Two esterases released from Mycobacterium smegmatis from the hydrolysis of long chain acyl-CoAs and Tween

An esterase activity hydrolyzing palmitoyl-CoA was released into the culture medium from Mycobacterium smegmatis. Although another esterase activity hydrolyzing Tween 20 (polyoxyethylene sorbitan monolaurate) was also found in the culture medium, the bulk of the esterase activity was retained in the cells. However, treatment of early-log phase cells with lysozyme to prepare ghosts released 80% of the Tween 20 hydrolyzing activity, indicating the localization of the esterase in the periplasmic space or the cell envelope fraction. The presence of two different esterases hydrolyzing palmitoyl-CoA and Tween 20, suggested by the above results, was confirmed by the separation of these esterases on phenyl-Sepharose column chromatography. Palmitoyl-CoA hydrolase (thioesterase) was purified 630-fold from lysozyme-treated supernatant fluid to homogeneity, by means of Sephadex G-100 gel filtration, and DEAE-cellulose, phenyl-Sepharose and Blue-Agarose column chromatographies. Its molecular weight was approximately 42,000. Tween hydrolase was partially purified 150-fold by the same purification procedure up to the step of phenyl-Sepharose chromatography and its molecular weight was found to be about 51,000. These activities were stable against heating at 60 degrees C and treatment with non-ionic detergents. Thioesterase hydrolyzed long chain acyl-CoAs (C12-C20), but not Tween 20-80 or beta-naphthyl acetate. On the other hand, Tween hydrolase hydrolyzed Tween 20-80 and beta-naphthyl acetate. On the other hand, Tween hydrolase hydrolyzed Tween 20-80 and beta-naphthyl acetate, but not acyl-CoAs. Both esterases hydrolyzed monoolein, but not diolein, triolein, or phosphatidylcholine.     

Affinity purification and some molecular properties of human liver alkaline phosphatase

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase (deaminating), EC 1.4.1.4) has been purified from Mycobacterium smegmatis CDC 46 using (NH4)2SO4 precipitation, negative adsorption on DEAE-cellulose, 2',5'-ADP-Sepharose affinity chromatography and Sephadex G-200. The enzyme was purified 1041.6-fold and the preparation was found to be homogeneous on column chromatography, polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. Alanine and threonine were identified as the N- and C-terminal amino acids of glutamate dehydrogenase from M. smegmastis. The enzyme kinetics and regulation of glutamate dehydrogenase activity by different nutritional factors has been studied. Initial velocity plots showed that the reaction mechanism of glutamate dehydrogenase from M. smegmatis followed an ordered sequential ter-bi mechanism.     

Factor X activating enzyme from Russell''s viper venom: isolation and characterization

The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).     

Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR)

Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.     

Chemotactic activity from rabbit peritoneal neutrophils. Lack of identity with N-acetyl-DL-phenylalanine beta-napthyl esterase

The chemotactic and N-acetyl-DL-phenylalanine beta-naphthyl esterase activities of rabbit peritoneal neutrophils are separable from each other by both DEAE cellulose and Sephadex G-100 column chromatography. Partially purified esterase obtained from DEAE-cellulose chromatography had molecular weight of 70 000. However, the partially purified fraction contained chemotactic activities with major activity in molecular weight of 28000 and minor activities in the molecular weights of 45000, 21900, 14500 and 10500. Esterase activity is inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate but chemotactic activity is not.     

Studies on almond emulsin beta-D-glucosidase. I. Isolation and characterization of a bifunctional isozyme

A beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme has been isolated from almond emulsin. The isolated enzyme is a glycoprotein and migrates as a single band on Sephadex G-200 filtration, CM 52 ion exchange chromatography, polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focussing. The glucosidase and galactosidase activities traverse together during Sephadex G-200 gel filtration. Polyacrylamide gels stained specifically for the 2 enzymes reveal that the two activities comigrate. The molecular weight of the isozyme has been found to be 135 180 +/- 770, and that of its protomers to be 65 150 +/- 650.     

