Detoxification of Mycotoxin Patulin by the Yeast Kluyveromyces marxianus YG-4 in Apple Juice.

Patulin (PAT) is a mycotoxin produced by Penicillium species, which often contaminates fruit and fruit-derived products, posing a threat to human health and food safety. This work aims to investigate the detoxification of PAT by Kluyveromyces marxianus YG-4 (K. marxianus YG-4) and its application in apple juice. The results revealed that the detoxification effect of K. marxianus YG-4 on PAT includes adsorption and degradation. The adsorption binding sites were polysaccharides, proteins, and some lipids on the cell wall of K. marxianus YG-4, and the adsorption groups were hydroxyl groups, amino acid side chains, carboxyl groups, and ester groups, which were combined through strong forces (ion interactions, electrostatic interactions, and hydrogen bonding) and not easily eluted. The degradation active substance was an intracellular enzyme, and the degradation product was desoxypatulinic acid (DPA) without cytotoxicity. K. marxianus YG-4 can also effectively adsorb and degrade PAT in apple juice. The contents of organic acids and polyphenols significantly increased after detoxification, significantly improving the quality of apple juice. The detoxification ability of K. marxianus YG-4 toward PAT would be a novel approach for the elimination of PAT contamination.

Optimal Extraction and Deproteinization Method for Mannoprotein Purification from Kluyveromyces marxianus.

Background: Mannoproteins, mannose-glycosylated proteins, play an important role in biological processes and have various applications in industries. Several methods have been already used for the extraction of mannoproteins from yeast cell-wall. The aim of this study was to evaluate the extraction and deproteinization of mannan oligosaccharide from the Kluyveromyces (K.) marxianus mannoprotein. Methods: To acquire crude mannan oligosaccharides, K. marxianus mannoproteins were deproteinized by the Sevage, trichloroacetic acid, and hydrochloric acid (HCL) methods. Total nitrogen, crude protein content, fat, carbohydrate and ash content were measured according to the monograph prepared by the meeting of the Joint FAO/WHO Expert Committee and standard. Mannan oligosaccharide loss, percentage of deproteinization, and chemical composition of the product were assessed to check the proficiency of different methods. Results: Highly purified (95.4%) mannan oligosaccharide with the highest deproteinization (97.33 ± 0.4%) and mannan oligosaccharide loss (25.1 ± 0.6%) were obtained following HCl method. Conclusion: HCl, was the most appropriate deproteinization method for the removal of impurities. This preliminary data will support future studies to design scale-up procedures.

Biological Properties of Yeast-based Mannoprotein for Prospective Biomedical Applications.

BACKGROUND: Natural products constitute more than half of all biomolecules lately being used in clinical settings. Mannoprotein derived from the yeast cell wall has found full biotechnological applications. OBJECTIVE: This study was intended to investigate the antioxidant, anticancer, and toxicological properties of Kluyveromyces marxianus mannoprotein (KM). METHODS: The KM extract was obtained through a sequence of operations, including centrifugation for cell isolation, precipitation with potassium citrate/sodium metabisulfite, and recovery and purification. Its antioxidant, growth inhibition, macrophage mitogenic, and toxic activities were evaluated for its future use in the biomedical field. RESULTS: Significant inhibitory effects of KM were obtained on reactive species. It showed antiproliferative activity against HeLa (human cervical adenocarcinoma) and MCF-7 (human breast cancer) cell lines with no toxic effects on HUVECs (human umbilical vein endothelial cells). The in vitro model of CHO-K1 (Chinese hamster ovary) cell lines did not show the cytotoxic and genotoxic of KM. Moreover, it enhanced macrophage activity in terms of nitric oxide (NO) production and viability. No sign of acute toxicity was found in BALB/c mice, and body weight remained unchanged in guinea pigs over three months. CONCLUSION: Comprehensive biological evaluations in this study are expected to expand the potential of KM as a natural material.

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

BACKGROUND: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. RESULTS: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. CONCLUSIONS: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

The antioxidant function of Sco proteins depends on a critical surface-exposed residue.

BACKGROUND: Besides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity. METHODS: Native and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays. RESULTS: A functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways. CONCLUSION: This study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology. GENERAL SIGNIFICANCE: These findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.

Structural insights into telomere protection and homeostasis regulation by yeast CST complex.

