）B3、地衣芽孢杆菌（B．1ichenitormis）B6、蜡样芽孢杆菌（B．cereus）B10和枯草芽孢杆菌（B．subtilis）B12的木聚糖酶基因片段，经克隆表达后获得4种工程茼经培养和IPTG诱导后进行SDS—PAGE电泳检测及酶特性分析、测序结果表明，4个基因的开放阅读框均为642bp。其中前3种木聚糖酶基因均为首次报道、4种重组木聚糖酶在55℃下的酶活分别为：36．16、3．85、7．22、98．98U／ml、、4种重组木聚糖酶的最适反应温度均在50～60℃，且枯草芽孢杆菌木聚糖酶最为耐热，、结果提示，枯草芽孢杆菌来源的木聚糖酶具有较高的酶活和较好的耐热性。

In this study, an enzyme activity comparasion was conducted to find the differenees among four kinds of xylanase gene expressions in E.coli BI21, providing the basis for factories to screen tile gene with high expression and heat resistance. A pair of primers was designed according to tile puh lished nucleotidc sequences of putative xylanase genes. With the primers, four target fragments of the β- 1,4 - endo-xylanase gene was amplified from Bacillus macerans （B.macerans） B3, the lichens Bacillus （B.lichenifonnis） B6, Bacillus cereus （B.cereus） B10 and Bacillus（B. subtilis） B12, the sequencing re.suits showed that the open reading frame of the four genes were all 642 bp. Three of which were first report- ed. The results of enzyme eharacteristics analysis showed that the fllur recombinant xylanase enzyme ac tivity at 55 ℃ was 36.16, 3.85, 7.22 and 98.98 U/ml, respectively, in which the bacillus suhlilis xylanase enzwne showed the highest enzyme activity. The heat resistance experiment results showed that the bacillus subtilis xylauase had a higher enzyme activity and better heat-resistance.