Home About us Contact
|
Home About us Contact | ||
|
|
||
Lipolytic Enzyme (lipolytic + enzyme)
Selected AbstractsA novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolaseFEBS JOURNAL, Issue 21 2000Thorsten Eggert A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p -nitrophenyl-esters of fatty acids with short chain lengths of ,,10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5,7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase. [source] Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organismJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008C. Roh Abstract Aims:, The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro-organisms. Methods and Results:, The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35·6 kDa. Typical residues essential for lipolytic activity such as penta-peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%, AAY45707) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7·5 and 10°C. At thermal stability analysis, lipo1 was more unstable at 40°C than 10°C. Conclusions:, An activity based strategy has been an effective method for fishing out a low-temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone-sensitive lipase family due to the enzyme's oxyanion hole by the sequence HGGG. Significance and Impact of the Study:, Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry. [source] Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2004Martin Zehl Abstract A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H]2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright © 2004 John Wiley & Sons, Ltd. [source] The lipase/acyltransferase from Candida parapsilosisFEBS JOURNAL, Issue 6 2002Molecular cloning, characterization of purified recombinant enzymes Candida parapsilosis has been previously shown to produce a lipase (i.e. able to catalyze efficiently the hydrolysis of insoluble lipid esters such as triacylglycerols) that preferentially catalyses transfer reactions such as alcoholysis in the presence of suitable nucleophiles other than water, even in aqueous media with high (> 0.9) water thermodynamic activity. The present work describes the cloning and the overexpression of the gene coding for this enzyme. Two ORFs (CpLIP1 and CpLIP2) were isolated. The deduced 465-amino-acid protein sequences contained the consensus motif (G-X-S-X-G) which is conserved among lipolytic enzymes. Only one of the two deduced proteins (CpLIP2) contained peptide sequences obtained from the purified lipase/acyltransferase. Homology investigations showed that CpLIP2 has similarities principally with 11 lipases produced by C. albicans (42,61%) and the lipase A from Candida antarctica (31%) but not with the other lipases sequenced so far. Both CpLIP1 and CpLIP2 were expressed in Saccharomyces cerevisiae, but only CpLIP2 coded for an active protein. The substrate specificity and the catalytic behavior of purified recombinant CpLIP2, with or without a C-terminal histidine tag, were not changed compared to those of the native lipase. [source] Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesisCELL BIOCHEMISTRY AND FUNCTION, Issue 5 2007Fatma M. Helmy Abstract The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38°C for 60,min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA2 action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed. Copyright © 2007 John Wiley & Sons, Ltd. [source] Calcium-Induced Membrane Microdomains Trigger Plant Phospholipase D ActivityCHEMBIOCHEM, Issue 17 2008Konstantin Kuppe Abstract Plant ,-type phospholipase D proteins are calcium-dependent, lipolytic enzymes. The morphology of the aggregates of their phospholipid substrate fundamentally defines the interaction between the enzyme and the surface. Here we demonstrate that the Ca2+ -induced generation of membrane microdomains dramatically activates ,-type phospholipase D from white cabbage. 500-fold stimulation was observed upon incorporation of 10 mol,% 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphate (POPA) into 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) vesicles in the presence of Ca2+ ions. Enhanced association of PLD,2 with phospholipid surfaces containing anionic components was indicated by lag phase analysis and film balance measurements. Differential scanning calorimetry showed that the POPA-specific activation correlates with the phase behavior of the POPC/POPA vesicles in the presence of Ca2+ ions. We conclude from the results that the Ca2+ -induced formation of POPA microdomains is the crucial parameter that facilitates the binding of PLD to the phospholipid surface and suggest that this effect serves as a cellular switch for controlling PLD activity. [source] | |||||||||||||