Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.     

Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711

A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth. It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml. The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O. At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O. EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective. The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+. The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively. It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.     

Mucin biosynthesis. Enzymic properties of human-tracheal epithelial GDP-L-fucose:beta-D-galactoside alpha-(1----2)-L-fucosyltransferase

The human-tracheal, epithelial alpha-(1----2)-L-fucosyltransferase that transfers L-fucose from GDP-L-fucose to an acceptor containing a beta-D-galactopyranosyl group at the nonreducing terminal was characterized. Optimal enzyme activity was obtained at pH 6.5. 20-30mM MnCl2 (or CaCl2), and 0.05% Triton X-100 or 0.5% Tween 20. Mg2+ and Ba2+ ions moderately enhanced the enzyme activity, whereas Fe2+, Co2+, Zn2+, and Cd2+ ions were inhibitory. The enzyme activity was inhibited by N-ethylmaleimide and nucleotides of guanine, inosine, xanthine, and uridine. However, ATP and dithiothreitol did not affect the enzyme activity. The apparent Michaelis constant for GDP-L-fucose, freezing point-depressing glycoproteins (expressed as Gal----GalNAc----Thr), and phenyl beta-D-galactopyranoside was 0.29, 5.70, and 25.4mM, respectively. Under alkali-borohydride conditions (0.05M NaOH-M NaBH4, 45 degrees, 20 h), an L-[14C]fucosyltrisaccharide was released from the product obtained by use of freezing point-depressing glycoprotein as the acceptor. The alpha-L anomeric configuration of the fucoside was determined by the release of L-[14C]fucose from the purified trisaccharide by Turbo cornutus alpha-L-fucosidase. The (1----2) linkage of the L-fucosyl group to the D-galactosyl residue was established by methylation technique (m.s.-g.l.c.). The present enzyme has properties similar to those of the human milk alpha-(1----2)-L-fucosyltransferase which is encoded by a secretor gene.     

Purification and properties of alpha-galactosidase from Klebsiella Sp. No. PG-2

alpha-Galactosidase has been purified from Klebsiella Sp. No. PG-2, a bacterium isolated from rat small intestine, using calcium phosphate gel, DEAE-cellulose column chromatography and gel filtration technique. About 130-fold increase in specific activity was observed, the pH optimum of 6.5-7.0 characterizes the enzyme as neutral alpha-galactosidase. The optimum temperature was 37 degrees C and the energy of activation was 11,856 cal/mole. Km values obtained for raffinose, mellibose, stachyose and p-nitrophenyl-alpha-D-galactopyranoside were 20.0, 6.6 33.3 and 4.0 mM respectively. The activity was inhibited by p-CMB; iodoacetate, Ag2+, Hg2+, Cu2+, Pb2+ and galactose. Examination of the enzyme activity indicated that the enzyme is cytosolic and is inducible in nature.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

Purification and some properties of beta-N-acetyl-D-glucosaminidase from viscera of green crab (Scylla serrata)

Beta-N-acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50 degrees C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45 degrees C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 +/- 0.012 mM and Vmax of 17.65 +/- 0.32 micromol/min at pH 5.8 and 37 degrees C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na+ and K+ have no effects on the enzyme activity; Mg2+ and Ca2+ slightly activate the enzyme, while Ba2+, Zn2+, Mn2+, Hg2+, Pb2+, Cu2+, and Al3+ inhibit the enzyme to different extents.     

Studies on phospholipases from Streptomyces. II. Purification and properties of Streptomyces hachijoensis phospholipase D.

1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate.     

CDP-diacylglycerol synthase activity in Clostridium perfringens.

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition of the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM, respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C.     

Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

Effect of temperature and pH on factor XIIIa from human placenta.

1. The activation of the native enzyme was achieved by a proteolytic procedure involving thrombin. 2. The pH profile was independent of the nature of the substrates assayed (casein or dimethylcasein plus putrescine). The optimum pH was between 7.6 and 7.9 and the pK values were 6.5/7 and 8.7/9. A cysteinyl residue appeared to be involved in the pH-dependence activity. 3. In the presence of calcium, the thermostability of enzyme was increased: the temperature at which enzyme lost half of its activity increased up to 7 degrees C. 4. The kinetics of the thermal deactivation of F XIIIa depended on the presence or absence of calcium. 5. In its presence the reaction obeyed second order kinetics, while in its absence, the kinetics were of first order. In the first case, the irreversible thermal deactivation could be described by a two-step mechanism (N----X----D) while in the second case, the deactivation followed the simple model (N----D). 6. Neither divalent cations like Sr2+, Ba2+, Mg2+, nor bovine serum-albumin and polyhydric alcohols were able to increase the thermostability of F XIIIa. 7. Thermal deactivation of F XIIIa did not appear linked to the redox state of enzyme, nor to the modification of SH groups. 8. We observed a good correlation between the loss of activity and the unfolding of the polypeptide chain of F XIIIa during heating. 9. The optimum temperature of F XIIIa activity was 40 degrees C at pH 8 and 45 degrees C at pH 7.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Calf thymus alkaline phosphatase. I. Properties of the membrane-bound enzyme.

