Expression and Characterization of Carotenoid Cleavage Oxygenases From and Capable of Biotransforming Isoeugenol and 4-Vinylguaiacol to Vanillin.

HsCCO and RbCCO from and were selected and characterized from five putative bacterial carotenoid cleavage oxygenase gene sequences, due to merits in expression solubility and catalytic properties. Both enzymes can convert 4-vinylguaiacol and isoeugenol to vanillin. HsCCO showed maximum activity at 40°C and pH 7.0 and was stable at pH 6.5-10 and temperature around 25°C, retaining over 90 and 80% of initial activity, respectively. RbCCO showed maximum activity at 35°C and pH 9.0 and was stable at pH 6-11 and temperatures of 25-30°C, retaining over 80% of initial activity. The kinetic constants of HsCCO for isoeugenol and 4-vinylguaiacol were 1.55 and 1.65 mM and were 74.09 and 27.91 nmol min mg, respectively. The kinetic constants of RbCCO for isoeugenol and 4-vinylguaiacol were 2.24 and 0.85 mM and were 76.48 and 19.96 nmol min mg, respectively. The transformed cells harboring converted isoeugenol and 4-vinylguaiacol at molar conversion yields of 80 and 55% and the maximum vanillin concentrations were up to 1.22 and 0.84 g L, respectively. Comparably, the molar conversion yields of the transformed cells harboring against isoeugenol 4-vinylguaiacol were 75 and 58%, and the maximum vanillin yields were up to 1.14 and 0.88 g L, respectively.