Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Cellulolytic complex of Aspergillus niger under conditions for citric acid production. Isolation and characterization of two β-(1→4)-glucan hydrolases

Summary Two -(14)-endoglucanases have been purified from industrial waste broth of Aspergillus niger grown under conditions which produce citric acid. Molecular weights for endoglucanase A were 43,000 and 25,000 for endoglucanase B. Both enzymes exhibited very similar properties: a rather broad pH optimum between pH 2 and 7 for CM-cellulose hydrolysis and an inability to degrade crystalline cellulose. The endoglucanases have a higher thermal stability at acid pH (up to 60°C) than at alkaline pH. They are inhibited by iodine, HgCl₂ and N-bromosuccinimide.     

Purification and characterization of a thermostable and acid-stable phytase from Pichia anomala

Phytase of Pichia anomala was purified to near homogeneity by a two-step process of acetone precipitation followed by anion exchange chromatography using DEAE-Sephadex. The enzyme had a molecular weight of 64 kDa. It was optimally active at 60 °C and pH 4.0. This enzyme was found to be highly thermostable and acid-stable, with a half life of 7 and 8 days at 60 °C and pH 4.0 respectively. At 80 °C, the half life of phytase could be increased from 5 to 30 min by the addition of materials such as sucrose, lactose and arabinose (10% w/v). The enzyme exhibited a broad substrate specificity, since it acted on p-nitrophenyl phosphate, ATP, ADP, glucose-6-phosphate besides phytic acid. The K _m value for phytic acid was 0.20 mM and V _max was 6.34 mol/mg protein/min. There was no requirement of metal ions for activity. SDS was observed to be highly inhibitory to phytase activity. Sodium azide, DTT, -mercaptoethanol, EDTA, toluene, glycerol, PMSF, iodo-acetate and N-bromosuccinimide did not show inhibitory activity. The enzyme was inhibited by 2,3-butanedione, indicating the involvement of arginine residues in catalysis. Phytase activity was not inhibited in the presence of inorganic phosphate upto 10 mM. The shelf life of the enzyme was 6 months at 4 °C and there was no loss in the activity on lyophilization. Very few studies have been done on purification of yeast phytases. This is the first report on purification and characterization of phytase from P. anomala. The enzyme is unique in being thermostable, acid-stable, exhibiting broad substrate specificity and in not requiring metal ions for its activity. The yeast biomass containing phytase appears to be suitable for supplementing animal feeds to improve the availability of phosphorus from phytates.     

Production,isolation and characterization of extracellular protease of an alkaliphilic strain of Arthrobacter ramosus,MCM B-351 isolated from the alkaline lake of Lonar,India

An extracellular protease was produced by Arthrobacter ramosus isolated from the alkaline lake of Lonar, Buldhana District of Maharashtra, India when grown on a synthetic medium of pH 10 containing casein. The optimum conditions for production were 3.0% initial casein concentration, 2% inoculum of 1 × 10⁸ cells/ml, pH 9.0, temperature 30 °C and shaken culture conditions. The protease was purified by ammonium sulphate precipitation followed by Sephadex G-100 chromatography. Two proteases viz. Arthro I and Arthro II, having molecular weights 21 and 11.4 kDa respectively were isolated. The Arthro II fraction had K _m 395 g/ml and V _max 10.55 g/min for azocasein. The maximum activity of enzyme was at 55 °C and pH 8. It was thermostable (up to 80 °C), alkali stable (pH 12) and stable in commercial detergent. The enzyme may contain a thiol group at the active site.     

Short communication: Purification and properties of a glucose-forming amylase of Lactobacillus brevis

An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K _m value for starch was 0.27 mg ml^-1 and its M _r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca²⁺, Mg²⁺, Na⁺ or K⁺ and inhibited by EDTA, KCN, citric acid and l-cysteine.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Production and properties of carboxymethylcellulase (endo-1,4-β-glucanase) fromCurvularia lunata

An extracellular carboxymethylcellulase (endo-1,4--glucanase) fromCurvularia lunata, grown at 30°C with an initial pH of 6.0, had optimal activity at pH 4.8 and 50°C. The enzyme was unstable above 50°C. The enzyme had aK _m for carboxymethylcellulose of 0.97 g/l and aV _max of 5.4 IU/ml.     

