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PubMed1989

Heparin decreases the degradation rate of hepatic lipase in Fu5AH rat hepatoma cells. A model for hepatic lipase efflux from hepatocytes.

The mechanism for the stimulation of hepatic lipase secretion by heparin was studied in cultured Fu5AH rat hepatoma cells. Quantitative immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radioactive enzyme; hepatic lipase protein mass was quantitated by ELISA. Addition of heparin to the medium resulted in a 2-fold increase in lipase secretion rate, whereas cell-surface-associated and intracellular lipase decreased by 76 and 20%, respectively. Rates of synthesis of hepatic lipase measured by incorporation of Trans 35S-label into enzyme protein were not different in control or heparin-treated dishes. In pulse-chase studies, it was estimated that the degradation rate constants for control and heparin-treated cultures were 0.51 +/- 0.09 and 0.14 +/- 0.13 h-1 for control and heparin-treated cultures, respectively. 52% of the synthesized enzyme was degraded in control cultures; addition of heparin to the culture medium reduced this figure to 11% of the synthetic rate. Equilibrium binding data of highly purified 125I-hepatic lipase to Fu5AH cells at 4 degrees C demonstrate the presence of a class of high-affinity binding sites. At 37 degrees C, cell-surface-bound 125I-hepatic lipase is internalized and either degraded or recycled to the medium. The half-intracellular residence times of hepatic lipase were 55 and 31 min in control and heparin-treated cultures, respectively. Radioactivity incorporated in the 55.4 kDa high-mannose-containing lipase and the mature 57.6 kDa species was measured as a means of locating the enzyme in the secretory pathway before or beyond the medial Golgi. The disappearance of the 55.4 kDa species from the cell is similar in control and heparin-treated cultures with half-intracellular residence times of 29 and 25 min, respectively. In contrast, the amount of radiolabeled 57.6 kDa species in control cells remained constant from 15 min to 2 h, whereas it decreased by 79% in heparin-treated cells. The above data demonstrate that the increase in hepatic lipase secretion is due to a decreased degradation rate with no change in synthetic rate and that heparin primarily affected the residence time of hepatic lipase in the medial Golgi-plasma membrane region.

Animals↗

PubMed1977

Are monoglyceride-lipase, triglyceride-lipase and phospholipase A of rat liver microsomes distinct protein entities?

1. Different extraction and purification techniques were employed for the separation of MG-lipase, TG-lipase and phospholipase A from rat liver microsomes. 2. Up to 60 per cent of the microsomal content of TG-lipase and phospholipase could be extracted with 1 M KCl or NaCl. MG-lipase was extracted more readily by detergents (e.g. emulphogen). 3. MG-lipase is more resistant to detergent and heat inactivation than TG-lipase and phospholipase A. It is retained, using the technique of affinity chromatography, on a column of CH-Sepharose coupled to monooleoylglycerol. In addition, MG-lipase was separated from TG-lipase by electrofocusing. 4. TG-lipase and phospholipase A were partially separated by gel filtration on Sephadex G-200 in the presence of 1 mM dithiothreitol and by chromatography on CH-Sepharose 4b. 5. On the basis of the present extraction and purification studies, it is concluded that mg-lipase is an enzyme protein distinct from TG-lipase and phospholipase A.

Animals↗

PubMed1996

The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase.

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.

Esters↗

PubMed1993

Decrease in contents of pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase in rats with streptozotocin-induced diabetes.

The changes in contents of pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase in rats with streptozotocin (STZ)-induced diabetes have been studied. The contents of pancreatic carboxyl ester lipase and phospholipase A2 decreased by 40% and 45%, respectively, 5 days after injection of STZ, whereas pancreatic lipase steadily increased to 100% over control. The content of lingual lipase decreased sharply by more than 90% 2 days after STZ injection, followed by a tendency to recover slightly. Insulin treatment at a dose abolishing the urine glucose in diabetic rats for 3 days restored the contents of pancreatic lipase, carboxyl ester lipase, and lingual lipase but not pancreatic phospholipase A2. The results indicate that lack of insulin action induces an anticoordinate change in gastrointestinal lipolytic enzymes, with decreases in pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase contents and an increase in pancreatic lipase content.