A membrane fraction from calf thymocytes was used to investigate molecular and catalytic properties of membrane-bound alkaline phosphatase (ortho-phosphoric-monoester phosphohydrolase EC 3.1.3.1). The principal findings were: 1. Solubilization of membranes with the non-ionic detergent Triton X-100 increases alkaline phosphatase activity by 30-40%. The enzyme activity elutes in a single peak (Stokes' radius = 7.7 nm) after chromatography in Sepharose 6B in the presence of Triton X-100. The activity also sediments as a single component of approx. 6.4 S during centrifugation in sucrose gradients containing Triton X-100. 2. Ion-exchange chromatography and isoelectric focusing in the presence of Triton X-100 indicate substantial charge heterogeneity. Two overlapping bands, a peak at pH 5.92 with a pronounced shoulder at pH 5.29, are apparent by isoelectric focusing. 3. The pH optimum for hydrolysis of p-nitrophenylphosphate (pNPhP) by the undissolved enzyme(s) is 9.57. Half-maximal activity occurs at pH 8.65 and ph 10.45. Triton X-100 has no effect on the pH profile. 4. Catalytic activity is affected by amines, especially analogues of ethanolamine. Diethanolamine exerts a unique stimulatory effect, but does not change the pH dependency. Increasing the concentration of diethanolamine from 0 to 1 M causes a 6-fold increase in Km and a 10-fold increase in the rate of hydrolysis of pNPhP. Glycine is inhibitory. 5. EDTA causes an irreversible loss of activity with t1/2 (1 mM EDTA, pH 8.2, 23 degrees C) = 3.5 h. Optimal activity is achieved in 0.1--1.0 mM Mg2+, although this does not cause the degree of activation reported to occur with the purified enzymes. Other divalent ions are inhibitory. Concentrations required to reduce activity to 50% of control are: Zn2+, 4.0 muM (no added Mg2+) and 30 muM (in the presence of 1 mM Mg2+); Mn2+, 0.25 mM (+/- Mg2+); Ca2+, 20 mM (+/- Mg2+). 6. Monovalent cations have little effect on activity. In the absence of added Mg2+, 50--150 mM Na+ is partially inhibitory, but markedly less so in the presence of 1 mM Mg2+. K+ has no significant effect. 7. Of the substrates tested, pNPhP (Km = 44 muM) was most rapidly hydrolyzed. Other substrates (rate relative to pNPhP) were alpha-naphthylphosphate (0.79), 2'-AMP (0.80), 5'-AMP (0.70), 3'-AMP (0.63), alpha-glycerophosphate (0.47) and glucose 6-phosphate (0.35). Phosphodiesterase activity was less than or equal to 10% of the phosphomonoesterase activity (for pNPhP) as evidenced by the lack of hydrolysis of bis(p-nitrophenyl)-phosphate and cyclic 3',5'-AMP. The ability of these substances to inhibit hydrolysis of pNPhP reflected their capacity as substrates, i.e. the most inhibitory were the most rapidly hydrolyzed.     

Purification and characterization of rat liver transglutaminase

1. Transglutaminase (EC 2.3.2.13) was purified from rat liver. 2. The enzyme was stable at 25 degrees C in the pH range of 6.0-9.0, with the optimum at pH 9.0. 3. The enzyme was inactivated after incubation for 20, 4 and 1 min at 44 degrees C, 52 degrees C, and 60 degrees C, respectively. 4. Activation energies were 30.4 kcal/mol for denaturation and 19.9 kcal/mol for substrate conversion to products. 5. The enzyme was inactivated by sulfhydryl modification with hydroxymercuribenzoate (99.1%) and N-ethylmalemide (78.5%). 6. Calcium, required for the activity, was replaced to a lesser extent, by Mg2+, Sr2+, Zn2+ and Mn2+ (31.8, 27.0, 24.6 and 3.5%). 7. Steady-state kinetics showed: Vmax = 10 microM-min-1, Km = 0.05 mM (N-dimethylated casein), kcat = 31.9 min-1 kcat/Km = 560 min-1 mM-1.     

诺卡氏菌形放线菌β-甘露聚糖酶的纯化和性质

产β－１,４－Ｄ甘露聚糖酶的诺卡氏菌形放线菌（Ｎｏｃａｒｄｉｏｆｏｒｍ ａｃｔｉｎｏｍｙｃｅｔｅｓ）菌株ＮＡ３－５４０,发酵培养７２ｈ,发酵液离心去菌体,上清经硫酸铵沉淀,９５％乙醇沉淀,ＣＭ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析、羟基磷灰石柱层析、ＤＥＡＥ－纤维素离子交换及Ｓｅｐｈａｄｅｘ Ｇ－１００分子凝胶过滤柱等步骤,β－甘露聚糖酶的比活提高了１３７倍,获得凝胶电泳均一的蛋白样品。经ＳＤＳ－ＰＡＧ     

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.     

Characterization of crystalline formate dehydrogenase from Candida methanolica

The crystalline formate dehydrogenase from Candida methanolica, which showed the highest specific activity (7.52 U/mg) so far reported, was characterized in detail. The enzyme is a dimer composed of identical subunits, each containing one SH group related to the catalytic activity. The molecular mass of the enzyme is about 82-86 kDa. The Km values were found to be 3.0 mM for formate and 0.11 mM for NAD+. Even if the enzyme was incubated at pH 6.5-9.5 or at 55 degrees C, the activity remained at 100%. Hg2+, Ni2+, NaCN, NaN3 and p-chloromercuribenzoate strongly inhibited the enzyme activity, while the enzyme showed relatively high resistance to various chelating agents. The amino acid composition and some other physicochemical properties of the enzyme were studied. Immunological studies revealed that formate dehydrogenases of methanol-utilizing yeasts immunologically more or less resemble each other, but differ from those of methanol-utilizing bacteria. Furthermore, yeast formate dehydrogenases can be immunologically classified into three types: (a) the Candida type, (b) the Torulopis/Hansenula/Pichia type and (c) the formaldehyde-resistant yeast type. For simple and large-scale preparation of the enzyme for practical use, treatment of cells of C. methanolica with the commercial cationic detergent, 'Benzalkonium' cation, is useful: the total and specific activities of the enzyme are 1.17-fold and 3.10-fold higher than those of the crude cell-free extract, respectively.