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.

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.

Production of antioxidant and ACE-inhibitory peptides from Kluyveromyces marxianus protein hydrolysates: Purification and molecular docking.

Kluyveromyces marxianus protein hydrolysates were prepared by two different sonicated-enzymatic (trypsin and chymotrypsin) hydrolysis treatments to obtain antioxidant and ACE-inhibitory peptides. Trypsin and chymotrypsin hydrolysates obtained by 5 h, exhibited the highest antioxidant and ACE-inhibitory activities. After fractionation using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC) techniques, two new peptides were identified. One fragment (LL-9, MW = 1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys showed significant ACE inhibitory activity (IC50 = 22.88 µM) while another peptide fragment (VL-9, MW = 1118 Da) with the amino acid sequence of Val-Leu-Ser-Thr-Ser-Phe-Pro-Pro-Lys showed the highest antioxidant and ACE inhibitory properties (IC50 = 15.20 µM, 5568 µM TE/mg protein). The molecular docking studies revealed that the ACE inhibitory activities of VL-9 is due to interaction with the S2 (His513, His353, Glu281) and S'1 (Glu162) pockets of ACE and LL-9 can fit perfectly into the S1 (Thr345) and S2 (Tyr520, Lys511, Gln281) pockets of ACE.

Cancer Chemopreventive, Antiproliferative, and Superoxide Anion Scavenging Properties of Kluyveromyces marxianus and Saccharomyces cerevisiae var. boulardii Cell Wall Components.

This study investigated the cancer chemopreventive, the antiradical, and the antiproliferative properties of polysaccharides extracts from cell wall of Saccharomyces boulardii and Kluyveromyces marxianus. ß-glucan, mannan, and chitin were also quantified to identify the most important extract responsible for these biological properties. Soluble and insoluble glucans as well as mannoprotein were extracted from cell wall using single hot-alkaline method. Superoxide anion scavenging (antiradical capacity), NAD(P)H: quinone reductase (QR) (EC 1.6.99.2) induction, and antiproliferative assays were done for the evaluation of biological properties of those extracts. The insoluble glucan from S. boulardii revealed the most relevant biological properties by increasing QR activity and exhibiting the highest growth inhibition against colorectal cancer cells. Moreover, high amount of glucan, high glucan/total sugars ratios, and low chitin/glucan ratios were shown to have an impact on enhancing cancer chemopreventive and antiproliferative properties. To our knowledge, this is the first study that demonstrates QR activity by yeast cell wall components in a dose-dependent manner.

Oxidative stress and antioxidant response in a thermotolerant yeast

Abstract Stress tolerance is a key attribute that must be considered when using yeast cells for industrial applications. High temperature is one factor that can cause stress in yeast. High environmental temperature in particular may exert a natural selection pressure to evolve yeasts into thermotolerant strains. In the present study, three yeasts (Saccharomyces cerevisiae, MC4, and Kluyveromyces marxianus, OFF1 and SLP1) isolated from hot environments were exposed to increased temperatures and were then compared with a laboratory yeast strain. Their resistance to high temperature, oxidative stress, and antioxidant response were evaluated, along with the fatty acid composition of their cell membranes. The SLP1 strain showed a higher specific growth rate, biomass yield, and biomass volumetric productivity while also showing lower duplication time, reactive oxygen species (ROS) production, and lipid peroxidation. In addition, the SLP1 strain demonstrated more catalase activity after temperature was increased, and this strain also showed membranes enriched in saturated fatty acids. It is concluded that the SLP1 yeast strain is a thermotolerant yeast with less oxidative stress and a greater antioxidant response. Therefore, this strain could be used for fermentation at high temperatures.

Purification of inulinases by changing the ionic strength of the medium and precipitation with alcohols

ABSTRACT The present study evaluated the purification of inulinase by changing the ionic strength of the medium by addition of NaCl and CaCl2 followed by precipitation with n-propyl alcohol or iso-propyl alcohol. The effects of the concentration of alcohols and the rate of addition of alcohols in the crude extract on the purification yield and purification factor were evaluated. Precipitation caused an activation of enzyme and allowed purification factors up to 2.4-fold for both alcohols. The purification factor was affected positively by the modification of the ionic strength of the medium to 0.5 mol.L-1 NaCl before precipitation with the alcohol (n-propyl or iso-propyl). A purification factor of 4.8-fold and an enzyme yield of 78.1 % could be achieved by the addition of 0.5 mol.L-1 of NaCl to the crude extract, followed by the precipitation with 50 % (v/v) of n-propyl alcohol, added at a flow rate of 19.9 mL/min.

Chemical structure, antiproliferative and antioxidant activities of a cell wall α-d-mannan from yeast Kluyveromyces marxianus.

