Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases

Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis. Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents. The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates. The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL). The resulting lipid-protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes. We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water. It was much higher when the protein was dissolved in aqueous ethanol. These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents.     

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.     

Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates).

The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.     

Lipolytic enzymes with improved activity and selectivity upon adsorption on polymeric nanoparticles

Nanostructured polystyrene (PS) and polymethylmethacrylate (PMMA) were used as carriers for the preparation of bioconjugates with lipolytic enzymes, such as Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL). Simple addition of the lipase solution to the polymeric nanoparticles under protein-friendly conditions (pH 7.6) led to the formation of polymer-enzyme bioconjugates. Energy filtered-transmission electron microscopy (EF-TEM) performed on immuno-gold labeled samples revealed that the enzyme preferentially binds to the polymer nanoparticles and that the binding does not affect the nanostructured features of the carriers. The studies performed on the activity of the bioconjugates pointed out that the lipases adsorbed onto polymeric nanoparticles show an improved performance in terms of activity and selectivity with respect to those shown by lipases adsorbed on the same non-nanostructured carriers. The residual activities of CRL and PCL immobilized on nanostructured PMMA and PS reached 60% and 74%, respectively. Moreover, we found that enantioselectivity and pH and thermal stability increase upon immobilization. These results highlight the fact that new protein conformers with improved enantioselectivity stabilized after adsorption on nanoparticles are obtained. On the basis of the chemical structures of the selected polymers and the slopes of the adsorption isotherms, a hydrophobic binding model for lipase/nanostructured polymers is suggested.     

Lipase-mediated hydrolysis of blackcurrant oil

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.     

Esterase EstA6 from Pseudomonas sp. CR-611 is a novel member in the utmost conserved cluster of family VI bacterial lipolytic enzymes

Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.     

Activity enhancement and stabilization of lipase from Pseudomonas cepacia in polyallylamine-mediated biomimetic silica

Triacylglycerol lipase from Pseudomonas cepacia and Fe(3)O(4) magnetic nanoparticles were encapsulated simultaneously within biomimetic silica through the catalysis of polyallylamine. The encapsulation efficiency reached 96% with an activity recovery of 51%. After 5 h at 37°C, the activities of the free and encapsulated lipases decreased by 77 and 16%, respectively. Addition of 10 and 15 mol% trimethylmethoxysilane to tetramethoxysilane during encapsulation doubled the lipase activity while inclusion of 50 and 60 mol% γ-(methacryloxypropyl)-trimethoxysilane tripled the activity. Thus, such encapsulation not only stabilized P. cepacia lipase but also could enhance the activity by varying silane additives.     

Selection of orlistat as a potential inhibitor for lipase from Candida species

Infections caused by Candida species manifest in a number of diseases, including candidemia, vulvovaginal candidiasis, endocarditis, and peritonitis. Candida species have been reported to possess lipolytic activity due to the secretion of lipolytic enzymes such as esterases, lipases and phospholipases. Extra-cellular hydrolytic enzymes seem to play an important role in Candida overgrowth. Candidiasis is commonly treated with antimycotics such as clotrimazole and nystatin. The antimycotics bind to a major component of the fungal cell membrane (ergosterol), forming pores that lead to death of the fungus. However, the secondary effects caused during such treatment have aroused a need to develop a treatment based on lipase inhibition. Nonetheless, no such lipase inhibitors for candidiasis treatment are currently available. Thus, we have performed a docking study with the natural inhibitor, orlistat or tetrahydrolipstatin. Our results have shown ten possible binding inhibitors to Candida rugosa lipase (CRL), out of which one possibility was selected, based on the weakest interatomic distance of 2.7 ?. Therefore, we propose the selection and design of a potential inhibitor candidate, orlistat for the treatment of candidiasis infections. However, this study has to be supported with in vitro and in vivo experiments to demonstrate the effectiveness of orlistat in lipase inhibition.     

