Bacterial tyrosinases

Tyrosinases are nearly ubiquitously distributed in all domains of life. They are essential for pigmentation and are important factors in wound healing and primary immune response. Their active site is characterized by a pair of antiferromagnetically coupled copper ions, CuA and CuB, which are coordinated by six histidine residues. Such a "type 3 copper centre" is the common feature of tyrosinases, catecholoxidases and haemocycanins. It is also one of several other copper types found in the multi-copper oxidases (ascorbate oxidase, laccase). The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of o-diphenols to o-diquinones (catecholase activity). The best-known function is the formation of melanins from L-tyrosine via L-dihydroxyphenylalanine (L-dopa). The complicated hydroxylation mechanism at the active centre is still not completely understood, because nothing is known about their tertiary structure. One main reason for this deficit is that hitherto tyrosinases from eukaryotic sources could not be isolated in sufficient quantities and purities for detailed structural studies. This is not the case for prokaryotic tyrosinases from different Streptomyces species, having been intensively characterized genetically and spectroscopically for decades. The Streptomyces tyrosinases are non-modified monomeric proteins with a low molecular mass of ca. 30kDa. They are secreted to the surrounding medium, where they are involved in extracellular melanin production. In the species Streptomyces, the tyrosinase gene is part of the melC operon. Next to the tyrosinase gene (melC2), this operon contains an additional ORF called melC1, which is essential for the correct expression of the enzyme. This review summarizes the present knowledge of bacterial tyrosinases, which are promising models in order to get more insights in structure, enzymatic reactions and functions of "type 3 copper" proteins in general.     

Bacterial pigments and their applications

Natural pigments sourced from ores, insects, plants and animals were the colorants used since prehistoric period. Synthetic dyes which took the place of natural pigments in the middle of 19th century still rule the field to the maximum extent in spite of its hazardous effect to humans, animals and environment. As an alternative to synthetic pigments, bacterial pigments due to their better biodegradability and higher compatibility with the environment, offer promising avenues for various applications. The industry is now able to produce some bacterial pigments for applications in food, pharmaceuticals, cosmetics and textiles. Extraction of bacterial pigments in relatively pure and concentrated forms is the main technological challenge. Optimization of fermentation process and the medium components are reported as key strategies for economic recovery of pigments. Research work needs to be carried out to formulate the fermentation media for each bacterial pigment on large scale by using economical and easily available sources for commercial process. Recent advances in synthetic biology, metabolic engineering efforts of bacteria will greatly expand the pigments that could be produced economically in sufficient amounts for industrial application. This review summarizes the current technology status and challenges, economics, novel strategies for production of bacterial pigments and metabolic engineering of bacteria with a focus on applications of bacterial pigments in food industry, pharmaceutical industry, dyeing as well as on other applications.     

Bacterial growth laws and their applications

Quantitative empirical relationships between cell composition and growth rate played an important role in the early days of microbiology. Gradually, the focus of the field began to shift from growth physiology to the ever more elaborate molecular mechanisms of regulation employed by the organisms. Advances in systems biology and biotechnology have renewed interest in the physiology of the cell as a whole. Furthermore, gene expression is known to be intimately coupled to the growth state of the cell. Here, we review recent efforts in characterizing such couplings, particularly the quantitative phenomenological approaches exploiting bacterial 'growth laws.' These approaches point toward underlying design principles that can guide the predictive manipulation of cell behavior in the absence of molecular details.     

Bacterial tyrosinases: old enzymes with new relevance to biotechnology

Tyrosinases are copper-containing dioxygen activating enzymes found in many species of bacteria and are usually associated with melanin production. These proteins have a strong preference for phenolic and diphenolic substrates and are somewhat limited in their reaction scope, always producing an activated quinone as product. Despite this fact they have potential in several biotechnological applications, including the production of novel mixed melanins, protein cross-linking, phenolic biosensors, production of l-DOPA, phenol and dye removal and biocatalysis. Although most studies have used Streptomyces sp. enzymes, there are several other examples of these proteins that are also of potential interest. For instance a solvent tolerant enzyme has been described, as well as an enzyme with both tyrosinase and laccase activities, enzymes with altered substrate preferences, an enzyme produced as an inactive zymogen as well as examples which do not require auxiliary proteins for copper insertion (unlike the Streptomyces sp. enzymes which do require such a protein). This article will summarise the reports on the biotechnological applications of bacterial tyrosinases as well as the current information available on the different types of this enzyme.     

