Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.     

Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device

A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA). Previous reports from our laboratory described the kinetic constants that characterize the reaction: K_m = 2.8 × 10^?3M, K_ia acetate = 5 × 10^?2M, and K_id deacetyl-7-ACA = 3.6 × 10^?2M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. where S_t = mg/ml 7-ACA, A_t = mg/ml acetate, D_t = mg/ml deacetyl-7-ACA. The predicted time course closely matched the time course measured experimentally. The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (>97%) hydrolysis of a 24 mg/ml 7-ACA solution. The esterase was immobilized by containment within an ultrafiltration device. With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7-ACA solutions containing 4 to 24 mg/ml 7-ACA. The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactivation occurred.     

Purification and characterization of two components of acid alpha-glucosidase from pig liver

Acid alpha-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the alpha-1,4 and alpha-1,6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-alpha-Glc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-alpha-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0 X 10(-4) M, 9.1 X 10(-3) M, 16.7 mg/ml, 6.7 X 10(-2) M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively. The Ki values of the larger component were 5.0 X 10(-2) M, 13.3 X 10(-3) M, and 3.2 X 10(-3) M, and those of the smaller component were 2.5 X 10(-2) M, 6.1 X 10(-3) M, and 4.7 X 10(-3) M, for erythritol, Tris, and turanose, respectively.     

The serine hydroxymethyltransferase of Plasmodium lophurae.

Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH4)2SO4 fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0.3.3 M (NH4)2SO4, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8-7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The Km was 4.3 X 10(-3) M for L-serine and 2.5 X 10(-4) M for tetrahydrofolate.     

A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1 % sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic β-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 °C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.     

Purification and characterization of a novel aminoacylase from Streptomyces mobaraensis

A novel aminoacylase was purified to homogeneity from culture broth of Streptomyces mobaraensis, as evidenced by SDS-polyacrylamide gel electrophoresis (PAGE). The enzyme was a monomer with an approximate molecular mass of 100 kDa. The purified enzyme was inhibited by the presence of 1,10-phenanthroline and activated by the addition of Co2+. It was stable at temperatures of up to 60 degrees C for 1 h at pH 7.2. It showed broad substrate specificity to N-acetylated L-amino acids. It catalyzed the hydrolysis of the amide bonds of various N-acetylated L-amino acids, except for Nepsilon-acetyl-L-lysine and N-acetyl-L-proline. Hydrolysis of N-acetyl-L-methionine and N-acetyl-L-histidine followed Michaelis-Menten kinetics with K(m) values of 1.3+/-0.1 mM and 2.7+/-0.1 mM respectively. The enzyme also catalyzed the deacetylation of 7-aminocephalosporanic acid (7-ACA) and cephalosporin C. Moreover, feruloylamino acids and L-lysine derivatives of ferulic acid derivatives were synthesized in an aqueous buffer using the enzyme.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Purification and properties of beta-mannosyltransferase that synthesizes Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol

