An Organic Solvent-tolerant Phenolic Acid Decarboxylase BLPAD Andits Application In The Bioproduction Of 4-vinyl Phenol Derivatives

A new phenolic acid decarboxylase gene(blpad) from Bacillus licheniformis was cloned and overexpressed in E. coli. The full-length blpad encodes a 166 amino acids polypeptide with a predicted molecular mass and pI of 19.521 KDa and 5.02, respectively. The recombinant BLPAD displayed maximum activity at 37?C and pH 6.0. This enzyme possesses a broad substrate specificity and is able to decarboxylate p-coumaric, ferulic, caffeic and sinapic acids at the relative ratios of specific activities 100:74.59:34.41: 0.29. Kinetic constants Km values towards p-coumaric, ferulic, caffeic and sinapic acids were 1.64, 1.55, 1.93 and 2.45 mM and Vmax values were 268.43, 216.80, 119.07 and 0.78 U mg-1, respectively. In comparison with other phenolic acid decarboxylases, BLPAD exhibited remarkable organic solvents tolerance and good thermal stability. BLPAD showed excellent catalytic performance in biphase organic/aqueous systems and efficiently converted p-coumaric and ferulic acids into 4-vinyphenol and 4-vinylguaiacol with the existence of toluene and cyclohexane. At 500 mM of p-coumaric and ferulic acids, the recombinant BLPAD produced a total 58.28 g L-1 4-vinyphenol and 56.33 g L-1 4-vinylguaiacol with the conversion yields 97.02% and 75.02%, respectively. The immobilization enzyme Ni-NTA-BLPAD can keep a percent conversion more than 90% when the concentration of the p-coumaric acid was at 50 and 100 mM after reused five times. After the molecular toxicity study of two-phase system,we found that the substrate concentration, residual organic solvents and products in the aqueous phase can inhibit the activity of BLPAD.The low yield and product concentration are the crucial drawbacks to the practical bioproduction of vinyl phenol derivatives using phenolic acid decarboxylases. These unusual properties make BLPAD a desirable biocatalyst for commercial use in bioconversion of hydroxycinnamic acids to vinyl phenol derivatives via enzymatic decarboxylation in a biphasic organic/aqueous reaction system,and according to the research results of the limitation of two-phase system and immobilization enzyme,we can make some design to reduce its adverse impact on our reaction and make it more favourable for industrial application.