Animals↗

PubMed1991

Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation.

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.

Adipose Tissue↗

PubMed1990

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).

Animals↗

PubMed1990

Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.

Animals↗

PubMed1987

Diacylglycerol breakdown in plasma membranes of bovine chromaffin cells is a two-step mechanism mediated by a diacylglycerol lipase and a monoacylglycerol lipase.

The recently identified diacylglycerol lipase activity in membranes of chromaffin cells from bovine adrenal medulla [24] is now shown to consist of two enzymes working in series. First the predominantly saturated fatty acid in the sn-1-position is split by a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.34). Subsequently the resulting sn-2-monoacylglycerol is split by a monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) which prefers sn-2-arachidonoyl-monoacylglycerol to sn-2-palmitoyl-monoacylglycerol. At pH 4.0 only the diacylglycerol lipase is active, whereas the monoacylglycerol lipase is irreversibly inactivated. At pH 6.0 both enzymes are active. Pretreatment of the membranes at pH 10 leads to the selective inactivation of the diacylglycerol lipase. Both enzymes are Ca2+- and calmodulin-independent and both are partially inhibited by p-bromophenacyl bromide, however, only at relatively high concentrations of the inhibitor. Chlorpromazine inhibits the diacylglycerol lipase to about the same extent as p-bromophenacyl bromide but the monoacylglycerol lipase is less sensitive. The specific diacylglycerol lipase inhibitor RHC 80267 (1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane) only interacts with the first step, i.e. the diacylglycerol lipase.

Acetophenones↗

PubMed1986

Bile salt-stimulated lipase in non-primate milk: longitudinal variation and lipase characteristics in cat and dog milk.

We report the presence of bile salt-stimulated lipase in milk collected from dog and cat. This enzyme has previously been found only in the milk of human and gorilla. Bile salt-stimulated lipase activity in individual dog milk specimens (range: 4.8-107.4 U/ml; 1 U = 1 mumol [3H]oleic acid released/min) was similar, while that in cat milk specimens (range: 2.2-16.9 U/ml) was lower than in human milk (range: 10-80 U/ml). Longitudinal patterns for bile salt-stimulated lipase activity differed depending upon the enzyme source: in dog milk, lipase activity was lowest in colostrum, while in cat milk, lipase activity was highest in colostrum and decreased at mid-lactation. In human milk, bile salt-stimulated lipase activity levels remain fairly constant throughout the first 3 months of lactation. Dog, cat and human milk bile salt-stimulated lipase activity had a neutral-to-alkaline pH optimum of 7.3-8.5, was stable at low pH (above 3.0 for at least 1 h), and was inhibited 95-100% by eserine (at concentrations greater than 0.6 mM). The lipase in the milk of the three species studied had an absolute requirement for primary bile salts (tauro- and glycocholate), and was inhibited by secondary bile salts (tauro- and glycodeoxycholate). These data are the first to report bile salt-stimulated lipase activity in milk from mammals other than the highest primates. Presence of this lipase in non-primate milk will permit the study of the factors that regulate the ontogeny, synthesis and secretion of the enzyme during pregnancy and lactation as well as its function in neonatal fat digestion.

Animals↗

PubMed1980

Evaluation of the roles of lipoprotein lipase and hepatic lipase in lipoprotein metabolism: in vivo and in vitro studies in man.