The beta-mannosyltransferase that adds mannose, from GDP-mannose, to GlcNAc-GlcNAc-pyrophosphoryl-dolichol, to form Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol was solubilized from pig aorta microsomal preparations, using 0.5% NP-40, and was purified about 116-fold using conventional methods. The purified enzyme was mostly free of alpha 1,3- or alpha 1,6-mannosyltransferase activities, since Man beta-GlcNAc-GlcNAc-PP-dolichol (PP = pyrophosphoryl) accounted for more than 95% of the product when enzyme was incubated with GDP-[14C]mannose and GlcNAc-GlcNAc-PP-dolichol. Very little Man-beta-GlcNAc-GlcNAc-PP-dolichol was formed when GDP-[14C]mannose was replaced by dolichol-phosphoryl-[14C]mannose, indicating that GDP-mannose was the mannosyl donor. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and was characterized by gel filtration as well as by susceptibility to beta-mannosidase and resistance to alpha-mannosidase. The partially purified enzyme could be stabilized by the addition of 20% glycerol and 0.5 mM dithiothreitol to the buffer, and could be kept in this solution for 5 or 6 days in ice. The enzyme was greatly stimulated by the addition of detergent (NP-40) with optimum activity being observed at 0.1%. However, no stimulation was seen with any phospholipid. The partially purified enzyme had a pH optimum of about 7.0, and showed an almost absolute requirement for Mg2+ with optimal activity occurring at about 5 mM Mg2+. Mn2+ and Ca2+ were only slightly active. The Km for GDP-mannose was about 5 X 10(-7) M and that for GlcNAc-GlcNAc-PP-dolichol about 1 X 10(-6) M. Beta-Mannosyltransferase activity was inhibited competitively by a variety of guanosine nucleotides with GDP and GDP-glucose being most active, but GTP, GMP, guanosine, and periodate-oxidized guanosine were also effective. The enzyme was strongly inhibited by p-chloromercuribenzenesulfonic acid and this inhibition was partially prevented by the addition of dithiothreitol.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part I: Cultivation of Pseudomonas syringae and partial purification and immobilization of 7-β-(4-carboxybutanamido) cephalosporanic acid acylase

The enzymatic transformation of 7-β-(4-carboxybutanamido)cephalosporanic acid (Gl-7-ACA) to 7-amino-cephalosporanic acid (7-ACA) is reported. The optimum conditions for cultivation of the producer strain Pseudomonas syringae, as well as the procedures for isolation, purification, and immobilization of the enzyme Gl-7-ACA acylase, are described. It is shown that when glutaraldehyde is used for immobilization of this enzyme, the yield of immobilization is low. After six hydrolyses of Gl-7-ACA to 7-ACA, the immobilized enzyme activity loss is less than 10%.     

Partial purification and characterization of an N2-guanine RNA methyltransferase from chicken embryos.

An N2-guanine RNA methyltransferase has been purified 1000-fold from chick embryo homogenates by phosphocellulose chromatography followed by chromatography on S-adenosylhomocystein-Sepharose. The enzyme was shown to methylate the G10 position of Escherichia coli B tRNAPhe and has a Km of 3X10(-7) M for tRNAPhe and 1.38 X 10(-6) M for S-adenosylmethionine. The molecular weight was estimated to be 77 000 by gel filtration and the pH optimum was 8.0 to 8.5. Magnesium ion was not required for activity but it stimulated the rate of methylation 1.5-fold with an optimum at 12 mM. Ammonium ion stimulated activity about twofold with an optimum at about 83 mM. Sodium and potassium ions above 0.1 M were inhibitory.     

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Purification and characterization of a non-kallikrein arginine esterase from dog urine

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Purification and Properties of Calmodulin-Lysine N-Methyltransferase from Rat Brain Cytosol

A S-adenosylmethionine:protein-lysine N-methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080-fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and phosphocellulose and octopus calmodulin-Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S-adenosyl-L-methionine were found to be 2.2 X 10(-8) M and 0.8 X 10(-6) M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10(-7) M Mn2+ and 10(-4) M Ca2+. HPLC of the acid hydrolysate of methyl-3H-labeled calmodulin showed the formation of epsilon-N-mono, epsilon-N-di, and epsilon-N-trimethyllysine. Reverse-phase HPLC of tryptic peptides of the methyl-3H-labeled calmodulin demonstrated that the labeled N-methyllysine lies in the 107-126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.     

Anacystis nidulans mutants resistant to aromatic amino acid analogues.

Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.     

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores.

Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg++ or Ca++. The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

The p-nitrophenyl phosphatase activity of ubiquitin from bovine erythrocytes

Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 X 10(-3) M. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 X 10(-3) M), molybdate (Ki = 1.2 X 10(-6) M), and phosphate (Ki = 1.4 X 10(-3) M). These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.     

Batch production of deacetyl 7-aminocephalosporanic acid by immobilized cephalosporin-C deacetylase

Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The K_m^app value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.