Purification of Streptomyces chromofuscus phospholipase D by hydrophobic affinity chromatography on palmitoyl cellulose

Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.     

Isolation of nonlabile human ceruloplasmin by chromatographic removal of a plasma metalloproteinase

Ceruloplasmin (EC 1.16.3.1) is a copper-containing alpha 2-glycoprotein and a member of the acute phase reactant family. Fragmentation of ceruloplasmin during purification and storage has hampered studies of its structure, but it has been shown to be a 132-kDa monomer. Combining two published chromatographic steps with additional gel filtration and fast protein liquid chromatography (FPLC) steps, we now report a procedure that yields a highly purified and nonlabile protein. Human plasma was subjected to QAE-Sephadex A-50 chromatography, precipitated with ammonium sulfate, and chromatographed on a hydroxyapatite column. The resulting protein was > 95% pure but highly unstable as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; at 37 degrees C the 132-kDa protein disappeared completely within 48 h. Further purification of ceruloplasmin by Sephadex G-50 chromatography and Mono Q FPLC yielded a protein that was essentially pure by multiple criteria and completely stable even after incubation at 37 degrees C for 4 weeks. When purified ceruloplasmin was reconstituted with fractions eluted from the Sephadex G-50 column, a single fraction induced proteolytic degradation. The degradation of ceruloplasmin by this fraction was inhibited by EDTA and 1,10-phenanthroline, indicating that a plasma metalloproteinase is responsible for degradation of ceruloplasmin.     

Purification of human factor IX by chromatography of a coagulation factor concentrate

A purification procedure for, and some properties of, coagulation factor IX are described. The coagulation factor concentrate used for the treatment of hemophilia B patients was employed as the starting material. The isolation procedure consists of chromatography in DEAE-cellulose, two chromatographies in hydroxyapatite gel and two gel filtrations in Sephadex G-200. Only trace amounts of factors II, VII and X were present in the final preparation and the specific activity of factor IX was 159 corresponding 10,300 times purification from plasma. The molecular weight was estimated to be 76,000 in gel filtration and 86,000 in sodium dodecyl sulfate disc gel electrophoresis. Three activity peaks with pIs 4.15, 4.25 and 4.40 were obtained by isoelectric focusing.     

Purification and properties of dipeptidyl amino-peptidase I from chicken liver (author's transl)

Dipeptidyl aminopeptidase I (E.S. 3.4.14.1) from chicken liver was purified by the following steps: homogenization at pH 5, thermic precipitation, acetone fractionation and Sephadex G-100, DEAE-cellulose and organomercurial-Sepharose column fractionations. The purified enzyme appears to be homogeneous by polyacrylamide gel electrophoresis at both pH 4.5 and 8.3 and has an isoelectric point of 5.7 +/- 0.05. The molecular weight of the enzyme reale 167,000 +/- 17,000 on the Sephadex G-150 column chromatography. The optimum pH for hydrolysis of Gly-Phe-p-nitroanilide (GPNA) and Gly-Phe-B-naphthylamide was 5.8. The value of Km for the hydrolysis of GPNA was estimated at 3.3 mM. The enzyme required halide ions for activity and was activated by thiol reagents (dithiothreitol, cysteine and 2-mercaptoethanol). Accordingly, DAP I was inhibited by thiol-blocking reagents (PCMB, IAA, Hg2+). The enzyme oxidation with oxygen current was fostered by chloride anion (50 nM); nevertheless the activity was recovered when cysteine was present in the incubation mixture; the latter, besides, seems to perform as enzyme protector.     

Partial purification and kinetic studies of exocellular proteinase from Trichophyton mentagrophytes var. erinacei

The dermatophyte Trichophyton mentagrophytes var. erinacei isolated from a patient with tinea cruris was cultured in peptone-glucose broth from which an exocellular proteinase was obtained. The enzyme was partly purified by Sephadex G-100 gel filtration. Its molecular weight was determined to be 33,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH was 8.5, the optimal temperature 35 degrees C. The proteolytic activity was specifically increased against casein and inhibited by phenylmethylsulphonyl fluoride. The enzyme was identified as alkaline serine proteinase.     

