Cladosporium tianshanense SL19来源的新型葡萄糖氧化酶的基因克隆及性质研究  

Gene Cloning and Characterization of a Novel Glucose Oxidase From Cladosporium tianshanense SL19

作　　者：葛建忠 尹亚昕 蒋肖 刘伟娜 姚斌[1] 罗会颖[1] GE Jianzhong;YIN Yaxin;JIANG Xiao;LIU Weina;YAO Bin;LUO Huiying(l.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture,Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China;College of Food Science and Engineering,Northwest A&F University,Yangling 712100,China)

机构地区：[1]中国农业科学院饲料研究所农业部饲料生物技术重点实验室,北京100081 [2]中国陕西省杨凌区西北农林科技大学食品科学与工程学院,陕西杨凌712100

出　　处：《中国农业科技导报》2019年第12期49-57,共9页Journal of Agricultural Science and Technology

基　　金：现代农业产业技术体系项目(CARS-41)

摘　　要：葡萄糖氧化酶(G0X)已广泛应用于各种工业生产中。目前,除了曲霉和青霉属来源的G0X夕卜,鲜有新型G0X被报道。经基因克隆和序列同源性分析,C/oz/osporium tianshanense SL19来源的葡萄糖氧化酶基因CtgoxB是一个新基因,该基因全长1707 bp,无内含子,编码568个氨基酸,N-端第1~16个氨基酸为信号肽,与已报道的GOX序列最高一致性为35%。CtgoxB在毕赤酵母中实现异源表达,但重组蛋白C/GOXB未检测到GOX活性。通过序列和结构分析,同源重组构建的突变体CzGOXB-Cl最终恢复了GOX活性。经SDSPAGE鉴定,重组蛋白CrGOXB-Cl表观分子量约为120 kDa,大于理论分子量(61.6 kDa)o经酶学性质测定,CzGOXB-Cl比活为123.8 U/mg,在pH 8.0和30咒条件下具有最高的酶活,且在10七时依然维持65%的酶活,具有嗜低温特性。CtGOXB-Cl在pH 6.0-9.0条件下保温1 h后,剩余酶活维持在80%以上,可以在中碱性条件下维持较高酶活,并且对表面活性剂SDS具有较强的抗性。这些性质表明CtGOXB-Cl在水产饲料和洗涤剂等行业中具有良好的应用潜力。Glucose oxidase ( GOX) is widely used in various industrial processes. Few new types of GOX have beenreported except from Aspergillus and Penicillium. A novel glucose oxidase gene CtgoxB from Cladosporiumtianshanense SL19 was obtained by gene cloning and sequence homology analysis. The full-length DNA fragment ofCtgoxB was 1 707 bp without intron, which encoded a protein of 568 amino acids with a signal peptide at N-terminal1 - 16 amino acid residues. The amino acids sequence encoded by CtgoxB shared the highest identity of 35% withreported GOXs. The activity could not be detected when CtgoxB gene was expressed in P. pastoris. The mutantCtGOXB-Cl was obtained according to the sequence and structural analysis by homologous recombination, whichrecovered the activity of glucose oxidase. The molecular weight of recombinant CtGOXB-Cl showed about 120 kDa bySDS-PAGE, which was larger than the theoretical molecular weight ( 61.6 kDa) . The purified recombinant glucoseoxidase CzGOXB-Cl exhibited maximal activity at pH 8.0 and 30 °C.. Its specific activity was 123.8 U/mg, and itretained about 65% activity at 10 七 with a psychrophilic characteristic. The CzGOXB-Cl was stable at pH 6.0~9.0and retained more than 80% of activity after incubating for 1 h, and maintained high enzyme activity under mediumalkalinecondition and was highly resistant against SDS. All these favorable enzymatic properties showed that C/GOXB-Cl was a good candidate for the aquatic feeds and detergent industries.

关 键 词：葡萄糖氧化酶 枝抱菌SL19 基因克隆 基因表达 性质研究 

分 类 号：Q78[生物学—分子生物学]