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

Abstract

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 °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.

Biotransformation of lignin into 4-vinylphenol derivatives toward lignin valorization

“…94 Mutants with an extended C-terminus of BaPAD improved its activity under acidic conditions, while the extended N-terminus helped to improve the resistance of the enzyme to alkalinity and heat. 94 The depolymerization of lignin is a crucial process for obtaining aromatic derivatives that are suitable for the biosynthesis of 4VPs. Ligninolytic enzymes, which are abundantly present in nature, play a significant role in biotransformation of lignin into valuable products.…”

“…93 Engineering of the N-and C-terminus of the enzyme is also a common strategy for protein engineering. 94 Mutants with an extended C-terminus of BaPAD improved its activity under acidic conditions, while the extended N-terminus helped to improve the resistance of the enzyme to alkalinity and heat. 94 The depolymerization of lignin is a crucial process for obtaining aromatic derivatives that are suitable for the biosynthesis of 4VPs.…”

“…94 Mutants with an extended C-terminus of Ba PAD improved its activity under acidic conditions, while the extended N-terminus helped to improve the resistance of the enzyme to alkalinity and heat. 94…”

A New Phenolic Acid Decarboxylase from the Brown-Rot Fungus Neolentinus lepideus Natively Decarboxylates Biosourced Sinapic Acid into Canolol, a Bioactive Phenolic Compound

“…A PAD from B. licheniformis has also been shown to display an anecdotic activity on SA, with relative ratios of specific activities of 100:75.6:34.4:0.3 for pCA, FA, CafA and SA, respectively [24]. Site-directed mutagenesis of wild-type B. pumilus and B. amyloliquefaciens PADs (originally inactive on SA) made it possible to obtain evolved enzymes able to decarboxylate SA, but this activity remained very low compared to the activity on FA and pCA [33,34].…”