Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.     

低温脂肪酶的研究现状与应用前景

低温脂肪酶在低温下仍保持高酶活,因此在应用中有着中温脂肪酶无法取代的优越性,而具有高活性的低温脂肪酶因其具有理论和应用上的双重意义成为了近年来的研究热点。本文从描述产低温脂肪酶的低温微生物特征入手,系统阐述了低温脂肪酶的来源、分类、特征、研究方法及最新进展,并简述了低温脂肪酶在食品、洗涤、制药以及低温环境修复等工业上的应用前景。     

微生物脂肪酶蛋白质工程*

微生物脂肪酶催化的化学反应具有严格的立体选择性、位点选择性等专一性,催化活性高而副反应少,催化反应不需要辅助因子等特点,因此广泛应用于工农业生产中的诸多领域。利用蛋白质工程技术,提高微生物脂肪酶的特异性、活性和稳定性,对提高微生物脂肪酶制剂产品的市场竞争能力,扩大微生物脂肪酶的应用领域,具有重要的意义。综述了蛋白质工程技术在微生物脂肪酶改性方面的应用现状、存在问题及前景。     

Geotrichum candidum produces several lipases with markedly different substrate specificities.

We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively. Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo-beta-N-acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.     

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.     

Fabrication and application of enzyme-incorporated peptide nanotubes

Enzyme engineering is a fast-growing field in the pharmaceutical and food markets. For those applications, various substrates have been examined to immobilize and stabilize enzymes. In this report, we examined peptide nanotubes as supports for enzymes. When a model enzyme, Candida rugosa lipase, was encapsulated in peptide nanotubes, the catalytic activity of nanotube-bound lipases was increased 33% as compared to free-standing lipases at room temperature. At an elevated temperature, 65 degrees C, the activity of lipases inside the nanotubes was 70% higher than free-standing lipases. The activity enhancement of lipases in the peptide nanotubes is likely induced by the conformation change of lipases to the open form (the enzymatically active structure) as lipases are adsorbed on the inner surfaces of peptide nanotubes.     

Lipase immobilization into porous chitoxan beads: activities in aqueous and organic media and lipase localization

Lipases were noncovalently immobilized in Chitoxan, a polyionic hydrogel obtained by complexation between chitosan and xanthan. The properties of free and immobilized lipases have been compared. In the aqueous medium, the activity was twice as high for immobilized lipases as for free lipases. Immobilized lipases in chitoxan were able to hydrolyze triacylglycerols in three distinct organic solvent media. At the microstructural level, lipases were not distributed uniformly in the chitoxan beads. Higher concentrations of lipase were found in the outer membrane-like layer of the beads, as compared with lower concentrations in the inner part of the beads.     

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.     

Towards the development of systems for high-yield production of microbial lipases

Microbial lipases are a versatile and attractive class of biocatalysts for a wide variety of applications. Lipases can be produced by bacteria, yeasts or filamentous fungi. Nevertheless, they are often not optimal for direct use in industrial conditions due to low yields, low specific activities and a limited spectrum of activities. Improvements in the productivity of lipases have been made by genetic manipulation of the cell factory production hosts and by optimizing production media and conditions. Advances in protein engineering technology, ranging from directed evolution to rational design, have also been able to tailor lipases to particular applications. This review describes various approaches used to improve lipase production and applications.     

Storage oil hydrolysis during early seedling growth

Storage oil breakdown plays an important role in the life cycle of many plants by providing the carbon skeletons that support seedling growth immediately following germination. This metabolic process is initiated by lipases (EC: 3.1.1.3), which catalyze the hydrolysis of triacylglycerols (TAGs) to release free fatty acids and glycerol. A number of lipases have been purified to near homogeneity from seed tissues and analysed for their in vitro activities. Furthermore, several genes encoding lipases have been cloned and characterised from plants. However, only recently has data been presented to establish the molecular identity of a lipase that has been shown to be required for TAG breakdown in seeds. In this review we briefly outline the processes of TAG synthesis and breakdown. We then discuss some of the biochemical literature on seed lipases and describe the cloning and characterisation of a lipase called SUGAR-DEPENDENT1, which is required for TAG breakdown in Arabidopsis thaliana seeds.     

