黑曲霉果胶酯酶在毕赤酵母中异源表达、性质分析及脱酯工艺优化  

Heterologous expression and properties analysis of Aspergillus niger pectinesterase in Pichia pastoris and process optimization of its de-esterification

作　　者：孙莹[1] 吴丹[1] 郑璞[1] 陈鹏程 SUN Ying;WU Dan;ZHENG Pu;CHEN Pengcheng(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2022年第21期17-23,共7页Food and Fermentation Industries

基　　金：国家轻工业技术与工程一流学科计划项目(LITE2018-04)。

摘　　要：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℃,K_(m)和V_(max)分别是13.8 mmol/L和9.04μmol/(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 K_(m) and V_(max)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.

关 键 词：果胶酯酶 毕赤酵母 异源表达 高密度发酵 酶学性质 脱酯 

分 类 号：TS201.2[轻工技术与工程—食品科学]