Engineering and linker-mediated co-immobilization of carotenoid cleavage oxygenase with phenolic acid decarboxylase for efficiently converting ferulic acid into vanillin

Abstract

The artificial cascade system with a coenzyme-independent decarboxylase and oxygenase was a very attractive approach for bioconversion of ferulic acid into vanillin. However, its potential was impeded by the low activity and thermostability of oxygenase. In this study, a combined strategy including the protein engineering of a carotenoid cleavage oxygenase from Thielavia terrestris (TtCCO) and its linker-mediated co-immobilization with a decarboxylase from Bacillus atrophaeus (BaPAD) was explored with aims to improve the catalytic efficiency and robustness of this artificial cascade system. We proved that engineering the enzyme by inserting an extra short peptide between E450 to E476 greatly affected TtCCO substrate specificity, catalytic activity, and thermostability. Among three variants, TtCCO2 displayed better thermostability and had 1.4-, 55- and 1.3-fold higher activity than TtCCO on 4-vinylguaiacol, resveratrol, and isoeugenol, respectively. The excellent protein and enzyme activity yield of immobilization was achieved by linker-mediated immobilization of 4lp-TtCCO2 @ mesoporous Y zeolite. A 100% conversion rate of ferulic acid and a 55% of the molar conversion yield of vanillin were obtained by co-immobilized 4lp-TtCCO2/4lp-BaPAD@mesoporous Y zeolite. This work demonstrated an effective engineering strategy for more efficient and robust oxygenases and the potential of this bi-enzyme cascade system for converting ferulic acid into vanillin.