Purification and characterization of an endo-polygalacturonase from a mutant of Saccharomyces cerevisiae.

An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.     

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42 kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50–60 °C, with some enzyme activity retained up to 80 °C. Its activation energy was 5.352 cal mol⁻¹. PGase I showed a higher affinity towards PGA than citric pectin (Km = 0.55 ± 0.02 and 0.72 ± 0.02 mg ml⁻¹, respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.     

Identification of a xylose reductase gene in the xylose metabolic pathway of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> NBRC1777

Kluyveromyces marxianus is thermotolerant yeast that is able to utilize a wider range of substrates and has greater thermal tolerance than most other yeast species. K. marxianus can assimilate xylose, but its ability to produce ethanol from xylose in oxygen-limited environments is poor. In the present study, the K. marxianus xylose reductase (KmXR) gene (Kmxyl1) was cloned and the recombinant enzyme was characterized to clarify the factors that limit xylose fermentation in K. marxianus NBRC1777. KmXR is a key enzyme in the xylose metabolism of K. marxianus, which was verified by disruption of the Kmxyl1 gene. The Km of the recombinant KmXR for NADPH is 65.67 μM and KmXR activity is 1.295 U/mg, which is lower than those of most reported yeast XRs, and the enzyme has no activity with coenzyme NADH. This result demonstrates that the XR from K. marxianus is highly coenzyme specific; combined with the extremely low XDH activity of K. marxianus with NADP⁺, the limitation of xylose fermentation is due to a redox imbalance under anaerobic conditions and low KmXR activity.     

Heterologous expression of a thermophilic esterase in <Emphasis Type="Italic">Kluyveromyces</Emphasis> yeasts

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

Effect of inulin on yeast inulinase production in sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K _m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA     

Purification and characterization of a pectin-releasing enzyme produced by Kluyveromyces wickerhamii

A pectin-releasing enzyme produced by Kluyveromyces wickerhamii IFO 1675 (PPase-W) was purified to homogeneity from a culture filtrate by cation-exchange and size-exclusion chromatographies. This enzyme had a molecular weight of 35,000 determined by both size exclusion chromatography and ultracentrifugal analysis, and of 40,000 by SDS-PAGE. It contained 2.4% sugar, and its isoelectric point was at pH 5.2. PPase-W catalyzed the release of highly polymerized pectin from various protopectins, and also showed endopolygalacturonase (endo-PGase) activity. The purified enzyme had optimum PGase activity at about pH 5.2 and 50°C and was stable in the range of pH from 4.0 to 7.0 and up to 50°C. The properties of PPase-W were compared with those of PPase-F from Kluyveromyces fragilis IFO 0288, and some differences were found. Also, some preliminary data dealing with the relationship between enzyme activities (PPase and endo-PGase) and protein structure are discussed.     

The use of the GDH gene for molecular identification and phylogenetic analysis of the yeast Kluyveromyces marxianus

Summary Our previous work showed that NADP⁺-dependent glutamate dehydrogenase from K. marxianus behaves similarly to its counterpart in S. cerevisiae. It suggested that the ammonia assimilation pathway might be different between K. marxianus and the genetic closed species K. lactis. In the present work, we analyzed the genetic similarity among the GDH gene family in K. marxianus and closed yeasts. Specific primers for GDH genes were designed based on the K. marxianus sequences deposited in the Génolevures Database. One of them, for the KmGDH2 gene, proved to be specific for K. marxianus DNA samples, which confirmed the molecular identification of environmental yeast isolates, and can be proposed for rapid screening of this yeast from environmental samples. The nucleotide sequence revealed that KmGDH2 belongs to the S. cerevisiae GDH1 gene family together with KlGDH gene.     

