Page 142 - 《精细化工》2023年第11期

第 40 卷第 11 期 精 细 化 工 Vol.40, No.11
2 023 年 11 月 FINE CHEMICALS Nov. 2023

生物工程
高酶活性 β-甘露聚糖酶定向进化及其

用于制备甘露寡糖

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陈 赟，高伟强，梁 琪，周晓雷，王丽丽，张春晓
（河北科技大学 食品与生物学院，河北 石家庄 050018）

摘要：为了提高 β-甘露聚糖酶的活性，采用易错 PCR 将来源于地衣芽孢杆菌（Bacillus licheniformis）KD-1 的 β-
甘露聚糖酶基因 manBl 进行定向进化，并在枯草芽孢杆菌（B. subtilis）中进行表达，以筛选酶活性提高的 β-甘
露聚糖酶突变体。筛选得到的突变体 ManBl(I91N/L211I)，其比酶活性为 15554.7 U/mg，是野生型 ManBl 的 4.1
倍，食品级表达的胞外酶产量达 17601.3 U/mL。分子动力学和分子对接结果表明，β-甘露聚糖酶在 2 个位点
（I91N/L211I）的突变，导致蛋白催化口袋柔性增加，与底物结合能力提高，进而提高了酶的催化效率。β-甘露
聚糖酶 ManBl(I91N/L211I)水解魔芋胶产物主要由甘露六糖、甘露三糖和甘露二糖组成。报道的 I91N/L211I 2 个
位点联合突变能够提高 β-甘露聚糖酶活性；ManBl(I91N/L211I)食品级表达为该酶的绿色安全应用奠定了基础。
关键词：β-甘露聚糖酶；枯草芽孢杆菌；定向进化；食品级表达；甘露寡糖；制备；生物工程
中图分类号：O636.1；TQ426.97；Q789 文献标识码：A 文章编号：1003-5214 (2023) 11-2454-09

Directed evolution of β-mannanase with higher enzymatic activity and
its application for mannooligosaccharide production

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CHEN Yun, GAO Weiqiang, LIANG Qi, ZHOU Xiaolei, WANG Lili, ZHANG Chunxiao
（College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang 050018, Hebei, China）

Abstract: To improve β-mannanase activity, β-mannanase gene from Bacillus licheniformis KD-1 was
amplified by error-prone PCR for directed evolution and further expressed in B. subtilis to screen mutants
with higher β-mannanase activity. It was found that the specific activity of mutant β-mannanase
ManBl(I91N/L211I) was 15554.7 U/mg, which was 4.1 times of wild-type ManBl, while the extracellular
yield of ManBl(I91N/L211I) expressed in B. subtilis at food-grade level reached 17601.3 U/mL. The data
from molecular dynamics and molecular docking showed that the mutation at two sites (I91N and L211I)
led to the enhancement of the catalytic pocket flexibility and the affinity with the substrate, thus improving
the catalytic efficiency of the enzyme. Furthermore, the β-mannanase ManBl(I91N/L211I) could hydrolyze
konjac glucomannan (KGM) into mannooligosaccharides (MOS) with different degree of polymerization
(DP) and main composition of mannohexaose, mannotriose, and mannobiose. The combined mutation of
I91N/L211I could increase β-mannanase activity. And the food-grade expression of ManBl(I91N/L211I)
laid the foundation for the green and safe application of the enzyme.
Key words: β-mannanase; Bacillus subtilis; directed evolution; food-grade expression; mannooligosaccharides;
preparation; bioengineering

β-甘露聚糖酶（EC 3.2.1.78）是一种能够随机 应用于多个领域，如作为饲料添加剂应用于动物养
[3]
水解甘露聚糖的 β-1,4-糖苷键的水解酶 [1-2] 。该酶可 殖中，通过降解抗营养物质提高饲料的利用率 ；

收稿日期：2022-12-29; 定用日期：2023-04-07; DOI: 10.13550/j.jxhg.20221182
基金项目：河北省重点研发计划项目（21372802D、19222906D）
作者简介：陈 赟（1995—），男，硕士生，E-mail：2428991358@qq.com。联系人：张春晓（1971—），女，副教授，E-mail：
cxzhang2009@163.com。