亮白曲霉乳糖酶基因在毕赤酵母中的高效分泌表达及酶学性质研究

Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties

将克隆的亮白曲霉 (Aspergilluscandidus)乳糖酶基因lacb′插入到毕赤酵母 (Pichiapastoris)高效表达载体pPIC9中 ,与分泌信号肽序列α_因子融合 ,通过同源重组将lacb′整合到酵母染色体上。通过SDS_PAGE检测和表达产物的酶活性筛选 ,得到重组转化子 ,证明乳糖酶获得有效分泌和高效表达。表达的乳糖酶为糖蛋白 ,表观分子量为 130kD ,脱糖基后的蛋白分子量下降为 110kD。经过 5L小罐高密度发酵 ,重组酵母中酶蛋白表达量为 6mg mL发酵液 ,每毫升发酵液中乳糖酶的活力为 36 0 0U ,高于目前国内外报道的水平。进一步研究了表达产物的酶学性质 ,该酶最适pH为 5 . 2 ,最适反应温度 6 0℃ ,比活性为 70 6 . 5± 2 . 6U mg ,Km为 1 7mmol L ,Vmax为 3. 3μmol min。与米曲霉ATCC 2 0 4 2 3的乳糖酶相比 ,该乳糖酶具热稳定性强、比活性高、pH范围宽等特点。

The lactase gene lacb’ from Aspergillus candidus was fused behind α-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosyled protein with an apparent molecular weight of 130kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6mg/mL with enzymatic activity of 3600U/mL in the 5L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60℃. The V max, K m and specific activity of the lactase are 3.3μmol/min, 1.7mmol/L and 706.5±2.6U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.