Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869

Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1, 2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1, 2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Kms of 92 and 143 microM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with kcat/Km values of 44.08 x 10(4) s-1 M-1 for the P25X enzyme and 39.34 x 10(4) s-1 M-1 for the P35X enzyme. Higher kcat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1, 2-dioxygenases was around 50 degrees C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.     

Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96

A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.     

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.     

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+.     

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.     

The characteristics and applications of recombinant cholesterol dehydrogenase

Mass production of an r-CDH derived from Nocardia species was made possible by gene technology. (Horinouchi et al., Applied and Environmental Microbiology, 57, 1386-1393 (1991)). However, the characteristics of the r-CDH have not been studied in detail and have not been improved enough for industrial use. We accordingly characterized both the native-CDH and the r-CDH prepared from Streptomyces lividans. Both CDHs were monomers with molecular masses of 37 kDa. The Km of r-CDH was 2.50 x 10(-3) M for cholesterol and 2.33 x 10(-4) M for NAD. The activators of CDHs were TritonX-100 and cholate. TritonX-405, Ag+, and Zn2+ inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37 degrees C, 60 min), while the r-CDH showed a residual activity of 87% (37 degrees C, 60 min). The r-CDH is an enzyme with high substrate specificity for cholesterol as well as native CDH and higher thermal stability than native CDH. We have developed a novel serum cholesterol assay using the r-CDH, which permits the direct measurement of cholesterol by measuring NADH reaction products. We conclude that this r-CDH enzyme is useful and can be used to measure cholesterol in a clinical chemistry setting.     

Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn

Syncephalastrum racemosum Cohn. produces an extracellular xylanase that was shown to potentially bleach pulp at pH 10 and 50 degrees C. The enzyme was found to be a dimer with an apparent molecular weight of 29 kDa as determined by SDS-PAGE. The optimum activity was found at two pH values 8.5 and 10.5; however the activity sharply decreased below pH 6 and above pH 10.5. The enzyme was stable for 72 h at pH 10.5 and at 50 degrees C. Kinetic experiments at 50 degrees C gave V(max) and K(m) of 1,400 U/ml min(-1) mg(-1) protein and 0.05 mg/ml respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by group II b metal ions like Zn2+, Hg2+, etc. Xylan completely protected the enzyme from being inactivated by N-bromosuccinimide.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

The influence of lysosomes on glycogen metabolism.

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Glucosidase II from control and ethanol-treated rats. Purification and properties

Liver glucosidase II from control and ethanol-treated rats was purified and its physical and catalytic properties studied. No significant variation was found in the purification and properties of the enzyme from either source (ethanol-treated and control rats), except for activity after storage. Glucosidase II was purified to electrophoretic homogeneity from liver microsomes by solubilization, protamine sulphate precipitation, and anion exchange (DEAE-Sephacel) and affinity (Con A-Sepharose-4B) chromatographies. The native enzyme molecule is a tetramer (Mr 425 +/- 10 kDa) with identical subunits (Mr 106 +/- 3 kDa). Km values, determined at pH 6.8 for the p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats, were 1.20 +/- 0.12 and 1.14 +/- 0.13 mM, respectively. The Arrhenius plot was linear, and the value for the apparent activation energy, calculated from this plot, was 56.64 kJ/mol. The p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats was inhibited to the same extent by NH4+, by the divalent cations Ca2+ and Mg2+, and by methanol, ethanol, 2-propanol, n-propanol, isobutanol and n-butanol.     

Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost.

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.     

Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile

An extracellular laccase-producing ascomycete was isolated from soil and identified as Paraconiothyrium variabile using rDNA sequence analysis. Typical laccase substrates including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol (DMP), and guaiacol were oxidized by the purified enzyme (designated as PvL). The molecular mass of PvL was 84 kDa and it showed a pI value of 4.2. The enzyme acted optimally at pH 4.8 and exhibited an optimum temperature of 50 °C. Using ABTS, PvL represented Km and Vmax of 203 μM and 40 μmol min(-1) mg(-1), respectively. After 24 h incubation at pH 4.8 and 4 °C, 80% of the initial activity of PvL remained. The enzyme was inhibited by Fe2+, Hg2+, and Mn2+, but induced by Cu2+. EDTA (10 mM), 1,4-dithiothreitol (DTT) (0.1 mM), and NaN3 (10 mM) were found to completely inhibit PvL. Sixty-eight percent of Malachite green was decolorized by 4 U/mL of PvL after 15 min incubation at 30 °C.     

棒曲霉Asp-195v产蛋白酶的酶学性质研究

对液体发酵的棒曲霉Asp-195v菌株所产蛋白酶的活力进行了研究,并通过分离纯化获得了电泳纯的酶蛋白。研究结果表明,该蛋白酶的最适反应温度为40℃,在30-50℃温度范围内相对活力可保持在70%以上;最适pH为7,pH稳定范围在4-8;Mn2+对该蛋白酶活力有明显的激活作用,K+、Ag+、Cu2+、Fe2+、Mg2+、Zn2+、Ca2+、Al3+和Fe3+离子则有明显的抑制作用,尤其是Hg2+和Pb2+对酶活的抑制作用更加强烈;其他试剂如葡萄糖、EDTA对酶活的抑制作用不明显,而蔗糖、SDS和Tween-20对酶活的抑制明显;以酪氨酸为底物采用双倒数作图法测得Vmax为30.40mmol/min,Km为97.53mmol/L。该酶的表观分子量为30.1kDa。     

Purification and characterization of beta-N-acetylhexosaminidase from rice seeds

N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).     

大豆异黄酮糖苷酶的纯化及性质研究

利用Absidiasp.R菌株,通过液体发酵的方法,得到了一种高活性的大豆异黄酮糖基水解酶。该酶经硫酸铵分级沉淀、DEAE-Cellocuse(DE-52)离子交换层析纯化,被纯化了11倍,收率为10.9%;经SDS-聚丙烯酰胺凝胶电泳测得该酶的分子量为53kD;该酶的最适反应温度为50℃;最适pH为5.0;温度低于60℃,pH在5.0~7.0范围内该酶较稳定,Co2 、Ca2 对该酶有激活作用;Ag 、Cu2 对该酶有抑制作用。当以染料木甙为底物时该酶的米氏常数(Km)为1.3×10-2mol/L。等电聚焦电泳测得其等电点为3.2。     

Purification and characterization of an intracellular beta-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.     

Purification and characterization of the FokI restriction endonuclease

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.