Application of a microtitre reader system to the screening of inulinase nulinase-producing yeasts

 Fourteen strains of yeast from genera Kluyveromyces, Candida, Debaryomyces and Schizosaccharomyces were investigated for inulinase production. In the first stage, the microtitre reader system SLT was used for the determination of enzyme activity and the evaluation of cellular growth. Different culture conditions were tested and four strains of Kluyveromyces were selected on the basis of enzyme activity and growth capacity at low pH and high temperature: K. marxianus CBS 6397, DSM 70792, ATCC 36907 and IZ 619. These strains were tested in greater volume using pH 4.0, 45^°C and inulin (10 g/l) as selection conditions. On the basis of results obtained, the strain K. marxianus ATCC 36907 was selected for inulinase production. Enzyme stability at low pH (4.0) as well as high temperature (50^°C) for 10, 30 and 60 min was also evaluated, but no significant difference in enzyme activity was observed. It could be demonstrated that the microtitre reader system is an excellent method for the screening of microorganisms. Received: 31 May 1995/Received revision: 20 September 1995/Accepted: 29 September 1995     

Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier

Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7.The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The yield of purified bioemulsifier from this strain of K. marxianus was 4–7% of the original dry cell weight. The purified product, at a concentration of 12 g l^–1, formed emulsions that were stable for 3 months when subjected to a range of pH (3–11) and NaCl concentrations (2–50 g l^–1). The composition of this mannoprotein was 90% carbohydrate (mannan) and 4–6% protein. These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source. Consequently K. marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.     

Molecular Characterization of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Agave fourcroydes</Emphasis> (Lem.) in Yucatan,Mexico

The molecular characterization of 14 strains of Kluyveromyces marxianus isolated from Agave fourcroydes (Lem.) in Yucatan, Mexico, was performed by AP-PCR analysis, PCR-RFLP of 5.8S-ITS, and complete NTS regions. A sequence analysis of the D1/D2 domain of the 26S rDNA was also carried out in six selected strains. The AP-PCR approach had the highest discrimination power for the molecular characterization of new henequen K. marxianus strains. PCR-RFLP of 5.8S-ITS regions did not reveal polymorphisms in this group of strains. The restriction enzyme digestion analysis of NTS region enables the separation among strains which coincides with ascospore shape groups. The molecular tools used in this article may be useful to confirm a preliminary screen of yeasts isolated from henequen without the use of growth characteristics or morpho-physiological tests.     

Heterologous expression of glucose oxidase in the yeast <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Background  

In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.     

The high fermentative metabolism of Kluyveromyces marxianus UFV-3 relies on the increased expression of key lactose metabolic enzymes

The aim of this work was to obtain insights about the factors that determine the lactose fermentative metabolism of Kluyveromyces marxianus UFV-3. K. marxianus UFV-3 and Kluyveromyces lactis JA6 were cultured in a minimal medium containing different lactose concentrations (ranging from 0.25 to 64 mmol l⁻¹) under aerobic and hypoxic conditions to evaluate their growth kinetics, gene expression and enzymatic activity. The increase in lactose concentration and the decrease in oxygen level favoured ethanol yield for both yeasts but in K. marxianus UFV-3 the effect was more pronounced. Under hypoxic conditions, the activities of β-galactosidase and pyruvate decarboxylase from K. marxianus UFV-3 were significantly higher than those in K. lactis JA6. The expression of the LAC4 (β-galactosidase), RAG6 (pyruvate decarboxylase), GAL7 (galactose-1-phosphate uridylyltransferase) and GAL10 (epimerase) genes in K. marxianus UFV-3 was higher under hypoxic conditions than under aerobic conditions. The high expression of genes of the Leloir pathway, LAC4 and RAG6, associated with the high activity of β-galactosidase and pyruvate decarboxylase contribute to the high fermentative flux in K. marxianus UFV-3. These data on the fermentative metabolism of K. marxianus UFV-3 will be useful for optimising the conversion of cheese whey lactose to ethanol.     

