Peptide antibiotic production through immobilized biocatalyst technology

The use of immobilized biocatalysts for producing known or new antibiotics is presented. An evaluation of the applicability of this concept in the fascinating field of peptide antibiotic bioconversions and fermentations is also given.The use of immobilized enzymes, organelles and cells to synthesize antibiotics as an alternative method to conventional fermentation is discussed. In vitro total enzymatic antibiotic synthesis is illustrated with the ‘multienzyme thiotemplate mechanism’ of Bacillus brevis, the producer of gramicidin S. Total synthesis of peptide antibiotics, based on immobilized living cells, has recently been demonstrated with penicillin, bacitracin, nisin and a few other antibiotics.As an industrial example of the use of enzymes or cells to convert peptide antibiotics into therapeutically useful derivatives, free and immobilized penicillin acylases, producing the penicillin nucleus 6-aminopenicillanic acid (6-APA), are reviewed as well as their potential to synthesize semisynthetic β-lactams (penicillins, cephalosporins).Acylases, acetylesterases and α-amino acid ester hydrolases acting on cephalosporin-compounds and yielding valuable intermediary or end products have also gained wide interest. Stereospecific enzymic side-chain preparations for semisynthetic penicillin and cephalosporin production have recently reached the industrial stage. Bioconversion possibilities with the novel β-lactam compounds are suggested.These examples of simple single-step, as well as complex multi-step, enzyme reactions point to the vast potential of immobilized biocatalyst technology in fermentation science, in organic synthesis and in biotechnological processes in general.     

Industrial relevance of thermophilic Archaea

The dramatic increase of newly isolated extremophilic microorganisms, analysis of their genomes and investigations of their enzymes by academic and industrial laboratories demonstrate the great potential of extremophiles in industrial (white) biotechnology. Enzymes derived from extremophiles (extremozymes) are superior to the traditional catalysts because they can perform industrial processes even under harsh conditions, under which conventional proteins are completely denatured. In particular, enzymes from thermophilic and hyperthermophilic Archaea have industrial relevance. Despite intensive investigations, our knowledge of the structure-function relationships of their enzymes is still limited. Information concerning the molecular properties of their enzymes and genes has to be obtained to be able to understand the mechanisms that are responsible for catalytic activity and stability at the boiling point of water.     

Biotechnology of non-Saccharomyces yeasts—the ascomycetes

Saccharomyces cerevisiae and several other yeast species are among the most important groups of biotechnological organisms. S. cerevisiae and closely related ascomycetous yeasts are the major producer of biotechnology products worldwide, exceeding other groups of industrial microorganisms in productivity and economic revenues. Traditional industrial attributes of the S. cerevisiae group include their primary roles in food fermentations such as beers, cider, wines, sake, distilled spirits, bakery products, cheese, sausages, and other fermented foods. Other long-standing industrial processes involving S. cerevisae yeasts are production of fuel ethanol, single-cell protein (SCP), feeds and fodder, industrial enzymes, and small molecular weight metabolites. More recently, non-Saccharomyces yeasts (non-conventional yeasts) have been utilized as industrial organisms for a variety of biotechnological roles. Non-Saccharomyces yeasts are increasingly being used as hosts for expression of proteins, biocatalysts and multi-enzyme pathways for the synthesis of fine chemicals and small molecular weight compounds of medicinal and nutritional importance. Non-Saccharomyces yeasts also have important roles in agriculture as agents of biocontrol, bioremediation, and as indicators of environmental quality. Several of these products and processes have reached commercial utility, while others are in advanced development. The objective of this mini-review is to describe processes currently used by industry and those in developmental stages and close to commercialization primarily from non-Saccharomyces yeasts with an emphasis on new opportunities. The utility of S. cerevisiae in heterologous production of selected products is also described.     

