Characterization of the ß-Glucosidase activity in indigenous yeast isolated from wine regions in China.

ß-glucosidase is a pivotal enzyme that hydrolyzes bound volatile aromatic compounds. However, the activity of ß-glucosidase in winemaking and the mechanism by which it affects the flavor and taste of wines have not been fully investigated. In this study, we profiled the characteristics of ß-glucosidase derived from wine-related yeasts isolated from different wine-making regions in China, and analyzed the enzyme activity from different parts of the cells under aerobic and anaerobic conditions. A total of 56 strains of wine-related yeasts producing ß-glucosidases were screened using the YNB-C medium (YNB 6.7 g L-1 , cellobiose 5 g L-1 , pH 5.0). We found that strain Clavispora lusitaniae C117 produced the highest enzyme activity (152.39 µmol pNP ml-1 h-1 ). In most strains, ß-glucosidase were located in whole cells (periplasmic space) and permeabilized cells (intracellular). The non-Saccharomyces species had the highest enzymatic activity in a strain-dependent manner. Under aerobic conditions, C. lusitaniae C117, Hanseniaspora guilliermondii A27-3-4, Metschnikowia pulcherrima F-1-6, and Pichia anomala C84 had the highest ß-glucosidase activity. We further investigated the ß-glucosidase activity during the wine fermentation and the effects of sugar, pH, temperature, and ethanol on the enzyme activities of P. anomala C84 and commercial Saccharomyces yeast strains RC212 and VL1. The presence of fructose, glucose, and sucrose strongly inhibited enzyme activity. Similarly, low pH and low temperature inhibited the activity of ß-glucosidase, whereas ethanol promoted enzyme activity. Our findings provide a theoretical basis on understanding the different yeast characteristics of ß-glucosidase and their potential application for further improving wine aroma complexity.

Basal catalase activity and high glutathione levels influence the performance of non-Saccharomyces active dry wine yeasts.

Non-Saccharomyces wine yeasts are useful tools for producing wines with complex aromas or low ethanol content. Their use in wine would benefit from their production as active dry yeast (ADY) starters to be used as co-inocula alongside S. cerevisiae. Oxidative stress during biomass propagation and dehydration is a key factor in determining ADY performance, as it affects yeast vitality and viability. Several studies have analysed the response of S. cerevisiae to oxidative stress under dehydration conditions, but not so many deal with non-conventional yeasts. In this work, we analysed eight non-Saccharomyces wine yeasts under biomass production conditions and studied oxidative stress parameters and lipid composition. The results revealed wide variability among species in their technological performance during ADY production. Also, for Metschnikowia pulcherrima and Starmerella bacillaris, better performance correlates with high catalase activity and glutathione levels. Our data suggest that non-Saccharomyces wine yeasts with an enhanced oxidative stress response are better suited to grow under ADY production conditions.

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugars.

BACKGROUND: α-Glucosidases are widely distributed enzymes with a varied substrate specificity that are traditionally used in biotechnological industries based on oligo- and polysaccharides as starting materials. According to amino acid sequence homology, α-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several α-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with α-(1→1), α-(1→3) and α-(1→6) glycosidic linkages from sucrose was described. RESULTS: In this study, genes codifying for α-glucosidases from the nectaries yeast M. gruessii and M. reukaufii were characterised and heterologously expressed in Escherichia coli for the first time. Recombinant proteins (Mg-αGlu and Mr-αGlu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-α-D-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the α-amylase family, in particular from glycoside hydrolases that belong to family GH13. The three acidic residues comprising the catalytic triad were identified and their relevance for the protein hydrolytic mechanism confirmed by site-directed mutagenesis. Recombinant enzymes produced oligosaccharides naturally present in honey employing sucrose as initial substrate and gave rise to mixtures with the same products profile (isomelezitose, trehalulose, erlose, melezitose, theanderose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-αGlu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, ~ 170 g/L, were the highest reported so far. CONCLUSIONS: Mg/Mr-αGlu were heterologously overproduced and their biochemical and structural characteristics analysed. The recombinant α-glucosidases displayed excellent properties in terms of mild reaction conditions, in addition to pH and thermal stability. Besides, the enzymes produced a rare mixture of hetero-gluco-oligosaccharides by transglucosylation, mainly isomelezitose and trehalulose. These compounds are natural constituents of honey which purification from this natural source is quite unviable, what make these enzymes very interesting for the biotechnological industry. Finally, it should be remarked that these sugars have potential applications as food additives due to their suitable sweetness, viscosity and humectant capacity.

