Über den Abbau von L-Äpfelsäure durch Hefen verschiedener Gattungen mit Malatenzym

Summary (1) The aerobic assimilation of malic acid is not a character of certain yeast genera or species as was shown by testing more than 300 different strains. Single strains of the following-species were found to grow on malic acid as the only carbon source: Candida pulcherrima, C. utilis, C. mycoderma, Torulopsis famata, Pichia membranaefaciens, P. wickerhamii, Hansenula capsulata, Trigonopsis variabilis , and Zygosaccharomyces chevalieri . (2) During fermentation C. pulcherrima and T. famata decompose up to 40% and C. utilis up to 80% of the L-malic acid that is present in the medium. (3) L-Malic acid is decomposed to CO 2 and the corresponding amounts of ethanol or pyruvate by cell fr…

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the ni…

Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidezed only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and red…

Die beim Abbau vonl-�pfels�ure durch Milchs�urebakterien entstehenden Isomeren der Milchs�ure

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H…

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000. Additional investigations of the primary toxin binding sites within the cell wall of sensitiv…

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme.

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme. 2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent. 3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese. 4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP. 5. The Km-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for…

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose…

Das Vorkommen von Malatenzym und Malo-Lactat-Enzym bei verschiedenen Milchs�urebakterien

1. Bei drei Stammen vonLactobacillus casei, die auf Apfelsaure als C-Quelle wuchsen, wurde durch Kultur mit Apfelsaure entweder Malatenzym oder durch Kultur mit Apfelsaure und Glucose Malo-Lactat-Enzym induziert. Zwei Stamme vonStreptococcus faecalis bildeten nur Malatenzym,Streptococcus lactis, der Glucose zum Wachstum braucht, nur Malo-Lactat-Enzym. 2. Durch aufeinanderfolgende Induktion wurden Zellen vonLactobacillus casei M40 mit Malatenzym und Malo-Lactat-Enzym erhalten. 3. Malatenzym und Malo-Lactat-Enzym unterscheiden sich im Induktionsverhalten, im pH-Optimum, in der Affinitat zum Substrat, in den Endprodukten und im Molekulargewicht.

Die Bildung höherer Alkohole bei Aminosäuremangelmutanten von Saccharomyces cerevisiae III. Höhere Alkohole als Nebenprodukte der Aminosäuresynthese

Summary The formation of higher alcohols as byproducts of the synthesis of threonine, isoleucine and valine was investigated using isogenic haploid and diploid mutant strains of Saccharomyces cerevisiae . Genetic markers were ade 2, leu 1. The growth rate, the formation of ethanol, the amino acid metabolism and the formation of higher alcohols were determined during fermentations in a synthetic medium. n-Propanol-1, 2-methylbutanol-l and isobutanol were synthesized parallel to ethanol formation during exponential and stationary growth phase. However, the formation of 3-methylbutanol-l from leucine was restricted to the exponential phase. A comparison of haploid and diploid strains revealed,…

Die Umsetzung von L-Äpfelsäure durchSaccharomyces cerevisiae bei der Gärung

Yeasts of the genusSaccharomyces are able to decompose L-malic acid partially, during and after fermentation, whereby ethanol and carbon dioxide are the end products. The decarboxylation of malic acid by yeast can be achieved with resting cells and cell free extracts.

Caseicin, a bacteriocin from Lactobacillus casei.

The intracellular bacteriocin caseicin 80 was purified from cell extracts of Lactobacillus casei strain B80. It is a thermolabile protein with an apparent molar mass of 42 kDa. As no plasmids were observed in the bacteriocinogenic strain it is assumed that caseicin is encoded by the bacterial chromosome. Using 14C-labelled precursors it was found that biosynthesis of DNA and proteins was influenced by caseicin but this inhibition is probably not the primary effect. The incorporation of fructose but not of glucose into cellular material was inhibited by caseicin.

