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 Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax 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.

中文翻译：

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产气肠杆菌B19属于糖苷水解酶家族1的新β-甘露聚糖酶的表达，同源性建模和酶促表征。

β-甘露聚糖酶可以通过内切糖苷酶的方式水解甘露聚糖的β-1,4糖苷键，生成甘露寡糖。目前，β-甘露聚糖酶已广泛应用于食品，医药，纺织，造纸和石油开采行业。β-甘露聚糖酶广泛存在于各种生物中，但是微生物是β-甘露聚糖酶的主要来源。相比于动植物，微生物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，耐酸碱性能以及底物特异性。因此，微生物β-甘露聚糖酶受到研究人员的高度评价。通过基因工程构建和蛋白质工程修饰的重组细菌已被广泛应用于生产β-甘露聚糖酶，在各个方面都比传统的微生物发酵更具优势。β-甘露聚糖酶基因（Man1E），从产气肠杆菌中克隆了编码731个氨基酸残基的蛋白。Man1E被归类为糖苷水解酶家族1。bSiteFinder预测显示，Man1E催化中心有8个基本残基，分别是Trp166，Trp168，Asn229，Glu230，Tyr281，Glu309，Trp341和Lys374。对Man1E的催化模块和碳水化合物结合模块（CBM）进行了同源建模。叠加分析和分子对接揭示了位于Man1E催化模块和Man1E的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。最佳反应条件为55°C和pH 6.5。该酶在60°C以下和pH 3.5–8.5范围内均显示出高稳定性。低浓度的Co2 +，Mn2 +，Zn2 +可激活β-甘露聚糖酶活性，Ba2 +和Ca2 +。Man1E对刺槐豆胶（LBG）的亲和力最高。LBG的Km和Vmax值分别为3.09±0.16 mg / mL和909.10±3.85μmol/（mL min）。异源表达和表征了一种新型的产自大肠杆菌的高活性β-甘露聚糖酶。该酶属于未报告的β-甘露聚糖酶家族（CH1家族）。它具有良好的pH和温度特性，并且对LBG和KGM具有出色的催化能力。该研究为将来的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。异源表达和表征。该酶属于未报告的β-甘露聚糖酶家族（CH1家族）。它具有良好的pH和温度特性，并且对LBG和KGM具有出色的催化能力。该研究为将来的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。异源表达和表征。该酶属于未报告的β-甘露聚糖酶家族（CH1家族）。它具有良好的pH和温度特性，并且对LBG和KGM具有出色的催化能力。该研究为将来的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。