An alpha-glucuronidase of Schizophyllum commune acting on polymeric xylan

The main alpha-glucuronidase (EC 3.2.1.131) of the fungus Schizophyllum commune was purified to homogeneity using standard chromatographic methods; anion exchange, hydrophobic interaction chromatography and gel filtration. The enzyme had a molecular mass of 125 kDa as determined by SDS-polyacrylamide gel electrophoresis and a pI value of 3.6 according to isoelectric focusing. The N-terminal amino acid sequence of the S. commune alpha-glucuronidase did not show any homology with other alpha-glucuronidases. It exhibited maximal activity at pH values from 4.5 to 5.5 and was stable for 24 h between pH 6 and 8 at 40 degrees C. The highest temperature at which the enzyme retained its full activity for 24 h at pH 5.8 was 40 degrees C. The alpha-glucuronidase of S. commune was able to remove almost all 4-O-methylglucuronic acid groups from water-soluble polymeric softwood arabinoglucuronoxylans. The action of the enzyme on birchwood acetyl-glucuronoxylan was limited due to the high amount of acetyl substituents. The degree of hydrolysis of partially soluble deacetylated glucuronoxylan did not exceed 50% of the theoretical maximum. However, together with a xylanase hydrolysing the xylan backbone the action of the alpha-glucuronidase of S. commune on glucuronoxylan was clearly enhanced. It was apparent that the enzyme was able to remove the 4-O-methylglucuronic groups mainly from soluble substrates.     

Gene cloning and characterization of alpha-glucuronidase of Bacillus stearothermophilus no. 236

The alpha-glucuronidase gene of Bacillus stearothermophilus No. 236 was cloned, sequenced, and expressed in Escherichia coli. The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34. The alpha-glucuronidase produced by a recombinant E. coli strain containing the aguA gene was purified to apparent homogeneity and characterized. The molecular weight of the alpha-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the alpha-glucuronidase therefore was dimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and 40 degrees C, respectively. The enzyme's half-life at 50 degrees C was 50 min. The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucopyranosyluronic)-D-xylobiose]. The alpha-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan. Nevertheless, synergism in the release of xylose from xylan was found when alpha-glucuronidase was added to a mixture of endoxylanase and beta-xylosidase.     

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

极端嗜热菌海栖热袍菌α-葡萄糖醛酸酶的高效表达和重组酶的纯化

α-葡萄糖醛酸酶作为木聚糖降解的限速酶之一,在木聚糖类半纤维素的生物转化中起着重要的作用。海栖热袍菌Thermotoga maritima是一个嗜极端高温的厌氧细菌,其产生的极耐热性酶类具有非常可观的工业应用前景。但热袍菌属Thermotoga的基因在大肠杆菌中的表达一般较困难。研究了T. maritima中的极耐热性α葡萄糖醛酸酶基因在大肠杆菌不同菌株中的表达水平及纯化技术。结果表明,稀有密码子AGA、AGG和AUA限制了该基因在大肠杆菌中的表达,在大肠杆菌BL21-CodonPlus(DE3)RIL可得到高效表达,重组蛋白表达量达20%,比酶活比野生菌株提高5倍;重组蛋白经热处理和金属Ni²⁺的亲和层析提纯后,达到了电泳纯,提纯倍数为5.1倍,收率为55.1%。对重组菌诱导表达条件的研究表明,营养丰富的TB培养基有助于重组菌的生长, 重组菌生长至OD₆₀₀为0.7～0.8时添加IPTG诱导5h后重组蛋白的表达量最高。     

A novel enzyme producing isoprimeverose from oligoxyloglucans of Aspergillus oryzae

An enzyme producing isoprimeverose from xyloglucan fragment oligosaccharides has been purified to the electrophoretically pure state from a commercial enzyme preparation of Aspergillus oryzae (Sanzyme 1000). The purified enzyme showed approximately 1,280-fold increase of the specific activity over the original preparation. The purified enzyme was shown to be an oligomeric protein consisting of two subunits, each of which had a molecular weight of 115,000. The enzyme showed the highest activity at pH 5.0 and 60 degrees C, and was stable in the pH range from 5 to 7 and at up to 50 degrees C. The isoelectric point of this enzyme was pH 3.9. The purified enzyme was highly specific for xyloglucan fragment oligosaccharides and split off isoprimeverose units from the non-reducing end of the backbone of the substrate.     

NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach

The gene (agu) encoding p-nitrophenyl alpha-D-glucuronopyranoside (pNP-GUA) hydrolyzing alpha-glucuronidase of the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified and characterized. The gene previously designated as putative alpha-glucosidase was found to code for a protein that had no alpha-glucosidase activity. It showed a rare activity profile with its ability to hydrolyze pNP-GUA, an activity not known in the alpha-glucuronidases from microbial sources. This is the first report on the occurrence of an alpha-glucuronidase which belongs to the family 4 of glycosyl hydrolases.     

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.     

Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius

Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.     

