Abstract
Background4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application.ResultsA phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min.ConclusionsThe extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable.
Highlights
Phenolic acid is a general term for a class of organic acids containing phenolic rings, which exist in plants and can resist pathogens [1, 2]
Li et al BMC Biotechnology (2021) 21:44 (Fig. 1) [5]. 4-vinyl guaiacol (3-methoxy-4-hydroxystyrene) and 4-vinyl phenol (4-hydroxystyrene) are 4-vinyl derivatives of phenolic acids characterized by unique odor and volatility, these can be used in food additives as well as flavor and fragrance industry [6]
Since Zago et al [8] cloned the phenolic acid decarboxylase from Bacillus pumilus and successfully expressed the gene in Escherichia coli and proved its decarboxylation effect on ferulic acid (FA), PAD genes were cloned from several strains, including Lactobacillus plantarum [9], Bacillus subtilis [10], and Enterobacter [11] corresponding genes, and research on a series of enzymatic properties was conducted [12]
Summary
A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; P-N had reduced affinity toward p-coumaric acid. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. The affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min
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