Abstract
HsCCO and RbCCO from Herbaspirillum seropedicae and Rhodobacteraceae bacterium 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 Km of HsCCO for isoeugenol and 4-vinylguaiacol were 1.55 and 1.65 mM and Vmax were 74.09 and 27.91 nmol min–1 mg–1, respectively. The kinetic constants Km of RbCCO for isoeugenol and 4-vinylguaiacol were 2.24 and 0.85 mM and Vmax were 76.48 and 19.96 nmol min–1 mg–1, respectively. The transformed Escherichia coli cells harboring HsCCO 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–1, respectively. Comparably, the molar conversion yields of the transformed E. coli cells harboring RbCCO against isoeugenol 4-vinylguaiacol were 75 and 58%, and the maximum vanillin yields were up to 1.14 and 0.88 g L–1, respectively.
Highlights
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important flavor additives worldwide
Isoeugenol was purchased from the Macklin Group (Shanghai, China); 4-vinylguaiacol was obtained from the LID Group (Shijiazhuang, China); vanillin was provided from SigmaAldrich
In a phylogenetic tree of carotenoid cleavage oxygenases, HsCCO and SeNCED are clustered in the up group of the evolutionary tree (Figure 1B), whereas, AvCCO, RbCCO, KsCCO, and RaCCO are clustered with CsO2 in the down group of the evolutionary tree (Figure 1B)
Summary
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important flavor additives worldwide. It is widely used in the food, pharmaceutical and the chemical industry. Natural vanillin is mainly derived from vanilla cells. Because the content of vanillin is low in vanilla cells and the cost of separation and extraction of vanillin is relatively high, natural vanillin accounts for only. 1% of global production (Priefert et al, 2001; Schwab et al, 2018). Chemical vanillin has become the first choice for industrial applications due to its low price. The chemical synthesis of vanillin causes environmental pollution issues during the production process and chemically produced vanillin has adverse health effects (Goutam and Pritam, 2018)
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