A single and continuous spectrophotometric assay for various lipolytic enzymes, using natural, non-labelled lipid substrates

A rapid, continuous spectrophotometric assay for measuring the amount and activity of several lipolytic enzymes is described. It is based on the metachromatic properties of the cationic dye safranine, and makes use of the fact that an adequate combination of a lipolytic enzyme with one of its substrates leads to a change in the net negative charge at the lipid/water interface, which is monitored by the absorbance change of safranine. Utilizing this method, most lipolytic enzymes can be detected in very low amounts (milliunit or less) in about 1 min without employing radiolabelled lipids or synthetic lipid analogues. Over a wide range of enzyme concentrations, there is a good linearity between the initial hydrolysis rate (determination by the safranine method) and the amount of enzyme. The versatility of the assay is illustrated by examples showing how phospholipase A2, triacylglycerol hydrolase, phospholipase D or phospholipase C (either general or phosphatidylinositol-specific) activities can be detected, either separately or sequentially. Due to its high sensitivity, simplicity, and rapidity, this assay should find its main application in monitoring column effluents during the purification steps of lipolytic enzymes.     

Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis

Background

New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB.

Methods

Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals.

Results

A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses.

Conclusion

These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.     

Screening for novel lipolytic enzymes from uncultured soil microorganisms

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C₄) but not p-nitrophenyl palmitate (C₁₆).     

Synthetic peptides as substrates for processing enzymes in the bovine pituitary

Synthetic peptides, based on sequences of proopiomelanocortin (POMC) cleaved in both the bovine anterior and intermediate pituitaries (-Phe-Pro-Leu-Gly-Phe-Lys-Arg-Glu-Leu-Thr-Gly-) and only in the intermediate lobe (-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-), were used as substrates for the enzymes that process POMC to active hormones in the anterior and intermediate lobes of the pituitary. Cleavage of these peptides at the dibasic pair of residues, the expected cleavage site, was observed with a lysate from bovine pituitary secretory granules. Cleavage occurred optimally at a pH between 4 and 5 and was inhibited with sulfhydryl reagents, pepstatin, and leupeptin. Little specificity for the nature of the basic residues at the cleavage site was observed. An additional cleavage, following glutamic acid residues, was also seen.     

4-Methylumbelliferyl substituted compounds as fluorogenic substrates for fungal extracellular enzymes

4-Methylumbelliferyl β-D-glucoside, 4-methylumbelliferyl β-D-galactoside and 4-methylumbelliferyl- N -acetyl-β- D -glucosaminide were used as substrates for screening for extracellular enzyme activity in fungal culture filtrates. Enzyme activity was observed as fluorescence under long wave ultraviolet light. The presence or absence of enzymic activity in fungal culture filtrates was compared to results obtained with the commercially produced API ZYM system.     

Formation of lipolytic enzymes by Brevibacterium linens

When Brevibacterium linens ATCC 9172 was grown in shake flasks, it produced a cell-associated lipase with a specific activity of 152 to 188 U g^–1 cells depending on the composition of the growth medium. There was no growth in media containing tributyrine as the sole carbon source. The cell-associated lipase had maximum activity at pH 8.0 and 37 °C and was strongly inhibited by 3,4-dichloroisocoumarin, an inhibitor specific for serine esterases. Cell-associated activity was released from the cells by treatment with lysozyme. The kinetics of lipase formation was closely related to the amount of biomass formed during growth.     

Mycobacterial lipolytic enzymes: A gold mine for tuberculosis research

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide with a strong impact in developing countries. Mycobacterium tuberculosis, the etiological agent of TB, has a high capacity to evade the host immune system and establish a chronic, asymptomatic and latent infection. In a latent TB infection, persistent bacilli are present in a non-replicating dormant state within host granulomas. During reactivation, bacilli start replicating again leading to an active TB infection that can be highly contagious. Mycobacterial lipids and lipolytic enzymes are thought to play important physiological roles during dormancy and reactivation. The role of lipolytic enzymes in the physiology of M. tuberculosis and physiopathology of the disease will be discussed in this review, with an emphasis on the secreted or cell wall-associated, surface exposed lipolytic enzymes characterized to date. Studies on the localization, enzymatic activity and immunological properties of these enzymes highlighted their possible usefulness as new diagnostic markers in the fight against TB.     

Solid state fermentation for the production of lipolytic fungal enzymes

The production of lypolitic moulds in solid state fermentation was studied.Aspergillus oryzae andRhizopus oryzae were grown on rice hulls using different media. Tween 80 and olive oil were employed as main carbon sources; the whole solid cultures were lyophilised and employed in heptane for catalysing the formation of various geranyl esters with molar conversions ranging from 40 to 95%, under optimised conditions, starting from 50 mM geraniol and equimolar amount of the acid.     

Gas phase transesterification reactions catalyzed by lipolytic enzymes

Porcine pancreatic lipase and Fusarium solani cutinase were used to catalyze transesterification reactions between methyl propionate, ethyl propionate, and a series of primary alcohols at high temperatures in a continuous packed-bed gas-solid reactor, in which the solid phase is composed of the enzyme and the substrates and products are in a gaseous form. In this type of system, enzyme activity was found to depend essentially on the water activity (A(w)) of the enzyme preparation.     

Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes

We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.     

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.     

9-Fluorenylmethoxycarbonyl-labeled peptides as substrates in a capillary electrophoresis-based assay for sirtuin enzymes

Sirtuins are the class III histone deacetylases that catalyze the deacetylation of acetyl-lysine residues of histones and other proteins using nicotinamide adenine dinucleotide (NAD⁺) as the cofactor. The reaction yields the deacetylated protein, nicotinamide, and 2’-O-acetyl-ADP-ribose. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides derived from the amino acid sequence of p53, Fmoc-KK(Ac)-NH₂, Fmoc-KK(Ac)L-NH₂, and Fmoc-RHKK(Ac)-NH₂, were characterized as substrates for two of the human sirtuins: SIRT1 and SIRT2. The deacetylation was monitored by a validated capillary electrophoresis assay. Efficient deacetylation by SIRT1 and SIRT2 was demonstrated for all three peptide substrates. The kinetics of the enzymatic reaction was determined with the Michaelis constants (K_m) varying between 16.7 and 34.6 μM for SIRT1 and between 34.7 and 58.6 μM for SIRT2. Resveratrol did not function as an activator for SIRT1 using the Fmoc-labeled peptides as SIRT substrates. The IC₅₀ values of sirtinol using the three peptide substrates were determined. Further sirtuin inhibitors were also evaluated.     

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.     

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.