Purification and properties of phenylalanine ammonia-lyase from leaf mustard

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the first enzyme in phenylpropanoid biosynthesis, catalyzes the elimination of ammonium ion from L-phenylalanine. In the present study, PAL was purified through ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-200 chromatography, and Q-Sepharose chromatography from the cytosolic fraction of leaf mustard (Brassica juncea var. integrifolia). It consists of 4 subunits, each having an estimated molecular weight of about 40,000 on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme are 9.0 and 45 degrees C, respectively. Its activity is inhibited by Zn2+ ion, and it is strongly activated by caffeic acid. The purified PAL seems to have some characteristics different from those obtained with other PALs.     

Purification and some properties of maleylpyruvate hydrolase and fumarylpyruvate hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn(2+) ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Evidence for cell surface and internal phospholipase activity in ascidian eggs

Glutaredoxin, also known as thioltransferase, was purified from Cryptococcus neoformans by procedures including DEAE-cellulose ion exchange chromatography, Q-Sepharose ion-exchange chromatography, and gel filtration on Sephadex G-50. Its purity was confirmed by SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 12,000 Da. The purified enzyme has a K(m) value of 1.03 mM with 2-hydroxyethyl disulfide as a substrate. The enzyme also utilizes L-sulfocysteine, L-cystine, and bovine serum albumin as substrates in the presence of reduced glutathione. The enzyme has K(m) values of 0.34-2.50 mM for these substrates. It was greatly activated by thiol compounds such as reduced glutathione, dithiothreitol, L-cysteine and beta-mercaptoethanol. It is partially inactivated at 60 degrees C or higher temperatures. It plays an important role in thiol-disulfide exchange in Cryptococcus neoformans.     

Kinetic competence of a phosphoryl enzyme intermediate in the glucose-1,6-p2 synthase-catalyzed reaction. Purification, properties, and kinetic studies

Glucose-1,6-P2 synthase of beef brain which catalyzes the formation of glucose-1,6-P2 and glycerate-3-P from glycerate-1,3-P2 and glucose-1-P has been purified 700-fold with an overall recovery of 19%. The purification procedure involves an ammonium sulfate fractionation of the crude extract, DE52 and hydroxylapatite column chromatography and isoelectric focusing. The isolated enzyme appears to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Its molecular weight is estimated to be about 70,000 by gel filtration on Sephadex G-200 which agrees with the value obtained by sodium dodecyl sulfate gel electrophoresis. A phosphoryl enzyme intermediate in the catalytic reaction is indicated by the following evidence: glycerate-1,3-P2[1-32P] labels the enzyme. The label is removed by acceptor substrates such as glucose-1-P. Using a rapid quenching device at 23 degrees and pH 8.0, the first order rate constant for phosphorylation of the enzyme is 20 s-1, compared with an overall rate with the best acceptor, glucose-1-P, of 19 s-1. Dephosphorylation by glucose-1-P is at 37 s-1. Mg2+ is required for both phosphoryl transfers and the overall reaction. In the complete reaction the fraction of enzyme that is phosphorylated depends on the concentrations of glycerate-1,3-P2 and the concentration and nature of the acceptor in a way that could be predicted from the steady state parameters, the Km values, and the kinetic constants observed for the single turnover. Reciprocal plots of initial rates as a function of both substrate concentrations are families of parallel lines. The 32P-labeled phosphoryl enzyme intermediate was found to be acid-stable and somewhat alkaline-labile. Phosphoserine was identified from the partial acid hydrolysate of a protease digest of [32P] phosphoryl enzyme by two-dimensional thin layer chromatography.     

