• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.489-498
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• 2013

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using $1.38{\times}10^9$ CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at $37^{\circ}C$, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and $70^{\circ}C$, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

• Microbiology and Biotechnology Letters
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• v.27 no.3
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• pp.184-190
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• 1999

To acquire an industrially useful biocatalyst for the enzymatic synthesis and production of various D-amino acid aminotransferase (D-AAT) activity. The enzyme activity was found from 110 strains of isolated thermophiles revealing its wide occurrence in thermophiles. Enzyme activity and thermal stability of the D-AAT producers were compared. Finally we have selected four thermophiles as producers of potent biocatalysts for the D-amino acid production; two thermophiles, Bacillus sp. Lk-1 and LK-2, having higher specific activity and two thermophiles, B. stearothermophilus KL-01 and Bacillus sp. KLS-01, having higher thermal stability than the D-AAT producers. Taxonomic and physiological characteristics of the four isolated thermophiles were described herein.

• Journal of Applied Biological Chemistry
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• v.42 no.3
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• pp.130-133
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• 1999

Lipoxygenase activities of three cultivars of black rice (Chindo, Suwon-415, Yongkeum-1) were determined using an oxygen polargraphic method to measure oxygen uptake. Studies at different pH levels revealed that the optimum pH was about pH 7.0 for Suwon-415 and pH 7.5 for Chindo and Yongkeum-1. The specific activities of Chindo, Suwon-415 and Yongkeum-1 at optimal pH were 41.0, 27.3, and 29.6 unit/mg-protein, respectively. In all the cultivars, there was an increase in the activity with increase in reaction temperature. Enzyme activity was tested at different concentrations of the substrate. The resulting $K_m$ and $V_{max}$ values of Chindo, Suwon-415, and Youngkeum-1 were 0.059, 0.050, and 0.066 mM and 2020, 2283, and 1616 unit/g-grain, respectively. Enzyme activity decreased at all storage temperatures(25, 4, and $-40^{\circ}C$).

• Microbiology and Biotechnology Letters
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• v.34 no.2
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• pp.121-128
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• 2006

The purified xylanase from Bacillus alcalophilus AX2000 was modified with various chemical modifiers to determine amino acid residues in the active site of the enzyme. Treatment of the enzyme with group-specific reagents such as carbodiimide or N-bromosuccinimide resulted in complete loss of enzyme activity. These results suggested that these reagents reacted with glutamic acid or aspartic acid and tryptophan residues located at or near the active site. In each case, inactivation was performed by pseudo first-order kinetics. Inhibition of enzyme activity by carbodiimide and N-bromosuccinimide showed non-competitive and competitive inhibition type, respectively. Addition of xylan to the enzyme solution containing N-bromosuccinimide prevented the inactivation, indicating the presence of tryptophan at the substrate binding site. Analysis of kinetics for inactivation showed that the loss of enzyme activity was due to modification of two glutamic acid or aspartic acid residues and single tryptophan residue.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.633-640
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• 2005

The purified xylanase from Bacillus pumilus TX703 was modified with various chemical modifiers to determine the active sites of the enzyme. Treatment of the enzyme with group-specific reagents such as carbodiimide or N-bromosuccinimide resulted in complete loss of enzyme activity. These results assumed that these reagents reacted with glutamic acid or aspartic acid and tryptophan residues located at or near the active site. In each case, inactivation was performed by pseudo first-order kinetics. Inhibition of enzyme activity by carbodiimide and W-bromosuccinimide showed non-competitive and competitive inhibition type, respectively. Addition of xylan to the enzyme solution containing N-bromosuccinimide prevented the inactivation, indicating the presence of tryptophan at the substrate binding site. Analysis of kinetics for inactivation showed that the loss of enzyme activity was due to modification of two glutamic acid or aspartic acid residues and single tryptophan residue.

