β-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of β-glucanases in probiotics. Here, β-glucanase genes were cloned from Bacillus licheniformis , Lactobacillus fermentum and L. johnsonii . β-glucanase genes, as blg , lfg and ljg isolated from B. licheniformis , L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that β-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of α-helices and β-strands in the three mutants were altered, which might result in β-glucanase activity increasement. Analysis of β-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30–50 ℃ than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about β-glucanase-encoding genes in L. fermentum and L. johnsonii . These findings provide an efficient way to improve the activity of β-glucanase.

中文翻译：

---

益生菌中 β-葡聚糖酶的定向突变以增强酶活性、热稳定性和 pH 稳定性

β-葡聚糖酶广泛应用于生物防治、酿造和饲料行业；然而，很少有关于益生菌中β-葡聚糖酶的研究。在这里，β-葡聚糖酶基因是从地衣芽孢杆菌、发酵乳杆菌和约氏乳杆菌中克隆的。β-葡聚糖酶基因，如从地衣芽孢杆菌、发酵乳杆菌和约氏乳杆菌中分离的blg、lfg和ljg，分别经原核表达获得重组菌株BL、LF和LJ。这些基因中的定向突变是通过顺序易错 PCR 引入的。结果表明，三个突变体 mblg、mlfg 和 mljg 中的 β-葡聚糖酶活性分别比 BL、LF 和 LJ 高 1.94、2.72 和 1.29 倍。突变位点分析显示 mblg 中 Ser370Gly 和 Leu395Phe 的取代；mlfg 中的 Arg169His 和 Asn302Ser；mljg 中的 Val132Met、Ser226Asn 和 Asp355Gly。空间结构预测显示三个突变体中α-螺旋和β-链的数量和位置发生了改变，这可能导致β-葡聚糖酶活性增加。β-葡聚糖酶特性的分析表明，突变株和野生型菌株之间的最适温度和 pH 值没有显着差异。然而，mlfg 和 mljg 在 30-50 ℃ 下表现出比野生型菌株更大的热稳定性，并且 mblg 与野生型菌株相比提高了 pH 稳定性。这是关于发酵乳杆菌和约氏乳杆菌中β-葡聚糖酶编码基因的首次报道。这些发现提供了一种提高β-葡聚糖酶活性的有效方法。β-葡聚糖酶特性的分析表明，突变株和野生型菌株之间的最适温度和 pH 值没有显着差异。然而，mlfg 和 mljg 在 30-50 ℃ 下表现出比野生型菌株更大的热稳定性，并且 mblg 与野生型菌株相比提高了 pH 稳定性。这是关于发酵乳杆菌和约氏乳杆菌中β-葡聚糖酶编码基因的首次报道。这些发现为提高β-葡聚糖酶的活性提供了一种有效的方法。β-葡聚糖酶特性的分析表明，突变株和野生型菌株之间的最适温度和 pH 值没有显着差异。然而，mlfg 和 mljg 在 30-50 ℃ 下表现出比野生型菌株更大的热稳定性，并且 mblg 与野生型菌株相比提高了 pH 稳定性。这是关于发酵乳杆菌和约氏乳杆菌中β-葡聚糖酶编码基因的首次报道。这些发现为提高β-葡聚糖酶的活性提供了一种有效的方法。