Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.     

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.     

Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2

In Saccharomyces cerevisiae, the alcohol dehydrogenase genes ADH1 and ADH5 are part of a duplicated block of genome, thought to originate from a genome-wide duplication posterior to the divergence from the Kluyveromyces lineage. We report here the characterization of Kluyveromyces marxianus ADH2 and the five genes found in its immediate downstream region, MRPS9, YOL087C, RPB5, RIB7 and SPP381. The order of these six genes reflects the structure of the ancestral S. cerevisiae genome before the duplication that formed the blocks including ADH1 on chromosome XV and ADH5 on chromosome II, indicating these ADH genes share a direct ancestor. On the one hand, the two genes found immediately downstream of KmADH2 are located, for the first, downstream ADH5 and, for the second, downstream ADH1 in S. cerevisiae. On the other hand, the order of the paralogs included in the blocks of ADH1 and ADH5 in S. cerevisiae suggests that two of them have been inverted within one block after its formation, and that inversion is confirmed by the gene order observed in K. marxianus.     

Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424.

Cell wall inulinase (EC 3.2.1.7) was purified from Kluyveromyces marxianus var. marxianus (formerly K. fragilis) and its N-terminal 33-amino acid sequence was established. PCR amplification of cDNA with 2 sets of degenerate primers yielded a genomic probe which was then used to screen a genomic library established in the YEp351 yeast shuttle vector. One of the selected recombinant plasmids allowed an invertase-negative Saccharomyces cerevisiae mutant to grow on inulin. It was shown to contain an inulinase gene (INU 1) encoding a 555-amino acid precursor protein with a typical N-terminal signal peptide. The sequence of inulinase displays a high similarity (67%) to S. cerevisiae invertase, suggesting a common evolutionary origin for yeast beta-fructosidases with different substrate preferences.     

Use of waste Chinese cabbage as a substrate for yeast biomass production

The possibility of using waste Chinese cabbage (Brassica campestris) as a substrate for microbial biomass production was investigated. The juice from waste Chinese cabbage contains relatively high amounts of reducing sugars suitable for yeast culture. The cell mass and protein content of four species of yeast, Candida utilis, Pichia stipitis, Kluyveromyces marxianus, and Saccharomyces cerevisiae, were determined when cultured in juice extracted from cabbage waste. Compared to YM broth containing the same level of sugar, all the strains except C. utilis showed higher total protein production in cabbage juice medium (CJM).     

Expression of the INU2 gene for an endoinulinase of Aspergillus ficuum in Saccharomyces cerevisiae

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase preparation free of an exoinulinase and an extracellular invertase in the culture medium. A recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony, when inulin was included in the medium.     

Regulation of bacterial photosynthesis genes by oxygen and light

When grown in the appropriate medium, several yeast species produce pectinases able to degrade pectic substances. It is mainly exocellular endopolygalacturonases that break pectins or pectate down by hydrolysis of alpha-1,4-glycosidic linkages in a random way. Biochemical characterisation of these enzymes has shown that they have an optimal pH in the acidic region and an optimal temperature between 40 and 55 degrees C. Their production by yeasts is a constitutive feature and is repressed by the glucose concentration and aeration. Pectic substances and their hydrolysis products are used as carbon sources by a limited number of yeasts and hence these enzymes must be involved in the colonisation of different parts of plants, including fruits. The first yeast pectic enzyme (encoded by the PSE3 gene) was cloned from Tichosporon penicillatum. Recently, a polygalacturonase-encoding gene from Saccharomyces cerevisiae has been cloned and overexpressed in several strains and the gene for an extracellular endopolygalacturonase from Kluyveromyces marxianus has also been described. Taking all the results together, the idea is now emerging that this type of yeast enzyme could offer an alternative to fungal enzymes for industrial applications.     