• Microbiology and Biotechnology Letters
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• v.21 no.5
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• pp.446-452
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• 1993

The cholesterol oxidase produced from Bacillus sphaericus was purified and characterized. Through a series of purification procedures including DEAE-Toyoperal 650C, Sephadex G-200 and DEAE-Sephadex A-50 column chromatography, the purified enzyme was shown to have a specific activity of 0.179 units/mg protein having 31.8 fold purification and final yield of 12%. The molecular weight of the enzyme was estimated to be 47kDa and 47.tkDa by Sephadex G-200 chromatography and SDS-PAGE. The optimum temperature and pH for the enzyme were 30C and 6.0, respectively. The activity of the purified cholesterol oxidase was inhibited by Fe2+ and Hg+.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.188-193
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• 1989

Gene for lactose catabolism in Lactobacillus casei SW-M1 was encoded by a 60Kb metabolic plasmid. A derivative of only 10kb, pPlac 15 of recombinant plasmid, was constructed by introducing into pBR322 and was cloned into E. coli using restriction endonuclease Pst I. A 10kb insery DNA in plasmid pBR322 was identified as a gene encoded phospho-$\beta$-galactosidase by the determination of enzyme activity. Phospho-$\beta$-galactosidase was apparently expressed in E. coli. The enzyme activities of cell-free extract from transformant E. coli HB101 carrying pPLac 15 DNA were not different from that of L. casei as a donor strain on the basis of enzyme properites. However, specific activity of phospho-$\beta$-galactosidase in the cloned strain with Lac $Y^{-}$ phenotype of E. coli HB101 was lower than that in L. casei strain.

• Journal of Life Science
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• v.12 no.5
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• pp.561-565
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• 2002

Pseudomonas sp. EL-041 was previously isolated from phenol-acclimated activated sludge. This bacterium was capable of degrading phenol and cometabolizing trichloroethylene (TCE). In this study, we report the identification of trichloroethylene- degrading enzyme in Pseudomonas sp. EL-041 by the investigation of enzyme activity and DNA sequencing of specific phenol oxygenase gene. As the results of experiment, trichloroethylene-degrading enzyme in Pseudomonas sp. EL-041 was monooxygenase and suspected to phenol hydroxylase.

• Korean Journal of Food Science and Technology
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• v.32 no.5
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• pp.1158-1167
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• 2000

To produce ${\beta}-cyclodextrin({\beta}-CD)$, a cyclodextrin glucanotransferase(CGTase) producing Aspergillus sp. CC-2-1 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. It was found that production of CGTase reached to the maximum when the wheat bran medium containing 0.1% albumin, 2% $(NH_4)_2S_2O_8$, 2% soluble starch and 0.2% $KH_2PO_4$ was cultured for 5 days at $37^{\circ}C$. The purity of CGTase was increased by 13.14 folds after DEAE-cellulose ion exchange chromatography and Sephadex G-100, G-150 gel filtration and the specific activity was 172.14 unit/mg. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. The molecular weight of CGTase was estimated to be 27,800 by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the CGTase activity were 9.0 and $80^{\circ}C$, respectively. The enzyme was stable in pH $8.0{\sim}11.0$ at $60{\sim}80^{\circ}C$. The activity of purified enzyme was activated by $K^+,\;Cu^{2+}$ and $Zn^{2+}$. The activity of the CGTase was inhibited by the treatment with 2,4-dinitrophenol and iodine. The result suggests that the purified enzyme has phenolic hydroxyl group of tyrosine, histidine imidazole group and terminal amino group at active site. The reaction of this enzyme followed typical Michaelis-Menten kinetics with the $K_m$ value of 18.182 g/L with the $V_{max}$ of 188.68 ${\mu}mole/min$. The activation energy for the CGTase was calculated by Arrhenius equation was 1.548 kcal/mol.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.189-196
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• 1992

A $\alpha$-l, 4-D-glucan maltohydrolase $(\beta$-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/mg. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64, 000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the $\alpha$-l, 4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and $\beta$-limited dextrin. This amylolytic enzyme displayed a temperature optimum at $45^\circ{C}$ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial $\beta$-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of from starch by $\beta$-amylase was greatly stimulated by pullulanase addition. These results differentiated from other $\beta$-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.