Purification and Biochemical Characterization of Polyphenol Oxidases from Embryogenic and Nonembryogenic Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) Cells

Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl₂, and FeCl₃ markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg²⁺, Ca²⁺, and Mn²⁺ and was moderately inhibited by Ba²⁺, Cu²⁺, and Zn²⁺. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.     

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Purification and Characterization of a Novel Collagenase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Col-J

The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn²⁺, Pb²⁺, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca²⁺ and Mg²⁺ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K _m and V _max of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.     

Purification and Biochemical Characterization of a Highly Thermostable Xylanase from <Emphasis Type="Italic">Actinomadura</Emphasis> sp. Strain Cpt20 Isolated from Poultry Compost

An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of 90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively. The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan. This enzyme obeyed the Michaelis–Menten kinetics, with the K _m and k _cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min⁻¹, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn²⁺, Ca²⁺, and Cu²⁺, it was, strongly inhibited by Hg²⁺, Zn²⁺, and Ba²⁺. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.     

Characterization of Cyclodextrin Glucanotransferase Produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.     

Purification and Characterization of Thermostable α-Galactosidase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis><Subscript>GR</Subscript>

An extracellular thermostable α-galactosidase producing Aspergillus terreus _GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 °C, respectively, for artificial substrate pNPαGal. α-Galactosidase from A. terreus _GR is found to be thermostable, as it was not inactivated after heating at 65 °C for 40 min. The K _m for pNPαGal, oNPαGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg²⁺, Ni²⁺, Ca²⁺, Co²⁺, and Mn²⁺ had no effect on enzyme activity, but Ag⁺, Hg²⁺, and Cu²⁺ have inhibited complete activity.     

Cloning,Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg⁻¹ at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K _m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na⁺, NH₄⁺, Mg²⁺, Fe²⁺, Fe³⁺, Co²⁺, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.     

Isolation and Characterization of a Novel Thermophilic-Organic Solvent Stable Lipase From <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis>

The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative molecular mass as 30 kDa. The enzyme expressed maximum activity at 60°C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80°C, respectively. A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely inhibited in the presence of Fe²⁺, Mn²⁺, EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl fluoride (PMSF), Na⁺, and Mg²⁺ and no significant effect was found in the presence of Ca²⁺ and Li⁺. Half of an activity was retained by Ba²⁺, Ag⁺, Hg⁺, Ni²⁺, Zn²⁺, and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C₈-C₁₂). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl esters (FAMEs) revealed 31.64 ± 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs, indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis and pharmaceutical industry.     

An Oxidant- and Organic Solvent-Resistant Alkaline Metalloprotease from Streptomyces olivochromogenes

Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the enzyme was estimated to be 51 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gelatin zymography. The activity was optimum at pH 7.5 and 50 °C and stable between pH 7.0 and 10.0. SOMP was stable below 45 °C and Ca²⁺ increased its thermostability. Ca²⁺ enhanced while Co²⁺, Cu²⁺, Zn²⁺, Mn²⁺, and Fe²⁺ inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity, suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents, and oxidizing agents, and the activity is enhanced by H₂O₂. The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological applications especially for organic solvent-based enzymatic synthesis.     

A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively. The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min, exhibiting a half-life of 8 min at 70 °C. The K _m and V _max for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination of Mg²⁺ and Co²⁺ or Zn²⁺ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.     

Glycosidases of turnip leaf tissues

Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure form. Myrosinase and disaccharase mol wt was 62.0 × 10³ and 69.5 × 10³ dalton, respectively, on the basis of gel filtration on Sephadex G-200. Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb²⁺, Ba²⁺, Cu²⁺ and Ca²⁺ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and sucrose. TheK _m andV _max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively. The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30 min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca²⁺, Ba²⁺, Cu²⁺, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose). TheK _m andV _max of disaccharase were 0.123 mM and 3.33 U, respectively.     

High Level Expression of an Acid-Stable Phytase from <Emphasis Type="Italic">Citrobacter freundii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²⁺–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K⁺, Ca²⁺, and Mg²⁺. These characteristics contribute to its potential application in feed industry.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Purification, characterization, and structural investigation of a new moderately thermophilic and partially calcium-independent extracellular α-amylase from Bacillus sp. TM1

A new α-amylase was extracted from a recently found strain of Bacillus sp. and purified by ion-exchange chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band for the purified enzyme with an apparent molecular weight of 59 kDa. The optimum temperature and pH range of the enzyme were 40–60°C and 4.5–7.5, respectively, and its activation energy was 1.974 kcal/mol. The K _m value for the enzyme activity on solubie starch was 4 mg/mL, and the T _m values obtained from the circular dichroism (CD) results of thermal unfolding were 78.7 and 80.2°C in the absence and presence of the calcium, respectively. The enzyme was almost completely inhibited by the addition of Fe³⁺, Mn²⁺, and Zn²⁺ and was activated by EDTA, Cr³⁺, and Al³⁺. Moreover, it was partially inhibited by Ca²⁺, Ba²⁺, Ni²⁺, and Co²⁺. Proteolytic digestion of the enzyme using trypsin combined with results from T _m using CD and irreversible thermoinactivation suggests that this enzyme can be considered a moderate thermophile with both mild flexibility and rigidity.     

