Beta-Galactosidase of Streptococcus cremoris H

Beta-galactosidase (Beta-D-galactosidegalactohydrolase, EC 3.2.1.23) of Streptococcus cremoris H was partially purified by ammonium sulfate fractionation. Only 10% of the protein was recovered as enzyme protein and more than 50% of the enzyme in the crude extract was lost. A 2.6-fold purification only was achieved. The enzyme was most active at 65°C and recorded an optimum pH at 7.0. Km and V_max with ortho-nitrophenyl beta-D galactopyranoside as the substrate were recorded as 0.384 mM and 12.6 μmoles/mg protein/min. Manganese ions activated the enzyme. The enzyme was strongly inhibited by Hg⁺⁺, Ca⁺⁺, Ni⁺⁺, and Ag⁺ as well as parachloromercuribenoate.     

Purification of New β-Galactosidase from Enterococcus faecium MTCC 5153 with Transgalactosylation Activity

A new β-galactosidase (β-gal) was purified from a lactic acid bacterial strain of Enterococcus faecium MTCC5153 by chromatographic techniques. The purified enzyme had a specific activity of 24.06 U/mg of protein with k _m and V_max values of 2 mM and 18.2 mM/min/mg of protein, respectively. The yield of purified β-gal was 10.65% and estimated molecular weight found to be ～90 kDa, consisting of two homodimeric subunits of 43kDa. The enzyme was stable in pH range of 8.0–9.0 with an optimum pH of 8 and the optimum temperature of 40°C. The enzyme was activated in the presence of metal ions such as Mg⁺², Mn⁺², Ca⁺², K⁺ and Na⁺ and was inhibited by Zn⁺², Co⁺² and Cu⁺². Chemical modifiers (N-bromosuccinamide and Diethylpyro carbonate) inactivated the enzyme indicating the role of tryptophan and histidine moieties for activity. The purified β-gal was able to synthesize oligosaccharides from lactose. This study suggests that the β-gal of Enterococcus faecium MTCC5153 could be applied in dairy industry for hydrolysis of lactose and to improve its digestibility. β-gal of probiotic cultures are of particular interest due to their transgalactosylation properties.     

THE CHARACTERISTICS OF SOYBEAN PHYTASE

Soybean phytase was extracted with 2% CaCl₂ and partially purified by ammonium sulphate fractionation followed by dialysis in 0.01 M tris-maleate buffer, pH 6.5. The enzyme showed an optimum pH of 4.8 and optimum temperature of 60°C. The phytase was partially inhibited at high substrate concentration, with an optimum substrate concentration at 20 mM and a K_m value of 2.4 × 10^-3 M. V_max was 0.22 μmole P_i liberated/min/mL enzyme. The inactivation and activation energies for the hydrolysis of phytic acid were approximately 47,000 cal/mole and 11,100 cal/mole, respectively. Enzyme activity was inhibited by about 25%, 23% and 22% in the presence of 10^-3 M Zn⁺⁺, Cu⁺⁺ and Hg⁺⁺, respectively, and was also decreased by about 85% in the presence of 10^-1 M N-ethylmaleimide and sodium fluoride. Reducing and chelating agents at concentrations up to 10^-1 M inhibited activity by about 50% and by more than 90%, respectively.     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

STABILITY AND ENZYMATIC PROPERTIES OF β-GALACTOSIDASE FROM KLUYVEROMYCES FRAGILIS

Kluyveromyces fragilis β-galactosidase purified to electrophoretic, chromatographic and immunochemical homogeneity was used. The enzyme specifically required potassium ions for stability; MnCl₂ increased the stability. The enzyme was maximally stable at pH 6.5 to 7.5; stability was markedly less at pH's below 6.5 and above pH 8.5 at 37°C. Temperature denaturation followed first order kinetics with an activation energy for denaturation of 56 kcal/mol. Maximum activity was achieved in the presence of 5mM KCl. In potassium phosphate buffer, the enzyme was further activated by Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺; Mn²⁺, at 0.1 mM, gave the highest activation. None of these ions activated the enzyme in Tris buffer and> 0.1 mM Zn²⁺ caused complete loss of activity. Activity was completely inhibited by ethylenediaminetetraacetate and partially restored by addition of MnCl₂. p-Chloromercuribenzoate caused rapid loss of activity which could be restored by dithiothreitol. Iodoacetamide, N-ethylmaleimide and sodium tetrathionate did not inactivate the enzyme. The enzyme was specific for β-galactosides. K_m's for o-nitrophenyl β-D-galactopyranoside and lactose were 2.72 and 13.9 mM, respectively, at pH 6.6 D-Galactono-1, 4-lactone was a good competitive inhibitor (K_i=0.17 mM). pH optimum for hydrolysis of o-nitrophenyl β-D-galactopyranoside was 6.2–6.4. V_max for this substrate was dependent on two ionizable groups of pK_a of 6.13 and 6.51 while V_max/K_m was dependent on two ionizable groups of pK_a of 6.39 and 7.23. Activation energy for hydrolysis of o-nitrophenyl β-D-galactopyranoside at pH 7.0 was 9.1 kcal/mol in the range 20–40°C.     

