Studies on Lipase from Candida cylindracea

The amino acid composition of the purified extracellular lipase from Candida cylindracea was determined by ion-exchange chromatography with the use of an automatic amino acid analyzer, and a high content of hydrophobic amino acid residues was found. The enzyme was a glycoprotein, in which mannose and xylose were contained as carbohydrate components. Physical properties of the enzyme were the sedimentation coefficient of 4.7 × 10^?13 (cm/sec.)/(dyne/g), the partial specific volume of 0.76 ml/g and the intrinsic viscosity of 0.085 dl/g, and its molecular weight was discussed.     

Lipase-catalyzed kinetic resolution of (R,S)-1-phenylethyl propionate in an enzyme membrane reactor

Enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow-fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10^?4, 1.3 × 10^?5 and 1.0 × 10^?5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide-6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β-cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.     

Studies on the Acid-protease of Paecilomyces varioti BAINIER TPR-220

Some physical and chemical properties were investigated of the crystalline acid-protease from Paecilomyces varioti BAINIER TPR-220. The sedimentation coefficient of the enzyme was calculated to be 2.7 × 10^?13 at 15°C and the molecular weight to be 37,300 by Archibald’s method. The isoelectric point of the enzyme protein was determined to lie at pH 3.8. The enzyme protein was consisted of 340 amino acid residues including only one residue of cysteine but excluding cystine. With the feature of amino acid composition of the protease acidic amino acids dominated over the basic ones.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.     

Purification and Some Properties of a Fungal Lipase Preparation Used for Milk Flavouring

Intracellular lipase of a strain of Rhizopus fungus which is effective for producing a milk flavor was purified and fractionated into two components, I and II, by DEAE Sephadex A-50 column chromatography. They both proved homogeneous by electrophoresis and ultracentrifugal analysis. The sedimentation coefficient was respectively calculated to be 5.8×10^?13 for lipase I, and to be 2.2×10^?13 for lipase II. From substrate specificity, it was found that lipase I was an ordinary lipase hydrolyzing olive oil and tributyrin favourably, while, II, rather, a special lipase having a high affinity towards tricaprylin. They, also, respectively had an apparent phospholipase activity on soy-lecithin and, clearing activity on chylomicron prepared from olive oil and human serum. Their mode of action, and the effect of metals and emulsifying agents on their activity are also presented.     

The inhibitory kinetics and mechanism of glycolic acid on lipase

Abstract

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people’s attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29?±?0.14?mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a K_I value of 19.61?±?0.26?mM. The inhibitory kinetics assay showed that the microscopic velocity constant k₊₀ of inhibition kinetics is 1.79?×?10^?3?mM^?1s^?1, and k_?0 is 0.73?×?10^?3 s^?1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.

Communicated by Ramaswamy H. Sarma     

Alanine Aminotransferase and Aspartate Aminotransferase in Leishmania tarentolae1

SYNOPSIS. Culture stages (promastigotes) of Leishmania tarentolae were tested for alanine aminotransferase (E.C.2.6.1.2) and aspartate aminotransferase (E.C.2.6.1.1.). Neither enzyme was detected in crude cell extracts. After starch block electrophoresis, however, both transaminase activities were found in proteins migrating toward the anode. Only one species of each enzyme was found. Using coupled enzyme assay systems, the following physical and kinetic properties were seen: 1) aspartate aminotransferase was inhibited by α-ketoglutarate concentrations above 1.68 × 10^?2 M and alanine aminotransferase was inhibited by concentrations higher than 1.34 × 10^?2 M; 2) the Michaelis constant (Km[α-ketoglutarate]) was 5.4 × 10^?4 M for aspartate aminotransferase and 3.0 × 10^?4 M for alanine aminotransferase; 3) maximum activity was found at ?pH 8.5 (broad range between pH 7.75–9.0) for aspartate aminotransferase whereas maximum activity for alanine aminotransferase was ?pH 7.2 (range between pH 7.0–7.5); 4) both enzymes lost half of their activity after 4 days at 8 C; 5) aspartate aminotransferase was most active at 35 C and completely inactivated at 59.5 C, alanine aminotransferase exhibited maximum activity at 29.5 C and was completely inactivated at 61 C; and 6) neither enzyme showed enhanced activity with added pyridoxal phosphate.     

Effect of Various Inhibitors on Lipase Action of Thermophilic Fungus Humicola lanuginosa S-38

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.     