Metabolism of the anaerobic formation of succinic acid bySaccharomyces cerevisiae

1. Succinic acid is formed in amounts of 0.2–1.7 g/l by fermenting yeasts of the genusSaccharomyces during the exponential growth phase. No differences were observed between the various species, respiratory deficient mutants and wild type strains. 2. At low glucose concentrations the formation of succinic acid depended on the amount of sugar fermented. However, the nitrogen source was found to be of greater importance than the carbon source. 3. Of all nitrogen sources, glutamate yielded the highest amounts of succinic acid. Glutamate led to an oxidative and aspartate to a reductive formation of succinic acid. 4. A reductive formation of succinic acid by the citric acid cycle enzymes was obs…

Propanediol-1,2-dehydratase and metabolism of glycerol of Lactobacillus brevis

While most strains of heterofermentative lactobacilli and strains of Leuconostoc species contained only traces of a dehydratase reacting with glycerol or propanediol-1,2, three strains of Lactobacillus brevis and one strain of L. buchneri that metabolized glycerol readily in the presence of glucose, contained propanediol-1,2 dehydratase (EC 4.2.1.28). This cobamide requiring enzyme from L. brevis B 18 was partially purified. It reacts with the substrates propanediol-1,2, glycerol and ethanediol-1,2 with the relative activities of about 3:2:1. This ratio remained unchanged throughout the purification procedure. The substrate affinities were measured: propanediol-1,2 K m=0.6 mM, glycerol K m=…

Das ?Malatenzym? von Lactobacillus plantarum und Leuconostoc mesenteroides

1. Das “Malatenzym” von induzierten Zellen von Lactobacillus plantarum B 38 wurde bis zu einer spezifischen Aktivitat von 170 u/mg Protein und von Leuconostoc mesenteroides 99 bis zu 17,5 u/mg angereichert. 2. Durch Gelfiltration, Chromatographie an Hydroxylapatit sowie Disk-Elektrophorese wurden aus L. plantarum Praparate gewonnen, die frei von Lactat-Dehydrogenase waren. 3. Fur das “Malatenzym” aus L. plantarum wurde ein Molekulargewicht von 150 000 und fur das Enzym aus Lc. mesenteroides von 130 000–140 000 ermittelt. Das Malatenzym (E.C. 1.1.1.38) aus Schizosaccharomyces pombe hat ein Molekulargewicht von 120 000–130 000. 4. Gereinigte Praparate von Malatenzym aus L. plantarum, die kein…

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a m…

Antibacterial polypeptides of Lactobacillus species

Twelve of 79 strains of the genus Lactobacillus, mainly isolated from plants or fermenting material, were found to inhibit at least one of the nine indicator strains of the species Lact. brevis, Pediococcus damnosus and Leucanostoc oenos. The antimicrobial activities from Lact. brevis B 37 and Lact. casei B 80 were caused by polypeptides detectable in the culture liquids. They are bacteriocins with a narrow antimicrobial spectrum. Brevicin 37 from Lact. brevis B 37 was active against many lactic acid bacteria and Nocardia corallina, whereas caseicin 80 from Lact. casei B 80 inhibits only one other strain of Lact. casei. Brevicin 37 is stable at 121dC, caseicin 80 is inactivated above 60dC, …

Regulation of the acetaldehyde concentration in culture medium during the fermentation of glucose by Saccharomyces cerevisiae

Die Konzentration an Acetaldehyd im Medium wahrend der anaeroben Vergarung von Glucose durch Saccharomyces cerevisiae weist in der logarithmischen Wachstumsphase die hochsten Werte auf. Die Induktion der Pyruvatdecarboxylase durch Glucose fordert die Akkumulation von Acetaldehyd, der ins Medium diffundiert. Hefestamme, die unterschiedlich viel Acetaldehyd bilden, unterscheiden sich in ihren Pyruvatdecarboxylaseaktivitaten. Diese engen Beziehungen zwischen Pyruvatdecarboxylase und Acetaldehydproduktion deuten auf die Kontrollfunktion der Pyruvatdecarboxylase bei der Acetaldehydakkumulation hin. Hohere Aktivitaten der Alkoholdehydrogenase verringern die Acetaldehydakkumulation, wodurch sich e…