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications

Tyrosinases are type-3 copper proteins involved in the initial step of melanin synthesis. These enzymes catalyse both the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphenols into reactive o-quinones, which evolve spontaneously to produce intermediates, which associate in dark brown pigments. In fungi, tyrosinases are generally associated with the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation. First characterized from the edible mushroom Agaricus bisporus because of undesirable enzymatic browning problems during postharvest storage, tyrosinases were found, more recently, in several other fungi with relevant insights into molecular and genetic characteristics and into reaction mechanisms, highlighting their very promising properties for biotechnological applications. The limit of these applications remains in the fact that native fungal tyrosinases are generally intracellular and produced in low quantity. This review compiles the recent data on biochemical and molecular properties of fungal tyrosinases, underlining their importance in the biotechnological use of these enzymes. Next, their most promising applications in food, pharmaceutical and environmental fields are presented and the bioengineering approaches used for the development of tyrosinase-overproducing fungal strains are discussed.     

Purification of hamster melanoma tyrosinases and structural studies of their asparagine-linked sugar chains

In cultured melanotic melanoma, a marked decrease of pigmentation has been found to be induced by the addition of tunicamycin [Y. Mishima and G. Imokawa (1983) J. Invest. Dermatol. 81, 106-114]. Since it appears that this impaired pigmentation arises from the loss of asparagine-linked sugar chains serving as a signal for transport of tyrosinase from GERL (Golgi-associated endoplasmic reticulum of lysosomes) to premelanosomes, tyrosinases from the membrane fraction of Greene's hamster melanoma have been purified, and the structures of their sugar chains have been analyzed. Two kinds of tyrosinases were purified by Triton X-100 solubilization; DEAE-cellulose, Sephadex G-200, and DEAE-Sephadex column chromatography; and preparative polyacrylamide gel electrophoresis. The two tyrosinases were separated by polyacrylamide gel electrophoresis, and both corresponded to Mr 69,000. Their asparagine-linked sugar chains were released by hydrazinolysis and analyzed. The sugar chains of the two tyrosinases were identical except for the sialic acid contents. One mole of each tyrosinase contained 1 mol of high-mannose-type sugar chains and 3 mol of complex-type sugar chains. The former chain has Man3 approximately 5 X GlcNAc2 and the latter has Man3 X GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their core structures. The complex-type sugar chains are composed of mono-, bi-, tri-, and tetraantennary sugar chains, with +/- Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----as their outer chains.     

Bacterial alginates: biosynthesis and applications

Alginate is a copolymer of β-d-mannuronic acid and α-l-guluronic acid (GulA), linked together by 1–4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers. Received: 14 March 1997 / Received revision: 7 May 1997 / Accepted: 11 May 1997     

Bacterial toxins and their application

The review deals with the structure of protein bacterial toxins, steps of the toxin molecule interaction with the target cell, molecular mechanisms of the toxic effect, as well as with the fields of application of toxins as research tools and as medicinal preparations.     

Bacterial quorum sensing: circuits and applications

Bacterial quorum sensing (QS) systems are cell density—dependent regulatory networks that coordinate bacterial behavioural changes from single cellular organisms at low cell densities to multicellular types when their population density reaches a threshold level. At this stage, bacteria produce and perceive small diffusible signal molecules, termed autoinducers in order to mediate gene expression. This often results in phenotypic shifts, like planktonic to biofilm or non-virulent to virulent. In this way, they regulate varied physiological processes by adjusting gene expression in concert with their population size. In this review we give a synopsis of QS mediated cell–cell communication in bacteria. The first part focuses on QS circuits of some Gram-negative and Gram-positive bacteria. Thereafter, attention is drawn on the recent applications of QS in development of synthetic biology modules, for studying the principles of pattern formation, engineering bi-directional communication system and building artificial communication networks. Further, the role of QS in solving the problem of biofouling is also discussed.     

