• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.270-276
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• 1998

Extracellular ${\alpha}$-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable ${\alpha}$-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at $60^{\circ}C$. It had a half-life of 35 min at $60^{\circ}C$. The enzyme was stable at the pH range of 4.5~7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable ${\alpha}$-glucosidase hydrolyzed the ${\alpha}$-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The $K_m$ (mM) for p-nitrophenyl-${\alpha}$-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the $V_{max}$(${\mu}mol{\cdot}min^-1$$mg^-1$) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.469-474
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• 1999

Thermoactinomyces sp. E79 produced two types of thermostable alkaline proteases extracellularly. A minor protease was separated from a major protease by using DEAE-column chromatography. This enzyme was purified to homogeneity by ammonium sulfate and DEAE-Sepharose ion-exchange chromatography. The purified minor protease showed different biochemical properties compared to the major protease. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 36 kDa. Its optimum temperature and pH for proteolytic activity against Hammarsten casein were $70^{\circ}C$ and 9.0, respectively. The enzyme was stable up to$75^{\circ}C$ and in an alkaline pH range of 9.0-11.0. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF) and $Hg^{2+}, indicating that the enzyme may be a cysteine-dependent serine protease. In addition, the enzyme cleaved the endoproteinase substrate, succinyl-Ala-Ala-Pro-Phe-p- nitroanilide, and the $K_m$ value for the substrate was 1.2 mM.

• BMB Reports
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• v.45 no.2
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• pp.91-95
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• 2012

Glutamate dehydrogenase from axenic bacterial cultures of a new microorganism, called GWE1, isolated from the interior of a sterilization drying oven, was purified by anion-exchange and molecular-exclusion liquid chromatography. The apparent molecular mass of the native enzyme was 250.5 kDa and was shown to be an hexamer with similar subunits of molecular mass 40.5 kDa. For glutamate oxidation, the enzyme showed an optimal pH and temperature of 8.0 and $70^{\circ}C$, respectively. In contrast to other glutamate dehydrogenases isolated from bacteria, the enzyme isolated in this study can use both $NAD^+$ and $NADP^+$ as electron acceptors, displaying more affinity for $NADP^+$ than for $NAD^+$. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. The enzyme was exceptionally thermostable, maintaining more than 70% of activity after incubating at $100^{\circ}C$ for more than five hours suggesting being one of the most thermoestable enzymes reported in the family of dehydrogenases.

• BMB Reports
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• v.30 no.4
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• pp.262-268
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• 1997

The thermophilic and thermostable alkaline phosphatase was purified to near homogeneity from the osmotic lysis of Thermus caldophilus GK24, The purified enzyme had an apparent molecular mass of 108, 000 Da and consisted of two subunits of 54,000 Da. lsoelectric-focusing analysis of the purified enzyme showed a pi of 7.3. The enzyme contained two Cys residues, and its amino acids composition was quite different from that of Thermus aquaticus YT-1 alkaline phosphatase and Escherichia coli alkaline phosphatase, The optimum pH and temperature of the enzyme were 11.0-11.5 and $80^{\circ}C$ respectively. The enzyme was stable in the pH range of 9.0-12.0 at $25^{\circ}C$ for 36 h. and the half-life at $80^{\circ}C$ (pH 11.0) was 6 h. The enzyme was activated by $MgCl_2$ and inhibited by EDTA. With ${\rho}-nitrophenyl\;phosphate\;({\rho}NPP)$ as the substrate, the enzyme had a Michaelis constant $(K_m) $of $3.6{\times}10^{-5}M$, The enzyme preferentially hydrolyzed the phosphomonoester bond of AMP in ribonucleotides and glycerophosphate.

• Food Science and Biotechnology
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• v.18 no.3
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• pp.655-660
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• 2009

Biotechnological phytase preparations are commercially available and are currently used in animal feeding. However, thermostability constraints, low yields, and the high cost of the enzyme have limited its use. This study represents a new perspective for the food enzyme market. The research screened thermostable yeast strains for their ability to produce phytase. The screening was carried out with a gradual increase in temperature ($30-48^{\circ}C$). Sixteen strains (1 strain identified as Saccharomyces cerevisiae) maintained the ability to produce phytase at $48^{\circ}C$ and their phytase activity was confirmed using 2 phytase assay methodologies. The yeast strains tested in this study seem to be potential efficient producers of phytase, indicating a possible new source of thermostable phytase of commercial interest, particularly that from S. cerevisiae.

• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.122-127
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• 1991

A bacterial strain NO. 32 which produced thermostable ${\alpha}$-amylase was isolated from soil and identified to genus of Bacillus. To enhance ${\alpha}$-amylase productivity, a successive mutation of Bacillus sp. No. 32 was attempted with treatment of N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The resulting mutant, Bacillus sp. No. 32H417, which is risistant to refampicin and deficient in spore formation, produced about 90-fold high level of ${\alpha}$-amylase when compared with parental strain. The properties of the enzyme for thermostability were investigated. The optimal temperature and pH for enzyme reaction were 95$^{\circ}C$ and pH6.5, respectively, in the presence of 0.3mM $Ca^{2+}$ as an effective stabilizer.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.53-56
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• 2000

A thermostable esterase from the hyper thermophilic archaeon Sulfolobus solfataricus was partially purified 590-fold with $16.2\%$ recovery. The partially purified esterase had a specific activity of $29.5\;{\mu}mol\;min^{-1}mg^{-1}$ when the enzyme activity was determined using p-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH for esterase were $75^{\circ}C$ and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating that S. solfataricus esterase can be used for the production of commercially important chiral drugs.

• Microbiology and Biotechnology Letters
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• v.9 no.2
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• pp.91-97
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• 1981

A thermophilic soil isolate Bacillus sp. Y-127 was selected for the production of thermostable amylase. The strain was used for the enzyme production and the thermostable amylase was characterized. The optimum cultural conditions for the enzyme production were 6$0^{\circ}C$ at pH 7.0 for 32 hours using a mineral medium containing 2% soluble starch and 0.2% yeast extract. The extra-cellular enzyme was purified about 123-folds with about 6% recovery. The purified enzyme was stable at pH between 4.0 and 7.0, and temperature up to 6$0^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.7 no.1
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• pp.37-42
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• 1997

A bacterial strain L1 producing a thermostable esterase was isolated from soil taken near a hot spring and identified as Bacillus stearothermophilus by its microbiological properties. The isolated thermostable esterase was purified by ammonium sulfate fractionation, ion .exchange and hydrophobic interaction chromatographies. The molecular weight of the purified enzyme was estimated to be 50,000 by SDS-PAGE. Its optimum temperature and pH for hydrolytic activity against PNP caprylate were $85^{\circ}C$ and 9.0, respectively. The purified enzyme was stable up to $70^{\circ}C$ and at a broad pH range of 4.0-11.5 in the presence of bovine serum albumin. The enzyme was inhibited by phenylmethylsulfonyl fluoride and diethyl p-nitrophenyl phosphate, indicating the enzyme is a serine esterase. The enzyme obeyed Michaelis-Menten kinetics in the hydrolysis of PNPEs and had maximum activity for PNP caproate ($C_6$) among PNPEs ($C_2-C_12$) tested.

• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.86-91
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• 1996

Thermostable $\beta$-mannanase from Bacillus sp. YA-14 was purified by acetone precipitation, CM-cellulose, Sephadex G-100 and hydroxyapatite column chromatography from culture supernatant. The final enzyme preparation appeared to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). $\beta$-Mannanase appeared to be a monomeric protein with a molecular weight of 67, 000 daltons. The optimal pH and temperature of the enzyme reaction were pH 6.0 and $75^{\circ}C$ , respectively. The enzyme was stable at a pH range of 6.0 to 9.0 and at temperatures between 45 and $85^{\circ}C$. The kinetic constants of $\beta$-mannanase as determined with a galactomannan (locust bean) as substrate were a Vmax of 25 unit/ml and a Km of 1.1 mg/ml. The enzyme had only limited activity on galactomannan substrate. It was suggested that mg $\beta$-mannanase activity is limited by the number of branched $\alpha$-galactose residues.