曲霉来源果胶裂解酶PnlF的克隆表达及酶学性质分析  

Cloning,expression and characterization of pectin lyase PnlF from Aspergillus

作　　者：宋晓慧 李悝悝 赵秒 李唐 尹恒[2] SONG Xiaohui;LI Kuikui;ZHAO Miao;LI Tang;YIN Heng(School of Textile and Engineering,Dalian Polytechnic University,Dalian 116023,China;Natural Products&Glycoconjugate Research Group,Liaoning Provincial Key Laboratory of Carbohydrates,Dalian 116023,China)

机构地区：[1]大连工业大学纺织与材料工程学院,辽宁大连116023 [2]中科院大连化学物理研究所辽宁省碳水化合物重点实验室大连市糖类农用制剂工程研究中心,辽宁大连116023

出　　处：《食品与发酵工业》2023年第1期25-32,共8页Food and Fermentation Industries

基　　金：大连市科技创新基金计划重点学科重大课题项目(2020JJ25CY017);陕西省重点研发计划项目(2020ZDLNY07-03)。

摘　　要：果胶裂解酶是一类通过β-消除机制降解果胶的多糖裂解酶,对果胶的利用具有重要意义。该研究利用真核表达载体,克隆表达了来自曲霉属Aspergillus costaricensis的酸性果胶裂解酶PnlF,经过亲和层析和凝胶过滤层析对该酶的发酵液进行了分离纯化,并对其酶学性质和降解果胶的产物进行了系统性研究。该研究成功实现了PnlF在毕赤酵母中的分泌表达,粗酶活力达到526 U/mg。PnlF的最适反应温度为50℃,最适反应pH为5.0;其酶活力在20~30℃及pH 5~6内稳定。Ca^(2+)和Mg^(2+)可以大幅度提高PnlF的活力;而Ag+和Cu^(2+)则会严重抑制其活力;此外SDS、TritonX-100、苯甲磺酰氟等化学试剂对该酶活性的抑制作用较强。Pectin lyases are a class of polysaccharide lyases that degrade pectin through aβ-elimination mechanism,and are of great significance to the utilization of pectin.In this study,the eukaryotic expression system was used to clone and express the pectin lyase PnIF from Aspergillus costaricensis.The broth of PnlF was isolated and purified by affinity chromatography and gel filtration chromatography,and its enzymatic properties as well as the degradation products from pectin through the PnlF activity were systematically investigated.The results showed that the secretory expression of PnIF in Pichia pastoris was successfully achieved,and the crude enzyme activity reached 526 U/mg.The optimum reaction temperature of PnlF was 50℃,and the optimum reaction pH was 5.0.The enzyme activity of PnlF was stable at 20-30℃and pH 5-6.Ca^(2+)and Mg^(2+)can strongly enhanced the activity of PnlF,while Ag+and Cu^(2+)can seriously inhibit its activity.In addition,chemical reagents such as SDS,TritionX-100 and PMSF have strong inhibitory effects on the enzyme activity.

关 键 词：果胶裂解酶 重组表达 分离纯化 酶学性质 产物分析 

分 类 号：TS201.25[轻工技术与工程—食品科学]