• Journal of Food Hygiene and Safety
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• v.26 no.1
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• pp.76-81
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• 2011

The protease produced by a Bacillus pumilus CN8 strain was purified by DEAE-Cellulose-52 ion exchange. It has a molecular weight of approximately 96,920 Dalton. In the present study, this protease showed strong activity over a broad range of pH (6.5-9.5) and temperature from $40^{\circ}C$ to $60^{\circ}C$, and the protease performed the maximal activity at pH 7.3 at $42^{\circ}C$. The effect of metal ions on protease activity showed that $K^+$ could slightly increase the protease activity, and other ions such as $Zn^{2+}$, $Fe^{2+}$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$ had no significant activation or inhibition to the protease (P> 0.05), and the more important is that $Cu^{2+}$, $Mn^{2+}$, $Sn^{2+}$, $Cd^{2+}$ had a strong inhibitory effect on the protease activity.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.20-27
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• 2011

The purification and characterization of a $Mn^{2+}$-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be $60^{\circ}C$. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. $Mn^{2+}$ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants ($H_2O_2$, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

• Journal of Microbiology and Biotechnology
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• v.32 no.1
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• pp.99-109
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• 2022

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9^T) production from Ornithinibacillus caprae L9^T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9^T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80℃) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70℃ and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag⁺, Ca²⁺ and Sr²⁺, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9^T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9^T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9^T protease in industrial applications, especially in leather processing.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.4
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• pp.636-641
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• 1995

Proteolytic activities were compared using three species involving in squid jeotkal fermentation and showing positive reaction upon casein test : Pseudomonas D2, Flavovacterium odoratum and Acinetobacter calcoaceticus. Pseudomonas D2 produced highest activity of protease at 72h when incubated in our own modified medium(polypeptone, 0.5% ; tryptone, 0.5% ; NaCl, 3% ; pH, 7.5). Thus, this specie was selected for the further study. The growth pattern was coincided with the production of protease. Thus purification of protease was proceeded by ethanol precipitation, sephadex G-100 gel filtration, and DEAE sepharose ion exchange chromatography. The purified protease showed highest activity at pH 7.0 and 5$0^{\circ}C$. The enzyme was very stable over the wide ragnes of the temperature ; even with one hour heat treatment at 7$0^{\circ}C$, the enzyme showed substantial amount of the activity toward casein. In addition, the enzyme was stable over the wide range of pH. Molecular weight of the protease was determined to be 17.4 kD by SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.667-667
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.677-684
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Journal of Microbiology and Biotechnology
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• v.21 no.2
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• pp.129-135
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• 2011

Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively.

• Journal of Life Science
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• v.23 no.2
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• pp.273-281
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• 2013

The purpose of this research was to investigate the production and characterization of alkaline protease from Micrococcus sp. PS-1 newly isolated from seawater. Micrococcus sp. PS-1 was grown in Luria-Bertani (LB) medium. Its optimal temperature and pH for growth were $30^{\circ}C$ and 7.0, respectively. The effect of nitrogen sources was investigated on optimal enzyme production. A high level of alkaline protease production occurred in LB broth containing 2% skimmed milk. The protease was purified in a 3-step procedure involving ultrafiltration, acetone precipitation, and dialysis. The procedure yielded a 16.43-purification fold, with a yield of 54.25%. SDS-PAGE showed that the enzyme had molecular weights of 35.0 and 37.5 kDa. Its maximum protease activity was exhibited at pH 9.0 and $37^{\circ}C$, and its activity was stable at pH 8.0-11.0 and $25-37^{\circ}C$. The protease activity was strongly inhibited by PMSF, EDTA, and EGTA. Taken together, the results demonstrate that the protease enzyme from Micrococcus sp. PS-1 probably belongs to a subclass of alkaline metallo-serine proteases.

• Korean Journal of Clinical Laboratory Science
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• v.44 no.4
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• pp.216-221
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• 2012

The fruiting body of Cordyceps is derived from the pupa or larva of insects infected by the entomopathogenic fungi Cordyceps. The fruiting body has been used as an anti-cancer and anti-inflammatory ingredient in traditional Chinese medicine. The biochemical characteristics of protease isolated from Cordyceps nutans were investigated in this study. The culturing period for production of protease by C. nutans was 10days. The acidity was pH 7.0, and the temperature was $25^{\circ}C$. The carbon and nitrogen sources for the production of the protease were glucose and yeast extract, respectively. The ratio of C/N was 2% glucose and 0.6% yeast extract. 0.06% $CuSO_4$ was used as the inorganic salt. The investigation into the acidity of the protease produced by C. nutans revealed that the optimal pH and temperature were pH 7.0 and $30^{\circ}C$. The stability of the protease was shown as pH 6.0~9.0 and $30{\sim}50^{\circ}C$. The investigation into the influence of the metal ions on the enzyme activation of C. nutans revealed that it was inhibited in $ZnSO_4$ and activated in $FeSO_4$.

• Korean Journal of Food Science and Technology
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• v.21 no.4
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• pp.569-574
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• 1989

These studies were conducted to investigate the purification and characterization of Kiwifruit protease, and the results obtained were as follows The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography and purified enzyme gave a single protein band on polyacrylamide gel electrophoresis The specific activity of purified enzyme was 30,10 units/mg protein and the yield was 7.48. The purified enzyme showed a high affinity for casein and hemoglobin. The optimal pH and temperature for enzyme activity were 7.0 and $45^{\circ}C$, respectively. The enzyme activity was strongly inhibited by $HgCl_2,\;MnSO_4$. However. the enzyme was activated by cysteine and EDTA. The Michaelis constant for casein was calculated to be 50.5mg/ml according to the Line weaver-Burk method, and its molecular weight was determied as 23,500 by polyacrylamide gel electrophoresis.