Pathways of Chlorophenol Formation in Oxidative Biodegradation of BHC

Hexose 1-phosphate uridylyltransferase (EC 2.7.7.12) was present constitutively in Bifidobacterium bifidum. The enzyme was purified to a homogeneous state from B. bifidum grown on a glucose medium and characterized. The molecular weight of the enzyme is about 110,000.The pH optimum of the enzyme was 7.5. The enzyme was very labile on the acidic side below pH 4.5. Thymidine diphosphate glucose could serve as a substrate with about 60% efficiency of UDP-glucose. The Km values for UDP-gtucose, galactose 1-phosphate (Gal-l-P), UDP-galactose and glucose 1-phosphate (Glc-1-P) were estimated to be 2.3×10^?5M, 5.0 × 10^?4M, 3.1 × 10^?5 M and 1.4 × 10^?4M, respectively. From these results the physiological roles of the enzyme were considered in relation to galactose metabolism in B. bifidum.     

Studies on Sugar-isomerizing Enzyme Purification,Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.

Glucose isomerase was purified by means of acetone fractionation, DEAE-cellulose column chromatography, DEAE-Sephadex column chromatography and crystallization. The purified enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis. The sedimentation coefficient, s_20,w, the diffusion coefficient, D_20,w, and partial specific volume of the enzyme were 8.0S, 4 × 10^?7cm²/sec and 0.69 ml/g, respectively. The molecular weight of the enzyme was estimated to be 157,000 from the sedimentation and diffusion measurements. The crystalline glucose isomerase contained cobalt and magnesium ions. The properties of the enzyme were also studied.     

Biochemical Studies on Rice Bran Lipase

Some chemical properties of the rice bran lipase were studied. The enzyme protein contained 14.98% nitrogen and consisted of 312 amino acid residues. It also contained a certain amount of lipid. The amino-terminal amino acids of the enzyme protein were shown to be glutamic acid and the carboxyl-terminal amino acids to be glycine and serine. The treatment of the enzyme protein with 8 m urea containing 1×10^?3m EDTA (ethyl-enediaminetetraacetic acid) seemed to cause dissociation of the subunits of the enzyme protein. From this observation and the results of the terminal amino acids analysis, it was presumed that the enzyme protein was composed of at least two types of subunits.     

Changes in Hypocholesterolemic Activity in Rats by Various Konnyaku Powder Treatments

Galactosylsucroses contained in soybeans are not digestible. Thus we wished to detect α-galactosidase (EC 3.2.1.22) in intestinal bacteria. The strain of E. coli in the title was found to produce considerably this enzyme adaptively. We could prepare rather pure solution of the enzyme from the sonicate of the strain. It was purified about 142-fold. It showed optimum pH and temperature at 6.8 and 37°C, respectively, with the substrate p-nitrophenyl-α-d-galactoside (PNPG). Dilute enzyme solutions were very unstable even at 0–5°C. However, concentrated solutions were considerably stable. The Michaelis constant (m) was 1.07 × 10^?4, 2.33 × 10^?3, and 3.65 × 10^?2 for PNPG, melibiose, and raffinose, respectively. The maximum velocity (mole/min/mg protein) was 2.72 × 10^?5, 2.67 × 10^?5, and 2.04×l0^?5, respectively for the same three substrates. This enzyme had a weak transferase action.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

Phospholipase A2 from Trypanosoma congolense: Characterization and haematological properties

Phospholipase A₂ was isolated from Trypanosoma congolense and purified to electrophoretic homogeneity. The enzyme appeared to exist in a dimeric form with subunit molecular weights of 16 500 and 18 000. It had a pH optimum of 6·8. Kinetic analysis with different substrates, showed that the enzyme had exceptional specificity for 1,2,dimyristoyl-sn-phosphatidylcholine and 1,2,dioleoyl-sn-phosphatidylcholine with K_m values of 1·85 × 10^?3 M and 2·12 × 10^?3 M respectively. The Arrhenius plot was linear with an activation energy of 5·8 kcal mol^?1. Inhibition studies with parahydroxymercuribenzoate and tri-butyltinoxide were positive thus implicating a thiol group at the catalytic site of the enzyme. The enzyme was stable to heat treatment and possessed haemolytic and anticoagulating properties.     

