Showing papers on "Lipase published in 1994"

Two conformational states of Candida rugosa lipase.

Abstract: The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.

Novel lipase, microorganism producing the lipase, process for producing the lipase, and use of the lipase

Abstract: A microorganism belonging to the genus Pseudomonas, an alkaline lipase produced thereby and having the following properties, a process for producing the lipase, and a detergent composition containing the lipase: (1) base: triolein emulsion, working pH: 3.5 to 12 and optimum pH: 10 to 11; (2) base: triolein emulsion, working temperature: 30 to 80 °C, and optimum temperature: 55 to 65 °C; (3) molecular weight determined by SDS-polyacrylamide gel electrophoresis: 31,000±2,000; and (4) isoelectric point determined by isoelectric polyacrylamide gel electrophoresis: 5.2±0.5. This lipase is highly stable to the components of a detergent, such as surfactant and protease, can be used together with protease when incorporated in a detergent, is reduced in the damage to activity caused by the components of a detergent, and can increase the detergency of a detergent.

New lipase variants for use in detergent applications

Abstract: Modified lipases are disclosed whereby at least the methionine at a position corresponding to position 21 in wild-type Pseudomonas pseudoalcaligenes lipase is substituted by another amino acid, which lipases exhibit a desired property change, in particular improved wash performance. Preferred embodiments are substitutions of said methionine by leucine, serine or alanine.

Lipase gene and variant lipase

Abstract: A lipase gene isolated from a chromosome DNA of Pseudomonas mendocian SD702; a variant lipase gene obtained by the variation of the above gene; and a variant lipase coded for by the above variant gene and having physical or chemical properties changed thereby. The invention gene serves to facilitate production and modification of lipase LP or a variant lipase thereof. The modification of lipase LP permits production of a variant lipase that has a new amino acid sequence and is more suitable to industrial and other application than the lipase LP, thus providing a lipase useful in the fields of detergent, food processing, papermaking and so forth.

Lipases : their structure, biochemistry and application

Abstract: 1. Sequence comparisons within the lipase family 2. A sequence analysis of lipases, esterases and related proteins 3. Lipases from plants 4. Three-dimensional structures of two lipases from filamentous fungi 5. Structural aspects of phospholipase C 6. Phospholipase A2: mechanism and structure 7. Structure and mechanism of human pancreatic lipase 8. Kinetics of triglyceride lipases 9. Gastric lipases: cellular, biochemical and kinetic aspects 10. Lipase inhibitors 11. Substrates for phospholipase C and sphigomyelinase 12. Isolation and purification of lipases 13. Industrial applications of lipases 14. Lipases and phospholipases in organic synthesis 15. Medical aspects of triglyceride lipases 16. Protein engineering of lipases Index.

Evidence that palmitic acid is absorbed as sn-2 monoacylglycerol from human milk by breast-fed infants.

Abstract: Milk fatty acids consist of about 20-25% palmitic acid (16:0), with about 70% of 16:0 esterified to the sn-2 position of the milk triacylglycerols. Hydrolysis of dietary triacylglycerols by endogeneous lipases produces sn-2 monoacylglycerols and free fatty acids, which are absorbed, reesterified, and then secreted into plasma. Unesterified 16:0 is not well absorbed and readily forms soaps with calcium in the intestine. The positioning of 16:0 at the sn-2 position of milk triacylglycerols could explain the high coefficient of absorption of milk fat. However, the milk lipase, bile salt-stimulated lipase, has been suggested to complete the hydrolysis of milk fat to free fatty acids and glycerol. These studies determined whether 16:0 is absorbed from human milk as sn-2 monopalmitin by comparison of the plasma triacylglycerol total and sn-2 position fatty acid composition between breast-fed and formula-fed term gestation infants. The human milk and formula had 21.0 and 22.3% of 16:0, respectively, with 54.2 and 4.8% 16:0 in the fatty acids esterified to the 2 position. The plasma triacylglycerol total fatty acids had 26.0 +/- 0.6 and 26.2 +/- 0.6% of 16:0, and the sn-2 position fatty acids had 23.3 +/- 3.3 and 7.4 +/- 0.7% of 16:0 in the three-month-old exclusively breast-fed (n = 17) and formula-fed (n = 18) infants, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a pancreatic lipase subfamily with new kinetic properties.

Abstract: Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phospholipases under physiological conditions. In fact, all RP2 lipases from other species might share phospholipase activity and fulfill new biological functions.

Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications

Abstract: A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent. © 1994 John Wiley & Sons, Inc.

