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Expression, characterization and structural profile of a heterodimeric β-galactosidase from the novel strain Lactobacillus curieae M2011381
Process Biochemistry ( IF 3.7 ) Pub Date : 2020-10-01 , DOI: 10.1016/j.procbio.2020.06.025
Jiaying Zhu , Jiaqi Sun , YaJie Tang , Jingli Xie , Dongzhi Wei

Abstract A heterodimeric β-galactosidase was discovered in the novel strain Lactobacillus curieae M2011381. The gene encoding the enzyme was expressed in Escherichia coli BL21 (DE3). The specific enzyme activities of the recombinant holoenzyme (LacLM) and large subunit (LacL) measured 11.4 U/mg and 3.8 U/mg, respectively. The kcat/Km values of LacLM and LacL were 740 mM−1 s−1 and 1.40 mM−1 s−1, respectively. LacLM showed maximum activity at pH 8.0 and 55 °C, and it could maintain its activity at a neutral pH and below 45 °C. LacLM displayed both hydrolysis and transgalactosylation activity on 200 g/L lactose. When LacLM was added to milk, the lactose was hydrolyzed after 6 h without galactooligosaccharide generation. The sequence alignment and homology modeling of the structures of the holoenzyme and subunits revealed that LacL has a catalytic domain with a catalytic dyad, Glu470 and Glu538, and small subunit LacM is a β-sheet domain with a conserved Trp294. The molecular docking of LacLM helped to illustrate the roles of both subunits in the reaction with lactose.

中文翻译:

来自新菌株乳酸杆菌 M2011381 的异二聚体 β-半乳糖苷酶的表达、表征和结构特征

摘要 在新菌株Lactobacillus curieae M2011381 中发现了异二聚体β-半乳糖苷酶。编码该酶的基因在大肠杆菌BL21 (DE3) 中表达。重组全酶 (LacLM) 和大亚基 (LacL) 的比酶活性分别为 11.4 U/mg 和 3.8 U/mg。LacLM 和 LacL 的 kcat/Km 值分别为 740 mM-1 s-1 和 1.40 mM-1 s-1。LacLM 在 pH 8.0 和 55 °C 时表现出最大活性,在中性 pH 和 45 °C 以下时仍能保持其活性。LacLM 在 200 g/L 乳糖上显示出水解和转半乳糖基化活性。当将 LacLM 添加到牛奶中时,乳糖在 6 小时后被水解,而没有低聚半乳糖生成。全酶和亚基结构的序列比对和同源性建模表明,LacL 具有催化结构域,具有催化二元组 Glu470 和 Glu538,而小亚基 LacM 是具有保守 Trp294 的 β-折叠结构域。LacLM 的分子对接有助于说明两个亚基在与乳糖反应中的作用。
更新日期:2020-10-01
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