| Cladosporium tianshanense SL19来源的新型葡萄糖氧化酶的基因克隆及性质研究 |
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| 引用本文： | 葛建忠,尹亚昕,蒋肖,刘伟娜,姚斌,罗会颖.Cladosporium tianshanense SL19来源的新型葡萄糖氧化酶的基因克隆及性质研究[J].中国农业科技导报,2019,21(12):49-57. |
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| 作者姓名： | 葛建忠 尹亚昕 蒋肖 刘伟娜 姚斌 罗会颖 |
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| 作者单位： | 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 |
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| 基金项目： | 现代农业产业技术体系项目（CARS-41）。 |
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| 摘 要： | 葡萄糖氧化酶(GOX)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GOX外,鲜有新型GOX被报道。经基因克隆和序列同源性分析,Cladosporium 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1 707 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CtGOXB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CtGOXB-C1最终恢复了GOX活性。经SDS-PAGE鉴定,重组蛋白CtGOXB-C1表观分子量约为120 kDa,大于理论分子量(61.6 kDa)。经酶学性质测定,CtGOXB-C1比活为123.8 U/mg,在pH 8.0和30℃条件下具有最高的酶活,且在10℃时依然维持65%的酶活,具有嗜低温特性。CtGOXB-C1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-C1在水产饲料和洗涤剂等行业中具有良好的应用潜力。
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| 关 键 词： | 葡萄糖氧化酶 枝孢菌SL19 基因克隆 基因表达 性质研究 |
Gene Cloning and Characterization of a Novel Glucose Oxidase From Cladosporium tianshanense SL19 |
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| GE Jianzhong,YIN Yaxin,JIANG Xiao,LIU Weina,YAO Bin,LUO Huiying.Gene Cloning and Characterization of a Novel Glucose Oxidase From Cladosporium tianshanense SL19[J].Journal of Agricultural Science and Technology,2019,21(12):49-57. |
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| Authors: | GE Jianzhong YIN Yaxin JIANG Xiao LIU Weina YAO Bin LUO Huiying |
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| Institution: | 1.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2.College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China |
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| Abstract: | Glucose oxidase (GOX) is widely used in various industrial processes. Few new types of GOX have been reported except from Aspergillus and Penicillium. A novel glucose oxidase gene CtgoxB from Cladosporium tianshanense SL19 was obtained by gene cloning and sequence homology analysis. The full-length DNA fragment of CtgoxB was 1 707 bp without intron, which encoded a protein of 568 amino acids with a signal peptide at N-terminal 1～16 amino acid residues. The amino acids sequence encoded by CtgoxB shared the highest identity of 35% with reported GOXs. The activity could not be detected when CtgoxB gene was expressed in P. pastoris. The mutant CtGOXB-C1 was obtained according to the sequence and structural analysis by homologous recombination, which recovered the activity of glucose oxidase. The molecular weight of recombinant CtGOXB-C1 showed about 120 kDa by SDS-PAGE, which was larger than the theoretical molecular weight (61.6 kDa). The purified recombinant glucose oxidase CtGOXB-C1 exhibited maximal activity at pH 8.0 and 30 ℃. Its specific activity was 123.8 U/mg, and it retained about 65% activity at 10 ℃ with a psychrophilic characteristic. The CtGOXB-C1 was stable at pH 6.0～9.0 and retained more than 80% of activity after incubating for 1 h, and maintained high enzyme activity under medium-alkaline condition and was highly resistant against SDS. All these favorable enzymatic properties showed that CtGOXB-C1 was a good candidate for the aquatic feeds and detergent industries. |
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| Keywords: | glucose oxidase Cladosporium tianshanense SL19 gene cloning gene expression characterization |
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