Properties of glutamate dehydrogenase from Beta vulgaris

Glutamate-NAD oxidoreductase, E.C. 1.4.1.3 (GDH), from seedlings of Beta vulgaris cv. Rota, Jahnsch Peragis Comp., was enzymatically characterized. This enzyme with molecular weight of 2.6 × 10⁵ has a pH optimum of around 8 for animation of α-KGA and around 9.5 for the desamination of glutamate. The apparent K_m for α-KGA is 6.7 × 10^?4M, for NH₃ 2.5 × 10^?3M, for NADH 3.2 × 10^?5M and for NAADPH 5.5 × 10^?4M. NAD¹ inhibits the reaction non-competitively when NADPH serves as substrate. The apparent K¹ is 4.5 × 10^?4M. The data are discussed on relation to the properties of GDH from other plant sources.     

Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

Purification and Characterization of the Cholinesterase of Pseudomonas aeruginosa A-16

The Cholinesterase of Pseudomonas aeruginosa A–16 was purified approximately 11,150-fold with an overall recovery of 15.2% and proved to be homogeneous by electrophoresis, ultracentrifugation and chromatography. The molecular weight of the enzyme was determined as approximately 30,000 by equilibrium centrifugation and gel filtration methods. The sedimentation coefficient, S_20,w was determined to be 3.3 S. Isoelectric focusing electrophoresis with carrier ampholite revealed that the enzyme had an isoelectric point around pH 8.1.

The purified Cholinesterase, which was considered to be an acetylcholinesterase from its substrate specificity, hydrolyzed acetylthiocholine and acetylcholine at the highest rates among the various esters tested.

The estimated values of Km at pH 7.5 and 25°C were 1.5 × 10^?4 m for acetylthiocholine and 1.9 × 10^?4 m for acetylcholine. The enzyme also hydrolyzed the acetyl and propionyl esters of several aliphatic and aromatic alcohols at a lower rate which was entirely dependent on the properties of the alcohol moiety of those esters.     

Enzymatic Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

Some enzymatic properties of Malbranchea β-xylosidase were investigated. The β- xylosidase activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, N-bromosuccinimide, p-chloromercuribenzoate and sodium laurylsulfate, while this activity was activated by Ca²⁺. The enzyme released xylose as the end product even from 10% xylobiose solution without forming any xylooligosaccharides. The enzyme well acted on aryl-β-d-xylosides, but showed no activity on alkyl-β-d-xylosides, and it was practically free from glucosidase activity. The Km and V_max values of this enzyme for xylobiose were calculated to be 2.86 × 10^?8 m and 34.5 μmoles/mg/min, respectively, and these values determined for phenyl-β-d-xyloside were 3.01 × 10^?8 m and 16.2 μmoles/mg/min, respectively.     

Effect of some Cardiac and Respiratory Drugs on Succinate-Cytochrome c Reductase

Succinate-cytochrome c reductase was inhibited in vitro and in vivo by phenobarbitone, aminophylline and neostigmine using both 2,6-dichlorophenolindophenol (DCIP) and cytochrome c (cyt c) as substrates. The enzyme was also activated by gallamine towards both substrates. In vitro, phenobarbitone and aminophylline inhibited the enzyme with respect to the reduction of DCIP and cyt c in a non-competitive manner with K_i values of 1.5 × 10^?5 and 5.7 × 10^?5 M, respectively. Moreover, neostigmine competitively inhibited the enzyme towards both substrates with K_i values of 1.36 × 10^?5 and 1.50 × 10^?5 M, respectively.     

Caratteristiche della fosfo-gluco-isomerasi di una pianta superiore

Abstract

PHOSPHOGLUCOISOMERASE FROM PEA COTYLEDONS. — 6-P-glucose iso-merase has been purified from pea cotyledons. A 70-fold purification has been obtained by means of acetone fractionation and two absorption-elution steps on calcium phosphate gel. The partially purified enzyme is free of interfering activities.

KM values of 2.5×10^?4 and 10^?4 been measured for glucose-6-P and fructose-6-P respectively. reaction, measured at pH 7.8 and 30° C., is 3.7 (Gl-6-PIFr-6-P).

The enzyme is not inhibited by p-chloro-mercurybenzoate up to 10^?3 M. Besides the substances already known to inhibit competitively the isomerase from animal tissues, the pea enzyme has been found to be competitively inhibited by ribose-5-P and by triosespho-sphates, the K₁, being respectively 7×10^?4 and 2.5×10^?4.

The properties of the pea enzyme are compared to those of animal tissues isomerase. The possible physiological significance of these properties is discussed.