Improving the catalytic characteristics of phenolic acid decarboxylase from Bacillus amyloliquefaciens by the engineering of N-terminus and C-terminus

Background 4-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. Results 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; 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 degrees C for 60 min. Conclusions The 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.

Automated summary

The study focused on improving the catalytic characteristics of phenolic acid decarboxylase for potential applications in food additives and the flavor and fragrance industry. Mutants with enhanced substrate affinities and turnover rates were created through site-directed mutagenesis of the wild-type enzyme. The extension of specific termini of the enzyme contributed to improved resistance to different environmental conditions and enhanced substrate binding.