Purification and Characterization of β-Glucosidase from Aspergillus niger

Aspergillus niger, an isolate of soil contaminated with effluents from cotton ginning mill was grown in Czapek-Dox medium containing sawdust, Triton-X 100 and urea for production of an extracellular β-glucosidase. β-Glucosidase enzyme was purified (86-fold) from culture filtrate of A. niger by employing ammonium sulphate precipitation and gel filtration on sephadex G-75. The molecular mass of the purified enzyme was estimated to be 95 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme had an optimal activity on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5.0. The K_m and V_max of the enzyme on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5 were 8.0 mM and 166 µmol/min/mg of protein, respectively. The enzyme could hydrolyze cellobiose and lactose but not sucrose. Heavy metals like Hg²⁺, Al³⁺, and Ag⁺ inhibited the activity, whereas Zn²⁺ and detergents such as Triton-X 100 and Tween-80 increased the activity at 0.01%. The enzyme activity increased in the presence of methanol and ethanol.     

Statistical optimization,partial purification,and characterization of coffee pulp β-glucosidase and its application in ethanol production

Extracellular β-glucosidase was produced using coffee pulp as a sole carbon source by Penicillium verrucosum by solid state fermentation and 897.36±59 U/g enzyme activity was obtained. Increase in 2.21-fold of enzyme activity on optimizing the bioprocess parameters by response surface methodology based on central composite rotatable design is illustrated. Maximum production level of 1,991.17 U/g was obtained with optimum values of pH 4.2, moisture 66.8%, and fermentation duration of 56 h. The enzyme was partially purified and the enzyme activity was optimum at 50°C temperature and at pH 6. The metal ions such as Mg²⁺, Zn²⁺, Ca²⁺, K⁺, detergents, and chelator such as EDTA were effective and further increased the β-glucosidase activity. On application of β-glucosidase for simultaneous saccharifiation and fermentation, 3.3% ethanol was obtained. Thus, this study provides insight on exploitation of P. verrucosum for synthesis of of β-glucosidase using coffee pulp which is available abundantly in coffee processing industries.

     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

A novel cyclodextrin glycosyltransferase from an alkalophilic Bacillus species: purification and characterization

Cyclodextrin glycosyltransferase (E.C. 2.4.1.19) of alkalophilic Bacillus sp. 7-12 was purified by ammonium sulfate precipitation, DEAE–cellulose column chromatography and Sepharose CL-6B column chromatography. The enzyme thus obtained consisted of a single band that did not dissociate into subunits by SDS–polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was determined to be 69,000 Da by SDS–PAGE. The enzyme was stable below 70 °C with an optimum activity at 60 °C, and was stable at a pH range of 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by MgCl₂, ZnCl₂, CuSO₄, Al₂(SO₄)₃, CoCl₂, AgNO₃, FeSO₄ and slightly inhibited by SnCl₂ and MnCl₂. CaCl₂, KCl, EDTA and DTT had no influence on the enzyme activity. For cyclodextrin production, up to 34% conversion to cyclodextrins was obtained from 10% starch. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.26:1:0.86.     

Purification and characterisation of a novel α‐L‐rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae

A novel α‐L‐rhamnosidase was isolated and purified from Aspergillus oryzae NL‐1. The enzyme was purified 13.2‐fold by ultrafiltration, ion exchange and gel filtration chromatography with an overall recovery of 6.4% and specific activity of 224.4 U/mg, and the molecular mass of its subunit was approximately 75 kDa. Its optimal temperature and pH were 65 °C and 4.5, respectively. The enzyme was stable in the pH range 3.5–7.0, and it showed good thermostability at higher temperatures. The K_M, kcat and kcat/K_M values were 5.2 mm , 1624 s^?1 and 312 s^?1 mm ^?1 using pNPR as substrates, respectively. Moreover, the enzyme exhibited transglycosylating activity, which could synthesise rhamnosyl mannitol through the reactions of transglycosylation with inexpensive rhamnose as the glycosyl donor. Our findings indicate that the enzyme has potential value for glycoside synthesis in the food industry.     

