Partial purification and characterization of sphingosine N-acyltransferase (ceramide synthase) from bovine liver mitochondrion-rich fraction

Sphingosine N-acyltransferase (ceramide synthase, E.C. 2.3.1.24) was solubilized from bovine liver mitochondrion-rich fraction with n-ocytl β-d-thioglucoside as the detergent and partially purified by sequential chromatography on columns of DE-32, shingosine affinity, and Sepharose CL-6B. The partially purified preparation migrated on SDS-polyacrylamide gel electrophoresis as two major protein bands of 62 and 72 kDa. The molecular mass of the enzyme estimated by gel filtration was 240–260 kDa, suggesting that the partially purified enzyme is present in a subunit form or simply has an aggregative nature. The specific activity of the final preparation for the condensation of sphingosine with stearoyl-CoA increased by 98.7-fold compared with the starting material. The optimal pH value for the ceramide synthesis was 7.5. The partially purified enzyme had an apparent K _m of 146 μM and a V _max of 11.1 nmol/min/mg protein for stearoyl-CoA. The K _m and V _max values toward sphingosine were 171 μM and 11.3 nmol/min/mg protein, respectively. Interestingly, sphinganine was also a good substrate for this enzyme, and the K _m and V _max values were 144 μM and 8.5 nmol/min/mg protein, respectively.     

Electrochemical investigations of sarcoma-related protein kinase inhibition

An electrochemical biosensor was developed for the determination of sarcoma (Src)-related protein kinase-catalyzed phosphorylation reactions in the presence of adenosine 5′-γ-ferrocenoyl triphosphate (Fc-ATP). The sensing platform is based on a highly specific amino acid sequence Glu-Gly-Ile-Tyr-Asp-Val-Pro (EGIYDVP), to which a Fc-PO₂ moiety can be transferred from Fc-ATP by the action of the Src kinase. The enzyme kinetics and kinase inhibition were investigated by square wave voltammetry (SWV). The kinetic parameters K_m and V_max were determined for Src protein kinase with respect to Fc-ATP co-substrate and were found to be 200 μM and 115 μA cm⁻² min, for phosphorylation of the EGIYDVP peptide substrate. Furthermore, the Src-catalyzed phosphorylation of Tyr was investigated in the presence of the small molecule inhibitors PP1, PP2, SU6656, and roscovitine. PP3 does not inhibit Src activity and was used as a control. The percent inhibition at half concentration, IC₅₀, values were determined for all inhibitors under the study and were estimated to be in the 5–30 nM range. The electrochemical study suggests that the increase in inhibition efficiency was in the order PP3 < SU6656 < roscovitine < PP2 < PP1.     

Characterization of phospholipase D activity in bovine photoreceptor membranes

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2³H]glycerol-3-phosphocholine (PC), or [¹⁴C]ethanol. Using [³H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [³H]phosphatidic acid and phosphatidylethanol [³H]PtdEt; whereas with [¹⁴C]ethanol or [³H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [¹⁴C]PtdEt or [³H]phosphatidylglycerol, respectively). The use of varying concentrations of [³H]PC and 400 mM of ethanol gave an apparent K _m value for PC of 0.51 mM and a V _max value of 111 nmol × h⁻¹ × (mg protein)⁻¹. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent K _m of 202 mM and a V _max value for ethanol of 125 nmol × h⁻¹ × (mg protein)⁻¹. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca²⁺ or Mg²⁺ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Effect of pressure on the extractive biocatalysis of ethanol

The effect of pressure on the esterification reaction of ethanol with water-immiscible organic acids, catalysed by a lipase from Mucor miehei (pH 4.5; 30°C), was studied through analysis of the kinetics and equilibrium parameters. An increase of the ethanol distribution between the aqueous and organic phases was observed by the addition of lipase and the increase of the pressure in the system. Furthermore, the enzyme showed high specificity for the acid substrate, esterifying preferentially long chain fatty acids (C₈-C₁₈). In the studies described oleic acid was used as substrate for the esterification reaction. A kinetic study with the free enzyme, showed that pressure affected the extraction system, increasing the maximum reaction rate (> V_max), the affinity (< K_m) and the specificity (> V_max/K_m = k_sp) of the enzyme to the substrate, probably due to the effect of pressure on the electrostatic interactions in biological systems. The enzyme operational stability, at 30°C, improved significantly with the increase of pressure, having lower values for the deactivation constant (k) (8.3 × 10^?3 h^?1) and higher values for the half-life times (t_1/2) (77 h) in comparison with those obtained under atmospheric pressure conditions (k = 2.3 × 10^?2h^?1; t_1/2 = 30 h).     

