A recombinant β-mannanase from Thermoanaerobacterium aotearoense SCUT27: Biochemical characterization and its thermostability improvement.
A recombinant β-mannanase from Thermoanaerobacterium aotearoense SCUT27: Biochemical characterization and its thermostability improvement.
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DOI：
10.1021/acs.jafc.9b06246
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发表时间：
2019-12
影响因子：
6.1
通讯作者：
Muzi Zhu;Ling Zhang;Fang Yang;Yaping Cha;Shuang Li;M. Zhuo;Shaobin Huang;Jianjun Li
中科院分区：
文献类型：
--
作者：
Muzi Zhu;Ling Zhang;Fang Yang;Yaping Cha;Shuang Li;M. Zhuo;Shaobin Huang;Jianjun Li
用刺槐豆胶（LBG）诱导嗜热厌氧杆菌（Thermoanaerobacterium aotearoense）SCUT 27表达β-甘露聚糖酶。GH 26 β-甘露聚糖酶的开放阅读框编码515个氨基酸的前体蛋白，并含有一个信号肽。在大肠杆菌中表达了不含信号肽的Man 25，比活为1286.2U/mg。此外，还建立了一种简便的琼脂平板法筛选β-甘露聚糖酶活性的方法.以LBG为底物纯化Man 25的最适温度为55 ℃。Man 25的催化活性和热稳定性对钙离子有很强的依赖性。通过对Man 25中Ca 2+结合位点的饱和诱变，获得了热稳定性最好的突变体ManM 3 -3（D143 A），其55 ℃半衰期是野生型的3.6倍。结果表明，在金属结合位点的诱变可能是一种有效的方法，以提高酶的热稳定性。
β-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 β-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without signal sequence (Man25) was overexpressed in Escherichia coli with the specific activity of 1286.2 U/mg. Moreover, a facile method for -mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate were 55 °C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 oC, compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.