摘要
为寻找新纤维素酶和纤维素酶基因资源,以羧甲基纤维素钠(CMC)改良的BHM培养基筛选菌株,经初筛和复筛筛选出目的菌株后,采用革兰氏染色法和16SrDNA基因片段比对法对筛选出的菌株进行鉴定;设计引物对P扩增其纤维素酶基因,连于pET-28a(+)载体构建原核表达载体并转入大肠杆菌BL21(DE3)进行表达,表达结果采用SDS-PAGE和测定表达产物活性的方法来检验。结果显示,本研究成功筛选出一株新的具有较高纤维素酶活的短小芽孢杆菌BY-1,该菌所产纤维素酶具有较好的热稳定性和酸碱稳定性,最适宜温度和pH分别为55℃和7.45,在此条件下最高酶活性为0.921 6μmol/(mL.min);此外,还成功克隆出1个1 851bp的纤维素酶新基因bglC-BY,具有GH9/CBM3纤维素酶结构,经IPTG诱导,SDS-PAGE图谱上于70ku有1个明显的蛋白条带,表达产物酶活性为0.541 3μmol/(mL.min)。本研究筛选出的短小芽孢杆菌内切纤维素酶活较高,具有一定的应用前景。
The aim of this study was to exploit new resources of cellulolytic bacteria and new cellulose genes for industrial processes.In this study,targeted bacteria were screened by CMC amended BHM medium and identified by Gram staining with 16S rDNA partial sequence alignment.After amplification of cellulase gene using primer pair P,it was ligated to pET-28a（＋） in corresponding sites to construct prokaryotic expression vector.Heterogeneous expression was conducted in E.coli BL21（DE3） and verified by SDS-PAGE and enzyme detection.The Bacillus pumilus BY-1 with premium enzyme activity was successfully screened.Under the optimum condition of 55 ℃ in temperature and 7.45 in pH,the crude enzyme,reached the maximum enzyme activity at 0.921 6 μmol/（mL·min）.Simultaneously,a new 1 851 bp cellulase gene bglC-BY with the structure of GH9/CBM3 was successfully cloned from this bacterial strain.After IPTG induction,SDS-PAGE verified the successful expression of bglC-BY with a mass of about 70 u and the enzyme activity was 0.541 3 μmol/（mL·min）.Thus,a potential new strain and a new endoglucanase gene with premium activity can be provided for industrial processes.
出处
《中国农业大学学报》
CAS
CSCD
北大核心
2012年第5期92-98,共7页
Journal of China Agricultural University
基金
国家科技支撑计划项目(2011BAD28B05-3)
陕西省科技统筹创新工程计划(2011KTCL02-09)
关键词
纤维分解菌
筛选
纤维素酶基因
克隆
表达
cellulolytic microbes； screening； cellulase gene； cloning； expression
