Cloning, Expression and Characterization of Phenolic Acid Decarboxylase from <i>Bacillus amyloliquefaciens</i> Q-426

doi:10.13523/j.cb.2202025

China Biotechnology

2022, Vol. 42

Issue (6): 20-29 DOI: 10.13523/j.cb.2202025

Cloning, Expression and Characterization of Phenolic Acid Decarboxylase from Bacillus amyloliquefaciens Q-426

REN Ming-jie¹,WANG Lu-lu²,SHEN Ji-hui¹,FAN Ruo-chen²,XU Yong-bin¹,ZHANG Li-ying¹,ZHENG Wei¹,QUAN Chun-shan^1,^2,^**(

)

1. Key Laboratory of Biotechnology and Bioresources of Ministry of Education, College of Life Seiences, Dalian Minzu University, Dalian 116600, China
2. School of Bioengineering, Dalian University of Technology, Dalian 116024, China

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Abstract

Objective: The preparation of 4-vinyl derivatives by biological decarboxylation has many advantages and promising prospects. In this study, the enzymatic properties of Bacillus amyloliquefaciens Q-426 phenolic acid decarboxylase (BaPAD-Q-426) were studied in detail to provide theoretical basis for its future application. Methods: In this study, the phenolic acid decarboxylase gene was cloned from Bacillus amyloliquefaciens. Using pET-28a (+) as vector, the recombinant plasmid was transformed into E.coli BL21 (DE3), to achieve high expression of BaPAD-Q-426. It was purified by Ni-NTA affinity chromatography, and the enzymatic properties were identified. Results: BaPAD-Q-426 maintained good pH stability in the range of pH 7.0~9.0, and the optimum pH was 8.0. The enzyme maintained high enzyme activity in the range of 25~40℃, and the optimum temperature was 35℃. After holding at 4℃ for 30 minutes, the enzyme still maintained more than 95% enzyme activity. K⁺ significantly promoted the enzyme activity of BaPAD-Q-426 with an increase of 60%. This enzyme was well tolerated in petroleum ethers and retained more than 50% of the enzyme activity in the presence of 40% petroleum ethers. The optimum substrate of BaPAD-Q-426 was ferulic acid, and its enzyme activity reached 19.5 IU/mL. Conclusion: Compared with phenolic acid decarboxylases from other sources, BAPAD-Q-426 has better stability at low temperature and has the strongest catalytic decarboxylation of ferulic acid in weakly alkaline environment.

Key words： Phenolic acid decarboxylase Bacillus amyloliquefaciens Enzyme characterization

Received: 18 February 2022 Published: 07 July 2022

ZTFLH:

Q786

Corresponding Authors: Chun-shan QUAN E-mail: mikyeken@dlnu.edu.cn

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	Ming-jie REN
	Lu-lu WANG
	Ji-hui SHEN
	Ruo-chen FAN
	Yong-bin XU
	Li-ying ZHANG
	Wei ZHENG
	Chun-shan QUAN

Cite this article:

REN Ming-jie,WANG Lu-lu,SHEN Ji-hui,FAN Ruo-chen,XU Yong-bin,ZHANG Li-ying,ZHENG Wei,QUAN Chun-shan. Cloning, Expression and Characterization of Phenolic Acid Decarboxylase from Bacillus amyloliquefaciens Q-426. China Biotechnology, 2022, 42(6): 20-29.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2202025 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I6/20

Fig.1 Agarose gel electrophoresis plot of the PCR amplification product of the phenolic acid decarboxylase geneM: DL2 000 DNA marker; 1: DNA of pET-28a(+)-BaPAD-Q-426

Fig.2 Construction of plasmid pET-28a(+)-BaPAD-Q-426M:DL10 000 DNA marker; 1: DNA of pET-28a(+)-BaPAD-Q-426; 2: pET-28a

Fig.3 Amino acid sequence alignment of phenolic acid decarboxylase and other decarboxylaseBaPAD-Q-426 is phenolic acid decarboxylase in this experiment. BAPAD is phenolic acid decarboxylase [Bacillus amyloliquefaciens DSM 7], LHPAD is a phenolic acid decarboxylase [Lactobacillus northsea JCM 18461] (BAP84656.1), LOPAD is phenolic acid decarboxylase [Lactobacillus oryzae JCM 18671] (GAK47549.1), BSPAS is phenolic acid decarboxylase [Bacillus subtilis] (WP_044429457.1), BHPAD is phenolic acid decarboxylase [Bacillus halotolerans] (WP_059292497.1), BCPAD is a phenolic acid decarboxylase[Bacillus coagulans 36D1] (AEP00765.1), PPPAD is phenolic acid decarboxylase [Pectobacterium] (ACX90155.1), DZPAD is phenolic acid decarboxylase [Dickeya zeae Ech586] (ACZ76892.1)^[1]

Fig.4 BaPAD-Q-426 protein purification by affinity chromatographyM: Protein marker; 1: Protein supernatants soft BaPAD-Q-426 after IPTG induction; 2: Protein precipitations soft BaPAD-Q-426 after IPTG induction; 3: Penetrating fluid; 4 - 6: Purified BaPAD-Q-426; 7: Concentrated BaPAD-Q-426

Fig.5 The optimal pH and stability of BaPAD-Q-426(a)Effect of pH on the activity of BaPAD-Q-426 (b)Stability of pH on the activity of BaPAD-Q-426

Fig.6 The optimal for temperature and stability of BaPAD-Q-426(a)Effect of temperature on the activity of BaPAD-Q-426 (b)Temperature stability of BaPAD-Q-426

Fig.7 Effect of metal ions on the enzyme activity of BaPAD-Q-426

Fig.8 Effect of organic solvent on the enzyme activity of BaPAD-Q-426

Fig.9 Effect of different volume scores organic solvent on the activity of BaPAD-Q-426(a)Methanol (b)Ethanol (c)Trichloromethane (d)Ligroin

Fig.10 Substrate specificity of recombinant BaPAD-Q-426

Fig.11 Chemical structure of substrates(a)p-Coumaric acid (b) Ferulic acid (c) Caffeic acid (d) Sinapic acid

Table 1 Comparsion of PAD with other decarboxylases


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