Krista M. Sumby

Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni

ABSTRACT We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4pNP-linked substrates.

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise l-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.

Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni

The study of gene expression and accurate quantitation of target genes in any organism depends on correct normalisation. Due to the increase in studies on Oenococcus oeni gene expression, there is a clear need for alternative reference genes in order to reliably measure expression levels. In this manuscript, we propose the approach of using multiple reference genes to provide a more robust basis for establishing a reference gene set. The identification and evaluation of a panel of nine reference genes, including the commonly used ldhD, for real-time PCR normalisation was performed in O. oeni. Expression levels of these reference genes were then measured by real-time qPCR in an independent set of O. oeni samples (n = 30). The nine genes were ranked according to their stability of gene expression measure (M) using geNorm to identify the most consistently expressed reference genes. This approach resulted in the identification of multiple reference genes that may be used for a screening and more robust normalisation of target gene expression measured by real-time RT-qPCR. Expression of esterase genes was then measured in these O. oeni samples in the presence of known esterase substrates. The results give an indication of how these genes may be involved in ester synthesis and hydrolysis in O. oeni.

Characterization of EstCOo8 and EstC34, intracellular esterases, from the wine-associated lactic acid bacteria Oenococcus oeni and Lactobacillus hilgardii

To clone and characterize two related intracellular esterases from Oenococcus oeni and Lactobacillus hilgardii under wine‐like conditions.

Ester synthesis and hydrolysis in an aqueous environment, and strain specific changes during malolactic fermentation in wine with Oenococcus oeni

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME-GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.

Application of directed evolution to develop ethanol tolerant Oenococcus oeni for more efficient malolactic fermentation

Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This study aimed to use directed evolution (DE) to produce a more robust strain of O. oeni having the ability to withstand high ethanol concentrations. DE involves an organism mutating and potentially adapting to a high stress environment over the course of extended cultivation. A continuous culture of O. oeni was established and exposed to progressively increasing ethanol content such that after approximately 330 generations, an isolate from this culture was able to complete MLF in high ethanol content medium earlier than its parent. The ethanol tolerance of a single isolate, A90, was tested to confirm the phenotype and its fermentation performance in wine. In order to investigate the genotypic differences in the evolved strain that led to the ethanol tolerance phenotype, the relative expression of a number of known stress response genes was compared between SB3 and A90. Notably, there was increase in hsp18 expression in 20% (v/v) ethanol by both strains with A90 exhibiting a higher degree of expression. This study is the first to use directed evolution for O. oeni strain improvement and confirms that this technique can be used successfully for the development of new candidate strains for the wine industry. This study also adds to the current knowledge on the genetic basis of ethanol tolerance in this bacterium.