Human digestive and metabolic lipases—a brief review

The major human lipases include the gastric, pancreatic and bile-salt-stimulated lipase that aid in the digestion and assimilation of dietary fats, and the hepatic, lipoprotein and endothelial lipase that function in the metabolism of lipoproteins. The triacylglycerol and phopholipase activities of these enzymes enable these varied functions. The lipase enzymes exhibit a high degree of sequence homology not only within but also across species. This and the diverse chromosomal location of their genes point to a multigenic family of enzymes involved in absorption and transport of lipids. Inactivation of lipolytic activity of microorganisms to control infection, inhibition of digestive lipase to control obesity, stimulation of metabolic lipase to reduce hyperlipidemia or procoagulant state, or use of pancreatic lipase supplement in the management of cystic fibrosis are examples of how lipase activity modulation can impact human health.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

Microwave-assisted rapid characterization of lipase selectivities

A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise.     

Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

脂肪酶的特异性折叠酶

以Pseudomonas aeruginosa为代表的Ⅰ.1亚家族脂肪醇和以Burkholderia cepacia为代表的Ⅰ.2亚家族脂肪酶具有优良的手性化合物拆分性能,在绿色化工领域具有良好的开发潜力及其应用前景.Ⅰ.1亚家族脂肪酶和Ⅰ.2亚家族脂肪醇均采用Ⅱ型分泌机制进行分泌,其分泌依赖于一种分子伴侣--脂肪酶的特异性折叠酶(lipase specific foldase,Lif)的协助.从Lif蛋白的分类、结构特点、介导对应脂肪酶折叠的特异性及机制、lif基因表达调控机制及尚待探讨的问题等方面系统介绍Lif蛋白的研究进展.     

Selection of pH buffers for use in conductimetric microbiological assays

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5° to 30°C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

The diversity of lipases from psychrotrophic strains of Pseudomonas : a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene ( lipA ) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens , the gene encodes a lipase of M_r 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M_r approximately 30 kDa comprising sequences from Ps. fragi , Ps. aeruginosa , Ps. fluorescens C9 and Burkholderia , and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling.

A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme.     

The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia.

The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined. The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase. In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation. The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181). This strain was identified after a limited screening for lipase activity. About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.     

Lipolysis: pathway under construction

PURPOSE OF REVIEW: The lipolytic catabolism of stored fat in adipose tissue supplies tissues with fatty acids as metabolites and energy substrates during times of food deprivation. This review focuses on the function of recently discovered enzymes in adipose tissue lipolysis and fatty acid mobilization. RECENT FINDINGS: The characterization of hormone-sensitive lipase-deficient mice provided compelling evidence that hormone-sensitive lipase is not uniquely responsible for the hydrolysis of triacylglycerols and diacylglycerols of stored fat. Recently, three different laboratories independently discovered a novel enzyme that also acts in this capacity. We named the enzyme 'adipose triglyceride lipase' in accordance with its predominant expression in adipose tissue, its high substrate specificity for triacylglycerols, and its function in the lipolytic mobilization of fatty acids. Two other research groups showed that adipose triglyceride lipase (named desnutrin and Ca-independent phospholipase A2zeta, respectively) is regulated by the nutritional status and that it might exert acyl-transacylase activity in addition to its activity as triacylglycerol hydrolase. Adipose triglyceride lipase represents a novel type of 'patatin domain-containing' triacylglycerol hydrolase that is more closely related to plant lipases than to other known mammalian metabolic triacylglycerol hydrolases. SUMMARY: Although the regulation of adipose triglyceride lipase and its physiological function remain to be determined in mouse lines that lack or overexpress the enzyme, present data permit the conclusion that adipose triglyceride lipase is involved in the cellular mobilization of fatty acids, and they require a revision of the concept that hormone-sensitive lipase is the only enzyme involved in the lipolysis of adipose tissue triglycerides.