Stereoselective lipases from Burkholderia sp., cloning and their application to preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for (R)-Metalaxyl

Two microbial strains (referred to as MC 16-3 and 99-2-1) that produce extracellular lipases were isolated from soil samples and identified as Burkholderia species. The lipases were partially purified by isopropyl alcohol precipitation and gave molecular weight of 33kDa. The lipases were characterized in terms of stereoselectivity with racemic methoxyethyl (R,S)-N-(2,6-dimethylphenyl)alaninate and the genes encoding the proteins have been identified by homology alignment of lipases reported belonging to I.2 subfamily and their complete DNA sequences were determined. The lipases will be useful for the preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for the synthesis of (R)-Metalaxyl, which is one of the best-selling fungicides.     

An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix

A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h⁻¹ g⁻¹, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the α-helical content of lipase increase after pretreatment.     

Lipase-Catalyzed Reactions for Modification of fats and other Lipids

Applications of lipase-catalyzed reactions, such as hydrolysis of fats for the production of fatty acids and esterification or interesterification of fats and other lipids for the preparation of diverse products in food and non-food industries, are reviewed. At present, the application of lipases in biotechnological processes seems to be economically feasible and appropriate mainly for the preparation of specific products of high commercial value, which cannot be prepared conveniently by chemical synthesis. For example, polyunsaturated fatty acids that can be used in dietetic products are prepared under mild conditions by hydrolysis of marine oils and certain plant oils with non-specific triacylglycerol lipases. Very long chain monounsaturated fatty acids (gadoleic, erucic and nervonic) that are of value in oleochemical industry can be prepared by partial hydrolysis of cruciferous oils with sn-1,3-specific lipases. Lipase-catalyzed esterification yields a variety of products, such as monoacylglycerols that are used as emulsifiers, and wax esters resembling jojoba oil which is used in cosmetics industry. Interesterification of fats with sn-1,3-specinc lipases affords specialty products, such as cocoa butter substitutes which are used in confectionary products and medium chain triacylglycerols that can be used in dietetic products. Phospholipase-catalyzed exchange of acyl moieties or bases of glycerophos-pholipids yields several products of biomedical interest.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.     

Lipase-Catalyzed Reactions for Modification of fats and other Lipids

Applications of lipase-catalyzed reactions, such as hydrolysis of fats for the production of fatty acids and esterification or interesterification of fats and other lipids for the preparation of diverse products in food and non-food industries, are reviewed. At present, the application of lipases in biotechnological processes seems to be economically feasible and appropriate mainly for the preparation of specific products of high commercial value, which cannot be prepared conveniently by chemical synthesis. For example, polyunsaturated fatty acids that can be used in dietetic products are prepared under mild conditions by hydrolysis of marine oils and certain plant oils with non-specific triacylglycerol lipases. Very long chain monounsaturated fatty acids (gadoleic, erucic and nervonic) that are of value in oleochemical industry can be prepared by partial hydrolysis of cruciferous oils with sn-1,3-specific lipases. Lipase-catalyzed esterification yields a variety of products, such as monoacylglycerols that are used as emulsifiers, and wax esters resembling jojoba oil which is used in cosmetics industry. Interesterification of fats with sn-1,3-specinc lipases affords specialty products, such as cocoa butter substitutes which are used in confectionary products and medium chain triacylglycerols that can be used in dietetic products. Phospholipase-catalyzed exchange of acyl moieties or bases of glycerophos-pholipids yields several products of biomedical interest.     

Variations among four commercial lipases from Chromobacterium viscosum in ester synthesis

The properties of four commercial lipases from Chromobacterium viscosum, CvL 1–4, in ester synthesis were investigated. Three lipases showed a high synthetic activity in esterification, with conversions of oleic acid as high as 86–95% in 24 h, whereas one (CvL 1) gave a poor result of only 11% with the same quantity of 9 mg crude lipase preparation. The elution profiles of the four lipases from Sephacryl S-100 HR differed and SDS-PAGE suggested that while CvL 1 lipase had two equivalent protein bands of molecular size 33 and 27 kDa, respectively, the other three lipases showed only one main protein band of 33 kDa. Isoelectric focusing revealed that all of the lipases contained several isoforms, but the proportions of the isoforms varied. Furthermore, both aggregated and single lipase forms obtained after gel filtration were able to catalyse ester synthesis, but the two lipases from CvL 1 showed lower synthetic activities than the others.     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.     

Deinking of soy bean oil based ink printed paper with lipases and a neutral surfactant

A screening of different strains of bacteria for the production of lipases which degrade the soy bean oil based binder component of newsprint ink was performed. Three strains were found to be efficient lipase producers and were selected for further investigations. The pH optimum, temperature stability and the optimum enzyme concentrations for the deinking of recovered paper printed with soy bean oil based ink were determined and compared with a commercially available porcine pancreas lipase preparation. A lipase preparation from Pseudomonas aeruginosa with a pH optimum and temperature stability suitable for an application in a deinking process and with a high deinking efficiency in combination with a neutral surfactant could be identified. The deinking effect of the lipases was caused by a partial degradation of the binder of the soy bean oil based inks thereby releasing the ink particles from the paper.     