Characterization of partially purified extracellular lipase fractions from Pseudomonas fragi CRDA 037

The purification of extracellular lipases from the culture medium of Pseudomonas fragi CRDA 037 was obtained by ammonium sulfate precipitation, followed by ion-exchange chromatography and then by size exclusion chromatography, and re-size exclusion chromatography, which resulted in two enzymatic fractions, FIVa′ and FIVb′. The fractions FIVa′ and FIVb′ had specific activities of 105.5 and 121.6 U/mg, respectively, with purification folds of 169.3 and 195.2, respectively, using triacetin as a substrate. The two purified fractions showed optimal activities at pH 9.5 and 10.0, respectively, at 80°C. Three bands were found in fraction FIVa′ and two bands in fraction FIVb′ by native polyacrylamide gel electrophoresis; these results indicated that homogeneity of the purified fractions was not achieved. The enzyme efficiency values, calculated as the ratio of V _max to K _m value for fractions FIVa′ and FIVb′, were 72.16 × 10⁻² and 38.15 × 10⁻², respectively. The lipase activity of fraction FIVa′ was more specific for the hydrolysis of fatty acid esters with fatty acid chain lengths of C12 to C18, whereas that of fraction FIVb′ showed a relatively broader range of specificity. The lipase activity of fraction FIVa′ showed higher specificity toward triacetin, tristearin, and tripalmitin as the substrate, whereas that of fraction FIVb′ exhibited higher affinity toward triacetin, trimyristin, and triolein. The effect of selected salts and detergents on the lipase activity of the purified fractions was also investigated. The lipase activity of the purified lipase fractions was completely inhibited by 10 mM of FeCl₂, FeCl₃, and Ellman’s reagent. However, 10 mM of CaCl₂ and EDTA activated the two purified lipase fractions by 20 to 50%.     

Site-directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis

A fragment of Staphylococcus epidermidis lipase gene (Lys-303 to Lys-688) was inserted into plasmid pET-20b(+). The resulting C-terminal His-tagged recombinant protein (43 kDa) was overexpressed in Escherichia coli BL21(DE3) as a highly active lipase and was purified with nickel-coupled resin. Putative catalytic sites were determined by site-directed mutagenesis. Mutant enzymes (S418C and H648K) lost enzyme activities, which strongly suggests that the proposed residues of Ser-418 and His-648 are involved in catalysis. Site-directed mutagenesis showed that in comparison with wild-type enzyme, the M419A and V649l enzymes showed a 2.0- and 4.0-fold increase in the k _cat/K _m′ respectively, but the M419l, M419Q, V649A, and V649L variants lost enzyme activities. The wild-type enzyme and the V649l mutant favored the hydrolysis of p-nitrophenyl esters of butyrate, but the M419A favored decanoate. The results suggested that the amino acid residues (Met-419 and Val-649), following the catalytic triad, could affect the substrate specificity and/or catalytic efficiency. This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists' Society, San Diego, CA.     

Some Biochemical Properties of Lipase from Bay Laurel (<Emphasis Type="Italic">Laurus nobilis</Emphasis> L.) Seeds

Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein⁻¹ in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. K _m and V _max values were determined as 0.975 g and 1.298 U mg protein⁻¹, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, and Mg²⁺ lightly enhanced bay laurel lipase activity.     

Study of a Lipase from Candida rugosa Diddens and Lodder

Lipasic system of Candida rugosa (CBS 613) strain was studied. The enzyme was purified in one step by hydrophobic chromatography. The properties of this lipase were determined. It is an oligomeric enzyme composed of five identical monomers of 46 kg · mol^?1. Its optimum reaction conditions are pH = 7 and temperature = 40°C. This enzyme presents a rapid thermal denaturation and then a more stable form. It is a cell-bound lipase which is induced by triacyl glycerols. This enzyme presents a high specificity for external positions on glycerol.     

Purification and characterization of an extracellular lipase from <Emphasis Type="Italic">Geotrichum marinum</Emphasis>

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V _max and K _m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na⁺, K⁺, Ca²⁺, and Mg²⁺ stimulated activity, but Na⁺, and K⁺ at 500 mM and Fe²⁺ and Mn²⁺ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

Trp89 in the lid ofHumicola lanuginosa lipase is important for efficient hydrolysis of tributyrin

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a V_max=6000±300 mmol·min⁻¹·g⁻¹ and an apparent K_m=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent K_m for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.     

