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Biocatalytic synthesis of flavor ester "pentyl valerate" using Candida rugosa lipase immobilized in microemulsion based organogels: effect of parameters and reusability.

ABSTRACT: Pentyl valerate was synthesized biocatalytically using Candida rugosa lipase (CRL) immobilized in microemulsion based organogels (MBGs). The optimum conditions were found to be pH 7.0, temperature of 37 °C, ratio of concentration of water to surfactant (Wo) of 60, and the surfactant sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) for MBG preparation. Although kinetic studies revealed that the enzyme in free form had high affinity towards substrates (K(m) = 23.2 mM for pentanol and 76.92 mM for valeric acid) whereas, after immobilization, the K(m) values increased considerably (74.07 mM for pentanol and 83.3 mM for valeric acid) resulting in a slower reaction rate, the maximum conversion was much higher in case of immobilized enzyme (~99%) as compared to free enzyme (~19%). Simultaneous effects of important parameters were studied using response surface methodology (RSM) conjugated with Box-Behnken design (BBD) with five variables (process parameters), namely, enzyme concentration, initial water content (Wo), solvent used for MBG preparation, substrate ratio and time, and response as the final product formation, that is, pentyl valerate (%). The MBGs were reused for 10 consecutive cycles for ester synthesis. Efficacy of AOT/isooctane as dehydrating agent for extracting excess water from MBGs was found to exert a positive effect on the esterification reaction.

SUBMITTER: Raghavendra T

PROVIDER: S-EPMC4100389 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Project description:BackgroundMarine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. However, since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud.ResultsBased on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48-79 % similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which has 316 amino acids with a predicted molecular weight of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity: 1389 U mg(-1)) at 45 °C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 °C, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90 % of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concentrations (up to 3.0 M). These results demonstrate a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concentrations in non-aqueous media.ConclusionsThis manuscript reports unprecedented alcohol tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high organic solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.

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Biocatalytic synthesis of flavor ester "pentyl valerate" using Candida rugosa lipase immobilized in microemulsion based organogels: effect of parameters and reusability.

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