The roles of lipoprotein lipase (LPL) and hepatic lipase in very low density lipoprotein (VLDL) and VLDL remnant metabolism were investigated by (1) in vivo studies where the kinetics of VLDL-apo B removal were measured in patients with non-functioning lipoprotein lipase systems, and (2) in vitro studies where the relative capacities of hepatic lipase and LPL to hydrolyse the triglyceride (TG) of different lipoprotein substrates was measured. The results indicated that VLDL-apo B removal was not impaired in patients with non-functional LPL, nor ws there any apparent abnormality in the conversion of VLDL-apo B to intermediate- (IDL) and low (LDL) density lipoprotein-apo B. Post-heparin plasma hepatic lipase activity against VLDL was normal in these subjects. Purified normal hepatic lipase had a similar Km for VLDL-TG hydrolysis (1.57 mmol/l) to that of LPL (1.49 mmol/l). However, at equal lipoprotein TG concentration, hepatic lipase had increasing activity with lipoproteins of decreasing particle size, in the order chylomicrons much less than VLDL of Sf 100-400 less than VLDL of Sf 60-100 less than VLDL of Sf 20-60 less than IDL. The mean contribution of hepatic lipase to VLDL-TG hydrolysis by post-heparin plasma was 35% in normal controls, but the contribution to IDL-TG hydrolysis was significantly higher (mean - 58%). It is concluded that hepatic lipase plays a significant role in VLDL and, especially, IDL metabolism, at least in patients with non-functioning lipoprotein lipase.

Adult↗

PubMedDate not supplied

Roles of lipoprotein lipase and hepatic triglyceride lipase in the catabolism in vivo of triglyceride-rich lipoproteins.

To define the roles, in vivo, of hepatic triglyceride lipase and lipoprotein lipase in the catabolism of triglyceride-rich lipoproteins, we investigated the relationship between the activities of the above enzymes in postheparin plasma and the fractional removal rates of very low density lipoproteins (VLDL) and VLDL remnant particles. In 22 patients, the fractional removal rates of VLDL and VLDL-remnant particles were determined from analyses of the disappearance of radioiodinated Sf 60-400 and Sf 12-60 lipoprotein B apoprotein. The maximal activities of hepatic triglyceride lipase and lipoprotein lipase were determined in plasma samples drawn 2-60 minutes after heparin injection (60 U/kg). A positive correlation was observed between the fractional removal rate of VLDL and postheparin plasma lipoprotein lipase activity (r = 0.65). When all 22 patients were considered together, no relationship was demonstrable between remnant fractional removal and postheparin plasma lipoprotein lipase activity. However, humans may be subdivided with respect to the way in which they catabolize remnants. In some, all remnant may be catabolized to form LDL. In others, some of the remnant may also be directly removed from the circulation. Those subjects in whom previous studies indicate that all remnant is converted to LDL demonstrated a positive correlation between remnant fractional removal rate and postheparin plasma lipoprotein lipase activity (n = 8, r - 0.83). No correlations between postheparin plasma hepatic triglyceride lipase activity and any of the fractional removal rates were found. These data are consistent with the following: 1) lipoprotein lipase plays a key regulatory role in the catabolism of triglyceride-rich lipoproteins; 2) this role applies only to those catabolic involving the formation of particles of higher density VLDL remnants and low density lipoprotein; and 3) hepatic triglyceride lipase plays no rate-limiting role in the catabolism of VLDL or VLDL-remnant particles.

Adult↗

PubMed1975

Serum-stimulated lipases (lipoprotein lipases). Immunological crossreaction between the bovine and the human enzymes.

A rabbit antiserum prepared against the serum-stimulated lipase (lipoprotein lipase) from bovine milk crossreacted with serum-stimulated lipases from human milk and from human postheparin plasma, but not with bile salt-stimulated lipase from human milk or with salt-resistant lipase from human postheparin plasma. Thus, the serum-stimulated lipase in bovine milk has immunological determinants in common with the serum-stimulated lipases in human milk and in human postheparin plasma. The time-courses for the appearance of serum-stimulated lipase and salt-resistant lipase activities in human plasma after heparin injection were different. The two activities were separated by heparin-Sepharose chromatography. After treatment of postheparin plasma with the antiserum only the salt-resistant lipase activity could be eluted from the column. Thus, these two enzyme activities in postheparin plasma reside in two different enzyme molecules.

Animals↗

PubMed2003

Structural insights into the lipase/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97A resolution.

