• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.282-282
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• 2003

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.

• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.56-61
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• 1997

An alkaline protease producing microorganism was isolated from korean traditional Meju and identified as Scopulariopsis brevicaulis. The optimum culture condition of Scopulariopsis brevicaulis for the production of alkaline protease was as follow: 2% soluble starch, 0.2$, tryptophan, 0.1% (NH$_{4}$) $_{2}$S$_{2}$O$_{8}$ 0.2% NaHPO$_{4}$, pH 7.5, 35$\CIRC $C. The optimum pH and temperature for the enzyme activity of alkaline protease producing Scopulariopsis brevicaulis were pH 9.0 and 50$\circ $C, respectively. The enzyme was relatively stable at pH 6.0~11.0 and at temperature below 40$\circ $C. The activity of the enzyme was inhibited by Hg$^{2+}$ whereas Cu$^{2+}$ gave rather activating effects on the enzyme activity. Phenylmethanesulfonyl fluoride inhibited the enzyme activity. This result indicates that serine is very important role in this enzyme. Km value for casein was 1.2410$^{4}$ M/L, V$_{max}$ value for casein was 25.99 $\mu $g/min. This enzyme hydrolyzed casein more rapidly than the hemoglobin.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• 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.

• Applied Biological Chemistry
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• v.35 no.4
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• pp.237-241
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• 1992

The protease from Halomonas sp. ES 10 was purified by methanol precipitation, gel filtration on Sephadex G-150 and G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The specific activity of purified enzyme was 1,014 units/mg protein, and the yield of the total activity from the culture filtrate was 7%. The optimal temperature and pH for the enzyme activity were $35^{\circ}C$, and pH 11.0, respectively. And the enzyme was stable in the range of $pH\;7.5{\sim}11.0$. The residual activity of the enzyme was 70%, when the enzyme was incubated at $50^{\circ}C$ for 40 min. The Km value of the enzyme was 7.4 mg/ml to milk casein. $Li^+$, $Ca^{2+}$, SDS and Tween 80 were appeared to activators, whereas $Hg^{2+}$ and EDTA to inhibitors. The addition of DFP and PMSF showed the relative enzyme activities of 63% and 107%, respectively, suggesting that the enzyme may not belong to serine type protease. When the alkaline protease was treated with 0.5 M and 1 M NaCl, the relative enzyme activities were 95% and 65%, respectively. This enzyme showed 20% and 15% higher enzyme activity than that of Aspergillus oryzae (Sigma Chemical Company product, P4755) in the presence of 0.5 M and 1 M NaCl.

• 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.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• Journal of the Korean Society of Food Science and Nutrition
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• v.14 no.4
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• pp.401-408
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• 1985

The yeast cell wall lytic action of the alkaline AL-protease, which was found out of the crude Zymolyase that a kind of yeast cell wall lytic $endo-{\beta}-1$, 3-glucanase produced from Arthrobacter luteus, was investigated with the viable cells of S. sake and it's cell wall preparation. AL-protease on the lysis of the viable yeast cells showed very low activities with the alone, but the lytic activities were highly increased with the combination of AL-protease and Zymolyase. On the stepwise treatment of the viable yeast cells with AL-protease and Zymolyase, the cells were lysed highly only by the course having a treatment with Zymolyase after pretreatment with AL-protease. Thus synergistic action of AL-protease was not observed with any some commercial enzymes, known as a type of alkaline and serine protease such as AL-protease, and was also found to be affected greatly by the culture conditions and species of the yeast tested. AL-protease caused the release of some peptide and a lot of sugar from the cell wall preparation, but could not lysed the cell wall more than 66%. Whereas Zymolyase could lysed the cell walls almost completely with alone. On the basis of these results, the synergistic action of AL-protease on the lysis of S. sake cells is hypothesized that at first AL-protease bind to the yeast cell surface layer consisting of mannan and protein, and then changes their conformation to facilitate the penetration of Zymolyase from the outside to the inside framework layer constituted of alkali insoluble ${\beta}-1,\;3-glucan$.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.1-6
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• 1996

A bacterial strain NS70 producing an alkaline protease was isolated from soil samples taken near a hot spring and identified as Bacillus licheniformis by its morphological and physiological properties and cellular fatty acid analysis. The isolated alkaline protease was purified by ammonium sulfate fractionation, DEAE-, CM-, and Phenyl-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 32, 000 Da by sodium dodecylsulfate polyacrylamide gel electrophoresis. Its optimal pH and temperature for proteolytic activity against Hammarsten casein were 12 and $65^{\circ}C$, respectively. The enzyme was stable at alkaline pH range from 6.0 to 12.0, and fairly stable up to $65^{\circ}C$. The enzyme was inhibited by phenylmethylsulfonyl fluoride but not by EDTA and N-ethylmaleimide indicating that the enzyme is serine protease. Enzyme activity was markedly inhibited by $Hg^{2+}$ and $Cu^{2+}$. Autolytic phenomena were observed on purified protease NS70 but autolysis was reduced by the addtion of $Ca^{2+}$ ion or bovine serum albumin.