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Abstract: Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.

Blueprint for a lipase support: Use of hydrophobic controlled-pore glasses as model systems.

Abstract: For the commercial exploitation of lipase biocatalysis to be successful, it is essential that effective supports are selected for lipase immobilization. In this study hydrophobic controlled-pore glasses have been used as model systems for the immobilization of Rhizomucor miehei lipase. The effect of pore diameter and surface chemistry on enzyme efficiency in a typical esterification reaction under essentially nonaqueous conditions has been examined. It has been found that pore diameters of at least 35 nm are needed for the lipase to be able to utilize the internal volume of the support particles in the immobilization process. Despite the small size of the substrates in the esterification reaction, even larger pores (>100 nm) are required for the lipase efficiency to become independent of pore diameter; below 100 nm lipase activity and efficiency are markedly reduced. It has also been shown that the chemical nature of the hydrophobic surface plays an important part in catalyst design. Although lipase will adsorb readily to a wide range of hydrophobic groups, the highest catalyst activities are obtained when the glass surface is derivatized to give long alkyl chains; the presence of unsaturated derivatives gonerally leads to a reduction in activity. (c) 1994 John Wiley & Sons, Inc.

Relationship between water activity and catalytic activity of lipases in organic media. Effects of supports, loading and enzyme preparation.

Abstract: The rates of synthesis of dodecyl decanoate in hexane have been measured as a function of water activity (aw), for various immobilised preparations of the lipases from Rhizomucor miehei and Candida rugosa. Only very large changes in the amount of enzyme adsorbed to the support affect the shape of the rate/aw profile; at the highest loadings the profiles tend to become somewhat flatter. A similar levelling can be obtained by pre-adsorbing an inert protein. The effect is probably due to adjacent protein molecules effectively replacing water; it does not simply reflect mass transfer or interfacial area limitation. The activity/aw profile was essentially the same with most supports tested: polypropylene, anion-exchange resin, celite, anion-exchange modified silica. A hydrophobic porous glass support reduced the rate somewhat at intermediate aw values with both enzymes; a polyamide material had this effect only with the lipase from Rh. miehei. The shape of the activity/aw profile was not affected by large differences in purity of the lipase preparation, but did differ between forms that probably differ in glycosylation. Overall, relatively few manipulations of the system can significantly affect the shape of the rate/aw profiles, which seem to be mainly an intrinsic property of the enzyme molecules used.

Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase.

Abstract: Lysosomal acid lipase (LAL) is a hydrolase essential for the intracellular degradation of cholesteryl esters and triacylglycerols. This report describes a multi-step procedure for the purification of LAL from human liver. After solubilization with non-ionic detergent, acid hydrolase activity was purified 17000-fold to apparent homogeneity by sequential chromatography on Concanavalin A Sepharose, carboxymethyl-cellulose, phenyl Superose, Mono S cation exchange and Superose 12 gel-filtration columns. This procedure yielded two silver-staining protein bands of 56 kDa and 41 kDa on SDS/PAGE. Size-exclusion chromatography of the 41-kDa protein indicated that the enzyme was catalytically competent as a monomer of approximately 38 kDa. When assayed in the presence of cholesteryl oleate or trioleoylglycerol, purified acid lipase had Vmax values of 4390 nmol fatty acid.min-1.mg protein and 4756 nmol fatty acid.min-1.mg protein-1, and apparent Km values of 0.142 mM and 0.138 mM, respectively. The purified enzyme was most active at low pH (4.5-5.0) and required non-ionic detergent and ethylene glycol for optimal stability. Incubation of the 41-kDa acid lipase with endoglucosaminidase H reduced the molecular mass by 4-6 kDa, demonstrating Asn-linked glycosylation with high-mannose oligosaccharides. Deglycosylation did not affect enzymic activity, indicating that carbohydrates are not required for LAL activity. Based on partial peptide sequence, an oligonucleotide was synthesized and utilized to isolate LAL cDNA clones from a human liver cDNA library. A full-length LAL cDNA contained 2626 nucleotides and coded for a predicted protein of 372 amino acids, preceded by a 27 residue hydrophobic signal peptide. Hepatic LAL differed from fibroblast acid lipase at the N-terminus and revealed extensive similarities with human gastric lipase and rat lingual lipase, confirming a gene family of acid lipases. Northern hybridization using the complete LAL cDNA as a radiolabeled probe indicated striking differences in mRNA expression among human tissues. LAL mRNA was most abundant in brain, lung, kidney and mammary gland. Placenta and HeLa cells expressed intermediate amounts of LAL mRNA, while RNA extracted from liver and heart showed low levels of expression.

