关键词: 漆酶, 基因克隆, 异源表达, 毕赤酵母

Abstract: Laccase is a copper-containing polyphenol oxidase, which has potential industrial value in the degradation of organic pesticide residues and lignin. In this study, the laccase gene Lac1 was isolated from a Humicola insolens Y1. The 1 803 bp DNA of Lac1 encoded a mature protein with 600 amino acids. The deduced amino acid sequence was aligned with available protein sequences held in the GenBank, the results showed that the highest similarity was 71% which was the polyphenol oxidase sequence from Chaetomium globosum CBS 148.51 (XP_001228806). The recombinant vector pPIC9r-Lac1 was constructed and transferred into Pichia pastris to integrate into the yeast genome and the recombinant laccase was secreted expression. It was confirmed by the PCR and SDS-PAGE. In 3 L fermentation, the expression of Lac1 protein reached 3.79 mg/mL, and the enzyme activity reached 1.3 U/mL. The analysis of enzymatic properties showed that the optimum temperature was 65℃, the optimum pH was 4.5, and the relative activity of enzyme was above 70% in pH 6～11.

Key words: Laccase, gene cloning, heterologous expression, Pichia pastoris