The β-Galactosidase Activity in <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> CBS6556 Decreases by High Concentrations of Galactose

In this paper we report on the effect of different concentrations of lactose and galactose in the production of β-galactosidase by Kluyveromyces marxianus CBS6556. The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the β-galactosidase activity. Therefore, maximum induction of β-galactosidase in K. marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM. Those informations can help to design low cost medium with higher β-galactosidase productivity by K. marxianus cells. Received: 8 August 2001 / Accepted: 15 October 2001     

Production, purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a pI of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn²⁺, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K_m and V_max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.     

Production,purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a pI of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn²⁺, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K_m and V_max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.     

Inulinase-hyperproducing strains of Kluyveromyces sp. isolated from aguamiel (Agave sap) and pulque

Summary Two strains of Kluyveromyces marxianus (A1 and A2) isolated from ‘aguamiel’ (agave sap) and one strain of K. lactis var. lactis (P7) isolated from ‘pulque’ (its fermented product), were studied to make a survey of inulinase production. The strains of K. marxianus A1 and A2 were the best producers of inulinase, giving up to 2.5 times more enzyme than the control hyperproducing strain K. marxianus CDBB-L-278, and showed lower catabolic repression than this. One strain isolated from pulque was identified as K. lactis var. lactis and was also an excellent inulinase producer, being the first strain of this species reported as such. These strains were very good inulinase producers and they had low susceptibility to catabolic repression probably because the source from which they were isolated was rich in sucrose and oligofructans. They can be used in the transformation of inulin to produce fructose and/or oligofructans.     

Characterization of Kluyveromyces marxianus as a potential feed additive for ruminants

Aim: The aim of this study was to find suitable yeast isolates as potential microbial feed additives for ruminants. Methods and Results: Yeast isolates from traditional fermented food (tapai) and home‐made wine were selected based on their tolerance to volatile fatty acids (VFA) mixture of acetic, propionic and butyric acids and to pH and temperature according to the rumen condition. The ability to grow in and produce ethanol was determined in yeast extract peptone glucose broth supplemented with a VFA mixture (VFA‐YEPG medium). Fifty‐five isolates showed OD_660nm values between 0·35–0·6, and 27 isolates showed ethanol production in the range of 0·17–0·30% (v/v). All selected isolates were identified as Kluyveromyces marxianus base on biochemical tests (BioLog kit; Biolog Inc., Hayward, CA) and molecular techniques. The best isolate in terms of ethanol production (K. marxianus WJ1) significantly (P < 0·01) improved in vitro apparent dry matter (DM) digestibility of alfalfa (Medicago sativa), guinea grass (Panicum maximum) and timothy (Phleum pretense) hay by rumen microbes. Conclusion: Yeast isolates from tapai and wine were able to grow in VFA‐YEPG medium, and K. marxianus WJ1 improved in vitro DM digestibility of plant substrates. Significance and Impact of the Study: This study indicated the possibility of using K. marxianus as a microbial feed additive.     

Lysinoalanine Formation in Gluten: A Conformational Effect

The production of pectolytic enzyme in the genus Kluyveromyces was investigated. The production of the enzyme was dependent on the strain, and some strains belonging to K. fragilis, K. Marxianus, and K. Wickerhamii produced this enzyme among 11 species (29 strains) of the genus Kluyveromyces. K. Fragilis IFO 0288 produced at least four endo-polygalacturonases which have different molecular weights. The dominant endo-polygalacturonase in the culture filtrate of the strain was purified and isolated as crystals. The purified enzyme was homogeneous based on analysis by polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme was a glycoprotein having an isoelectric point around pH 5.6. The sedimentation coefficient (s_2o,w) was 3.77S, and the molecular weight was around 33,000. The enzyme contained aspartic acid (asparagine), serine,threonine, and glycine at relatively high levels. The enzyme showed the highest activity around pH 5.0 and was stable at pH 5.0 up to 30°C. With the enzyme, and activity which releases highly polymerized pectin from various protopectins (protopectinase activity) was found.