Genes and derived amino acid sequences of S-layer proteins from mesophilic, thermophilic, and extremely thermophilic methanococci

Cells of methanococci are covered by a single layer of protein subunits (S-layer) in hexagonal arrangement, which are directly exposed to the environment and which cannot be stabilized by cellular components. We have isolated S-layer proteins from cells of Methanococcus vannielii ( T(opt.)=37 degrees C), Methanococcus thermolithotrophicus ( T(opt.)=65 degrees C), and Methanococcus jannaschii ( T(opt.)=85 degrees C). The primary structure of the S-layer proteins was determined by sequencing the corresponding genes. According to the predicted amino acid sequence, the molecular masses of the S-layer proteins of the different methanococci are in a small range between 59,064 and 60,547 Da. Compa…

Wine Fermentation

Currently wineries are facing new challenges due to actual market demands for creation of products exhibiting more individual flavors[...]

Cell Wall Structures of Mesophilic, Thermophilic and Hyperthermophilic Archaea

How to Deal with Uninvited Guests in Wine: Copper and Copper-containing Oxidases

Copper is one of the most frequently occurring heavy metals in must and wine. It is introduced by pesticides, brass fittings, and as copper sulphate for treatment of reductive off-flavors. At higher concentrations, copper has harmful effects on the wine. It contributes to the oxidation of wine ingredients, browning reactions, cloudiness, inhibition of microorganisms, and wine fermentation. Last but not least, there is also a danger to the consumer. At present, some physicochemical methods exist to reduce the copper content in must and wine, but they all have their shortcomings. A possible solution is the biosorption of metals by yeasts or lactobacilli. Copper can also reach must and wine in…

Proteinaceous Surface Layers ofArchaea: Ultrastructure and Biochemistry

The cell walls of the Archaea are composed of different polymers such as glutaminylglycan, heterosaccharide, methanochondroitin, pseudomurein, protein, glycoprotein, or glycocalyx. The S-layer glycoprotein of Halobacterium salinarum was the first glycoprotein discovered in bacteria and archaea. Initially, the novel cell wall structures were viewed as curiosities, and their taxonomic significance was not realized until the concept of the Archaea was published. At this time, the results of cell wall studies supported the new view of the phylogeny of the Bacteria and Archaea. Many archaea possess proteinaceous surface layers (S layers), which form two-dimensional regular arrays. The chemical s…

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains ofSaccharomyces cerevisiaeand interspecies hybrids

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the p…

Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany

Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no dis…

Yeast Mixtures and Saccharomyces Hybrids: Suitable Tools for Performing More Sophisticated Must Fermentations

It can be concluded from the early development of viniculture that the yeast Saccharomyces cerevisiae is one of the oldest domesticated organisms. The wine producing techniques have been further developed empirically over generations. Despite this long time of wine production, our knowledge about the microbiological and biochemical background of the transformation of must into wine is relatively recent. Fundamental investigations about must transformation into wine were carried out at the end of the eighteenth and in the course of the nineteenth century. Today a relatively broad knowledge exists about the diversity of wine-related microorganisms. Nevertheless, sluggish or stuck fermentation…

The Potential of the Yeast Debaryomyces hansenii H525 to Degrade Biogenic Amines in Food

Twenty-six yeasts from different genera were investigated for their ability to metabolize biogenic amines. About half of the yeast strains produced one or more different biogenic amines, but some strains of Debaryomyces hansenii and Yarrowia lipolytica were also able to degrade such compounds. The most effective strain D. hanseniii H525 metabolized a broad spectrum of biogenic amines by growing and resting cells. Degradation of biogenic amines by this yeast isolate could be attributed to a peroxisomal amine oxidase activity. Strain H525 may be useful as a starter culture to reduce biogenic amines in fermented food.

Antioxidant capacity of phenolic compounds on human cell lines as affected by grape-tyrosinase and Botrytis-laccase oxidation.

Phenolic components (PCs) are well-known for their positive impact on human health. In addition to their action as radical scavengers, they act as activators for the intrinsic cellular antioxidant system. Polyphenol oxidases (PPOs) such as tyrosinase and laccase catalyze the enzymatic oxidation of PCs and thus, can alter their scavenging and antioxidative capacity. In this study, oxidation by tryosinase was shown to increase the antioxidant capacity of many PCs, especially those that lack adjacent aromatic hydroxyl groups. In contrast, oxidation by laccase tended to decrease the antioxidant capacity of red wine and distinct PCs. This was clearly demonstrated for p-coumaric acid and resverat…

Molecular Methods for Identification of Wine Microorganisms and Yeast Development

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. Nevertheless, most of the environmental microorganisms are graded as “yet not cultivable” because the nutritional requirements are unknown or they could not be isolated due to the fact that fast-growing strains overgrow other microorganisms of a microbiota. In addition to plating techniques, isolation without cultivation and analysis of microbes could be performed by micromanipulation techniques or the application of optical tweezers followed by the utilization of PCR-based technologies.

