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.

Key words: glucose oxidase, Cladosporium tianshanense SL19, gene cloning, gene expression, characterization

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。

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