微生物脂肪酶的重组表达

微生物脂肪酶在传统和新型工业催化领域中的应用越来越广泛与深入。作为脂肪酶规模化制备主要途径的高效重组表达,为脂肪酶催化剂的最终形成及工业催化奠定了坚实的技术基础。概述并讨论了微生物脂肪酶重组表达的最新策略和发展趋势,阐述密码子优化、融合共表达、杂合启动子、同源高效表达、细胞表面展示和表达文库的高通量筛选等技术特点及其表达应用现状,指出细胞表面展示表达和表达文库高通量筛选体系为脂肪酶重组表达注入强劲活力;在此基础上对几种代表性微生物脂肪酶的重组表达进展作一综述,为微生物脂肪酶的重组表达研究及工业生产提供借鉴。     

Properties of lipases from Oospora lactis

The procedure of isolation and purification of lipases from the fungus Oospora lactis have been developed. The existence of two different lipases has been demonstrated, one of which (Mr = 43000) is localized in the periplasmic space and can be liberated into the external medium in an unchanged form, while the other (Mr = 40000) is tightly bound to the membranes and can be solubilized by detergent treatment. The most essential properties of the lipases are discussed and a detailed analysis of the functional and physicochemical properties of extracellular lipase is given.     

Technical methods to improve yield,activity and stability in the development of microbial lipases

Lipases are ubiquitous biocatalysts that catalyze various reactions in organic solvents or in solvent-free systems and are increasingly applied in various industrial fields. In view of the excellent catalytic activities and the huge application potential, more than 20 microbial lipases have been realized in large-scale commercial production. The potential for commercial exploitation of a microbial lipase is determined by its yield, activity, stability and other characteristics. This review will survey the various technical methods that have been developed to enhance yield, activity and stability of microbial lipases from four aspects, including improvements in lipase-producing strains, modification of lipase genes, fermentation engineering of lipases and downstream processing technology of lipase products.     

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.     

Catalytic properties and potential applications of Bacillus lipases

Up to date more than 70 lipases from the Bacillus and Geobacillus genera have been isolated, but for most of them only basic biochemical properties have been reported. In general, Bacillus lipases are easily produced and display high tolerance toward organic solvents, proving them useful in the synthesis of esters for food industry, cosmetics and biodiesel production. Many lipases preserve their activity at extreme temperatures and pH, and in the presence of surfactants, hydrogen peroxide, sodium hypochlorite, and therefore they can be applied in laundry formulations. Bacillus lipases display diverse selectivity to the chain length of the acid, and few enzymes show positional specificity. Several enzymes can be applied in the production of enantiopure compounds for the pharmaceutical industry due to their remarkable enantioselectivity. The immobilization experiments with Bacillus lipases, though a limited number, illustrate the vast possibilities for optimization of the properties of the biocatalysts for a particular application. The paper summarizes available experimental data on Bacillus and Geobacillus lipases and identifies areas for further research.     

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.     

Enzymatic resolution of racemic ibuprofen by surfactant-coated lipases in organic media

Summary Surfactant-coated lipases have been utilized as a biocatalyst for the resolution of racemic ibuprofen. S-(+)-ibuprofen was selectively transferred to the ester form by Mucor javanicus or Candida rugosa lipase. The enzymatic activity of upases in organic media was remarkably enhanced by coating with a nonionic surfactant. The reaction rates of the coated lipases were increased around 100-fold that of the powder lipases.     

Staphylococcal lipases: biochemical and molecular characterization

To date, the nucleotide sequences of nine different lipase genes from six Staphylococcus species, three from S. epidermidis, two from S. aureus, and one each from S. haemolyticus, S. hyicus, S. warneri, and S. xylosus, have been determined. All deduced lipase proteins are similarly organized as pre-pro-proteins, with pre-regions corresponding to a signal peptide of 35 to 38 amino acids, a pro-peptide of 207 to 321 amino acids with an overall hydrophilic character, and a mature peptide comprising 383 to 396 amino acids. The lipases are secreted in the pro-form and are afterwards processed to the mature form by specific proteases. The pro-peptide of the S. hyicus lipase is necessary for efficient translocation and for protection against proteolytic degradation. Despite being very similar in their primary structures the staphylococcal lipases show significant differences in their biochemical and catalytic properties, such as substrate selectivity, pH optimum and interfacial activation. The lipase from S. hyicus is unique among the staphylococcal and bacterial lipases in that it has not only lipase activity, but also a high phospho-lipase activity. All staphylococcal lipases are dependent on Ca(2+), which is thought to have a function in stabilizing the tertiary structure of the lipases. Evidence exists that staphylococcal lipases like other bacterial lipases, possess a lid-like domain that might be involved in the interfacial activation of these enzymes.     

Purification of lipases.

Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.     

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.