郭明遗^1，2*，马梓婷¹，孙 悦¹，邓 艳¹，何龙羽¹，吴华昌¹，张宿义^3，4

（1.四川旅游学院食品学院，四川成都 610100；

2.四川旅游学院烹饪科学四川省高等学校重点实验室，四川成都 610100；

3.泸州老窖股份有限公司，四川泸州 646000；

4.国家固态酿造工程技术中心，四川泸州 646000）

摘要：以高产酯化酶毕赤酵母（Pichia farinosa）为研究对象，利用双水相萃取体系，在单因素试验基础上，选择聚乙二醇（PEG）分子质量、PEG质量分数、磷酸盐质量分数为主体因素，以比酶活力为指标进行结果分析，采用正交设计优化酯化酶提取工艺条件：在25 mL 4%毕赤酵母细胞混悬液中添加1∶1（m/m）甘露聚糖酶和β-葡聚糖酶双酶破壁剂，使其用量达1.2 mg/g菌体，破壁温度为55 ℃，双水相体系中NaCl质量分数为1%，PEG分子质量为800，PEG质量分数为29%，磷酸盐质量分数为8%，在此条件下酯化酶比酶活力为3 050 U/g。酶学特性结果显示，该酯化酶最适反应温度35 ℃，最适pH 6.0时酯化酶酶活力最高，金属离子Zn^2＋可提高酯化酶活性，Na^＋影响较小，Mg^2＋、Mn^2＋、Cu^2＋均有一定的抑制作用，且Cu^2＋抑制作用最明显。本研究对毕赤酵母功能微生物酯化酶在酿酒领域的开发应用具有重要的参考价值。

关键词：毕赤酵母；酯化酶；聚乙二醇；双水相；酶学性质

中图分类号：TS261   文献标识码：A   文章编号：1674-506X（2023）05-0010-0007

Optimization of Esterases Extraction from Pichia farinosa by Two-enzymes with Aqueous Two-phase System and Research on the Enzymatic Properties

GUO Mingyi^1，2*, MA Ziting¹, SUN Yue¹, DENG Yan¹, HE Longyu¹, WU Huachang¹, ZHANG Suyi^3，4

（1.College of Food Science and Technology, Sichuan Tourism University, Chengdu Sichuan 610100, China;

2.Cuisine Science Key Laboratory of Sichuan Province, Sichuan Tourism University, Chengdu Sichuan 610100, China;

3.Luzhou Laojiao Co., Ltd., Luzhou Sichuan 646000, China;

4.National Research Center of Solid-State Brewing, Luzhou Sichuan 646000, China）

Abstract：A high yield esterase strain of Pichia farinosa was selected as the research strain. Using aqueous two-phase extraction system, based on the results of single-factor tests, three factors including polyethylene glycol (PEG) molecular mass, PEG mass fraction, phosphate mass fraction were identified as main variables that affect enzyme activity. Analyzing results using enzyme activity as an indicator, the extraction conditions of esterases from Pichia farinosa were optimized by orthogonal design technique. The results showed that the optimum extraction parameters were as follows: mannanase and β-glucanase 1∶1 (m/m) adding into 25 mL 4% cell suspension of Pichia farinosa to the amount of 1.2 mg/g cells, cell wall disruption temperature 55 ℃, NaCl mass fraction 1% in aqueous two-phase system, PEG molecular mass 800, PEG mass fraction 29%, phosphate mass fraction 8% . Under these conditions,  the enzyme activity was 3 050 U/g. The enzymatic properties of esterase showed that the enzyme activity was the highest at the optimum reaction temperature of 35 ℃ and the pH value of 6.0. The metal ions Zn^2＋ increased the esterase activity，Na^＋ had insignificant effect，Mg^2＋, Mn^2＋,  Cu^2＋ had inhibitory effect to a certain degree, and the inhibitory effect of Cu^2＋ was the most obvious. This research has important reference value for the development and utilization of Pichia farinosa esterase of functional microorganisms in baijiu brewing field.

Keywords：Pichia farinosa; esterase；polyethylene glycol（PEG）; aqueous two-phase system; enzymatic property

doi：10.3969/j.issn.1674-506X.2023.05-002

收稿日期：2023-03-09

基金项目：四川省自然科学基金项目（2022NSFSC1676）；烹饪科学四川省高等学校重点实验室资助项目（PRKX2021Z03）；四川

省级大学生创新创业训练计划项目（S202111552169）；四川旅游学院大学生科研项目（2023XKZ13）

作者简介：郭明遗（1986-），男，博士，高级工程师。研究方向：食品微生物发酵。

*通信作者

引用格式：郭明遗,马梓婷,孙悦,等.基于双酶辅助双水相提取毕赤酵母酯化酶工艺优化及其酶学特性研究[J].食品与发酵科技,2023,59(5):10-16.