特异腐质霉来源漆酶基因的克隆及其在毕赤酵母中的表达

漆酶是一种含铜的多酚氧化酶,在有机农药残留和木质素的降解方面具有潜在的工业价值。从特异腐质霉Y1(Humicola insolens Y1)中分离到了漆酶基因Lac1,其全长1 803 bp,编码600个氨基酸,与NCBI蛋白数据库比对结果表明,与来源于Chaetomium globosum CBS 148.51(XP_001228806)的多酚氧化酶序列相似性最高为71%,表明是一个新的漆酶基因。将其克隆入毕赤酵母表达载体pPIC9r中,通过PCR和SDS-PAGE检测证实基因已经整合入酵母基因组中且能分泌表达。在3 L发酵罐中,重组Lac1蛋白表达量达到3.79mg/mL发酵液,酶活性达到1.3 U/mL发酵液。酶学性质分析结果显示,漆酶的最适作用温度为65℃,最适反应pH为4.5,且在pH 6～11的范围内酶的相对活力均在70%以上。

Cloning of a Novel Laccase Gene From Humicola insolens Y1 and Its Heterologous Expression in Pichia pastoris

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.