A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

Extracellular Protease from Pseudomonas marinoglutinosa: Some Properties and Its Action on Fish Actomyosin

A bacterium isolated from Indian mackerel (Rastrelliger kanagurta) and identified as Pseudomonas marinoglutinosa, was found to produce appreciable amounts of extracellular protease when grown in nutrient medium. This enzyme which degraded several proteins, was found to be most active against mackerel myofibrillar proteins. The optimum temperature and pH range for enzyme activity were 50°C and 7–8 respectively. Treatment of mackerel actomyosin with the protease at 0–2°C for 4 days resulted in degradation of the protein as assessed by release of tyrosine, loss in Mg⁺⁺-dependent ATPase activity and changes in SDS-polyacrylamide gel electrophoretic patterns.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Characteristics and Novel Features of Thermostable α-Amylase Produced by Bacillus licheniformis M27 under Solid State Fermentation

The purified α-amylase from Bacillus licheniformis M27, produced under solid state fermentation technique, showed novel characteristics as compared to those reported by other workers for the purified α-amylases from B. licheniformis obtained by submerged fermentation process. Some of the novel features of the characteristics of the enzyme from B. licheniformis M27 include two peaks for pH optima at 6.5–7.0 and 8.5–9.0, gradual loss of activity to about 86% between pH 7.0–7.5 followed by rise to full activity between 7.5–8.5, temperature optimum at 85–90°C at pH 7.0 and 9.0, sharp fall in stability at acidic pH values and the thermostability response which is more similar to the enzyme from other species of Bacillus. The moleculuar weight of the enzyme was found to be 19,500 ± 500 and 56,000 ± 2,000 when determined by gel filtration and SDS PAGE, respectively. The activation energy is 20.4 times lower than that reported for the enzyme from another strain of B. licheniformis. It also showed differences in the contents of amino acids such as serine, proline and methionine.     

Purification,properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr‐Sepharose®‐PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as K_m = 0.098 mg ml⁻¹ and V_max = 3.86 µmol min⁻¹ ml⁻¹ of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry     

Winged bean lipoxygenase—Part 2: Physicochemical properties

Winged bean lipoxygenase (linoleate: oxygen oxidoreductase EC 1.13.11.12) isoenzymes FI and FII were isolated and purified according to the method of Truong et al. (1980).FI and FII were both highly specific for linoleic acid. They exhibited optimal activity at pH 6·0 and 5·8, respectively at 30°C. An activation energy of 4·5 kcal mol^?1 was calculated for this lipoxygenase within the temperature range of 30–50°C.At 0·075% Tween 20, FI and FII had K_m values for linoleic acid of 0·44 and 0·37 × 10^?3M, respectively, compared to 0·4 × 10^?3M for the crude enzyme. Maximal activity was obtained at 1·6 × 10^?3M. Higher levels of Tween 20 inhibited the lipoxygenase activity.Both isoenzymes had identical average molecular weight of 80 000 daltons by gel filtration and SDS gel electrophoresis.FI and FII isoenzymes were strongly inhibited by Hg⁺⁺, Mn⁺⁺, Mg⁺⁺ and Fe⁺⁺⁺ and activated by Zn⁺⁺, Co⁺⁺ and Fe⁺⁺. A difference in the degree of inhibition or activation was observed between FI and FII response. Ca⁺⁺ inhibited both FI and FII but the former was more sensitive to Ca⁺⁺. KCN also inhibited the two isoenzymes.Among the antioxidants tested, butylated hydroxytoluene and butylated hydroxyanisole most effectively inhibited both FI and FII at only 10^?6M. Sulphydryl reagents such as iodoacetamide and dithiothreitol have little effect on the lipoxygenase isoenzyme activity.The lipoxygenase isoenzymes were more stable at neutral pH. The enzyme in the crude extract and especially in situ was more stable to heat treatment.     

Purification and Characterization of β-amylase from Bacillus polymyxa No. 26-1

An extracellular β-amylase, which was easily adsorbable onto raw corn starch, was purified 22.5-fold from a new isolate of Bacillus polymyxa No 26–1 with a M_r of 53 kDa and pI of 9.1. The optimum temperature was 45°C and pH 5.5 for raw corn starch. Thermal stability at 40°C and pH stability at 5.0–8.5 were shown. The degradation ofraw starch by β-amylase was greatly stimulated by pullulanase addition. Scanning electron micrographs revealed that starch granule degradation by the enzyme alone occured at the equatorial grooves of lecticular granules. Corn starch granules hydrolyzed by β-amylase had large holes on granule surfaces.     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

PURIFICATION AND CHARACTERIZATION OF α‐GALACTOSIDASE FROM PEANUTS1

Alpha‐galactosidase was characterized in two peanuts market types, Runner and Spanish. The enzyme was purified 54 fold using ammonium sulfate precipitation, anion exchange chromatography and Size Exclusion High‐performance Liquid Chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicated that the enzyme has a molecular weight of 30, 000 Da, and isoelectric focusing showed a pI of 5.2. The optimum temperature and pH were 50C and 6.0, respectively. The enzyme had a K_m of 0.221 mM when p‐nitrophenyl α‐D‐galactopyranoside (PNPG) was used as a substrate and 80.8 mM when raffmose was a substrate. Raffmose and galactose were found to be competitive inhibitors when PNPG was the substrate: K_i values were 25.4 and 189, respectively. The enzyme was very sensitive to Hg⁺⁺, Ag⁺⁺ and to a lesser extent to Cu⁺⁺. However, ethylcne diamine tetraacetic acid did not have an effect indicating no requirement for cations. The two peanut types tested showed identical enzyme activities.