Purification, characterization, kinetic properties, and thermal behavior of extracellular polygalacturonase produced by filamentous fungus Tetracoccosporium sp

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30°C in an orbital shaker at 160 rpm for 48h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40°C, and the K _m and V _max values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 μmol/min, respectively. Ag⁺, Co²⁺, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al³⁺, Ba²⁺, Ca²⁺, Fe²⁺, Fe³⁺, Ni²⁺, Mg²⁺, Mn²⁺, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t _1/2 of 63 min at 60°C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min⁻¹, and 35.18 mL/(min·mg), respectively. The activation energy (ΔE ^#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters ΔG ^#, ΔH ^#, and ΔS ^# were also calculated, revealing a potential application for the industry.     

Purification and Characterization of a Novel Heparin Degrading Enzyme from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> (MTCC-8654)

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/μg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 °C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co²⁺, Mn²⁺ ions, and reducing agents (β-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K _m and V _max as 2.2 × 10⁻⁵ M and 30.8 mM min⁻¹, respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.     

Purification and Characterization of a Hyperthermostable and High Maltogenic α-Amylase of an Extreme Thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>

The purified α-amylase of Geobacillus thermoleovorans had a molecular mass of 26 kDa with a pI of 5.4, and it was optimally active at 100 °C and pH 8.0. The T _1/2 of α-amylase at 100 °C increased from 3.6 to 5.6 h in the presence of cholic acid. The activation energy and temperature quotient (Q ₁₀) of the enzyme were 84.10 kJ/mol and 1.31, respectively. The activity of the enzyme was enhanced strongly by Co²⁺ and Fe²⁺; enhanced slightly by Ba²⁺, Mn²⁺, Ni²⁺, and Mg²⁺; inhibited strongly by Sn²⁺, Hg²⁺, and Pb²⁺, and inhibited slightly by EDTA, phenyl methyl sulfonyl fluoride, N-ethylmaleimide, and dithiothreitol. The enzyme activity was not affected by Ca²⁺ and ethylene glycol-bis (β-amino ethyl ether)-N,N,N,N-tetra acetic acid. Among different additives and detergents, polyethylene glycol 8000 and Tween 20, 40, and 80 stabilized the enzyme activity, whereas Triton X-100, glycerol, glycine, dextrin, and sodium dodecyl sulfate inhibited to a varied extent. α-Amylase exhibited activity on several starch substrates and their derivatives. The K _m and K _cat values (soluble starch) were 1.10 mg/ml and 5.9 × 10³ /min, respectively. The enzyme hydrolyzed raw starch of pearl millet (Pennisetum typhoides) efficiently.     

A Temperature and Salt-Tolerant <Emphasis Type="SmallCaps">l</Emphasis>-Glutaminase from Gangotri Region of Uttarakhand Himalaya: Enzyme Purification and Characterization

Purification and characterization of halotolerant, thermostable alkaline l-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. l-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0–11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. l-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K _m and V _max values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived l-glutaminase reported so far.     

Grass Degrading β-1,3-1,4-d-glucanases from Bacillus subtilis GN156: Purification and Characterization of Glucanase J1 and pJ2 Possessing Extremely Acidic pI

Purification of β-1,3-1,4-glucanase from the cell-free culture fluid of Bacillus subtilis GN156 by affinity chromatography of epoxy-activated sepharose 6B and ultrafiltration technique resulted in homogeneous J1 and partially purified pJ2 enzymes. The molecular weight and pI of J1 were 25 kDa and 3.5, respectively, while those for J2 were 90 kDa and 3.6, respectively. Both β-1,3-1,4-glucanase J1 and pJ2 had optimum pH values of 6–6.5 and an optimum temperature of 60°C. Both enzymes were not inhibited by Li²⁺ but were inhibited significantly by Ca²⁺, Cu²⁺, Mn²⁺ and Zn²⁺. However, J1 was slightly inhibited by Fe²⁺, while pJ2 was inhibited by Mg²⁺ as well. They were highly specific to only barley β-glucan. K _m and V _max values of J1 were 1.53 mg/ml and 8,511 μU/ml.min, respectively, while those for pJ2 were 4.36 mg/ml and 7,397 μU/ml.min, respectively. Degradation of barley β-1, 3-1,4-glucan resulted in four different oligosaccharides with 1,3 linkages triose, tetrose, pentose and a high molecular weight (HMW) with 1,3 linkage estimated from their mobilities. The quantitative degradation by the crude enzyme after of incubation yielded in descending order: triose, pentose and tetrose, while that of J1 in descending order yielded: pentose, triose and tetrose. The pJ2 showed low activity yielding a degradation pattern in descending order: pentose, triose, tetraose and a HMW polysaccharide.