产耐热木聚糖酶细菌的分离鉴定及酶易错PCR致突变条件优化  

Isolation & Identification of a Heat-Resistant Xylanase-Producing Bacterial Strain & Optimization of the Enzyme Error-Prone PCR Mutagenic Conditions

作　　者：赵超[1,2,3] 张宁宁[3] 梅凡[1,3] 艾超[1,3] 阮灵伟[4] 黄一帆[1,2] 刘斌[1,2,3] 

机构地区：[1]福建农林大学食品科学学院,福建福州350002 [2]国家菌草工程技术研究中心,福建福州350002 [3]福建农林大学生物能源研究所,福建福州350002 [4]国家海洋局第三海洋研究所海洋生物遗传资源重点实验室,福建厦门361005

出　　处：《微生物学杂志》2013年第6期6-12,共7页Journal of Microbiology

基　　金：国家自然科学基金(41306181;31370146);中国博士后科学基金(2013M530303);福建省教育厅科技项目(JB12055);三亚市院地科技合作项目(2013YD76);国家海洋局海洋生物遗传重点实验室开放研究基金(HY201301);福建农林大学青年教师科研基金重点项目(2012XJJ03)

摘　　要：从福建省永泰县温泉采集样品中筛选到1株产耐热木聚糖酶嗜热菌株TC-W7,并获得该木聚糖酶基因。在此基础上,采用易错PCR技术在木聚糖酶基因中引入突变,研究Mg2+浓度、Mn2+浓度、dTTP/dCTP浓度等条件对突变率的影响。通过形态特征、生理生化试验及16S rRNA序列相似性比对分析,初步鉴定菌株TC-W7为土壤芽胞杆菌(Geobacillus),菌株TC-W7在最适温度75℃和pH 8.2条件下,其木聚糖酶活力为215.83 U/mL,Triton X-100和DDT能显著增强该酶的活性。在Mg2+浓度为20μmol/L,Mn2+浓度为0.80μmol/L,dTTP/dCTP浓度为0.30 mmol/L的致突变条件下,碱基突变率为0.98%。Geobacillus sp.TC-W7产木聚糖酶具有较好的耐热和耐碱等工业应用特性,对该酶易错PCR致突变条件优化结果,可用于后续木聚糖酶的耐热定向进化。A heat-resistant xylanase-producing bacterial strain TC-W7 from samples collected in a hot spring in Yong-tai County, Fujian Province was screened and obtained xylanase gene of the strain. Based on these an error-prone PCR （ Ep-PCR） technique was adopted to introduce mutation in the xylanase gene, to study the effects of the concentration such as Mg2＋, Mn2＋ and dTTP/dCTP and other conditions on the mutation rate. It was initially identified that strain TC-W7 belonged to Geobacillus through morphology features, physiological and biochemical tests as well as 16S rRNA＆amp;nbsp;sequence comparative analysis. Under the most suitable temperature 75℃ and pH 8. 2, the activity of xylanase was at 215. 83 U/mL, Triton X-100 and DDT could remarkably increase the activity of xylanase. The base mutation rate was at 0. 98% under the mutagenic conditions of 20. 0 μmol/L Mg2＋, 0. 80 μmol/L Mn2＋ and 0. 30 mmol/L dTTP/dCTP. The xylanase-producing Geobacillus sp. TC-W7 had a fine heat and alkali resistance and other industry appli-cable features. The results of Ep-PCR mutagenic conditions optimization of the enzyme can be used for the following-up heat-resistance oriented evolution of the xylanase.

关 键 词：耐热木聚糖酶 土壤芽胞杆菌 分离鉴定 

分 类 号：Q939.97[生物学—微生物学]