果胶酯酶作为一种食品酶制剂,因其能够将高酯果胶的甲酯基脱去生成低酯果胶而受到广泛关注,但目前国内尚无自主研发的商品果胶酯酶。为了促进果胶酯酶的应用,该研究合成了密码子优化后的黑曲霉果胶酯酶基因,并实现了其在毕赤酵母X33中成功表达。3 L发酵罐高密度发酵96 h后,得到的发酵液上清液中果胶酯酶的活力为85.12 U/mL,是摇瓶水平的7倍,也是目前果胶酯酶表达的最高水平。对重组果胶酯酶进行分离纯化和酶学性质研究,结果表明,重组果胶酯酶的最适pH和最适温度分别是pH 5.0和55 ℃,Km和Vmax分别是13.8 mmol/L和9.04 μmol/(L·min)。该酶的温度稳定性较好,在55 ℃处理40 min,剩余活力仍保持在60%以上;其pH耐受范围广,在pH 3.5~6.5下处理120 min,仍保持60%以上的剩余活力。以高酯果胶为底物,对该果胶酯酶的脱酯条件进行优化,优化得到的脱酯工艺参数为:30 g/L果胶、加酶量65.4 U/g、脱酯温度50 ℃、初始pH 5.5、脱酯时间60 min。在此条件下果胶的酯化度从70.8%降至13.6%。该研究重组表达得到的果胶酯酶具有良好的pH稳定性和热稳定性,在低酯果胶的工业生产方面具有良好的应用前景。
Pectinesterase, as a food enzyme, has attracted attention because it can remove the methyl ester group of high methoxyl pectin to produce low methoxyl pectin. In order to promote the application of pectinesterase, a codon optimized pectinesterase gene from Aspergillus niger was synthesized and expressed in Pichia pastoris X33. After 96 h high density fermentation in 3 L fermenter, the enzyme activity of pectinesterase in the supernatant of recombinant fermentation broth was 85.12 U/mL, which was 7 fold of the level of shaking flask. The optimum pH and temperature of recombinant pectinesterase were 5.0 and 55 °C, and the Km and Vmax were 13.8 mmol/L and 9.04 μmol/(L·min), respectively. The pectinesterase was stable at 55 ℃ for 40 min, and the residual activity remained above 60%. The pH tolerance range of the pectinesterase was wide, and more than 60% of residual enzyme activity was still maintained at pH 3.5-6.5 for 120 min. The optimal de-esterification conditions were optimized as follows: 30 g/L pectin concentration, 65.4 U/mL pectinesterase, 50 ℃, initial pH 5.5, 60 min. Under the condition, the esterification degree of pectin decreased from 70.8% to 13.6%. The recombinant pectinesterase obtained in this study has good pH stability and thermostability, and has potential application in the industrial production of low methoxyl pectin.
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