Purification and characterization of GDP-L-Fuc: N-acetyl beta-D-glucosaminide alpha1-->6fucosyltransferase from human blood platelets

c-6-L-Fucosyltransferase (alpha1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man alpha1,3 antenna was substituted with GlcNacbeta1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP-L-fucose were 29 and 28 microM, respectively. The optimum pH of the enzyme was 6.0. The activity of alpha1,6FucT was abolished in the presence of beta-mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.     

Successful removal of large adrenal pheochromocytoma on the right side with liver mobilization

An extracellular lipase, active on water-insoluble triacylglycerols, has been isolated from Penicillium cyclopium. The purified enzyme has a molecular mass of 29 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. It hydrolyzes emulsions of tributyrin, trioctanoin, and olive oil at the same rate as pancreatic lipase and shows very low activity against partial acylglycerols (monooctanoin and dioctanoin) and methyl esters. It is stable at 35 degrees C for 60 min and has maximal activity in a pH range of 8-10. Hydrolysis of triacylglycerols by P. cyclopium lipase is inhibited by detergents such as Triton X-100. Comparison of the sequence of the 20 first amino acid residues of P. cyclopium triacylglycerol lipase with other Penicillium lipases indicates a high homology with previously characterized lipases produced by P. expansum and P. solitum which are enzymes of comparable size and substrate specificity. Conversely, homology between P. cyclopium lipase and P. simplicissimum lipase, a nonspecific lipolytic enzyme, is low. Penicillium cyclopium triacylglycerol lipase shows no homology with P. camembertii lipase which is specific to monoacylglycerol and diacylglycerol.     

O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Subunit structure

The molecular weight of O-acetylserine (OAS)-O-acetylhomoserine (OAH) sulfhydrylase purified from yeast was estimated to be about 200,000 by Sephadex G-200 gel chromatography in various buffers. The S20, w value of this protein was determined to be about 9.0 by sucrose density gradient centrifugation. The calculated molecular weight based on this value was similar to that estimated by gel chromatography. Treatment with 1% sodium dodesylsulfate (SDS) or 6 M urea dissociated the enzyme into 4 subunits; these had a molecular weight estimated to be 51,000 by SDS-poly-acrylamide gel electrophoresis and to be 57,000 by Sephadex G-100 gel chromatography in the presence of 6 M urea and 0.5% beta-mercaptoethanol. The 4 subunits appeared to be identical, based on the symmetric subunit elution pattern from a Sephadex column, a single peptide band on SDS-polyacrylamide gel, and the detection of histidine as the sole N-terminal amino acid in the native enzyme. Since dissociation into the subunits occurred without the use of reducing agents, the association of the subunits seems to require no disulfide linkage. One mole of the subunit contained one mole of sulfhydryl group which appeared to be buried inside the molecule. Partial restoration of the catalytic activity was observed when the urea-denatured enzyme was dialyzed to remove urea, especially in the presence of reducing agents such as dithiothreitol. The urea-denatured enzyme showed a tendency in the absence of reducing agents to form a subunit dimer linked by a disulfide bond between the cystine residues exposed by denaturation. The amino acid composition of the enzyme was determined; it contained one half-cystine residue per subunit, and the content of acidic residues was much higher than that of basic residues. Based on these findings, the subunit structure of the enzyme is discussed.     

Purification and partial characterization of acid phosphatase from Candida lipolytica

Non-specific acid phosphatase from Candida lipolytica cells was purified 111-fold by chromatography on DEAE-cellulose and gel filtration on Sephadex G-100 and Sepharose 4B. The enzyme is a glycoprotein containing 67% neutral sugars. The molecular mass of the highly purified acid phosphatase was found to be approximately 95 kDa by both SDS-PAGE and gel filtration. The pH and temperature optima were 5.8 and 55 degrees C, respectively. The enzyme was stable at pH values between 3.5 and 5.5 and at temperatures up to 60 degrees C. The purified phosphatase had a Km value of 3.64 mM for p-nitrophenyl phosphate and showed broad substrate specificity.