An alternative, banana peel-based medium used to investigate the catalytic properties of peroxidase from a fungus, Inonotus sp SP2, recently isolated in southern Chile

Recycling industrial wastes is one of the major goals of bioengineering research. Agricultural wastes are often rich in natural sources of organic and inorganic compounds. The present study investigates the use of banana peel waste as a non-conventional alternative to nitrogen-enriched glucose media for a white rot fungus (WRF), Inonotus sp. SP2, recently isolated in southern Chile. WRF are known to produce biodegrading enzymes, such as peroxidases, that can have industrial and biotechnological applications. To that end, the metabolic characteristics and catalytic properties of peroxidases produced by Inonotus sp. SP2 were compared between glucose and banana peel-based growth mediums. The results establish that this strain of WRF produces high concentrations of a Mn⁺²-dependent peroxidase, with greater enzymatic activity in extracellular fluid and crude enzyme extracted from fungus grown in banana peel and glucose media, respectively. H₂O₂ has an inhibiting effect that is greater for enzymes produced in glucose media, and greater biomass can be obtained in banana-peel based media. This demonstrates that banana peel is a suitable and more cost-effective alternative to conventional glucose-based media for the production of biodegradative enzymes, such as peroxidase. Unlike other strains of WRF, the metabolic characteristics of Inonotus sp. SP2 demonstrate that it enters secondary metabolism with the production oxidative enzymes after both carbon and nitrogen sources are depleted. This suggests that with further investigation, this strain of WRF may be useful in industrial applications that require the biodegradation of nitrogen and carbon-based wastes and recalcitrant compounds.     

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.     

Microbial transglutaminase and its application in the food industry. A review

The extremely high costs of manufacturing transglutaminase from animal origin (EC 2.3.2.13) have prompted scientists to search for new sources of this enzyme. Interdisciplinary efforts have been aimed at producing enzymes synthesised by microorganisms which may have a wider scope of use. Transglutaminase is an enzyme that catalyses the formation of isopeptide bonds between proteins. Its cross-linking property is widely used in various processes: to manufacture cheese and other dairy products, in meat processing, to produce edible films and to manufacture bakery products. Transglutaminase has considerable potential to improve the firmness, viscosity, elasticity and water-binding capacity of food products. In 1989, microbial transglutaminase was isolated from Streptoverticillium sp. Its characterisation indicated that this isoform could be extremely useful as a biotechnological tool in the food industry. Currently, enzymatic preparations are used in almost all industrial branches because of their wide variety and low costs associated with their biotechnical production processes. This paper presents an overview of the literature addressing the characteristics and applications of transglutaminase.     

Characterization of a thermotolerant laccase produced by Streptomyces sp. SB086

Laccases have become desirable enzymes for application in many industrial processes. Nowadays, most of these enzymes are obtained from fungi. Among prospective studies for bacterial laccase genes, some have included actinomycetes, but only a few studies have characterized the enzyme produced. Thus, we have isolated a laccase-producing actinomycete from forest soil under restoration process and further aimed to characterize its produced enzyme. The isolate SB086 was assigned to the Streptomyces genus by a combination of phenotypical, chemical and phylogenetic properties. Our data indicate that the bacterium produces a thermotolerant laccase. The maximum activity was obtained in the pH range 4.0–5.0 and at 50 °C in reaction mixture containing 5 mM CuSO₄; thermal stability was noted at 60 °C and 70 °C—a well-desired characteristic for industry. The active enzyme presented a high molecular mass (over 100 kDa) and was less sensitive to inhibition by metal ions than generally described for bacterial laccases. Our findings support in silico data of bacterial laccase secretion, and reinforce the view that actinomycetes may be a rich source of laccase for industrial application.     