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9′

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9 was found to be the best lipase producer. The maximum lipase activity was 68 units mL^–1 culture medium on the fifth day of incubation at pH 6.0 and 20°C. Therefore, P. chrysogenum 9 was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30°C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The K_m and V_max were calculated to be 2.33 mM and 22.1 units mL^–1, respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca²⁺. The P. chrysogenum 9 lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.     

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65°C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K _m for p-nitrophenylphosphate and fructose-6-phosphate of 370 M and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and -glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Hydration of 3-cyanopyridine to nicotinamide by crude extract nitrile hydratase

Summary The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K _m =28 mM;K _p =36 mM;K _s =155 mM;V _m =5.8 mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.     

Purification and partial characterization ofd-(−)-lactate dehydrogenase fromLactobacillus helveticus CNRZ 32

Summary d-(–)-Lactate dehydrogenase (LDH) was purified to homogeneity from a cell-free extract ofLactobacillus helveticus CNRZ 32. The native enzyme was determined to have a molecular weight of 152 000 and consisted of four identical subunits of 38 000. This enzyme was NAD dependent fructose 1,6-diphosphate (FDP) and ATP independent. It was most active on pyruvate followed by -hydroxypyruvate as substrates. TheK _m values for pyruvate andd-(–)-lactate were 0.64 and 68.42 mM respectively, indicating that the enzyme has a higher affinity for pyruvate. The enzyme activity was completely inhibited byp-chloromercuribenzoate (1 mM) and partially by iodoacetate, suggesting the involvement of the sulfhydryl group (-SH) in catalysis. Optima for activity by the purified enzyme were pH 4.0 and 50–60°C. Limited inhibition ofd-(–)-LDH was observed with several divalent cations. Additionally, HgCl₂ was observed to strongly inhibit enzyme activity. The purified enzyme was not affected by dithiothreitol or any of the metal chelating agents examined.     

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 g of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (M_r = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.     

Purification and some properties of superoxide dismutase from<Emphasis Type="Italic"> Deinococcus radiophilus</Emphasis>, the UV-resistant bacterium

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg^–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H₂O₂. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian     

Purification and characterization of β-galactosidase from a strain of Bacillus coagulans

-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3×10⁵ and an optimal temperature of 55°C. The optimal pH at 30°C is 6.0 whereas at 55°C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The K_m with ONPG as substrate is 4.2–5.6mm and with lactose is 50mm. The K_i for inhibition by galactose is 11.7–13.4mm and for dextrose is 50mm. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55°C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55°C. An overall purification of 75.3-fold was achieved.     

Tetrahydromethanopterin-dependent serine transhydroxymethylase from Methanobacterium thermoautotrophicum

Serine transhydroxymethylase of Methanobacterium thermoautotrophicum has been purified to within 95% of homogeneity. Activity was strictly dependent on tetrahydromethanopterin, tetrahydrofolate being unable to serve as the acceptor C₁ units from l-serine. The native protein has a molecular weight of about 102,000 daltons. The enzyme shows maximal activity at 60°C, has a pH optimum of 8.1, and required pyridoxal-5-phosphate and Mg²⁺ for optimal activity.     

Isolation and characterization of glutathione-S-transferase isozyme Q3 from the northern quahog,Mercinaria mercinaria

Three glutathione-S-transferase (GST) isozymes (Q1, Q2, and Q3) from the northern quahog (Mercinaria mercinaria) were purified and separated with a combination of affinity and ion exchange chromatography. SDS-PAGE analysis of the separated quahog GSTs indicated there are four distinct subunits of the enzyme with molecular masses ranging between 23 and 27 kDa. The electrophoretic analysis in combination with GST information from literature indicates that among the quahog GST isozymes, there is a single homodimer and two heterodimers. Enzymatic kinetic analysis of the homodimeric quahog GST (Q3) using 1-chloro-2,4-dinitrobenzene and glutathione as reactants resulted in V _max and K _m values of 33.2 mol min^–1 mg^–1 and 0.40 mM, respectively. A pH profile analysis of the Q3 GST indicates that the optimum catalytic pH is 7.6. The Q3 isozyme composes about 28% of the ion exchange purified GSTs but accounts for only 9% of the total GST enzymatic activity (25 mol min^–1 mg^–1). An analysis investigating the dependence of the Q3 GST activity on temperature resulted in a retention of enzymatic activity (50–30% at temperature extremes from –13°C to 100°C), suggesting a unconventional role for the Q3 GST in quahog metabolism.