School of Biological Science and Bioengineering, South China University of Technology, Guangzhou, 510006, China.
Microb Cell Fact. 2020 Jul 14;19(1):142. doi: 10.1186/s12934-020-01399-w.
β-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects.
A β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co, Mn, Zn, Ba and Ca. Man1E showed the highest affinity for Locust bean gum (LBG). The K and V values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively.
A new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.
β-甘露聚糖酶能够以内切糖苷酶的方式水解甘露聚糖中的β-1,4 糖苷键，生成甘露低聚糖。目前，β-甘露聚糖酶已广泛应用于食品、医药、纺织、造纸和石油开采等行业。β-甘露聚糖酶广泛存在于各种生物中，然而微生物是β-甘露聚糖酶的主要来源。与动植物来源的β-甘露聚糖酶相比，微生物来源的β-甘露聚糖酶具有更宽的 pH 范围、温度范围和更好的热稳定性、耐酸碱性以及底物特异性。因此，微生物来源的β-甘露聚糖酶受到了研究人员的高度重视。通过基因工程构建的重组菌，并经过蛋白质工程修饰，已广泛应用于β-甘露聚糖酶的生产，在各个方面都比传统的微生物发酵具有更多的优势。
从产气肠杆菌中克隆得到了一个编码 731 个氨基酸残基的β-甘露聚糖酶基因（Man1E）。Man1E 被分类为糖苷水解酶家族 1。bSiteFinder 预测表明，Man1E 催化中心有 8 个必需残基，分别为 Trp166、Trp168、Asn229、Glu230、Tyr281、Glu309、Trp341 和 Lys374。利用同源建模对 Man1E 的催化模块和碳水化合物结合模块（CBM）进行了建模。叠加分析和分子对接揭示了位于 Man1E 催化模块和 Man1E CBM 中的残基。重组酶成功表达、纯化，并通过 SDS-PAGE 检测到约 82.5 kDa。该酶的最适反应条件为 55°C 和 pH 6.5。该酶在 60°C 以下表现出较高的稳定性，在 pH 3.5-8.5 范围内具有良好的稳定性。低浓度的 Co、Mn、Zn、Ba 和 Ca 能激活 β-甘露聚糖酶的活性。Man1E 对卢卡豆胶（LBG）表现出最高的亲和力。LBG 的 K 和 V 值分别为 3.09±0.16 mg/mL 和 909.10±3.85 μmol/（mL·min）。
从产气肠杆菌中异源表达和表征了一种新型高活性的β-甘露聚糖酶。该酶属于一个未报道的β-甘露聚糖酶家族（CH1 家族）。它具有良好的 pH 和温度特性，对 LBG 和 KGM 具有优异的催化能力。本研究为进一步应用和分子修饰以提高其催化效率和底物特异性奠定了基础。