�pfels�urestoffwechsel bei Saccharomyces

1. Aus Saccharomyces cerevisiae St. 79 konnte durch Protamin-und Ammoniumsulfatfallung sowie durch Chromatographie an DEAE-Cellulose ein Malatenzym [l-Malat: NAD(P) Oxidoreduktase, decarboxylierend, E.C. 1.1.1.38 oder 40] angereichert und von Malat-Dehydrogenase (l-Malat: NAD Oxidoreduktase, E.C. 1.1.1.37) weitgehend abgetrennt werden. 2. Neben Mn++-Ionen benotigt das Malatenzym der Hefe NAD oder NADP, bei einem optimalen pH-Wert von 7,5. Es ist spezifisch fur l-Malat, d-Malat wird nicht umgesetzt. Die Enzympraparate decarboxylierten Oxalessigsaure bei Abwesenheit von NAD. 3. Die Km Werte von Malatenzym sind fur l-Malat 5 · 10-2 M, fur NAD 5 · 10-4 M und fur Mangan 1,4 · 10-4 M. 4. Ein Zusa…

Killer toxins in new isolates of the yeasts Hanseniaspora uvarum and Pichia kluyveri

From various habitats (plant material, fruits, soil), yeasts belonging to the species of Pichia kluyveri and Hanseniaspora uvarum were isolated that showed killer activity. According to the activity spectrum against other yeasts these strains belonged to 11 different groups that were distinguishable from the killer strains K1-K10. The isoelectric points of the killer proteins were in the range of pH 3.5–3.9, the activity optimum was observed at pH 4.2–4.6. Above pH 5 and above a temperature of 25–35°C the killer proteins were inactivated.

Acetaldehyd als Indicator f�r die Regulation von Atmung und G�rung bei der aeroben Verg�rung von Glucose durch Saccharomyces cerevisiae

Wahrend der aeroben Vergarung von Glucose wurde die Konzentration von Acetaldehyd im Garmedium uber den gesamten Garablauf bei mehreren Stammen von Saccharomyces cerevisiae verfolgt. Die Aldehydkonzentration weist bei Glucosekonzentrationen zwischen 5 und 20% zwei Maxima auf. Damit ist der Konzentrationsverlauf von Acetaldehyd aerob wesentlich anders als bei der anaeroben Garung, mit nur einem meist niedrigen Maximum. 10-3 M Azid hemmt die Bildung von Acetaldehyd ganz oder weitgehend. Das deutet auf die Funktion bzw. Synthese der Cytochrome, die in Gegenwart von Sauerstoff offensichtlich auch bei hohen Glucosekonzentrationen nicht vollstandig reprimiert werden. Der durch die Atmung bedingte…

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28.

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannos…

Caseicin 80: purification and characterization of a new bacteriocin from Lactobacillus casei

When grown in complex or synthetic media, Lactobacillus casei B 80 synthesizes a mitomycin C-inducible polypeptide with very specific bactericidal activity against the sensitive strain Lactobacillus casei B 109. The amount of secreted bacteriocin in the culture solution was low, about 1 mg/l. The bacteriocin which we called caseicin 80, was also detectable in cell extracts, although only 2% of the total activity was retained intracellularly. Caseicin 80 was concentrated by ultrafiltration and purified by cation exchange chromatography with Cellulose SE-23 and Superose. The molecular weight was in the range of M r=40,000–42,000 and the isoelectric point was pH 4.5.

2-Oxoglutarate decarboxylase ofLeuconostoc oenos

InLeuconostoc oenos, the typical organism of the malolactic fermentation of wine, a 2-oxoglutarate decarboxylase was detected. This inducible enzyme decarboxylates 2-oxoglutarate but not pyruvate. The resulting succinaldehydate is rapidly reduced to 4-hydroxybutyrate or oxidized to succinate in further reactions. 2-Oxoglutarate decarboxylase is thiamin-diphosphate-dependent; the pH optimum is at 5.3 and theK m value for 2-oxoglutarate is 1.8 mmol/L.