Exoenzymes of Wine Microorganisms

The production of wine from grape juice is predominantly the result of enzymatic reactions. The enzymes originate from the grape itself, from epiphytic fungi like Botrytis cinerea colonizing the grape surface and finally from yeasts and bacteria growing in the must until termination of alcoholic fermentation. Especially nonSaccharomyces yeasts, also called “wild” yeasts, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora, Kluyveromyces, and Metschnikowia produce and secrete several enzymes (esterases, glycosidases, lipases, glucanases, proteases, cellulases, etc.) to the periplasmatic space and the medium where they may interact w…

Biosorption of copper by wine-relevant lactobacilli

Must and wine may be contaminated with elevated copper concentrations by the use of fungicides or in course of the vinification process. Hitherto only a few practicable and harmless procedures exist to reduce an excess of copper from must and wine. For this reason we investigated the biosorption of copper by eight wine-relevant Lactobacillus species. Both, living and heat-inactivated cells revealed a significant degree of Cu adsorption. It was shown that Cu binding correlated positively with an increasing pH value of the environment. The highest binding capacity of the tested lactic acid bacteria was found for L. buchneri DSM 20057 with a maximum of 46.17 μg Cu bound per mg cell in deionize…

Laccases of Botrytis cinerea

Phenolic compounds significantly affect the color, odor and taste of wine. Due to their presumptive beneficial impact on human health, polyphenols in red wine have gained increasing public and scientific interest. Wine phenols are extremely sensitive to oxygen and are easily converted to brownish oxidation products accompanied by loss of nutritive values of the wine. Enzymatic oxidation takes place under the influence of polyphenoloxidases including tyrosinases and laccases. The latter are produced by the phytopathogenic fungus Botrytis cinerea and enter the must with contaminated berries. Although uncontrolled action of Botrytis-laccase has a dramatic impact on wine quality, the oxidative …

Laccases: structure, reactions, distribution

Laccases (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that use molecular oxygen to oxidize various aromatic and non-aromatic compounds by a radical-catalyzed reaction mechanism. The enzymes are involved in the pathogenicity, immunity and morphogenesis of organisms and in the metabolic turnover of complex organic substances such as lignin or humic matter. Owing to their high non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. Until recently, laccases were only found in eukaryotes (fungi, higher plants, insects), but now there is strong evidence for their widespread distribution in prokaryotes and the fi…

An Exemplary Model Study for Overcoming Stuck Fermentation during Spontaneous Fermentation with the Aid of a Saccharomyces Triple Hybrid

Sluggish or stuck fermentations cause significant financial losses for winemakers each year. In order to investigate the reasons for problems during spontaneous fermentation of Riesling must in a well-known German vineyard of the lower Moselle, yeast strains involved in must fermentation were identified during winemaking in the two years 2011 and 2012. Identification of the yeast isolates was performed by applying analyses of the ITS-1-5.8-ITS2 region and restriction fragment analyses of different gene sequences. It revealed that Saccharomyces (S.) bayanus and not Saccharomyces cerevisiae was the main fermenting yeast. Either S. bayanus finished the fermentation or led to stuck fermentation…

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been prove…

Properties of Halococcus salifodinae, an Isolate from Permian Rock Salt Deposits, Compared with Halococci from Surface Waters

Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl), whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing …

Effect of carboxymethyl cellulose on tartrate salt, protein and colour stability of red wine

Background and Aims Recent studies have confirmed a long-term effect of carboxymethyl cellulose (CMC) for tartrate salt stabilisation in white wine. It has been argued that CMC is not only less effective in red wine but also interacts with proteins and polyphenols generating turbidity and change in colour. In order to explain these effects, we studied in detail the impact of CMC on haze formation and colour stability of red wine. Methods and Results The influence of CMC concentration was tested with ten samples of red wine produced from several grape cultivars. The haze-forming material was analysed by sodium dodecyl-sulfate polyacrylamide electrophoresis and the protein composition by high…

Copper-Containing Oxidases: Occurrence in Soil Microorganisms, Properties, and Applications

Copper is an essential trace element in living systems, where it is used as a cofactor in many enzymatic redox reactions and oxygen transport. The copper-containing oxidases tyrosinase and laccase are widely distributed in soil microorganisms. Recently, progress has been made with respect to structural and catalytic data for the tyrosinase of the soil bacterium Streptomyces. The formation of eumelanins—dark polymers with multiple functionality (e.g., UV protection, immune defense)—is a well-established task of tyrosinases. An abundance of information exists on the structures and biochemical properties of fungal laccases. Many studies have demonstrated a physiological role for them in the de…

Microbial Degradation of 2,4,6-Trinitrotoluene In Vitro and in Natural Environments

2,4,6-Trinitrotoluene (TNT) is a nitroaromatic explosive that was released into soil and water ecosystems mainly due to its massive use during the two world wars.

