Gene Cloning and Enzymatic Properties of an Intracellular Maltogenic Amylase from <i>Bacillus</i> sp. B110

LIU Xia; DAI Longhua; HUANG Zhen; WU Xiaohua; WANG Fei

doi:10.13386/j.issn1002-0306.2022080183

Volume 44 Issue 10

May 2023

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Science and Technology of Food Industry > 2023 > 44(10): 123-129. > DOI: 10.13386/j.issn1002-0306.2022080183

LIU Xia, DAI Longhua, HUANG Zhen, et al. Gene Cloning and Enzymatic Properties of an Intracellular Maltogenic Amylase from Bacillus sp. B110[J]. Science and Technology of Food Industry, 2023, 44(10): 123−129. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022080183.

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

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

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Received Date: August 17, 2022
Available Online: March 08, 2023

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Abstract

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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