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

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