Roles for glutathione transferases in plant secondary metabolism

Plant glutathione transferases (GSTs) are classified as enzymes of secondary metabolism, but while their roles in catalysing the conjugation and detoxification of herbicides are well known, their endogenous functions are largely obscure. Thus, while the presence of GST-derived S-glutathionylated xenobiotics have been described in many plants, there is little direct evidence for the accumulation of similarly conjugated natural products, despite the presence of a complex and dichotomous metabolic pathway which processes these reaction products. The conservation in glutathione conjugating and processing pathways, the co-regulation of GSTs with inducible plant secondary metabolism and biochemical studies showing the potential of these enzymes to conjugate reactive natural products are all suggestive of important endogenous functions. As a framework for addressing these enigmatic functions we postulate that either: (a) the natural reaction products of GSTs are unstable and undergo reversible S-glutathionylation; (b) the conjugation products of GSTs are very rapidly processed to derived metabolites; (c) GSTs do not catalyse conventional conjugation reactions but instead use glutathione as a cofactor rather than co-substrate; or (d) GSTs are non-catalytic and function as transporter proteins for secondary metabolites and their unstable intermediates. In this review, we describe how enzyme biochemistry and informatics are providing clues as to GST function allowing for the critical evaluation of each of these hypotheses. We also present evidence for the involvement of GSTs in the synthesis of sulfur-containing secondary metabolites such as volatiles and glucosinolates, and the conjugation, transport and storage of reactive oxylipins, phenolics and flavonoids.     

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.     

Integrated butanol recovery for an advanced biofuel: current state and prospects

Butanol has recently gained increasing interest due to escalating prices in petroleum fuels and concerns on the energy crisis. However, the butanol production cost with conventional acetone–butanol–ethanol fermentation by Clostridium spp. was higher than that of petrochemical processes due to the low butanol titer, yield, and productivity in bioprocesses. In particular, a low butanol titer usually leads to an extremely high recovery cost. Conventional biobutanol recovery by distillation is an energy-intensive process, which has largely restricted the economic production of biobutanol. This article thus reviews the latest studies on butanol recovery techniques including gas stripping, liquid–liquid extraction, adsorption, and membrane-based techniques, which can be used for in situ recovery of inhibitory products to enhance butanol production. The productivity of the fermentation system is improved efficiently using the in situ recovery technology; however, the recovered butanol titer remains low due to the limitations from each one of these recovery technologies, especially when the feed butanol concentration is lower than 1 % (w/v). Therefore, several innovative multi-stage hybrid processes have been proposed and are discussed in this review. These hybrid processes including two-stage gas stripping and multi-stage pervaporation have high butanol selectivity, considerably higher energy and production efficiency, and should outperform the conventional processes using single separation step or method. The development of these new integrated processes will give a momentum for the sustainable production of industrial biobutanol.     

Biotechnological potential of pectinolytic complexes of fungi

Plant cell wall-degrading enzymes, such as cellulases, hemicellulases and pectinases, have been extensively studied because of their well documented biotechnological potential, mainly in the food industry. In particular, lytic enzymes from filamentous fungi have been the subject of a vast number of studies due both to their advantages as models for enzyme production and their characteristics. The demand for such enzymes is rapidly increasing, as are the efforts to improve their production and to implement their use in several industrial processes, with the goal of making them more efficient and environment-friendly. The present review focuses mainly on pectinolytic enzymes of filamentous fungi, which are responsible for degradation of pectin, one of the major components of the plant cell wall. Also discussed are the past and current strategies for the production of cell wall-degrading enzymes and their present applications in a number of biotechnological areas.     

Sirtuins: not only animal proteins

Sirtuins are proteins belonging to the group of NADH-dependent deacetylase and mono-ADP-ribosyltransferase enzymes. Sirtuins have been discovered for the first time in yeasts, subsequent studies have shown their presence in bacteria, plants and animals. These enzymes are frequently called longevity enzymes due to the fact that they are part of genetic apparatus involved in aging control. In animals, sirtuins are key regulators of cell defense in response to stress caused by many metabolic processes; they are also involved in the regulation of cell division, metabolism, gene silencing and genetic material repair as well as apoptosis. Thus far, only several well-known research teams have been studying plant proteins resembling animal sirtuins. Considering the fact how essential functions sirtuins play in other organisms, it is extremely interesting to understand their role in plants, especially that the knowledge about them is still limited. It is believed that the function of sirtuins in Arabidopsis thaliana is associated with mitochondrial energy metabolism. Possibly they may also control the synthesis of auxins or proteins involved in their transport, or they may be responsible for regulating cellular response to auxin action. In rice, sirtuins are necessary for the protection against genomic instability and cell damage that guarantee their growth. They also take part in a defensive response against Pseudomonas syringae. They may also be involved in the ripening of fruits. Moreover, their functions are associated with photosynthetic activity and aging of leaves.     