Cell wall polysaccharides from filamentous fungi and yeasts have been reported as antioxidant and antiproliferative polymers. Thus, we evaluated these activities from cell wall polysaccharides from Kluyveromyces marxianus CCT7735. By using a centrifugal filter, a 203kDa α-d-mannan (KMM-5) was obtained. KMM-5 exhibited no effect on HeLa cells and a weak antiproliferative activity against Hep-G2 cells. In addition, at higher concentrations, it presented a cytotoxicity to the normal cell line, 3T3. However, KMM-5 showed copper- and iron-chelating abilities, the latter of which presented improved activity. By using 2D-NMR COSY, HSQC edited and HMBC experiments, a structure arrangement was proposed. The main chain was formed by 6)-α-d-Manp-(1→6) units substituted at the 2-O-position by non-reducing terminals α-d-Manp-(1→2) and by a branched tetrasaccharide. The latter was formed by an internal 2)-α-d-Manp-(1→2) unit with linked to it a 2,3)-α-d-Manp-(1→2) unit substituted at the 2-O-position by a non-reducing terminal α-d-Manp-(1→2), and at the 3-O-position by a non-reducing terminal α-d-Manp-(1→3). In conclusion, we considered K. marxianus CCT7735 a source of natural and renewable polysaccharides with pharmacological properties.

Characterization of Extracellular Yeast Peptide Factors and Their Stress-Protective Effect on Probiotic Lactic Acid Bacteria.

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia li- polytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus,'and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcusjaponicus subsp. casei R. In BS-treated cells of the tester bacteria, a pro- tective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RE: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pro- nounced than the protector effect, with the CFU number I to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row: C. utilis > K. lactis > Y lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recepient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one, for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.

Mutations on the N-terminal edge of the DELSEED loop in either the α or ß subunit of the mitochondrial F1-ATPase enhance ATP hydrolysis in the absence of the central γ rotor.

F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)ß(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)ß(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)ß(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and ß subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the ß subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the ß subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or ß subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)ß(3) subcomplex.

Cytosolic proteome of Kluyveromyces lactis affected by the multidrug resistance regulating transcription factor KlPdr1p.

Multidrug resistance (MDR), a ubiquitous phenomenon conserved from bacteria to humans, causes serious problems in the treatment of human cancers and infections of bacterial and fungal origin. The development of MDR in yeast is frequently associated with gain-of-function mutations in the Zn(2)Cys(6) transcription factors activating the expression of several plasma membrane exporters. In the aerobic yeast Kluyveromyces lactis the Zn(2)Cys(6) transcription factor KlPdr1p is involved in the control of multidrug resistance. The aim of the present study was to identify the changes in K. lactis proteome of the Klpdr1Δ deletion mutant compared with the wild-type expressing the KlPDR1 gene from a multicopy plasmid. A total of 15 differentially expressed proteins, out of 20 spots with different intensities detected, were identified. In the Klpdr1Δ deletion mutant, the increase in the abundance of proteins involved in carbohydrate metabolism (mainly glycolysis/gluconeogenesis) was observed. Most of the proteins overexpressed in the wild type strain containing the KlPDR1 gene on multicopy plasmid were involved in the stress defence and redox homeostasis. The results indicate a close connection between MDR and oxidative stress response associated with the post-translational mechanisms regulating the levels of active forms of proteins involved in K. lactis MDR.

Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a ß-propeller protein family.

ß-propellers that bind polyphosphoinositides (PROPPINs), a eukaryotic WD-40 motif-containing protein family, bind via their predicted ß-propeller fold the polyphosphoinositides PtdIns3P and PtdIns(3,5)P(2) using a conserved FRRG motif. PROPPINs play a key role in macroautophagy in addition to other functions. We present the 3.0-Å crystal structure of Kluyveromyces lactis Hsv2, which shares significant sequence homologies with its three Saccharomyces cerevisiae homologs Atg18, Atg21, and Hsv2. It adopts a seven-bladed ß-propeller fold with a rare nonvelcro propeller closure. Remarkably, in the crystal structure, the two arginines of the FRRG motif are part of two distinct basic pockets formed by a set of highly conserved residues. In comprehensive in vivo and in vitro studies of ScAtg18 and ScHsv2, we define within the two pockets a set of conserved residues essential for normal membrane association, phosphoinositide binding, and biological activities. Our experiments show that PROPPINs contain two individual phosphoinositide binding sites. Based on docking studies, we propose a model for phosphoinositide binding of PROPPINs.

The Kluyveromyces lactis CPY homologous genes: cloning and characterization of the KlPCL1 gene.

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.

Evaluation of Kluyveromyces marxianus as a source of yeast autolysates.