Isolation of a highly active preparation of beta-D-galactosidase

Methods for isolation and purification of beta-galactosidase from Bacillus subtilis, st. IBP-101 are described. The bacterial cells were disrupted by different procedures such as freezing and thawing with subsequent autolysis at 37 degrees C, disrupting in a French press DKM-3 or in ultrasonic disintegrators UZDN-1 (USSR) and Soniprep-150. It is shown that the specific activity and yield of the enzyme depends to a great extent on the disrupting procedure used. The best results were obtained in case of sonication. The preparation was purified by precipitation with ammonium sulphate (25-75% saturated) and chromatography on DEAE-cellulose and DEAE-Sephadex. The purified enzyme had a specific activity of 3155 units per mg protein. The molecular weight of the homogeneous according to gel polyacrylamide electrophoresis preparation was 215,000, as estimated by gel filtration, and 105,000, as estimated by SDS gel electrophoresis. The enzyme retains the activity in the presence of Na+, Mn2+ or Mg2+ ions or the thiolic reagents, dithiothreitol or 2-mercaptoethanol. The pH optimum of the enzyme activity is 6.3 and it is stable in water solutions at pH from 6 to 9 and can be lyophilized. The given preparation of beta-galactosidase has a high affinity for synthetic substrates such as o- and p-nitrophenyl-beta-D-galactopyranosides and 4-methylumbelliferyl-beta-D-galactopyranoside.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification and characterization of a novel alpha-glucuronidase from Aspergillus niger specific for O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid

A new alpha-glucuronidase that specifically hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-alpha-D-glucosyluronic acid alpha-D-glucoside, O-alpha-D-glucosyluronic acid beta-D-glucosiduronic acid, O-alpha-D-glucosyluronic acid-(1-->2)-beta-D-fructosiduronic acid, p-nitrophenyl-O-alpha-D-glucosiduronic acid, methyl-O-alpha-D-glucosiduronic acid, or 6-O-alpha-(4-O-alpha-D-glucosyluronic acid)-D-glucosyl-beta-cyclodextrine. Furthermore, it showed no activity on alpha-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-alpha-(1-->2)-xylooligosaccharides, derived from xylan, a supposed substrate of alpha-glucuronidases.The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS-PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0-3.5 and at 40 degrees C. It was stable in pH 2.0-4.5 and below 30 degrees C. It hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid to produce alpha- and beta-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

PURIFICATION AND CHARACTERIZATION OF AN ALKALINE CELLULASE PRODUCED BY Bacillus subtilis (AS3)

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6–10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.     

Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.     

Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin

An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe7-Phe8 residues was purified from the membrane-bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP-hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy-terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X-100 in 20 mM Tris-HCI pH 7.8. After ion exchange chromatography (DEAE-Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G-50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex 75 HR 10/30) using a new preparative system, AKTA-purifier, controlled by UNICORN software version 2.20.     

Human liver alpha-L-fucosidase. Purification, characterization, and immunochemical studies.

Human liver alpha-L-fucosidase has been purified 6300-fold to apparent homogeneity with 66% yield by a two-step affinity chromatographic procedure utilizing agarose epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all six isoelectric forms of the enzyme were purified. Polyacrylamide gel electrophoresis of the purified alpha-L-fucosidase demonstrated the presence of six bands of protein which all contained fucosidase activity. The purified enzyme preparation was found to contain only trace amounts of seven glycosidases. Quantitative amino acid analysis was performed on the purified fucosidase. Preliminary carbohydrate analysis indicated that only about 1% of the molecule is carbohydrate. Gel filtration on Sepharose 4B indicated an approximate molecular weight for alpha-L-fucosidase of 175,000 +/- 18,000. High speed sedimentation equilibrium yielded a molecular weight of 230,000 +/- 10,000. Sodium dodecyl sulfate polyacrylamide gels indicated the presence of a single subunit of molecular weight, 50,100 +/- 2,500. The enzyme had a pH optimum of 4.6 with a suggested second optimum of 6.5. Apparent Michaelis constants and maximal velocities were determined on the purified enzyme with respect to the 4-methylumbelliferyl and the p-nitrophenyl substrates and were found to be 0.22 mM and 14.1 mumol/mg of protein/min and 0.43 mM and 19.6 mumol/mg of protein/min, respectively. Several salts had little or no effect on fucosidase activity although Ag+ and Hg2+ completely inactivated the enzyme. Antibodies made against the purified fucosidase were dound to be monospecific against crude human liver supernatant fluids and the pure antigen. No cross-reacting material was detected in the crude liver supernatant fluid from a patient who died with fucosidosis.     

MOLECULAR CHARACTERIZATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS AND SOME IMMUNOLOGICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME

Choline acetyltransferase from bovine brain caudate nucleus has been purified to a specific activity of 25–30 μmol ACh formed per min and mg protein. Disc electrophoresis at pH 9.5 of the purified enzyme showed two protein bands localized close to each other. We were not able to show if ChAT was linked to one or both bands. In SDS disc electrophoresis the enzyme preparation showed one major and one minor protein band with molecular weights of 69,000 and 34,000, respectively. Heterogeneity of the enzyme preparation was also demonstrated by immunodiffusion and immunoelectrophoresis. After ammonium sulphate precipitation no aggregation of the enzyme could be detected by gelfiltration on Ultrogel AC-34 whilst a high molecular weight fraction was occasionally observed by gelfiltration on Sephadex G-200. The enzyme was, however, separated into two molecular forms (A and B) on CM-Sephadex chromatography. Both molecular forms had the same S²_20w but differed in heat stability and affinity for acetyl-CoA. Both forms were inactivated by an antibody preparation raised against either a purified preparation of ChAT, or A and B separately. The highly purified enzyme preparation was inactivated more than 98% by immunoprecipitation. The antibody crossreacted with ChATs from several mammalian species, but only slightly with ChAT from pigeon. The results of binding studies with affinity columns, suggest that the enzyme contains a hydrophobic lobe and a dinucleotide fold, and that a free purine rather than a free ribosyl ring of acetyl-CoA is important for the binding of the substrate to the active site. The hydrophobic lobe may be the same as the dinucleotide fold.     

Purification and characterization of a small size protease from Bacillus sp. APR-4

A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.     

Pseudomonas aeruginosa A2 elastase: purification, characterization and biotechnological applications

An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34 kDa. Temperature and pH with highest activity were 60 °C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 40 °C with an enzyme/substrate (E/S) ratio of 5 U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes.