Development of simple, scalable protease production from Botrytis cinerea

Abstract: Heat haze-forming proteins are stable during winemaking and are typically removed via adsorption to bentonite. Proteolytic degradation is an alternative method to prevent wine-haze and offers the opportunity to reduce the environmental impacts and labor cost of the process. Herein, we describe the development of a production system for Botrytis cinerea proteases for the enzymatic degradation of heat haze-forming proteins. The effect of culture medium on the secretion of glucan by B. cinerea was investigated and methods to inactivate B. cinerea laccase in liquid culture medium were assessed. Protease production by B. cinerea was scaled up from 50 mL in shake flasks to 1 L in bioreactors, resulting in an increase in protease yield from 0.30 to 3.04 g L−1. Glucan secretion by B. cinerea was minimal in culture medium containing lactose as a carbon source and either lactic or sulfuric acid for pH control. B. cinerea laccases were inactivated by reducing the pH of culture supernatant to 1.5 for 1 h. B. cinerea proteases were concentrated and partially purified using ammonium sulfate precipitation. SWATH-MS identified aspartic acid protease BcAP8 amongst the precipitated proteins. These results demonstrate a simple, affordable, and scalable process to produce proteases from B. cinerea as a replacement for bentonite in winemaking. Key points: • Isolates of B. cinerea that produce proteases with potential for reducing wine heat-haze forming proteins were identified. • Media and fermentation optimization increased protease yield tenfold and reduced glucan secretion. • Low pH treatment inactivated laccases but not proteases. Graphical abstract: [Figure not available: see fulltext.]</p

Grape and Wine Proteins: Their Fractionation by Hydrophobic Interaction Chromatography and Identification by Chromatographic and Proteomic Analysis

A method to fractionate grape and wine proteins by hydrophobic interaction chromatography (HIC) was developed. This method allowed the isolation of a thaumatin-like protein in a single step with high yield and >90% purity and has potential to purify several other proteins. In addition, by separating HIC fractions by reverse phase HPLC and by collecting the obtained peaks, the grape juice proteins were further separated, by SDS-PAGE, into 24 bands. The bands were subjected to nanoLC-MS/MS analysis, and the results were matched against a database and characterized as various Vitis vinifera proteins. Moreover, either directly or by homology searching, identity or function was attributed to all of the gel bands identified, which mainly consisted of grape chitinases and thaumatin-like proteins but also included vacuolar invertase, PR-4 type proteins, and a lipid transfer protein from grapes.11 page(s

Structural studies on three Vitis vinifera thaumatin-like proteins and their hazing potential in white wines

White wines generally contain a relatively low concentration of pathogenesis-related (PR) proteins of grape (Vitis vinifera), namely, thaumatin-like proteins (TLPs) and chitinases. These proteins play roles in the defence mechanism of plants against pathogens, and can cause protein haze in white wine unless removed prior to bottling. TLPs are, after the chitinases, the second most abundant proteins found in wines. It has been demonstrated that different isoforms of TLPs have different hazing potential and aggregation behaviour, but an explanation on why this occurs is lacking. In this work we present the elucidation of the molecular structures of three isoforms of grape TLPs purified from Sauvignon blanc grape juice. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62\ub0C). Interestingly, protein F2/4JRU was found to be heat unstable thus forming haze, while this was not the case for I/4L5H. From the comparison of the three structures we attributed these differences in properties to the conformation of a single loop and the amino acid composition of its flanking regions. The presence of a disulfide bridge in this loop is probably the key for the unfolding/refolding behaviour of this area, and the fact that white wines are typically produced in reducing conditions and with SO2 added to prevent faults due to oxidation is likely to exacerbate haze formation. The availability of structural information on haze forming proteins could inform research into alternative wine stabilisation solutions as the development of target proteases for the degradation of haze-forming proteins, resulting in an important change in the winemaking stabilization practices

Effect of pH and alcohol on perception of phenolic character in white wine

The in-mouth perception of textures of white wine arising from the interactions among white wine phenolics, pH, and alcohol level was evaluated. Phenolics were extracted from white wines and added back to white wines that were adjusted to different pH and ethanol concentrations within wine realistic ranges. Adding phenolics to a white wine at pH 3.3 significantly increased its astringency, but the same addition did not contribute to the higher astringency elicited by the same wine when adjusted to pH 3.0. Higher phenolics generally increased bitterness and viscosity, but the effect depended on the source of the phenolics. Wines with added phenolics were generally perceived to be hotter, and significantly so when the wine was low in alcohol. The combined effect of phenolic content and alcohol concentration on astringency and bitterness was additive, suggesting that alcohol directly contributes to these attributes in white wines. Overall, the tastes and textures produced by white wine phenolics were more pronounced in wines with lower alcohol levels.5 page(s

Roles of proteins, polysaccharides, and phenolics in haze formation in white wine via reconstitution experiments