Studies on Two Exoenzymes Which Lyse Wine-Spoiling Bacteria

Microorganisms play an important role in the conversion of grape juice into wine. Different species of yeast are mainly responsible for the production of ethanol. Lactic acid bacteria also occur regularly in must or wine. They are mostly undesirable due to their capacity to produce wine-spoiling compounds. Especially strains of Lactobacillus brevis are able to produce biogenic amines as well as precursors of ethyl carbamate and different off-flavours (N-heterocycles, volatile phenols). By excessive formation of acetic acid some lactobacilli may even induce slow/stuck grape juice fermentations. In conventional winemaking, additions of sulphite or lysozyme are used to inhibit the growth of sp…

Solution Properties and Potential Biological Applications of Zwitterionic Poly(ε-N-methacryloyl-l-lysine)

Poly(e-N-methacryloyl-l-lysine) (PMALys) was synthesized by free radical polymerization yielding a zwitterionic polymer with Mw = 721 000 g mol–1. The polymer dissolves in pure water as well as in aqueous salt solution up to 5 M NaClO4 and over wide range of pH values (1.3 ≤ pH ≤ 12.7) as single chains without any sign for aggregate formation. The zwitterionic polymer shows an expanded random coil structure at and close to isoelectric conditions and further expands upon addition of acid and base, respectively. The polymer fulfills four major prerequisites for a promising nano carrier in potential biomedical applications: (1) It is biocompatible, indicated by a low cytotoxicity. (2) It does …

Microbial Enzymes: Relevance for Winemaking

Production of wine from grape juice is predominantly the result of complex enzymatic reactions. The primary bioconversion of grape sugar to ethanol and CO2 by the yeast Saccharomyces cerevisiae is catalyzed by cytosolic enzymes. In spontaneous must fermentations, also strains of Saccharomyces bayanus or interspecies hybrids may dominate, probably because of better adaptation to specific environmental conditions. In addition, various enzymes released into the must influence the final composition, color, and sensory properties of wines. These enzymes originate from the grape itself, from epiphytic fungi like Botrytis cinerea, and from yeasts and bacteria associated with vineyards and wine cel…

The yeastWickerhamomyces anomalusAS1 secretes a multifunctional exo-β-1,3-glucanase with implications for winemaking

A multifunctional exo-β-1,3-glucanase (WaExg2) was purified from the culture supernatant of the yeast Wickerhamomyces anomalus AS1. The enzyme was identified by mass spectroscopic analysis of tryptic peptide fragments and the encoding gene WaEXG2 was sequenced. The latter codes for a protein of 427 amino acids, beginning with a probable signal peptide (17 aa) for secretion. The mature protein has a molecular mass of 47 456 Da with a calculated pI of 4.84. The somewhat higher mass of the protein in SDS–PAGE might be due to bound carbohydrates. Presumptive disulphide bridges confer a high compactness to the molecule. This explains the apparent smaller molecular mass (35 kDa) of the native enz…

Cell Envelopes of Methanogens

Methanogens play an important role in the global carbon cycle, because they are involved in the last step of anaerobic degradation of organic material to methane. Although the first report on methane emanation from aquatic muds was given by Alessandro Volta in the year 1776, the first methanogen was not obtained in pure culture before 1947. Special culture techniques had to be developed for growing the strict anaerobic methanogenic isolates. The methanogens were the first species of the archaeal domain (Archaea) detected. Their unique biochemical and genetic properties have stimulated basic investigations of this microbial group in the last three decades. The methanogenic Archaea possess a …

Primary Structure of Selected Archaeal Mesophilic and Extremely Thermophilic Outer Surface Layer Proteins

The archaea are recognized as a separate third domain of life together with the bacteria and eucarya. The archaea include the methanogens, extreme halophiles, thermoplasmas, Sulfate reducers and Sulfur metabolizing thermophiles, which thrive in different habitats such as anaerobic niches, salt lakes, and marine hydrothermals systems and continental solfataras. Many of these habitats represent extreme environments in respect to temperature, osmotic pressure and pH-values and remind on the conditions of the early earth. The cell envelope structures were one of the first biochemical characteristics of archaea studied in detail. The most common archaeal cell envelope is composed of a single cry…

Molecular organization of selected prokaryotic S-layer proteins.

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-)protein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequen ces of hyperthermophilic archaeal species exhibit some characteristic signatures. Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sulf…