Lipases from the genus Rhizopus: Characteristics,expression, protein engineering and application

Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications.     

Production, purification, characterization, and applications of lipases

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing.     

Bacterial lipases: an overview of production,purification and biochemical properties

Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.     

Cold active microbial lipases: some hot issues and recent developments

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.     

Recombinant microbial lipases for biotechnological applications

Lipases, mainly of microbial origin, represent the most widely used class of enzymes in biotechnological applications and organic chemistry. Modern methods of genetic engineering combined with an increasing knowledge of structure and function will allow further adaptation to industrial needs and exploration of novel applications. Production of such tailored lipases requires their functional overexpression in a suitable host. Hence, this article describes the functional heterologous production of commercially important microbial lipases. Based on the knowledge of different lipases' substrate binding sites, the most suitable lipase for a particular application may be selected.     

Burkholderia cepacia lipase is a promising biocatalyst for biofuel production

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far‐UV circular dichroism (CD) and intrinsic fluorescence, displays a T_m above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.     

Lipases for biotechnology

Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.     

Solvent tolerant Pseudomonads as a source of novel lipases for applications in non-aqueous systems

Abstract

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous biocatalysts known to catalyze the hydrolysis of water insoluble triglycerides in aqueous medium and carry out the reverse reaction (synthesis) under organic solvent rich medium. Microbial lipases have received a great deal of attention in the field of food technology, pharmaceutical sciences, chemical and detergent industries due to their stability, selectivity, mild operation conditions and broad substrate specificity. Despite these advantages, low activity and stability displayed in organic medium has restricted their commercial application in organic synthesis. Researchers have explored alternative ways to modify the enzymes making them suitable for use in non-conventional media. In this context, harvesting lipases from “Solvent Tolerant Microbes” has recently become an attractive approach. These microbes are able to grow in the presence of high concentrations of organic solvents, generally known to have detrimental effect on microorganisms. Such microbes survive through novel adaptation mechanisms and secretion of solvent stable enzymes having efficient functionality in solvent-rich media. These enzymes could be useful for bioconversion in non-conventional media. In the current review, this approach is described with an emphasis on characteristics, applications and genetic aspect of lipases from the genus Pseudomonas.     

Potential for industrial products from the halophilic <Emphasis Type="Italic">Archaea</Emphasis>

The halophilic Archaea are a group of microorganisms that have not been extensively considered for biotechnological applications. This review describes some of the enzymes and products and the potential applications of this unique group of microorganisms to various industrial processes. Specifically, the characteristics of the glycosyl hydrolases, lipases and esterases, proteases, biopolymers and surfactants, as well as some miscellaneous other activities will be described.     

Lipase promiscuity and its biochemical applications

Lipases are the most widely used class of enzymes in organic synthesis. Availability of large number of commercial preparations, their broad specificity and relatively better stability (as compared to other enzymes) in media containing organic solvents have all been contributing factors for this. This review has a sharp focus on their specificity. The recent results with catalytic promiscuity have shown that lipases are even more versatile than thought so far. These results have also prompted workers to rationalize the classification of specificity in terms of substrate promiscuity, condition promiscuity and catalytic promiscuity. The review also attempts to recast the known information on specificity of lipases in the context of enzyme promiscuity. Lipases can exhibit regiospecificity, specificity in terms of fatty acids, nature of the alcohol, and stereospecificity (distinction between sn-1 and sn-3 position on the triglyceride). Lipases show varied stability toward presence of organic solvents, extreme pH conditions and ionic liquids. In low water media, condition promiscuity in terms of esterification, transesterification and interesterification has been extensively studied. The catalytic promiscuity is being increasingly observed for CC bond formation reactions. Finally, the beneficial consequences of this promiscuous behavior in biotechnology sectors are also discussed.     

The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications

Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.     

Biocatalytic synthesis of ascorbyl esters and their biotechnological applications

Ascorbyl fatty acid esters act both as antioxidants and surfactants. These esters are obtained by acylation of vitamin C using different acyl donors in presence of chemical catalysts or lipases. Lipases have been used for this reaction as they show high regioselectivity and can be used under mild reaction conditions. Insolubility of hydrophilic ascorbic acid in non-polar solvents is the major obstacle during ascorbic acid esters synthesis. Different strategies have been invoked to address this problem viz. use of polar organic solvents, ionic liquids, and solid-phase condensation. Furthermore, to improve the yield of ascorbyl fatty acid esters, reactions were performed by (1) controlling water content in the reaction medium, (2) using vacuum to remove formed volatile side product, and (3) employing activated acyl donors (methyl, ethyl or vinyl esters of fatty acids). This mini-review offers a brief overview on lipase-catalyzed syntheses of vitamin C esters and their biotechnological applications. Also, wherever possible, technical viability, scope, and limitations of different methods are discussed.     

Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential

Myceliophthora thermophila syn. Sporotrichum thermophile is a ubiquitous thermophilic mould with a strong ability to degrade organic matter during optimal growth at 45?°C. Both genome analysis and experimental data have suggested that the mould is capable of hydrolyzing all major polysaccharides found in biomass. The mould is able to secrete a large number of hydrolytic enzymes (cellulases, laccases, xylanases, pectinases, lipases, phytases and some other miscellaneous enzymes) employed in various biotechnological applications. Characterization of the biomass-hydrolyzing activity of wild and recombinant enzymes suggests that this mould is highly efficient in biomass decomposition at both moderate and high temperatures. The native enzymes produced by the mould are more efficient in activity than their mesophilic counterparts beside their low enzyme titers. The mould is able to synthesize various biomolecules, which are used in multifarious applications. Genome sequence data of M. thermophila also supported the physiological data. This review describes the biotechnological potential of thermophilic mould, M. thermophila supported by genomic and experimental evidences.     

Directed evolution of Bacillus licheniformis lipase for improvement of thermostability

Lipases catalyze the hydrolysis of carboxylic acid esters and owing to their vast substrate specificity, they have many industrial applications. Due to the demand of thermostable lipases in industrial applications, we have enhanced the thermostability of lipase from Bacillus licheniformis RSP-09. The thermostable mutant lipases of Bacillus licheniformis RSP-09 were isolated following two rounds of directed evolution using error-prone PCR. The best mutant lipases obtained after first and second round of error-prone PCR were purified and characterized. The mutant lipases showed increased thermostability and retained catalytic function. The best mutant lipase (eP-231-51) showed 13.5-fold increase in percentage thermal stability (% remaining activity after incubation of purified enzyme at 60 °C for 1 h) than wild-type lipase. Also, this mutant lipase (ep-231-51) showed 30% improved catalytic efficiency compared with the wild-type which is due to significant decrease in K_m and marginal increase in k_cat. In addition, the thermostable mutant lipases have shown resistance to hydrophobic organic solvents. The role of mutations in the best mutant lipases of second round i.e. eP-231-51 (Asp72Gly, Asp61Gly, Tyr129His, and Thr101Pro) and eP-231-137 (Leu49Arg, Thr101Pro, Asp72Gly), that led to thermostability have been postulated after the comparison of molecular models of wild-type and mutated enzymes.     

Isolation and characterization of some moderately halophilic bacteria with lipase activity

Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group of biocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from the Maharla salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 ± 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from the Maharla lake.     

Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.

Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Nematodes from saline and freshwater lakes of the Vestfold Hills,East Antarctica,including the description of <Emphasis Type="Italic">Hypodontolaimus antarcticus</Emphasis> sp. n.

The invertebrate fauna of many Antarctic ice-free areas, even those close to permanent research stations, can be poorly known. Here we describe some nematodes from freshwater and saline, marine-derived lakes of the Vestfold Hills, East Antarctica. The freshwater lakes contained the widespread East Antarctic endemic species, Plectus frigophilus Kirjanova, 1958. The saline lakes were inhabited by two recently described species, Halomonhystera halophila Andrássy, 2006 and Halomonhystera continentalis Andrássy, 2006, and by a new species described in this report, Hypodontolaimus antarcticus sp. n. Originally marine but now brackish Highway Lake contained a nematode fauna with both freshwater and marine-derived components. The nematode fauna of Antarctica now consists of 54 named species, 22 of which are found in East Antarctica.     

Esterification of phenolic acids catalyzed by lipases immobilized in organogels

Lipases from Rhizomucor miehei and Candida antarctica B were immobilized in hydroxypropylmethyl cellulose organogels based on surfactant-free microemulsions consisting of n-hexane, 1-propanol and water. Both lipases kept their catalytic activity, catalyzing the esterification reactions of various phenolic acids including cinnamic acid derivatives. High reaction rates and yields (up to 94%) were obtained when lipase from C. antarctica was used. Kinetic studies have been performed and apparent kinetic constants were determined showing that ester synthesis catalyzed by immobilized lipases occurs via the Michaelis–Menten mechanism.     

Correcting Misperceptions about the History of <Emphasis Type="Italic">Castanea</Emphasis> Stands in <Emphasis Type="Italic">Satoyama</Emphasis> in Japan

Correcting Misperceptions about the History of Castanea Stands in Satoyama in Japan. Mistaken ideas about the naturalness of past and present landscapes are widespread in diverse cultures and in the scientific literature, and many of these ideas are only now being seriously challenged by current research (e.g., Erickson 2006; Fairhead and Leach 1996; Hall 1998; Ramankutty and Foley 1999; Willis et al. 2004). For example, the chestnut, Castanea crenata, has long been an important tree in Japanese culture, which has been cultivated, among other things, for its much loved edible nut and its valuable timber. Today, the widely-held view in Japan, which also appears in the scholarly and popular literature, is that in the past Castanea stands covered a large area throughout Japan, and these stands only disappeared because of economic development, especially in association with railway construction. Otaru, Hokkaido, is one of the places where people believe Castanea stands covered a large area and were deforested only recently. Local people in Otaru believe that the stand in Temiya Park has existed since the Jomon Period. For a more accurate historical perspective on Japanese forestation, we have performed pollen analysis to clarify the timing of the introduction of the Castanea tree into Otaru region and to reveal the history of this specific Castanea stand in Temiya Park. The results indicate that Castanea was first found in Otaru region 7100 B.P., but that it was not cultivated extensively until recently. Based on our study, and on data from this area dating to the late 19th century, we concluded instead that the Castanea stand we studied in Temiya Park, Otaru, was established after the mid-20th century. We believe the results of this study are applicable to Castanea stands in other parts of Japan as well.