Biocatalytic Properties of Lipase from Walnut Seed (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K _m and V _max values were determined as 48 mM and 23.06 × 10⁻³ U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Ni²⁺ ions, while Mg²⁺ and Zn²⁺ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.     

PC and PE synthesis: mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1)

The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual-specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP-choline or CDP-ethanolamine to DAG to form PC and PE. We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1. A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides. The CEPT1 enzyme displayed an apparent K _m of 36 μM for CDP-choline and 4.2 mol% for di-18∶1 DAG with a V _max of 14.3 nmol min⁻¹ mg⁻¹. When CDP-ethanolamine was used as substrate, the apparent K _m was 98 μM for CDP-ethanolamine and 4.3 mol% for di-18∶1 DAG with a V _max of 8.2 nmol min⁻¹ mg⁻¹. The preferred diradylglycerol substrates used by CEPT1 with CDP-choline as the phosphobase donor were di-18∶1 DAG, di-16∶1 DAG, and 16∶0/18∶1 DAG. A major difference between previous emulsion-based assay results and the mixed micelle results was a complete inability to use 16∶0(O)/2∶0 as a substrate for the de novo synthesis of platelet-activating factor when the mixed micelle assay was used. When CDP-ethanolamine was used as the phosphobase donor, 16∶0/18∶1 DAG, di-18∶1 DAG, and di-16∶1 DAG were the preferred substrates. The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1. The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC₅₀ values of 40 μM.     

Kinetic data and substrate specificity of a keratinase from Chryseobacterium sp. strain kr6

BACKGROUND: Keratinases are important enzymes for biotechnological processes involving keratin hydrolysis. In this work substrate specificity and kinetic properties of a keratinase from Chryseobaterium sp. were investigated. RESULTS: The optimal conditions for activity of purified keratinase with respect to pH, temperature and sodium chloride concentration were established using factorial design and surface response techniques. The optimum conditions for keratinase activity were pH from 7.4 to 9.2, temperature from 35 °C to 50 °C and NaCl concentration from 50 to 340 mmol L^?1, having azocasein as substrate. Subsequently, the kinetic parameters for this substrate were determined to be K_m = 0.75 mg mL^?1 and V_max = 59.5 U min^?1. The K_i value for 1,10‐phenanthroline was estimated at 0.78 mmol L^?1. The enzyme specificity was evaluated over different synthetic and insoluble substrates. The protease exhibited specificity with selectivity for hydrophobic and positively charged residues. In relation to the insoluble substrates, the enzyme hydrolyzed preferably chicken nails. CONCLUSIONS: This enzyme effectively hydrolyzes insoluble keratin substrates. The knowledge of keratinase properties is an essential step in the development of biotechnological processes involving keratin hydrolysis. Copyright © 2008 Society of Chemical Industry     

Esterification of polyunsaturated fatty acids by various forms of immobilized lipase from Rhizomucor miehei

Ethyl esterification specificity of a lipase from Rhizomucor miehei for polyunsaturated fatty acids (PUFA) was compared at 1 and 100 mM to study molecular recognition of PUFA. The chemical shift of methylene adjacent to carboxyl groups in the nuclear magnetic resonance spectrum of docosahexaenoic acid (DHA) in ethanol moved to a lower magnetic field as the concentration of DHA increased, suggesting that the degree of dissociation of DHA decreased. Specificity constants or apparent second-order rate constants (V _max/K _m or catalytic power) for 1 mM esterification by immobilized lipases were higher than the native lipase. Immobilized hydrophobic carrier of low mass transfer resistance for the esterification substrate may improve maximal velocity and affinity for the substrate. Higher specificity constants for 1 mM substrates were observed using immobilized lipases fixed on an anion exchange resin with glutaraldehyde and on a cation exchange carrier with carbodiimide. Activity yields measured with 1 mM PUFA substrate were high. For the substrates at a concentration of 100 mM, higher specific constants with these bifunctional reagents were not observed but higher activity yields were found.     

Activation of the phospholipase A1 activity of lipoprotein lipase by apoprotein C-II

The effect of apo very low density lipoprotein (apo VLDL) and apoprotein C-II on the phospholipase A₁ activity associated with lipoprotein lipase (E.C.3.1.1.3) was studied using purified bovine milk lipoprotein lipase. The enzyme degraded¹⁴C phosphatidylcholine (PC) to¹⁴C 2-acyl lysophosphatidylcholine at a rate of 0.28±0.01 nmol/min/ml and triolein at a rate of 20.3±0.4 nmol/min/ml in mixed emulsions of PC and triolein. The phospholipase activity and triacylglycerol lipase activity were both increased by the addition of apo VLDL and apoprotein C-II. After maximal activation, the rate of PC degradation was 1.19±0.02 nmol/min/ml and triolein degradation 64.4±0.4 nmol/min/ml. Activation of phospholipase A₁ activity and triacylglycerol lipase activity occurred in parallel.     

