Molecular and biochemical characterisation of esterases from oenococcus oeni and their potential for application in wine.

Abstract

This study examined the complex array of ester synthesis and hydrolysis activities in whole cells by cloning, heterologous expression, partial purification, and biochemical characterisation of EstA2, EstB28 and EstCOo8 esterase proteins from O. oeni alongside EstC34, an esterase from Lactobacillus hillgardii. With a view to applying these esterases to wine, enzyme function under frequently encountered harsh physicochemical conditions in wine was examined. EstB28, EstCOo8 and EstC34 showed highest activity with pNPacetate (C₂) and pNP-butanoate (C₄). The highest activity of EstA2 was observed with pNP-hexanoate (C₆). All enzymes retained at least some activity under conditions relevant to winemaking. However, only EstB28 and EstA2 showed an increase in activity above 10% ethanol. EstB28 and EstA2 also had a higher relative activity to EstCOo8 and EstC34. Once characterisation of these esterases showed that they should retain at least partial activity under wine-like conditions, EstA2 and EstB28 were assayed for activity in two separate wine samples by SPME-GCMS. Studies were also conducted to determine activity on natural substrates. Synthesis and hydrolysis of ethyl esters with acyl chain lengths of between C₂-C₈ was measured by SPME-GCMS. All four esterases where found to have the ability to synthesise and hydrolyse ethyl esters under optimal conditions (pH 5.0). Observed strain-specific differences in whole cell ester hydrolysis were also investigated. Firstly wine MLF trials were conducted in Chardonnay and Cabernet Sauvignon wines to determine if these strain specific differences could be translated into hydrolysis ability in wine. Eight strains of LAB were initially used in this study and strains with the lowest activity against pNP-linked ester substrates (Ooeni2, Ooeni3, Ooeni6, Lac34 and Lac40) were compared to the strains with the highest activity (Ooeni8, Ooeni12 and Ooeni28). In an effort to better understand the O. oeni strain specific differences in esterase activity real-time qPCR was carried out on an independent set of samples (n = 30) using strain Ooeni28 (‘high’ activity against pNP-linked ester substrates). Expression of esterase genes was measured in the presence of known esterase substrates. Both estA2 and estA7 showed an increase in expression in the presence of ethanol and butyric acid, whereas estB28 and estC expression increased in the presence of ethyl butyrate. However, when the same experiment was repeated with Ooeni2 (‘low’ activity) there was little change in the expression of the characterised esterase genes. Further investigation is required to determine if this is response is related to the phenotype of ‘low’ activity. Finally esterase genes were also sequenced from strains demonstrating ‘low’ and ‘high’ esterase hydrolysis activity to establish if there are any differences in predicted protein sequences amongst these strains. All strains sequenced had a homologue of EstA2, EstA7 and EstB28. Based on EstA2 and EstB28 sequences the strains with ‘high’ whole cell activity can be separated from strains with „low‟ whole cell activity through single nucleotide polymorphisms (SNP). This study will improve the understanding of the functioning of LAB esterases in wine conditions and the reason for strain specific differences in activity, with a view towards modulating and controlling the impact of LAB in ester profile modifications during the MLF.