4-Vinylguaiacol (4-VG) is widely used in the pharmaceutical, cosmetic and food industries and has important commercial value.At present, it is mainly synthesised by chemical methods, which are not environmentally friendly and have by-products.The biological synthesis of 4-VG by non-oxidative decarboxylation of ferulic acid (FA) catalyzed by phenolic acid decarboxylase (PAD) is considered to be a promising method.However, the inhibition effect of FA and 4-VG leads to a low conversion rate, low product concentration, and no reusability, which limits the practical application of this method.In this study, the phenolic acid decarboxylase (Bapad) from Bacillus atrophaeus was displayed on the surface of S.cerevisiae (SD-Bapad) and expressed in Pichia pastoris to compare its enzymatic properties and tolerance to high concentrations of substrates, products and organic solvents.Subsequently, a two-phase reaction system was used to produce 4-VG in a 5 L bioreactor using SD-Bapad.Results showed that SD-Bapad retained 54.7% of the relative enzyme activity after incubation at 55 ℃ for 1 h, and retained more than 65% of the relative enzyme activity after incubation at pH 3 and 10 for 1 h, while the free enzyme was almost inactivated, and showed stronger tolerance under high concentration of substrates, products and organic solvents.In addition, SD-Bapad still maintained 63% enzyme activity after repeated use in the organic / water two-phase system 6 times.Finally, FA was efficiently converted to 4-VG in a 5 L reactor using SD-Bapad as a whole-cell catalyst.Efficient conversion of FA to 4-VG yielded approximately 157.4 g/L (1 047.9 mmol/L) within 20 hours, the yield was about 7.9 g/(L/h), and the molar conversion rate was about 84.8%.Results showed that the surface display technology of S.cerevisiae could significantly improve the stability and reusability of Bapad, which provided a new way and potential solution for industrial biological production of 4-VG.
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