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.     

Characterization of three distinct forms of lipolytic enzyme in a commercialCandida lipase preparation

Summary Three distinct forms of lipolytic enzyme were identified in a commercialCandida lipase preparation. Two of these lipases (lipases A & C) were isolated and characterized. Lipase A had a higher optimal reaction pH and a better thermal stability than those of lipase C. Lipase A and C displayed different acyl chain length specificity on the lipolysis of p-nitrophenol esters.     

Proteolytic Activity in Commercial Triacylglycerol Hydrolase Preparations

The proteolytic activity of 34 commercial lipase preparations (CLP) was determined using a labeled casein substrate. Only three CLP were free from proteolytic activity. Porcine pancreatic lipases exhibited levels of proteolytic activity comparable to or greater than that of a reference porcine trypsin. Bacterial lipases contained up to 10% of the proteolytic activity of commercial trypsin. Proteolytic activities in lipases from fungal species were present at low levels (<1% of the activity in trypsin). Among preparations of fungal origin, lipases from Aspergillus niger and Mucor javanicus were highest in proteolytic activity; Aspergillus oryzae and Pseudomonas cepacia lipases were lowest. Proteins in CLP were separated by non-denaturing PAGE; between 4 and 17 protein bands in the range &#104 6.5- &#83 200 kDa were observed. With the exception of a single pair of Rhizomucor miehei lipases, the distribution of apparent molecular weights (AMW) was unique to each preparation. Bands of caseinolytic activity in commercial lipases were visualized by applying a zymographic technique. CLP contained between 0 (P. cepacia lipases) and 6 (porcine pancreas lipase and Rhizopus oryzae lipase) discrete proteolytic bands. Common themes of proteolytic AMW emerged, including 21-23 kDa and 30-35 kDa bands.     

Biospecific chromatography of lipolytic enzymes and methods for determining their activity

The review concerns application of affinity chromatography for isolation of phospholipases and lipases, as well as the methods for determining their activities. Main emphasis is laid on the preparation of biospecific supports with lipid ligands as well as on development of new methods for assaying lipolytic activity.     

Determination of the quantitative stereoselectivity fingerprint of lipases during hydrolysis of a prochiral triacylglycerol

We propose a method for characterizing quantitatively the stereoselectivity of lipases during hydrolysis of triacylglycerols. Although it is of general applicability, we demonstrate it specifically for sn-1,3-regiospecific lipases. In this case the method generates a "stereoselectivity fingerprint" that consists of ratios of the specificity constants for the various reactions that produce and consume the 1,2-sn- and 2,3-sn-diacylglycerols. We use the method to determine the stereoselectivity fingerprint of several lipases during the hydrolysis of the prochiral substrate triolein. Our method opens up the possibility of correlating quantitative fingerprints with structural information, in the quest to elucidate the mechanisms underlying the stereoselectivity of lipases.     

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.     

Preparation of an optically pure secondary alcohol of synthetic pyrethroids using microbial lipases

Summary Enzyme-catalysed hydrolysis of esters of 4-hydroxy-3-methyl-2-(2-propynyl)-cyclopent-2-enone (HMPC) was examined for the preparation of the optically pure alcohol moiety of synthetic pyrethroids. Among microorganisms and lipases tested, some bacterial lipases hydrolysed the ester of HMPC with high enantioselectivity and high reaction rate. Arthrobacter lipase gave the optically pure (R)-HMPC at 50% hydrolysis in a two-liquid phase reaction system of water and the insoluble substrate. The hydrolysis proceeded even at a substrate concentration of 80w/v%. The enantioselectivity was not changed with the chain length of the acid moiety of the esters. By combination of the enzymatic resolution with a chemical inversion of the (R)-alcohol, an efficient proess was developed for the total conversion of racemic HMPC to (S)-HMPC, which is an important alcohol for preparation of an insecticidallyactive synthetic pyrethroid.Biological preparation of an optically active alcohol. Part I     

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.     

Biocatalytic synthesis of optically active tertiary alcohols

The enzymatic preparation of optically pure tertiary alcohols under sustainable conditions has received much attention. The conventional chemical synthesis of these valuable building blocks is still hampered by the use of harmful reagents such as heavy metal catalysts. Successful examples in biocatalysis used esterases, lipases, epoxide hydrolases, halohydrin dehalogenases, thiamine diphosphate-dependent enzymes, terpene cyclases, -acetylases, and -dehydratases. This mini-review provides an overview on recent developments in the discovery of new enzymes, their functional improvement by protein engineering, the design of chemoenzymatic routes leading to tertiary alcohols, and the discovery of entirely new biotransformations.     

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.