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance. Despite extensive studies, only structural information of individual domains of CST is available; the architecture of CST still remains unclear. Here, we report crystal structures of Kluyveromyces lactis Cdc13-telomeric-DNA, Cdc13-Stn1 and Stn1-Ten1 complexes and propose an integrated model depicting how CST assembles and plays its roles at telomeres. Surprisingly, two oligonucleotide/oligosaccharide-binding (OB) folds of Cdc13 (OB2 and OB4), previously believed to mediate Cdc13 homodimerization, actually form a stable intramolecular interaction. This OB2-OB4 module of Cdc13 is required for the Cdc13-Stn1 interaction that assembles CST into an architecture with a central ring-like core and multiple peripheral modules in a 2:2:2 stoichiometry. Functional analyses indicate that this unique CST architecture is essential for both telomere capping and homeostasis regulation. Overall, our results provide fundamentally valuable structural information regarding the CST complex and its roles in telomere biology.

Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of ß-Galactosidase from Kluyveromyces lactis.

Here, we report the effect of polyethylene glycol (PEG6000)-induced molecular crowding (MC) on the catalytic activity and thermal stability of Kluyveromyces lactis ß-galactosidase (ß-Gal). The ß-Gal-catalyzed hydrolysis of o-nitrophenyl-ß-d-galactopyranoside followed a Michaelian kinetics at [PEG6000] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG6000). Compared with dilute solutions, in the MC media, ß-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (e.g., 45 °C) and by the slower inactivation kinetics. Considering the effects of water thermodynamic activity on the reaction kinetics and protein structure and the effect of the exclusion volume on protein conformation, we suggest that changes in the protein oligomerization state and hydration could be the responsible for the behavior observed at the highest MC levels assayed. These results could be relevant and should be taken into account in industrial food processes applying ß-Gal from K. lactis.

Whole-Cell Biocatalysis in Seawater: New Halotolerant Yeast Strains for the Regio- and Stereoselectivity Reduction of 1-Phenylpropane-1,2-Dione in Saline-Rich Media.

The application of green chemistry concepts in catalysis has considerably increased in recent years, and the interest in using sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and Schwanniomyces for catalytic processes run in this medium. In the present work, we select other yeasts based on their halotolerance to widen the scope of this strategy. We consider them for the monoreduction of 1-phenylpropane-1,2-dione, a well-characterized reaction that produces acyloin intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this reaction is possible. The best ones were obtained for Saccharomyces cerevisiae FY86 and Kluyveromyces marxianus, for which acyloins with different stereochemistry were obtained with good to excellent enantiomeric excess.

Structural basis for the evolution of cyclic phosphodiesterase activity in the U6 snRNA exoribonuclease Usb1.

U6 snRNA undergoes post-transcriptional 3' end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3' end of U6 and, in humans, leaves a 2',3' cyclic phosphate that is recognized by the Lsm2-8 complex. Saccharomycescerevisiae Usb1 has additional 2',3' cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3' phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5'-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n - 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.

Probiotic properties and fatty acid composition of the yeast Kluyveromyces lactis M3. In vivo immunomodulatory activities in gilthead seabream (Sparus aurata).

The aim of this study was to analyze the probiotic potential, fatty acid composition and immunostimulant activities of Kluyveromyces lactis M3 isolated from a hypersaline sediment. For this purpose, K. lactis M3 resistance to different pH, salinities and bile, as well as its antioxidant capability were assayed. Furthermore, total fatty acid composition of the yeast was determined where the dominant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. K. lactis M3 showed no cytotoxic effects on peripheral blood leukocytes. During an in vivo experiment in gilthead seabream (Sparus aurata), dietary K. lactis M3 supplemented at 0.55 or 1.1% of the basal diet enhanced bactericidal activity against Vibrio parahaemolyticus N16, V. harveyi Lg 16/00, and V. anguillarum CECT 43442 compared to fish fed commercial diet (control group). Finally, nitric oxide production, peroxidase activity and skin mucus lectin union levels strongly increased in fish fed K. lactis M3 with respect to the control group. The results suggested that the yeast K. lactis M3 had exhibited high antioxidant capability, and its dietary administration at 0.55 or 1% basal diet had immunostimulant activity for gilthead seabream. For all these reasons, it should be considered an appropriate probiotic candidate for the aquaculture fish industry.