Structural and mechanistic aspects of carotenoid cleavage dioxygenases (CCDs)

Carotenoid cleavage dioxygenases (CCDs) comprise a superfamily of mononuclear non-heme iron proteins that catalyze the oxygenolytic fission of alkene bonds in carotenoids to generate apocarotenoid products. Some of these enzymes exhibit additional activities such as carbon skeleton rearrangement and trans-cis isomerization. The group also includes a subfamily of enzymes that split the interphenyl alkene bond in molecules such as resveratrol and lignostilbene. CCDs are involved in numerous biological processes ranging from production of light-sensing chromophores to degradation of lignin derivatives in pulping waste sludge. These enzymes exhibit unique features that distinguish them from other families of non-heme iron enzymes. The distinctive properties and biological importance of CCDs have stimulated interest in their modes of catalysis. Recent structural, spectroscopic, and computational studies have helped clarify mechanistic aspects of CCD catalysis. Here, we review these findings emphasizing common and unique properties of CCDs that enable their variable substrate specificity and regioselectivity.This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Rio Tinto as a niche for acidophilus enzymes of industrial relevance

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.     

微生物嗜盐酶盐适应性的分子结构基础研究

由高盐环境中生长的微生物里分离出的嗜盐酶在高盐度下仍然具有催化活性,工业上具有良好的应用前景。一些嗜盐酶已被克隆纯化出来,它们的分子结构特点也已经被广泛研究。该文从嗜盐酶的蛋白质序列和结构特征等方面综述了嗜盐酶嗜盐的分子结构基础研究进展,分析了存在的问题并对未来工作提出了展望。研究嗜盐酶盐适应性的分子基础,可以为新的功能蛋白的发展和鉴定提供依据。     

Extremophiles as a source of novel enzymes for industrial application

Extremophilic microorganisms are adapted to survive in ecological niches such as at high temperatures, extremes of pH, high salt concentrations and high pressure. These microorganisms produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. Some of the enzymes from extremophiles have already been purified and their genes successfully cloned in mesophilic hosts. In this review we will briefly discuss the biotechnological significance of extreme thermophilic (optimal growth 70–80 °C) and hyperthermophilic (optimal growth 85–100 °C) archaea and bacteria. In particular, we will focus on selected extracellular-polymer-degrading enzymes, such as amylases, pullulanases, cyclodextrin glycosyltransferases, cellulases, xylanases, chitinases, proteinases and other enzymes such as esterases, glucose isomerases, alcohol dehydrogenases and DNA-modifying enzymes with potential use in food, chemical and pharmaceutical industries and in environmental biotechnology. Received: 14 August 1998 / Received revision: 17 February 1999 / Accepted: 19 February 1999     

Protein engineering of bacterial alpha-amylases

alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas.     

Microbial Diversity in Natural Asphalts of the Rancho La Brea Tar Pits

Bacteria commonly inhabit subsurface oil reservoirs, but almost nothing is known yet about microorganisms that live in naturally occurring terrestrial oil seeps and natural asphalts that are comprised of highly recalcitrant petroleum hydrocarbons. Here we report the first survey of microbial diversity in ca. 28,000-year-old samples of natural asphalts from the Rancho La Brea Tar Pits in Los Angeles, CA. Microbiological studies included analyses of 16S rRNA gene sequences and DNA encoding aromatic ring-hydroxylating dioxygenases from two tar pits differing in chemical composition. Our results revealed a wide range of phylogenetic groups within the Archaea and Bacteria domains, in which individual taxonomic clusters were comprised of sets of closely related species within novel genera and families. Fluorescent staining of asphalt-soil particles using phylogenetic probes for Archaea, Bacteria, and Pseudomonas showed coexistence of mixed microbial communities at high cell densities. Genes encoding dioxygenases included three novel clusters of enzymes. The discovery of life in the tar pits provides an avenue for further studies of the evolution of enzymes and catabolic pathways for bacteria that have been exposed to complex hydrocarbons for millennia. These bacteria also should have application for industrial microbiology and bioremediation.     

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.