Variability within the Candida rugosa Upases family

Abstract: Several fungi secrete lipase isozymes differing in biochemical properties and in some cases in substrate specificity. In the yeast Candida rugosa, a family of related genes encodes for multiple lipase proteins, highly homologous in sequence but partially different in the regions interacting with the substrate molecule. Analysis of these substitutions performed on the basis of multiple alignments and using a 3-D model of the enzyme, allows identification of a restricted number of amino acids possibly involved in substrate specificity of Candida lipases.

Enzymatic Esterification by Surfactant‐Coated Lipase in Organic Media

Abstract: Surfactant-coated lipases have been prepared with a synthesized surfactant. Preparation conditions to obtain a suitable surfactant-coated lipase were investigated. The enzymatic activity of the lipase in an organic solvent significantly increased with the coating of the surfactant. The esterification rate from the surfactant-coated lipase was much higher than that from the powder lipase. An aliphatic solvent showed higher activity than did alcohol, aromatic, and chloric solvents. Among them, isooctane gave the highest activity. The reactivity of the surfactant-coated lipase depends on the pH of the aqueous solution in the preparation and on the buffer solution. Surfactant-coated lipase prepared in the middle pH range using phosphate buffer showed high enzymatic activity. The surfactant-coated lipase was thermostable at high temperature compared to the native lipase. A kinetic study enabled a ping-pong bi-bi reaction mechanism with alcohol inhibition to be suggested. From the kinetic analysis, it was found that an alcohol substrate inhibits enzymatic esterification by lipase. The reaction rate of the coated lipase was about 100 times that of the powder lipase.

Purification and Characterization of an Alkaline Lipase from Pseudomonas fluorescens AK102

Abstract: An extracellular, novel alkaline lipase produced by Pseudomonas fluorescens AK102 was purified by ultrafiltration, ammonium sulfate precipitation, and DEAE-Toyopearl 650M and Phenyl-Toyopearl 650M column chromatographies. The purified enzyme was homogeneous on SDS-PAGE. The molecular weight was estimated to be about 33,000 by SDS-PAGE. The isoelectric point was pH 4.0 by isoelectric focusing. The pH stability was 4 to 10 and the optimum pH was 8 to 10. The optimum temperature was 55 degrees C and the enzyme was stable below 50 degrees C. The enzyme unspecifically liberated short chain to long chain fatty acids from p-nitrophenyl esters, methyl esters, and triglycerides. In the presence of an anionic surfactant, the enzyme was characteristically stable. These results suggested that the enzyme can be used as a home laundry product ingredient.

Development of a new bacillus carboxyl esterase for use in the resolution of chiral drugs

Abstract: We have screened a new enzyme for the resolution of R, S-naproxen enantiomers. The enzyme is free of lipase activity, and possesses a very high sterospecificity on S-naproxen [2-(6-methoxy-2-naphthyl)-propionic acid] esters and esters of related drugs. The primary structure of the enzyme, determined from the nucleotide sequence, shows limited homology with the catalytic site of lipases. The gene coding for the steroselective carboxylesterase has been cloned and expressed in Bacillus subtilis. Using a multicopy vector and an additional strong promoter an efficient production process was developed. The enzyme was shown to be sensitive to very high concentrations of the products formed during the reaction it catalyses. To increase the resistance of the enzyme, lysine residues thought to be responsible for this phenomnon were replaced through site-directed mutagenesis. Enzymes with improved stability were obtained. An explanation is given in terms of a model in which a reaction of the acid moiety of naproxen with free lysine NH2 groups is a major cause of inactivation.

Hepatic lipase may act as a ligand in the uptake of artificial chylomicron remnant-like particles by isolated rat hepatocytes.

Abstract: Active and heat-inactivated hepatic lipase stimulated to a statistically comparable extent the uptake of chylomicron remnant-like particles by isolated rat hepatocytes by 3-fold and 2.3-fold respectively and, likewise, their binding to hepatic plasma membranes by 5-fold and 4-fold respectively. Hepatic lipase may facilitate uptake of these particles, not only as a lipolytic enzyme, but also as a ligand anchored to extracellular glycosaminoglycans.

The Pseudomonas fluorescens lipase has a C‐terminal secretion signal and is secreted by a three‐component bacterial ABC‐exporter system

Abstract: Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N-terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide-independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C-terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three-component bacterial ABC-exporter systems.