栓菌420漆酶C基因的克隆、高效表达及重组酶的染料脱色潜能

Cloning, sequence analysis and expression of anthranilate synthetase gene in Corynebacterium pekinense

摘要:

根据漆酶铜结合保守区氨基酸序列设计简并引物，从新型漆酶合成菌株栓菌420（Trametes sp. 420）基因组DNA扩增得到一新的漆酶同工酶基因（lacC）片段，应用长距离反向PCR技术获得其两端侧翼序列。克隆得到的lacC序列长3640bp，包括长2263bp的开放读码框及5′和3′-非编码区。lacC cDNA序列长1560bp，编码一519aa的多肽。推导的LacC蛋白序列内部存在有10个潜在的N-糖基化位点和4个铜原子结合区。将不含自身信号序列的lacC cDNA以pPIC9载体为媒介克隆到表达载体pPIC9K上，转化毕赤酵母（Pichia pastoris）GS115细胞。重组菌在含有0.3mmol/L CuSO4和0.8%丙氨酸的BMM培养基中20℃培养9d，重组漆酶（rLacC）产量达到1.62×104U/L。用发酵粗酶液对终浓度50mg/L的染料进行脱色实验，结果表明，6U/L的rLacC对测试的三甲基类和偶氮类染料具有良好的脱色作用，小分子介体ABTS和HBT能够提高rLacC对染料的脱色效率和脱色速度。

Abstract:

A new laccase gene (lacC) was cloned from the genomic DNA isolated from Trametes sp. 420, a new laccase-producing fungus, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR (LD-IPCR) was used to amplify the flanking sequences of the gene. The lacC DNA sequence obtained was 3640 base pairs (bp), including the entire open reading frame (2263bp) and the 5′-and 3′-noncoding regions. The lacC cDNA sequence is 1560bp, encoding a 519 amino acid protein. The deduced peptide sequence of LacC contains ten putative N-glycosylation sites and four conserved copper-binding regions. The lacC cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The positive transformant was cultured at 20℃ in BMM medium containing 0.3mmol/L CuSO₄ and 0.8% alanine, with the yield of the recombinant laccase rLacC being 1.62×10₄ U/L after a 9-day cell growth. Furthermore, the crude enzyme was used to decolorize several synthetic dyes at a final concentration of 50mg/L. The results showed that rLacC (6U/L) possessed the valuable ability to decolorize dyes of triarylmethane and azo types tested. The presence of low molecular weight redox mediators of ABTS and HBT increased the efficiency and velocity of dye decolorization significantly.