Characterisation of intracellular esterases from Oenococcus oeni and Lactobacillus hillgardii and their potential for application in wine

Abstract

Oenococcus oeni is the lactic acid bacterium (LAB) most typically used in winemaking to carry out malolactic fermentation (MLF). Alongside this, LAB can metabolise precursors present in wine during the MLF and as a consequence alter its chemical composition and quality. Aroma compounds such as esters and the quantities in which they occur can play a particularly important role in determining wine quality (Sumby et al. 2010). Esters are primarily formed during fermentation by yeast after which LAB are capable of modifying ester concentration during MLF. Ester hydrolysis and synthesis can be catalysed by esterases, which have greatest specificity for water-soluble short-chain esters.

With a view towards understanding and enhancing the role of LAB in ester profile modifications during MLF, we report the cloning, heterologous expression, partial purification, and biochemical characterisation of EstA2, EstB28, and EstCOo8 esterases from O. oeni and EstC34 esterase from Lactobacillus hillgardii. Enzyme function under the harsh physicochemical conditions frequently encountered in wine was examined to evaluate their potential applicability in this context. The influence of pH (3.0 to 8.0), temperature (10 to 60°C) and ethanol (2 to 22% (vol/vol)) on esterase activity was determined. All retain at least partial activity under wine like conditions of pH, temperature and ethanol (Sumby et al, 2009; 2012, 2013a). Substrate specificity trials were conducted in which eight different pNP-linked ester substrates. This activity was also confirmed using natural substrates (Sumby et al, 2013b). Substrate specificity of the enzymes was shown to vary, thereby suggesting possible applications in wine for targeted ester removal (Figure 1).

Having established that EstA2 and EstB28 should retain at least partial activity in wine, they were assayed for activity in two separate wines by using SPME-GCMS to monitor the appearance or disappearance of esters. EstB28 and EstA2 demonstrated duel hydrolysis and synthesis activity in wine and are the first LAB esterases demonstrated to retain activity in wine (Table 1). Once the esterase enzymes were characterised the basis for previously observed strain specific differences in ester hydrolysis by whole cells was investigated through wine MLF trials. Ester concentration changes in wine were dependent on the strain conducting the MLF. Further investigations will allow for a more informed choice of MLF strain.