Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.     

Production and characterization of a thermostable glucoamylase from Streptosporangium sp. endophyte of maize leaves

Thermostable amylolytic enzymes are currently investigated to improve industrial processes of starch degradation. Streptosporangium sp. an endophytic actinomycete isolated from leaves of maize (Zea mays L.) showed glucoamylase production, using starch-Czapek medium, and the highest rate was obtained in the initial growth phase, after incubation for 24 h at pH 8.0. Maximum glucoamylase activity (158 U mg(-1) protein) was obtained at pH 4.5 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C for 30 min with total inhibition at 100 degrees C. Extracellular enzyme from Streptosporangium sp. was purified by fractionated precipitation with ammonium sulphate. After 60% saturation produced 421 U mg(-1) protein, and yield was 74% with purification 2.7 fold. The enzyme produced by Streptosporangium sp. has potential for industrial applications.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.     

Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus.

A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.     

The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis]

After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours.     

Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257

A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7. 3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.     

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.     

嗜热菌的耐热L—乳酸脱氢酶的研究

About 200 strains of extreme thermophilic bacteria were isolated from hot springs in Guandong province. A strain, HG25, was found to produce thermostable intracellular L-lactate dehydrogenase (EC. 1.1.1.27). It has the characteristic of Thermus sp. The cells were gram-negative, non-sporulating, nonmotile, aerobic rods containing yellow pigment. The optimum temperature for growth was between 65 degrees C to 75 degrees C, the maximum 85 degrees C, and minimum 40 degrees C. The generation time at the optimum was about 80 min. Starch was not hydrolyzed. Acid was not produced from glucose. The G+C content in DNA was 62-65 mol% (Tm). As the properties of strain HG25 is similar to those of Thermus aquaticus and T. thermophilus HB 8 belonging to the genus Thermus. The thermostable L-lactate dehydrogenase was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. For pyruvate reduction, the optimum temperature of the enzyme was 60 degrees C and pH 8.0. After incubation in 0.1 mol/L phosphate buffer pH 7.4 at 70 degrees C for 10 min, the enzyme retained about 85% of its original activity. The half-live time (t1/2) at 85 degrees C was 10 min.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus.

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.     

On the activation of bovine chymotrypsinogen A. Preparation of alanine-neochymotrypsinogen and its activation to alpha-chymotrypsin

Alanine-neochymotrypsinogen was prepared by incubating 20 parts bovine pancreas chymotrypsinogen A with one part alpha-chymotrypsin in a solution containing 1 M (NH4)2SO4, 0.1 M sodium acetate, 0.05 M Tris buffer (pH 8.0) and 0.5 mg/ml soybean trypsin inhibitor. Optimal yields of NH2-terminal alanine were obtained after 60 h incubation at 4 degrees C. Ala-neochymotrypsinogen was isolated from the reaction mixture by affinity chromatography and ion-exchange chromatography on carboxymethyl-cellulose. As expected, the purified preparation was enzymatically inactive and, compared to chymotrypsinogen, had one additional NH2-terminal group identified as alanine. Ala-neochymotrypsinogen was activated by incubating with trypsin at a zymogen : trypsin ratio of 30 : 1 in 0.1 M phosphate buffer, pH 7.6 at 4 degrees C for 1 h. The fully active, stable species was identified as alpha-chymotrypsin.     

Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.     

Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2

A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile.     

Light-scattering studies of the alpha-ketoglutarate dehydrogenase complex from escherichia coli. I. Characterization of the self-association of the complex

The self-association of Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) purified by a column Chromatographic technique, was characterized by light-scattering photometry. The complex adopts a solution conformation somewhat larger than that observed in the electron microscope. The evidence suggests a nonideal indefinite self-association model for KGDC in KCl, phosphate buffer. The KGDC monomer has a molecular charge of about -3 x 10(2) at neutral pH. The self-association is promoted by increasing KCl concentrations, pH (in the range from 6.3 to 7.4) and temperature (from 20 to 30 degrees C). The effects of pH changes suggest a release of protons during the self-association and a minor 'preferential' interaction of phosphate ions. For the association of one monomer to the aggregate at neutral pH and 25 degrees C. DeltaG degrees = -7.8 kcal mol(-1). DeltaH degrees = 24 kcal mol(-1) and DeltaS degrees = 1.1 x 10(2) cal mol(-1) K(-1). These data indicate that hydrophobic interactions drive the association. Thermodynamically, the self-association of KGDC is a complex phenomenon and may serve to stabilize the enzyme complex in solution.     

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).