Biochemical Characterization of Purified Esterase from Soybean (Glycine max) Seed

Alkaline esterase (carboxylic‐ester hydrolases; EC 3.1.1.1) extracted from germinated soybean seeds (Glycine max) was purified approximately 3.6 times by chromatography in a DEAE‐cellulose anion exchange column and filtration in Sephadex G100 gel. The molecular mass of the enzyme was estimated at 45 kDa by gel electrophoresis (SDS‐PAGE). The purified enzyme showed a specific activity of 5.6 U mg^?1 using p‐nitrophenyl butyrate as substrate. The esterase showed optimal activity at 47 °C in moderately alkaline pH, low stability in temperatures higher than 50 °C, and high stability at pH values between 6 and 9.5. The Ca²⁺ and Co²⁺ ions proved to have a positive effect on enzyme activity; however, Hg²⁺ completely inhibited esterase activity. Using p‐nitrophenyl butyrate as substrate, the enzyme showed a K_m of 0.39 mM, V_max of 31.5 mM mg^?1 min^?1 and k_cat 7.60 × 10⁶ s^?1. Regarding substrate affinity, the enzyme showed greater activity for substrates containing short‐chain fatty acids, especially p‐nitrophenyl acetate. Such characteristics give the enzyme great potential for application in the production of low molecular weight esters, in the food industry, and in chemical products. This enzyme is another new member of the family of lipases and esterases from vegetable seeds with high activity and stability in alkaline pH.     

Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169

Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni²⁺. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter V_max of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.     

Detergent Compatible Extracellular Lipase from Streptomyces cellulosae AU‐10: A Green Alternative for the Detergent Industry

Enzymes can decrease the environmental and economic load of detergent products by reducing the amount of chemicals used in detergents and by allowing washing at ambient temperatures. In this study, Streptomyces cellulosae AU‐10 (GenBank accession number: MG780240) lipase was purified 7.08‐fold with 68% yield using an aqueous 2‐phase system. The Streptomyces sp. AU‐10 lipase showed maximal activity at pH 9.0 and 40 °C. Hundred percent activities were measured in the pH range from 9.0 to 11.0 for 1 h. The enzyme was also highly stable at 30–50 °C. The values of K_m and V_max were calculated as 0.34 mM and 0.83 mM min^?1, respectively. The lipase has high hydrolytic activity for olive oil and sunflower oil. The effect of ethylenediamine tetraacetic acid on the enzyme has shown that the lipase is a metalloenzyme. The activity increased in the presence of Fe²⁺, Cu²⁺, and various boron compounds. The enzyme has shown a good stability not only with surfactants but also with oxidizing agents. In addition, activities in the presence of Omo, Ariel, Tursil, Pril, and Fairy were measured as 108.8%, 115.6%, 98.35%, 140.4%, and 107.6%, respectively. Considering its remarkable ability, the S. cellulosae AU‐10 lipase can be considered as a potential additive in the detergent industry.     

Production and Characteristics of an Enantioselective Lipase from Burkholderia sp. GXU56

The lipase production of Burkholderia sp. GXU56 was influenced by carbon and nitrogen sources, inorganic salts, initial pH of the medium and cultivation temperature. The maximum lipase production was 580.52 U/mL and reached 5 times the level of the basic medium in the optimum medium at pH 8.0, 32 °C, 200 rpm and 40–48 h. The lipase was purified 53.6 fold to homogeneity and the molecular weight was 35 KDa on SDS‐PAGE. The optimum pH and temperature of the lipase were 8.0 and 40 °C, respectively, and it was stable in the range of pH 7–8.5 and at temperatures below 45 °C. The lipase activity was strongly inhibited by Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Fe³⁺ ions and SDS, while it was stimulated by Li⁺ and Ca²⁺ ions and in presence of 0.1 % CTAB, 0.1 % Triton X‐100 and 10 % DMSO. K_m and V_max of the lipase were calculated to be 0.038 mmol/L, and 0.029 mmol/L min^–1, respectively, with PNPB as the substrate. The GXU56 lipase showed enantioselective hydrolysis of (R,S)‐methyl mandelate to (R)‐mandelic acid, which is an important intermediate in the pharmaceutical industry.     

Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). V_max values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The V_max values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.