Residual proteins in finished wines can aggregate to form haze. To obtain insights into the mechanism of protein haze formation, a reconstitution approach was used to study the heat-induced aggregation behavior of purified wine proteins. A chitinase, four thaumatin-like protein (TLP) isoforms, phenolics, and polysaccharides were isolated from a Chardonnay wine. The same wine was stripped of these compounds and used as a base to reconstitute each of the proteins alone or in combination with the isolated phenolics and/or polysaccharides. After a heating and cooling cycle (70 °C for 1 h and 25 °C for 15 h), the size and concentration of the aggregates formed were measured by scanning ion occlusion sensing (SIOS), a technique to detect and quantify nanoparticles. The chitinase was the protein most prone to aggregate and the one that formed the largest particles; phenolics and polysaccharides did not have a significant impact on its aggregation behavior. TLP isoforms varied in susceptibility to haze formation and in interactions with polysaccharides and phenolics. The work establishes SIOS as a useful method for studying wine haze.8 page(s

White wine taste and mouthfeel as affected by juice extraction and processing

The juice used to make white wine can be extracted using various physical processes that affect the amount and timing of contact of juice with skins. The influence of juice extraction processes on the mouthfeel and taste of white wine and their relationship to wine composition were determined. The amount and type of interaction of juice with skins affected both wine total phenolic concentration and phenolic composition. Wine pH strongly influenced perceived viscosity, astringency/drying, and acidity. Despite a 5-fold variation in total phenolics among wines, differences in bitter taste were small. Perceived viscosity was associated with higher phenolics but was not associated with either glycerol or polysaccharide concentration. Bitterness may be reduced by using juice extraction and handling processes that minimize phenolic concentration, but lowering phenolic concentration may also result in wines of lower perceived viscosity.7 page(s

Proteomic analysis indicates massive changes in metabolism prior to the inhibition of growth and photosynthesis of grapevine (Vitis vinifera L.) in response to water deficit

Background Cabernet Sauvignon grapevines were exposed to a progressive, increasing water defict over 16 days. Shoot elongation and photosynthesis were measured for physiological responses to water deficit. The effect of water deficit over time on the abundance of individual proteins in growing shoot tips (including four immature leaves) was analyzed using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS). Results Water deficit progressively decreased shoot elongation, stomatal conductance and photosynthesis after Day 4; 2277 proteins were identified by shotgun proteomics with an average CV of 9% for the protein abundance of all proteins. There were 472 out of 942 (50%) proteins found in all samples that were significantly affected by water deficit. The 472 proteins were clustered into four groups: increased and decreased abundance of early- and late-responding protein profiles. Vines sensed the water deficit early, appearing to acclimate to stress, because the abundance of many proteins changed before decreases in shoot elongation, stomatal conductance and photosynthesis. Predominant functional categories of the early-responding proteins included photosynthesis, glycolysis, translation, antioxidant defense and growth-related categories (steroid metabolism and water transport), whereas additional proteins for late-responding proteins were largely involved with transport, photorespiration, antioxidants, amino acid and carbohydrate metabolism. Conclusions Proteomic responses to water deficit were dynamic with early, significant changes in abundance of proteins involved in translation, energy, antioxidant defense and steroid metabolism. The abundance of these proteins changed prior to any detectable decreases in shoot elongation, stomatal conductance or photosynthesis. Many of these early-responding proteins are known to be regulated by post-transcriptional modifications such as phosphorylation. The proteomics analysis indicates massive and substantial changes in plant metabolism that appear to funnel carbon and energy into antioxidant defenses in the very early stages of plant response to water deficit before any significant injury.22 page(s

Thermal stability of thaumatin-like protein, chitinase, and invertse isolated from sauvignon blanc and semillon juice and their role in haze formation in wine

A thermal unfolding study of thaumatin-like protein, chitinase, and invertase isolated from Vitis vinifera Sauvignon blanc and Semillon juice was undertaken. Differential scanning calorimetry demonstrated that chitinase was a major player in heat-induced haze in unfined wines as it had a low melt temperature, and aggregation was observed. The kinetics of chitinase F1 (Sauvignon blanc) unfolding was studied using circular dichroism spectrometry. Chitinase unfolding conforms to Arrhenius behavior having an activation energy of 320 kJ/mol. This enabled a predictive model for protein stability to be generated, predicting a half-life of 9 years at 15 °C, 4.7 days at 30 °C, and 17 min at 45 °C. Circular dichroism studies indicate that chitinase unfolding follows three steps: an initial irreversible step from the native to an unfolded conformation, a reversible step between a collapsed and an unfolded non-native conformation, followed by irreversible aggregation associated with visible haze formation

Comparison of the hydrophobicity of proteins F2/4JRU and I/4L5H.

<p>Hydrophobicity plot based on Kyte & Doolittle's scale for the alignment of proteins F2/4JRU (blue) and I/4L5H (red) obtained with AlignMe software <a href="http://www.bioinfo.mpg.de/AlignMe/AlignMe.html" target="_blank">http://www.bioinfo.mpg.de/AlignMe/AlignMe.html</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113757#pone.0113757-Stamm1" target="_blank">[56]</a>. Window size used for analysis: 9 residues.</p