The yeast Candida rugosa produces several closely related extracellular lipases that differ in their substrate specificity. Here, we report the crystal structure of the isoenzyme lipase 2 at 1.97A resolution in its closed conformation. Lipase 2 shows a 79.4% amino acid sequence identity with lipase 1 and 82.2% with lipase 3, which makes it relevant to compare these three isoenzymes. Despite this high level of sequence identity, structural comparisons reveal several amino acid changes affecting the flap (residue 69), the substrate-binding pocket (residues 127, 132 and 450) and the mouth of the hydrophobic tunnel (residues 296 and 344), which may be responsible for the different substrate specificity and catalytic properties of this group of enzymes. Also, these comparisons reveal two distinct regions in the hydrophobic tunnel: a phenylalanyl-rich region and an aliphatic-rich region. Whereas this last region is essentially identical in the three isoenzymes, the phenylalanyl content in the first one is specific for each lipase, resulting in a different environment of the catalytic triad residues, which probably tunes finely their lipase/esterase character. The greater structural similarity observed between the monomeric form of lipase 3 and lipase 2 concerning the above-mentioned key residues led us to propose a significant esterase activity for this last protein. This enzymatic activity has been confirmed with biochemical experiments using cholesteryl [1-14C]oleate as substrate. Surprisingly, lipase 2 is a more efficient esterase than lipase 3, showing a twofold specific activity against cholesteryl [1-14C]oleate in our experimental conditions. These results show that subtle amino acid changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties.

Amino Acid Sequence↗

PubMed2000

Identification of a second lipase gene, gehD, in Staphylococcus epidermidis: comparison of sequence with those of other staphylococcal lipases.

The identification and molecular characterization of a previously unidentified lipase, gehD, from the human cutaneous commensal Staphylococcus epidermidis is reported. A lipase-GehC-deficient but otherwise isogenic mutant of S. epidermidis 9 was constructed by allele replacement. However, the mutant was found to retain 50% of the wild-type lipase activity in liquid culture. Rescreening of a genomic library revealed the presence of a second lipase gene, gehD, which was subsequently mapped and sequenced. In common with other staphylococcal lipases, GehD appeared to be translated as a 650-700 amino acid precursor which is processed post-translationally to an extracellular mature lipase of 360 amino acids with a size of approximately 45 kDa. Comparison of the amino acid sequence of GehD with those of other staphylococcal lipases revealed a high level of conservation between the mature lipase domains of different species. By hybridization studies, both gehC and gehD genes were found to be present in S. epidermidis isolates from both clinical and non-clinical backgrounds, but neither hybridized to DNA isolated from other staphylococcal strains. Construction of a phylogenetic tree and calculation of amino acid sequence homologies between mature lipases, however, suggested that the lipases of S. epidermidis may be more closely related to those of Staphylococcus aureus than to each other.

Amino Acid Sequence↗

PubMed1992

Sequence of horse pancreatic lipase as determined by protein and cDNA sequencing. Implications for p-nitrophenyl acetate hydrolysis by pancreatic lipases.

The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p-nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p-nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C-terminal domain (336-449) of human lipase reveals that this hydrolysis is the result of the superimposition of independent events; a specific linear hydrolysis occurring at the active site of lipase, a fast acylation depending on the presence of Lys373 and a non-specific hydrolysis most likely occurring in the C-terminal domain of the enzyme. This finding definitely proves that pancreatic lipase bears only one active site and raises the question of a covalent catalysis by pancreatic lipases. Moreover, based on sequence comparison with the above-mentioned pancreatic lipases, three residues located in the C-terminal domain, Lys349, Lys398 and Lys419, are proposed as possible candidates for lipase/colipase binding.

Amino Acid Sequence↗

PubMed2001

Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30 degrees C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml. OD (optical density) (28 microg/ml. OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.

ATP-Binding Cassette Transporters↗

PubMed2001

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator.

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Bacterial Proteins↗

PubMed2000

Production of extracellular lipases by Penicillium cyclopium purification and characterization of a partial acylglycerol lipase.

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.

Amino Acid Sequence↗