<i>Bacillus</i> sp. B110胞内麦芽糖淀粉酶基因克隆与酶学特性

刘霞; 戴隆华; 黄珍; 吴小花; 王飞

doi:10.13386/j.issn1002-0306.2022080183

Bacillus sp. B110胞内麦芽糖淀粉酶基因克隆与酶学特性

Gene Cloning and Enzymatic Properties of an Intracellular Maltogenic Amylase from Bacillus sp. B110

摘要

摘要: 目的：对菌株Bacillus sp. B110的胞内麦芽糖淀粉酶BMAL进行基因克隆、异源表达、纯化及酶学性质研究，为后期开发新的淀粉加工用酶打下基础。方法：使用PCR技术对Bacillus sp. B110的胞内麦芽糖淀粉酶bmal基因序列进行全长克隆，异源表达，使用Ni²⁺-NTA进行纯化，再对其酶学特性进行测定，使用序列分析工具BioEdit、MEGA等对其氨基酸序列进行分析，使用AlphaFold2对其三级结构进行预测分析。结果：BMAL基因全长1770 bp，编码一个589氨基酸残基的蛋白。重组酶rBMAL经Ni²⁺-NTA亲和层析纯化后，SDS-PAGE电泳结果显示其分子量大小为63 kDa。氨基酸序列分析和三维建模表明BMAL与来源于B.subtilis 168和B.subtilis SUH4-2的麦芽糖淀粉酶有较高的一致性，且BMAL具有一个麦芽糖淀粉酶所独有的N端结构域以及由Asp328-Glu357-Asp424三个氨基酸残基所构成的催化中心。重组酶rBMAL最适反应温度为45 ℃，最适反应pH为6.0。重组酶rBMAL在30 ℃条件下保藏7 h残留酶活为60%，但在60 ℃条件下保藏2 h残留酶活力下降98%，说明BMAL对热敏感。重组酶rBMAL在4 ℃，pH7.0~9.5保藏12 h活性稳定。当存在1 mmol/L的金属离子Mg²⁺时，重组酶rBMAL活力提高36%，而Ni²⁺、Fe³⁺、Co²⁺、Cu²⁺、Zn²⁺、Al³⁺、Ca²⁺对重组酶rBMAL有抑制作用，酶活力减少85%~48%。有机溶剂和化学试剂甲醇、乙醇、丙酮、异腈、EDTA和SDS对重组酶rBMAL有较强的抑制作用，酶活力减少至32.3%~64.8%。底物特异性实验结果证实BMAL最适底物为环精糊。结论：Bacillus sp. B110的胞内麦芽糖淀粉酶BMAL具有良好的催化特性和pH稳定性，在面包烘焙工业上具有潜在的应用价值。

Abstract: Objective: To study enzymatic properties of intracellular maltogenic amylase (BMAL) in Bacillus sp. B110, purification, gene cloning, heterologous expression and purification were performed. It laid a foundation for later research on the development of new starch processing enzymes. Method: The full-length of BMAL gene was amplified by PCR and over-expressed and heterologous expressed in Escherichia coli. The recombinant enzyme was purified with nickel affinity chromatography (Ni²⁺-NTA), and its enzymatic characteristic were determined. The amino acid sequence of BMAL was analyzed by sequence analysis tools BioEdit and MEGA tools, and the three-dimensional model was predicted by alpha fold2. Results: The nucleotide sequence analysis revealed an open reading frame (ORF) of 1770 bp encoded a putative protein of 589 aa residues. The recombinant enzyme was purified with nickel affinity chromatography (Ni²⁺-NTA). SDS-PAGE analysis of the purified enzyme revealed that the molecular mass of BMAL was 63 kDa. The primary structure of BMAL was similar to those of MAases from B. subtilis 168 and B. subtilis SUH4-2, such as possession of an extra domain at its N-terminal and had a catalytic triad Asp328-Glu357-Asp424. The purified recombinant enzyme rBMAL had an optimum temperature of 45 ℃ and an optimum pH around 6.0. The rBMAL retained about 60% activity after 7 h of incubation at below 30 ℃, but it lost 98% of the original activity after 2 h of incubation at 60 ℃. These results revealed that the rBMAL was inthermostability. The activity of rBMAL was stable in pH7.0~9.5 at 4 ℃ for 12 h. In the presence of 1 mmol/L metal ion Mg²⁺, the activity of rBMAL increased by 36%, while 1 mmol/L Ni²⁺, Fe³⁺, Co²⁺, Cu²⁺, Zn²⁺, Al³⁺, Ca²⁺ inhibited the activity of rBMAL, which decreased by 85%~48%. The recombinant enzyme activity was inhibited by methanol, acetone, ethanol, acetonitrile, EDTA and SDS, and the enzyme activity was decreased to 32.3%~64.8%. Conclusion: The intracellular maltose amylase BMAL from Bacillus sp. B110 had high catalytic capacity and pH stability, which had potential application in bread baking industry.