<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

doi: 10.13386/j.issn1002-0306.2022080183

刘霞^1,,
戴隆华²,
黄珍²,
吴小花¹,
王飞^2,*, ,

1.
南昌理工学院新能源与环境工程学院，江西南昌 330044
2.
江西农业大学生物科学与工程学院，江西南昌 330045

基金项目: 国家自然科学基金项目（31560031）；江西省教育厅科学技术研究项目（GJJ160387）。

详细信息

作者简介:
刘霞（1983−），女，硕士，副教授，研究方向：食品微生物，E-mail：25963535@qq.com

通讯作者:
王飞（1976−），男，博士，副教授，研究方向：微生物资源与蛋白质工程，E-mail：wangfei179@163.com

中图分类号: Q933
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出版历程
- 收稿日期: 2022-08-18
- 刊出日期: 2023-05-15

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

LIU Xia^1
,,
DAI Longhua²,
HUANG Zhen²,
WU Xiaohua¹,
WANG Fei^{2,*
, ,}

1.
Department of New Energy and Environmental Engineering, Nanchang Institute of Technology, Nanchang 330044, China
2.
College of Bioscience and Bioengineering, Jiangxi Agriculture University, Nanchang 330045, China

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。
- Bacillus sp. B110 /
- BMAL /
- 麦芽糖淀粉酶 /
- 基因克隆 /
- 酶学特性
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.
- Bacillus sp. B110 /
- BMAL /
- maltogenic amylase /
- gene cloning /
- enzymatic properties

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图 1 麦芽糖淀粉酶基因BMAL的克隆

注：M1：DNA分子量标准；1：PCR产物。

Figure 1. Cloning of the BMAL gene

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图 2 纯化BMAL的SDS-PAGE分析

注：1.蛋白分子质量标准；2.总蛋白；3. 50 mmol/L咪唑洗脱液；4. 50 mmol/L咪唑洗脱液；5. 200 mmol/L咪唑洗脱液。

Figure 2. SDS-PAGE analysis of the purified BMAL

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图 3 邻接法构建BMAL和其它来源环糊精酶的系统发育树

Figure 3. Phylogenetic tree of BMAL and related CDases obtained from different sources constructed by the neighbor-joining method

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图 4 BMAL的三维结构图

注：a.BMAL飘带图；b.BMAL与底物麦芽六糖分子对接；c.BMAL与潘糖分子对接图；d.活性中心。

Figure 4. 3D structure of BMAL

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图 5 pH和温度对重组酶rBMAL的影响

注：a. rBMAL的最适反应pH；b. rBMAL在不同pH缓冲液中的稳定性；c. rBMAL的最适反应温度； d.rBMAL的温度稳定性。

Figure 5. Effects of pH and temperature on the recombinant rBMAL

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表 1 金属离子和化学试剂对重组酶rBMAL酶活力的影响

Table 1. Effect of metal ions and chemical reagents on the activity of rBMAL

金属离子（1 mmol/L）	相对酶活（%）	化学试剂	相对酶活（%）
CK	100±1.22^b	CK	100.00±6.94^a
Al³⁺	34.04±0.32^de	Methanol	32.33±3.75^f
Fe³⁺	23.29±3.10^f	Ethanol	55.43±4.67^e
Zn²⁺	36.80±1.82^d	Acetone	58.96±4.45^de
Co²⁺	15.00±3.01^g	Isopropanol	54.22±0.82^e
Cu²⁺	26.15±3.35^f	Acetonitrile	67.00±3.21^c
Ni²⁺	31.14±4.60^e	SDS	80.80±2.43^b
K⁺	56.10±1.81^c	Tween 80	98.53±3.64^a
Mn²⁺	98.91±1.08^b	TritonX-100	97.94±4.56^a
Mg²⁺	136.77±0.41^a	EDTA	63.82±3.37^cd
Ca²⁺	52.23±1.82^c
注：采用邓肯氏新复极差法检验进行统计分析，不同小写字母表示差异显著，P<0.05。

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表 2 重组酶rBMAL的动学常数

Table 2. Apparent kinetic constants of rBMAL

底物	单位酶活（U·mg⁻¹）	K_m （mg·mL⁻¹）	k_cat （s⁻¹）	k_cat/K_m （mL·mg⁻¹·s⁻¹）
可溶性淀粉	32.5	20.2	143.33	7.10
直链淀粉	24	15.89	119.07	7.49
普鲁兰糖	68.2	3.35	302.09	90.18
β-环糊精	85	2.85	374.85	131.53

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