Transglutaminases: recent achievements and new sources

Transglutaminases are a family of enzymes (EC 2.3.2.13), widely distributed in various organs, tissues, and body fluids, that catalyze the formation of a covalent bond between a free amine group and the γ-carboxamide group of protein or peptide-bound glutamine. Besides forming these bonds, that exhibit high resistance to proteolytic degradation, transglutaminases also form extensively cross-linked, generally insoluble, protein biopolymers that are indispensable for the organism to create barriers and stable structures. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have also been explored, but in a much lesser extent. Our group has identified a transglutaminase in the oomycete Phytophthora cinnamomi, which is able to induct defense responses and disease-like symptoms. In this mini-review, we report the achievements in this area in order to illustrate the importance and the versatility of transglutaminases.     

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.     

Prospects for an HIV Vaccine: Conventional Approaches and DNA Immunization

Abstract

Microbial transglutaminase is an important enzyme in food processing for improving protein properties by catalyzing the cross-linking of proteins. Recently, this enzyme has been shown to exhibit wider potential application in tissue engineering, textiles and leather processing, site-specific protein conjugation and wheat gluten allergy reduction. The production of microbial transglutaminase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering during the last three decades. More recently, studies on the biological mechanism of transglutaminase synthesis have further contributed towards the understanding of microbial transglutaminase production by Streptomyces. This will further facilitate improving the production of recombinant microbial transglutaminase. In this paper, we will review the progress in bioprocess engineering and genetic engineering in microbial transglutaminase production. We will highlight our understanding of the biological mechanisms of microbial transglutaminase synthesis, including biotechnological approaches used based on these biological mechanisms as a way of improving transglutaminase production.We address in addition the future research needs for microbial transglutaminase production.     

Microbial transglutaminase: An overview of recent applications in food and packaging

Abstract

The glue of proteins, microbial transglutaminase (MTG) has been adopted in the food processing industries for its broad enzymatic action. Microorganisms such as Streptoverticillium and Streptomyces are the major sources, to decrease the cost of manufacturing animal origin transglutaminase. The net % increase of its demands in the food processing is estimated at 21.9% per year. In fact, MTG is consumed by most food industries, spanning the meat, dairy, seafood and fish, plant proteins, edible film preparation and more. It used to improve gelation and change foaming, emulsification, viscosity, consistency and water-holding capacity properties. This paper presents an overview of the literature that described and explored the recent microbial origins, production media and applications of microbial transglutaminase.     

Recent trends in industrial microbiology

The majority of current biotechnological applications are of microbial origin, and it is widely appreciated that the microbial world contains by far the greatest fraction of biodiversity in the biosphere. Because of their biotech impact, numerous efforts are being undertaken worldwide, with an ultimate goal to deliver new usable substances of microbial origin to the marketplace. However, the direct isolation of microbes always revealed that the majority are not amenable to be cultured and no representatives for many major microbial phyla have been thus far characterized. Therefore, the knowledge on new microbes and/or genomic information thereof, or from their communities, will pose an enormous potential to provide industry with novel products and processes based on the use of microbial resources, and contribute to and extend the basic mechanistic knowledge on the functioning of organisms. The present review highlights some examples and advances in the exploration of the genetic reservoir of (un)cultured microbes for industrial applications.     

Development of GRAS strains for nutraceutical production using systems and synthetic biology approaches: advances and prospects

Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.     

Microbial transglutaminase—a review of its production and application in food processing

Transglutaminase (EC 2.3.2.13) catalyses an acyl-transfer reaction in which the -carboxamide groups of peptide-bound glutaminyl residues are the acyl donors. The enzyme catalyses in vitro cross-linking in whey proteins, soya proteins, wheat proteins, beef myosin, casein and crude actomysin refined from mechanically deboned poultry meat. In recent years, on the basis of the enzyme's reaction to gelatinize various food proteins through the formation of cross-links, this enzyme has been used in attempts to improve the functional properties of foods. Up to now, commercial transglutaminase has been merely obtained from animal tissues. The complicated separation and purification procedure results in an extremely high price for the enzyme, which hampers a wide application in food processing. Recently studies on the production of transglutaminase by microorganisms have been started. The enzyme obtained from microbial fermentation has been applied in the treatment of food of different origins. Food treated with microbial transglutaminase appeared to have an improved flavour, appearance and texture. In addition, this enzyme can increase shelf-life and reduce allergenicity of certain foods. This paper gives an overview of the development of microbial transglutaminase production, including fermentation and down-stream processing, as well as examples of how to use this valuable enzyme in processing foods of meat, fish and plant origin.     