Inulase-secreting strain of Saccharomyces cerevisiae produces fructose

The gene encoding inulase of the yeast Kluyveromyces marxianus (INU1Km) was cloned and expressed in the inulin-negative yeast Saccharomyces cerevisiae. Cells of S. cerevisiae transformed with the INU1Km gene have acquired extracellular inulase activity and were able to grow in the medium with inulin as a sole carbon source. The S. cerevisiae strain was constructed that is capable of heterologous expression of secreted K. marxianus inulase and is defective in fructose uptake due to null-mutations of the hexokinase structural genes HXK1 and HXK2. When grown in inulin-containing media, this strain is capable of accumulating at least 10% glucose-free fructose in the culture liquid.     

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.     

Ethanol production from fodder beets

Various yeasts such as two strains of Saccharomyces cerevisiae, Saccharomyces diastaticus, and Kluyveromyces marxianus were investigated for their ability to ferment fodder beet juice to alcohol. Juice extracted from fodder beet roots without any additives was used as a fermentation substrate. The fermentation kinetic parameters were determined and compared for each species of yeast tested. The best species for fodder beet juice fermentation was chosen and products obtained by fermentation of one hectare of fodder beet plants are given.     

Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae

Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography. Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay. The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537-2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Comparison of enzymatic antioxidant defence systems in different metabolic types of yeasts

The enzymatic defence system in the 2 yeasts Kluyveromyces marxianus and Rhodotorula glutinis, differing in their mode of oxygen uptake and energy generation, was characterized and compared with the well-studied facultatively fermentative Crabtree-positive Saccharomyces cerevisiae strain. Twofold higher superoxide dismutase (SOD) and catalase activities were detected in K. marxianus and R. glutinis when cells were cultured on glucose. Further increases of 10%-15% in SOD activity and 30%-50% in catalase were measured in all studied yeasts strains after transfer to media containing ethanol. An evaluation of the ratio of Cu/Zn SOD / Mn SOD was performed as a measure of the oxidative metabolism. A 20% decrease was observed when the respiratory source of energy was ethanol, with the lowest ratio being observed for the oxidative type of K. marxianus yeasts. Electrophoretic analysis revealed that all tested strains possess active Cu/Zn and Mn SODs. A reverse electrophoretic mobility pattern of K. marxianus and R. glutinis SOD enzymes was observed in comparison with the same couple in S. cerevisiae. The investigation of electrophoretic profile of catalase enzymes showed that alongside their different taxonomic status and fermentative capacity, all tested strains possess 2 separate catalases. The role of antioxidant enzymes in preventing oxidant-induced cytotoxicity (treatment with hydrogen peroxide, paraquat, and menadione) was shown.     

Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).     

Iron enriched yeast biomass--a promising mineral feed supplement

Yeast biomass enriched with iron could represent a new and safer solution for prevention from anaemia development. Such an iron source is less toxic and has better absorbability in organisms. The purpose of our research was the determination of the most suitable iron source in the cultivation medium for the yeast Saccharomyces cerevisiae, regarding good growth and iron accumulation in cells. Iron(III) citrate, iron(III) chloride, iron(III) nitrate and Fe-EDTA complex were used. The uptake of the chosen iron compound, Fe(III) citrate, by the yeasts Candida intermedia and Kluyveromyces marxianus was also investigated. Different growth behaviour of the three yeast strains in the presence of Fe(III) citrate was observed. The highest amounts of accumulated iron in S. cerevisiae, C. intermedia and K. marxianus biomass were about 13, 20 and 34mgFeg(-1)dry wt., respectively. To optimise the accumulation of iron in K. marxianus and to characterise iron enriched yeast biomass, further experiments are needed.     

一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

Overexpression, purification and properties of alcohol dehydrogenase IV from Saccharomyces cerevisiae

We have purified ADHIV, a novel alcohol dehydrogenase (ADH) isozyme in the yeast Saccharomyces cerevisiae, after increasing the normally low amount of ADHIV protein in laboratory strains. This was done by overexpression of the structural gene (ADH4) on a 2micro-based multicopy vector. Characterization of the purified enzyme revealed a dimeric structure as well as a different substrate specificity and pH profile as compared to other alcohol dehydrogenase isozymes. On the other hand, we could demonstrate that ADHIV is activated by zinc ions, like the other yeast alcohol dehydrogenase isozymes, and not by ferrous ions, like a structurally similar alcohol dehydrogenase from the bacterium Zymomonas mobilis.     

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.