Screening Of Carotenoid Cleavage Oxygenases And Its Application In The Production Of Vanillin

Vanillin(4-hydroxy-3-methoxybenzaldehyde)is one of the most important flavor additives in the world.It is widely used in the food,pharmaceutical and the chemical industry.Natural vanillin production is minimal,but the chemical synthesis of vanillin causes serious environmental pollution and chemically produced vanillin has adverse health effects.Therefore,biosynthesis of vanillin is very popular.In recent years,genetic engineering and enzyme catalysis were used to product vanillin.Carotenoid cleavage oxygenases have the ability to catalyze aromatic compounds to vanillin.Search for bacterial genomic sequences by BLASTP found five genes of carotenoid cleavage oxygenases(HsCCO?RbCCO?KsCCO?AvCCO and Ra CCO).E.coli BL21(DE3)was used for genes cloning and expressing hosts.Preliminary enzyme activity measurement of all five carotenoid oxygenases revealed that HsCCO and RbCCO displayed much higher activity than others.It was also found that the solubility and optimum pH of carotenoid cleavage oxygenases may be related to amino acid sequences.HsCCO showed maximum activity at 40?and pH 7.0 and was relatively stable at pH 6.5-10 and temperature around 25°C.RbCCO showed maximum activity at 35?and pH 9.0 and was relatively stable at pH 6-11 and temperature 25?to 30?.The kinetic constants K_m of HsCCO for isoeugenol and 4-vinylguaiacol were 1.55 m M and 1.65 m M and V_max was 74.09nmol min^-1 mg^-1 and 27.91 nmol min^-1 mg^-1.The kinetic constants K_m of RbCCO for isoeugenol and 4-vinylguaiacol were 2.24 m M and 0.85 m M and V_max was 76.48 nmol min^-1 mg^-1 and 19.96nmol min^-1 mg^-1.The ability of HsCCO to convert 4-vinylguaiacol was slightly higher than RbCCO.5 m M Ca²⁺can assist HsCCO to convert isoeugenol.HsCCO and RbCCO were less tolerant to organic solvents.The transformed E.coli cells harboring HsCCO converted isoeugenol and 4-vinylguaiacol at molar conversions of 80%and 55%and the maximum vanillin concentrations were up to 1.22g L^-1 and 0.84 g L^-1,respectively.The transformed E.coli cells harboring HsCCO converted isoeugenol and 4-vinylguaiacol at molar conversions of 75%and 58%and the maximum vanillin concentrations were up to 1.14 g L^-1 and 0.88 g L^-1,respectively.The research can help to further understand characterization of carotenoid cleavage oxygenases,and provide possibility for the biosynthesis of vanillin.