Properties and applications of microbial transglutaminase

Some properties and applications of the transglutaminase (TGase) referred to as microbial TGase (MTGase), derived from a variant of Streptomyces mobaraensis (formerly classified as Streptoverticillium mobaraense), are described. MTGase cross-linked most food proteins, such as caseins, soybean globulins, gluten, actin, myosins, and egg proteins, as efficiently as mammalian TGases by forming an -(-glutamyl)lysine bond. However, unlike many other TGases, MTGase is calcium-independent and has a relatively low molecular weight. Both of these properties are of advantage in industrial applications; a number of studies have illustrated the potential of MTGase in food processing and other areas. The crystal structure of MTGase has been solved. It provides basic structural information on the MTGase and accounts well for its characteristics. Moreover, an efficient method for producing extracellular MTGase has been established using Corynebacterium glutamicum. MTGase may be expected to find many uses in both food and non-food applications.     

Chitosan-whey protein edible films produced in the absence or presence of transglutaminase: analysis of their mechanical and barrier properties

Chitosan-whey protein edible films with different protein concentrations were prepared in the absence or presence of microbial transglutaminase as cross-linking agent. The films prepared in the presence of the enzyme showed low solubility at a wide range of pH, a lower degree of swelling, and good biodegradability following protease treatments. The presence of transglutaminase induced also an enhancement in film mechanical resistance and a reduction in their deformability. Finally, the barrier efficiency toward oxygen and carbon dioxide was found to be markedly improved in the cross-linked films which showed also a lower permeability to water vapor. Some potential practical applications of transglutaminase-treated chitosan-whey protein films are suggested.     

微生物谷氨酰胺转胺酶的表达及分子改造研究进展

微生物谷氨酰胺转胺酶具有催化蛋白质和某些非蛋白物质交联的功能,被广泛应用于食品、医药及纺织等领域.为提高该酶的产量及建立相应的分子改造平台,上世纪90年代日本味之素公司便开展了微生物谷氨酰胺转胺酶重组菌构建的研究.目前,该酶已在多个表达系统中实现活性表达,部分重组菌较野生菌的产酶能力有显著提高.近年来,谷氨酰胺转胺酶的分子改造研究也取得了初步进展,酶的催化活力、热稳定性及底物专一性得到提升.文中对上述研究中涉及的蛋白质表达及改造策略进行了简要的总结及分析,并指出相关研究的发展趋势.     

Transglutaminase-mediated macromolecular assembly: production of conjugates for food and pharmaceutical applications

Various strategies have been explored in the last 20 years to modify the functional properties of proteins and, among these, protein/polymer conjugation resulted one of the most successful approaches. Thus, the surface modification of polypeptides of potential industrial interest by covalent attachment of different macromolecules is nowadays regarded as an extremely valuable technique to manipulate protein activities. Protein derivatives with a number of either natural or synthetic polymers, like different polysaccharides or polyethylene glycol, have been obtained by both chemical and enzymatic treatments, and in this context, the crosslinking enzyme transglutaminase is attracting an increasing attention as a simple and safe means for protein processing in vitro. In this short review, we summarized the most significant experimental findings demonstrating that a microbial form of the enzyme is an effective tool to obtain several biopolymer-based conjugates potentially useful for both food and pharmaceutical applications.     

Isolation,screening, and optimization of bacterial strains for novel transglutaminase production

Transglutaminases are a class of transferases known to form isopeptide bond between glutamine and lysine residues in a protein molecule. Increasing demand for transglutaminase in food and other industries and its low productivity have compelled researchers to isolate and screen micro-organisms with potential to produce it. In the present investigation around 200 isolates were screened for extracellular secretion of microbial transglutaminase (MTGase). Isolate B4 showed enzyme activity of 1.71?±?0.2?U/mL followed by isolate C2 which showed 1.61?±?0.17?U/mL activity, comparable with the activity of industrially used microbial strains. Biochemical analysis along with 16S r-RNA sequencing revealed these isolates (B4 and C2) to be Bacillus nakamurai and a variant of Bacillus subtilis, respectively. Amongst the various production media screened, a medium containing starch and peptone was found best for MTGase production. Correlation between growth, enzyme production, and sugar utilization was also studied and maximum enzyme production was obtained after 48 to 60?hr. Highest MTGase titer (3.95?±?0.03?U/mL for B4 and 2.65?±?0.17?U/mL for C2) was obtained by optimization of parameters. The enzyme was characterized for temperature and pH optima, pH and thermal stability, and effect of metal ions, suggesting its potential use in future applications.     

Characterization of biosurfactants and their use in pollution removal – State of the Art. (Review)

Surface-active compound of biological origin (biosurfactants) have only been described in the past few decades. With the advantage of biodegradability and production on renewable resources, biosurfactants have been gaining prominence and their applications are becoming wider. So far, literature contains mixed reports on the successes of the applications of biosurfactants and their economical viability. They remain compounds which are not very well understood, yet, with several important applications. The target industries for biosurfactant use are the petroleum remediation industries and environmental conservation agencies. These industries, however, seem reluctant to use them for fear of dealing with microbes or microbial products. This includes cleaning up oil spills from the environment, remediation of metal-contaminated soils or waste streams, mobilizing heavy oil sludge and enhanced oil recovery. The importance of biosurfactants, their production, characteristics and limited successes and applications in oil pollution remediation and oil storage tank cleaning are discussed.     

Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC

Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC^L177Q) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial.     

药用级微生物谷氨酰胺转胺酶的纯化工艺研究

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高 选择性和高载量地捕获,再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。     

Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment

Aerobic granular sludge can be classified as a type of self-immobilized microbial consortium, consisting mainly of aerobic and facultative bacteria and is distinct from anaerobic granular methanogenic sludge. Aerobic granular technology has been proposed as a promising technology for wastewater treatment, but is not yet established as a large-scale application. Aerobic granules have been cultured mainly in sequenced batch reactors (SBR) under hydraulic selection pressure. The factors influencing aerobic granulation, granulation mechanisms, microbial communities and the potential applications for the treatment of various wastewaters have been studied comprehensively on the laboratory-scale. Aerobic granular sludge has shown a potential for nitrogen removal, but is less competitive for the high strength organic wastewater treatments. This technology has been developed from the laboratory-scale to pilot scale applications, but with limited and unpublished full-scale applications for municipal wastewater treatment. The future needs and limitations for aerobic granular technology are discussed.     

Aerobic granular sludge: characterization,mechanism of granulation and application to wastewater treatment

Aerobic granular sludge can be classified as a type of self-immobilized microbial consortium, consisting mainly of aerobic and facultative bacteria and is distinct from anaerobic granular methanogenic sludge. Aerobic granular technology has been proposed as a promising technology for wastewater treatment, but is not yet established as a large-scale application. Aerobic granules have been cultured mainly in sequenced batch reactors (SBR) under hydraulic selection pressure. The factors influencing aerobic granulation, granulation mechanisms, microbial communities and the potential applications for the treatment of various wastewaters have been studied comprehensively on the laboratory-scale. Aerobic granular sludge has shown a potential for nitrogen removal, but is less competitive for the high strength organic wastewater treatments. This technology has been developed from the laboratory-scale to pilot scale applications, but with limited and unpublished full-scale applications for municipal wastewater treatment. The future needs and limitations for aerobic granular technology are discussed.     

Use of methylumbeliferyl-derivative substrates for lipase activity characterization

Lipases and esterases have been recognized as very useful biocatalysts because of their wide-ranging versatility in industrial applications, their stability, low cost, and non-requirement for added cofactors. The physical properties of lipidic substrates, typically water insoluble, have determined a great difficulty in studying lipolytic enzymes. A method for fast and simple detection of lipolytic activity, based on the use of 4-methylumbelliferone (MUF)-derivative substrates was developed. The system has been used for the detection of lipase activity either from microbial colonies, cell culture suspensions, or from proteins separated on SDS-polyacrylamide or isoelectric focusing gels. The use of MUF-derivative substrates has also been extended to the quantitative determination of lipolytic activity from a variety of assays including optimum pH and temperature determination, growth dependency, kinetics or stability studies, or residual activity quantification after treatment with potential inhibitors. The method has shown to be a useful tool for the characterization of a variety of lipases from microbial origin, including those cloned in heterologous hosts.     

Modifications and applications of the <Emphasis Type="Italic">Acinetobacter venetianus</Emphasis> RAG-1 exopolysaccharide,the emulsan complex and its components

Since its discovery in the late 1970s, emulsan has been the subject of significant interest for fundamental biosynthesis and structure–function relationships as well as for its potential industrial applications. These studies initially examined the emulsification properties of the compound, while more recent efforts have focused on potential biomedical applications. As a result of this change of focus, it became necessary to more completely characterize the structure of the emulsan molecule and to develop a more reproducible purification process. We review previous studies with emulsan and explain how prior notions were recently shown to be incorrect through the development of a new purification process. More recent genetic modification of the relevant operon is also reviewed. Finally, the potential applications for the new purified polymer will be discussed.     

Enzymatic modification of self-assembled peptide structures with tissue transglutaminase

A de novo peptide that self-assembles into fibrillar structures and serves as a substrate for the cross-linking enzyme tissue transglutaminase was developed (Ac-QQKFQFQFEQQ-Am). Congo red staining, circular dichroism, and FTIR spectroscopy showed that this 11-amino acid peptide produced predominantly beta-sheet structures. TEM with negative staining and quick-freeze deep etch (QFDE) TEM showed that the peptide structures were composed of a highly entangled fibrillar network. These beta-sheet fibrillar nanostructures were then covalently coupled to pendant amine-containing biomolecules via tissue transglutaminase. MALDI-TOF mass spectrometry and HPLC were utilized to monitor the extent of the transglutaminase modification of the peptide, showing that as many as five glutamines in the peptide were reactive via transglutaminase for covalent conjugation. This strategy, based on the post-assembly modification of a self-assembling peptide, has potential applications for tailoring supramolecular structures for drug delivery, tissue engineering, or other biomedical applications.