Glycosylation and adhesiveness differentiate larval Ceratitis capitata tyrosinases

Larval Ceratitis capitata phenoloxidases (POs) from hemocytes, serum, integument, and fat body were analyzed. Two types of PO were recorded: the tyrosinase type found in hemocytes, serum, integument, and fat body and the laccase type found in integument. Tyrosinase from all larval tissues and integumental laccase as well, showed similarity in molecular weight (93 KDa), activation by Escherichia coli at 5 mM Ca²⁺, and reactivity to antibodies raised against serum tyrosinase. However, the enzymes differed with respect to their glycosylation and adhesiveness. The serum and integumental enzyme forms contain concanavalin A reacting material, whereas hemocyte and integumental tyrosinase(s) are adhesive. These differences in enzyme forms, although not influencing their substrate specificity, seem to give advantages to performing their function, i.e., the adhesive enzyme form facilitates the adherence to E. coli cell wall and hemocyte surface (unpublished data) while the glycosylated form facilitated the secretion into serum. © 1994 Wiley-Liss, Inc.     

Polypeptide composition of two fungal tyrosinases

The presence of two distinct molecular structures for tyrosinase in fungi is confirmed. The enzyme from Agaricus bisporus is acidic and comprises two dissimilar subunits which aggregate to form a tetramer. This tetramer constitutes the majority both in the resting and functional states. In Neurospora crassa, tyrosinase is slightly basic and contains only one subunit, similar in size to the larger subunit of the Agaricus enzyme. In the resting state Neurospora tyrosinase is distributed among a number of forms, from the monomer to the tetramer. In this case it was possible to show that a species smaller than the tetramer, probably the monomer, was fully active.     

Comparison of the characteristics of fungal and plant tyrosinases

Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, -casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic l-tyrosine and diphenolic l-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein -casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink -casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink -casein. In the reaction conditions studied, TrT showed the best ability to crosslink -casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.     

细菌全基因组关联研究的方法与应用

随着测序技术的发展和全基因组序列的不断积累,全基因组关联研究(genome-wide association study, GWAS)在人类复杂疾病研究中取得了丰硕成果,10余年间发现了数以万计的疾病风险因子。同样,GWAS也为探索细菌表型的遗传机制提供了新的工具。自2013年第一项细菌GWAS(bacterial GWAS, BGWAS)工作发表以来,目前已有10多项相关研究报道,分别揭示了细菌宿主适应性、耐药性及毒力等表型的遗传机制,极大加深了人们对细菌遗传、进化及传播等方面的认识。本文对目前BGWAS的研究方法、应用成果及存在的问题进行了总结,并对BGWAS的研究前景进行了展望,旨在为微生物学领域开展BGWAS研究提供参考。     

Bacterial volatiles and their action potential

During the past few years, an increasing awareness concerning the emission of an unexpected high number of bacterial volatiles has been registered. Humans sense, intensively and continuously, microbial volatiles that are released during food transformation and fermentation, e.g., the aroma of wine and cheese. Recent investigations have clearly demonstrated that bacteria also employ their volatiles during interactions with other organisms in order to influence populations and communities. This review summarizes the presently known bioactive compounds and lists the wide panoply of effects possessed by organisms such as fungi, plants, animals, and bacteria. Because bacteria often emit highly complex volatile mixtures, the determination of biologically relevant volatiles remains in its infancy. Part of the future goal is to unravel the structure of these volatiles and their biosynthesis. Nevertheless, bacterial volatiles represent a source for new natural compounds that are interesting for man, since they can be used, for example, to improve human health or to increase the productivity of agricultural products.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

Bacterial endophytes: recent developments and applications.

Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes appear to originate from the rhizosphere or phyllosphere; however, some may be transmitted through the seed. Endophytic bacteria can promote plant growth and yield and can act as biocontrol agents. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. There is increasing interest in developing the potential biotechnological applications of endophytes for improving phytoremediation and the sustainable production of nonfood crops for biomass and biofuel production.