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts.

Metschnikowia reukaufii is a widespread yeast able to grow in the plants' floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l-1 , which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1→1), α-(1→3) and α-(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l-1 , the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l-1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.

The expression, secretion and activity of the aspartic protease MpAPr1 in Metschnikowia pulcherrima IWBT Y1123.

Protease-secreting yeasts have broad biotechnological potential for application to various industrial processes, including winemaking. However, this activity is influenced by the yeast response to environmental factors such as nitrogen and protein sources, as are found in grape juice. In this study, the wine-relevant yeast Metschnikowia pulcherrima IWBT Y1123, with known protease-secreting ability, was subjected to different nitrogen-containing compounds to monitor their impact on protease secretion and activity. Protease activity increased above basal levels for haemoglobin-containing treatments, indicating an inductive influence of proteins. On the other hand, treatments containing both haemoglobin and assimilable nitrogen sources led to a delayed increase in protease activity and protein degradation, suggesting a nitrogen catabolite repression mechanism at work. Protease activity and expression were furthermore evaluated in grape juice, which revealed increased expression and activity levels over time as promising results for further investigations into the impact of this yeast on wine properties.

Characterization of gelatinase produced by Antarctic Mrakia sp.

In the present study, 20 psychrotolerant yeast species isolated from the soils of King George Island in the sub-Antarctic region were evaluated for the production of extracellular gelatinase, an enzyme with high potential for applications in diverse areas, such as food and medicine. The production of extracellular gelatinase was confirmed in the yeasts Metschnikowia sp., Leucosporidium fragarium, and Mrakia sp., the last one being the yeast in which the highest gelatinase activity was detected. The enzyme was purified from cultures of Mrakia sp., and the effect of different physical-chemical factors on its activity was determined. The gelatinase produced by Mrakia sp. would correspond to a protein of relative molecular weight (rMW) 37,000, which displayed the highest activity at 36°C, pH 7.0, 10 mM CaCl 2 , and 5 mM ZnSO 4 .

Monitoring the impact of an aspartic protease (MpAPr1) on grape proteins and wine properties.

The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.

Metschnikowia persici sp. nov., A Novel Protease-Producing Yeast Species from China.

Three yeast strains, named as FHL-A, FHL-B, and FHL-C, were isolated from peach fruit surfaces collected from different regions in the North of China highly produced protease and were presented as single separate group in the genus Metschnikowia by sequence comparisons of 26S rRNA gene D1/D2 domain and internal transcribed spacer (ITS) region. BLASTn alignments on NCBI showed that the similarity of 26S rRNA gene sequences of the three strains to all sequences of other yeasts accessed into the GenBank/EMBL/DDBJ and other database was very low (≦93%). The phylogenetic tree based on the D1/D2 region of 26S rRNA gene sequences revealed that three strains are most closely related to Metschnikowia koreensis KCTC 7828T (AF257272.1) (sequence similarity: 93.0%) and Metschnikowia reukaufii CBS9709T (AJ716113.1) (sequence similarity: 93.0%). However, the strains are distinguished from M. koreensis by its non-assimilation of galactose, ribitol, and D-xylose, and by its growth at 37 °C or in vitamin-free medium, and are notably different from M. reukaufii by its non-assimilation of galactose, D-xylose, D-arabinose, and D-ribose, and by its growth at 35 °C or in vitamin-free medium. The strain FHL-B formed asci in V8 juice sporulation medium for 3 weeks. Therefore, the name Metschnikowia persici is proposed for the novel species, with FHL-B (= CBS12815T = CFCC 3578T) as the type strain.

Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima.

BACKGROUND: MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+ . Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax ) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions. CONCLUSION: Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.

Selection and use of pectinolytic yeasts for improving clarification and phenolic extraction in winemaking.

Pectinase enzymes have shown a considerable influence in both, sensitive and technological properties of wines. They can help to improve clarification process, releasing more color and flavor compounds entrapped in grape skin, facilitating the liberation of phenolic compounds. This work aims to find yeasts that, because of their native pectinases, can be applied on combined fermentations with Saccharomyces cerevisiae obtaining significant benefits over single-inoculated traditional fermentations. 462 yeast strains isolated from wineries were identified and tested for several enzymatic activities of recognized interest for enology industry. Considering the 7 identified species, only Aureobasidium pullulans, Metschnikowia pulcherrima and Metschnikowia fructicola showed polygalacturonase activity. Because of its interest in winemaking, due to its reported incidence in wine flavor, the impact of M. pulcherrima as a source of pectinolytic enzymes was analyzed by measuring its influence in filterability, turbidity and the increase on color, anthocyanin and polyphenol content of wines fermented in combination with S. cerevisiae. Among the strains screened, M. pulcherrima NS-EM-34 was selected, due to its polygalacturonase activity, for further characterization in both, laboratory and semi-industrial scale assays. The kinetics concerning several metabolites of enological concern were followed during the entire fermentation process at microvinification scale. Improved results were obtained in the expected parameters when M. pulcherrima NS-EM-34 was used, in comparison to wines fermented with S. cerevisiae alone and combined with other pectinolytic and non-pectinolytic yeasts (A. pullulans and Lachancea thermotolerans, respectively), even working better than commercial enzymes preparations in most parameters. Additionally, M. pulcherrima NS-EM-34 was used at a semi-industrial scale combined with three different S. cerevisiae strains, confirming its potential application for red wine improvement on the mentioned sensorial and technological properties.

A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must.

The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches.

Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl ß-d-N,N',N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases.

An ene reductase from Clavispora lusitaniae for asymmetric reduction of activated alkenes.

A putative ene reductase gene from Clavispora lusitaniae was heterologously overexpressed in Escherichia coli, and the encoded protein (ClER) was purified and characterized for its biocatalytic properties. This NADPH-dependent flavoprotein was identified with reduction activities toward a diverse range of activated alkenes including conjugated enones, enals, maleimide derivative and α,ß-unsaturated carboxylic esters. The purified ClER exhibited a relatively high activity of 7.3 U mg(prot)⁻¹ for ketoisophorone while a remarkable catalytic efficiency (k(cat)/K(m)=810 s⁻¹ mM⁻¹) was obtained for 2-methyl-cinnamaldehyde due to the high affinity. A series of prochiral activated alkenes were stereoselectively reduced by ClER furnishing the corresponding saturated products in up to 99% ee. The practical applicability of ClER was further evaluated for the production of (R)-levodione, a valuable chiral compound, from ketoisophorone. Using the crude enzyme of ClER and glucose dehydrogenase (GDH), 500 mM of ketoisophorone was efficiently converted to (R)-levodione with excellent stereoselectivity (98% ee) within 1h. All these positive features demonstrate a high synthetic potential of ClER in the asymmetric reduction of activated alkenes.

Identification and partial characterization of extracellular aspartic protease genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384.

The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomyces cerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence.

Production of carbonyl reductase by Metschnikowia koreensis.

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.

Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli.

The SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6x His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54kDa. The optimal temperature and pH of the purified rSAP6 were 40 degrees C and 3.4, respectively. The enzyme was stable below 45 degrees C and between pH 2.6 and 5.0. The results show that Mn(2+) had an activating effect on the enzyme, while Cu(2+), Mg(2+), Zn(2+) and Ag(+) acted as inhibitors of the enzyme. However, Ca(2+) had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.

Cloning and characterization of a novel aspartic protease gene from marine-derived Metschnikowia reukaufii and its expression in E. coli.

Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.