Cells of Kluyveromyces marxianus FII 510700 and Saccharomyces cerevisiae CBS 1907 were autolysed in phosphate buffer, pH 4.5, for a maximum of 10 days to compare chemical changes that occur in the carbohydrate, protein, amino acid and nucleic acid content. Approximately 2.2-3% carbohydrate, 9.5-12% protein, 0.6-1.0% DNA and 6-7% RNA were recovered in the autolysates. The main amino acids were beta-alanine, phenylalanine, cysteine, methionine, glutamic acid and isoleucine. No significant differences in the yeast autolysates of K. marxianus and S. cerevisiae were observed. Consequently, K. marxianus produced from lactose-based media has potential as a source of yeast autolysates used in the food industry.

Producción de biomasa de Kluyveromices fragilis en suero de leche desproteinizado / Production of Kluyveromices fragilis biomass in deproteinized milk whey

The milk whey from a mature cheese factory deproteinised by acid thermic coagulation (pH 4.5 and 90 degrees C), provides a good culture media for the production of Kluyveromices fragilis biomass. The optimal experimental conditions for the maximal production of biomass were established by using fermenters with different capacity and design. For lactose concentration of 15 g/l, pH 4.5, 30 degrees C and aireation between 0.25 and 1 VVM, the duplication time was below two hours and 98 per cent of the lactose was consumed. The obtained yield in dried weight was between 36 and 49 per cent (g biomass/g lactose). The biomass (without broken cell) contain 46 per cent protein on dry base and showed an "in vitro" digestibility of 65 per cent. The organic mass decreased 80 per cent after 12 hour of fermentation. This process eliminates a polluting agent and simutaneously, produces a biomass that could have industrial use as a protein complement in feeds.

Purification of microbial (beta)-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning

This work investigated the partitioning of (beta)-galactosidase from "Kluyveromyces fragilis" in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-(beta)-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme (beta)-galactosidase from "Kluyveromyces fragilis" was developed. In the first step, a system composed of 6(per cent) PEG 4000-APGP and 8(per cent) dextran 505 was used, where (beta)-galactosidase was strongly partitioned to the top phase (K = 2.330). In the second step, a system formed of 13(per cent) Peg-APGP and 9(per cent) phosphate salt was used to revert the value of the partition coefficient of (beta)-galactosidase (K = 2.0E-5) in order to provide the purification and recovery of 39(per cent) of the enzyme in the bottom salt-rich phase

The HIS4 gene from the yeast Kluyveromyces lactis.

The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb.

Yeast heat shock transcription factor N-terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy.

The structure and dynamics of the N-terminal activation domains of the yeast heat shock transcription factors of Kluyveromyces lactis and Saccharomyces cerevisiae were probed by heteronuclear 15N[1H] correlation and 15N[1H] NOE NMR studies. Using the DNA-binding domain as a structural reference, we show that the protein backbone of the N-terminal activation domain undergoes rapid, large-amplitude motions and is therefore unstructured. Difference CD data also show that the N-terminal activation domain remains random-coil, even in the presence of DNA. Implications for a "polypeptide lasso" model of transcriptional activation are discussed.

Solution structure of the Kluyveromyces lactis LAC9 Cd2 Cys6 DNA-binding domain.

The Zn2Cys6 DNA-binding domain has been identified by sequence homology in approximately forty fungal proteins, including the K. lactis LAC9 transcriptional activator. Using 1H NMR spectroscopy, we have determined the solution structure of a cadmium-substituted form of the LAC9 DNA-binding domain. We have complemented this approach by applying a series of 113Cd-1H NMR experiments, including several novel heteroTOCSY-based techniques. The DNA-binding domain forms a core of two alpha-helix/extended strand segments around the Cd2 binuclear cluster, with a network of amide proton-cysteinyl S gamma hydrogen bonds stabilizing the cluster. Comparison with other Zn2Cys6 domain structures provides insight into the common structural elements used in metal coordination and DNA binding.

Chemical composition of a mixture of single-cell protein obtained from Kluyveromyces fragilis and whey proteins

De la fermentación de suero de leche entera se obtuvo un producto consistente en una mezcla de biomasa de Kluyveromyces fragilis y proteínas coaguladas del suero. El producto tuvo una composición similar a la de productos lavados de que se informa en la literatura, con un alto contenido de proteína cruda y un bajo contenido de cenizas. Asimismo, acusó tambiém un alto contenido de aminoácidos azufrados y de triptofano, los que usualmente son limitantes en al biomasa de levadura. El contenido de lisina fue inexplicablemente más bajo de lo esperado resusltando ser el aminoácido limitante. La calificación química de la protéina fue 91%. Del producto de biomasa com proteínas de suero se obtuvo un concentrado proteínico con un rendimento de 80%. El contenido de proteína del aislado fue de 75% y el contenido de ácidos nucleícos se redujó en 90.8%. Los restos de pared celular también se redujeron considerablemente