• Journal of the Korean Society of Food Science and Nutrition
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• v.13 no.2
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• pp.193-204
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• 1984

The enzymatic properties of the alkaline AL-protease, which had been prepared from the crude zymolyase of Arthrobzoter luteus, was investigated together with its active amino acid residue. Complete inactivaton of the proteolytic activity of AL-protease by either DFP or PMSF was simultaneously accompanied by the loss of its lytic effect on the lysis of yeast cell wall. In the reaction, AL-protease showed the pattern of inactivation to decrease very slowly, as compared to that of chymotrypsin, and that enzyme and DFP were found to react with a molar ratio of 1 : 1. The preparation of AL-protease exhibited no hydrolytic activity in any substrates of polysaccharases, playing a significant role in the lysis of yeast cell wall. The optimum pH and temperature of AL-protease was pH 10.5 and $65^{\circ}C$, respectively. It also showed stability in the pH range from 5 to 11 and at the temperature below $65^{\circ}C$. Through the identification of the amino acid residue in the active site of the $^{32}P$-diisopropylph-osphorylated(DIP) AL-protease modified specifically with $^{32}P$-labeled DFP, AL-protease was found to be a DFP-sensitive which has a mole of active serine residue involved in its proteolytic activity per mole of the enzyme.

• Korean Journal of Microbiology
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• v.34 no.3
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• pp.82-86
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• 1998

An alkaline proteinase secreted from Yarrowia lipolytica 504D was purified by salting-out and column chromatography. The molecular weight of the purified enzyme was about 32,000 Da estimated by SDS-PAGE. The optimal condition for the activity of the enzyme was at pH 9.5 and $42^{\circ}C$ The enzyme was stable up to $45^{\circ}C$ and at the range of pH 4-10. Because the enzyme was inhibited by PMSF as well as EDTA, EGTA, and phenan-throlin, it is uncertain whether the enzyme is serine proteinase or metalloproteinase. However, almost all metal salts tested did not increase the enzyme activity, and Ca salt restored the activity of the enzyme inactivated by EDTA. Therefore, the purified enzyme seems to be an serine proteinase (E.C. 3.4.21.14).

• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.6
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• pp.811-814
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• 1993

Properties of a protease purified from Sarcodon asparatus(Berk.) S. Ito have been investigated. The enzyme displays as a glycosylated serine protease. The sequence for the 21 amino acids of the N-terminal side in the enzyme was determined by automated sequence analysis. The sequence was V-T-T-K-Q-T-N-A-P-W-G-L-G-N-I-S-T-T-N-K-L-.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.87-92
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• 2005

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.544-549
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• 1995

The alkaline elastase is an extracellular serine protease of the alkalophilic Bacillus strain Ya-B. To increase the gene copy number and the production level of the alkaline elastase Ya-B, we designed, on the B. subtilis chromosome, a gene amplification of the 10.6 kb repeating unit containing amyE, aleE (alkaline elastase Ya-B gene) and tmrB. The aleE was inserted between amyE and tmrB, and B. subtilis APT119 strain was transformed with this amyE-aleE-tmrB-junction region fragment. As a result, we succeeded in obtaining tunicamycin-resistant (Tm$^{r}$) transformants (Tf-1, Tf-2) in which the designed gene amplification of 10.6 kb occurred in chromosome. The transformants showed high productivity of $\alpha $-amylase and alkaline elastase Ya-B. The copy number of the repeating unit (amyE-aleE-tmrB) was estimated to be 25, but plasmid vector (pUC19) was not integrated. The amplified aleE of chromosome was more stable than that of plasmid in absence of antibiotics.

• Journal of Microbiology
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• v.41 no.1
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.534-540
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• 1995

Alkalophilic coryneform bacteria TU-19 isolated from soil extracellularly produced at least three proteases (Protease I, II, and III). Investigating the cultural conditions related to the enzyme production of this bacterial cell, the optimum pH and temperature were 10.0 and $30^{\circ}C$, respectively. In order to purify these enzymes from the 2 day culture broth ammonium sulfate fractionation, gel filtration and QAE-Sephadex column chromatography were performed step by step. And then these three proteases were purified to near homogeneity by judging from SDS-PAGE pattern, and had the molecular weights of 120, 80, and 45 kilodaltons, respectively. The optimum pH and temperature for the enzyme activity of Protease I and II were 10.5 and $45^{\circ}C$, respectively, and Protease II were 11.0 and $50^{\circ}C$. And the enzymes were completely inhibited by PMSF suggesting serine protease, but not affected by pCMB. 1,10-phenanthroline, IAA, and EDTA.

• Journal of Life Science
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• v.11 no.1
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• pp.42-46
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• 2001

Subtilisin YaB, produced by alkalophilic Bacillus strain YaB, is an extracellular alkaline serine protease having 55% homology to subtilisin BPN'. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine the role of pro-sequence for the secretion of subtilisin YaB, we have studied the expression, in Bacillus subtilis, of a mutant preprosubtilisin YaB in which active site Ser214 is substituted with Cys. The use of a six protease-deficient strain, WB600, was required for its efficient production. The prosubtilisin YaB, thus produced, was indeed secreted into the culture medium and was processed to its mature form upon treatment with exogenously added active subtilisin YaB. From these results, we have concluded that the processing of pro-sequence is not essential for the secretion of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.181-186
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• 2000

MAPI (microbial alkaline protease inhibitor) was isolated from cultrue broth of Streptomyces chromofuscus SMF28. The Ki values of MAPI for the representative serine proteases such as chymotrypsin and proteinase K were 0.28 and $0.63{\;}\mu\textrm{M}$, respectively, and for the cysteine proteases cathepsin B and papain were 0.66 and $0.28{\;}\mu\textrm{M}$, respectively. These data indicate that MAPI is not a potent selective inhibitor of serine or cysteine proteases. Progress curves for the inhibition of three proteases by MAPI exhibithe characteristic patterns; MAPI exhibited slow-binding inhibition of cathepsin B. It was rapidly associated with chymotrypsin before the addition of substrate and then reactivation of MAPI-inhibited enzyme was investigated in the presence of substrate. On the other hand, MAPI-proteinase K interaction was typical for those classical inhibitors. When MAPI was incubated with trypsin, there was an extensive reduction in the ingibitory activities of MAPI corresponding to 66.5% inactivation of MAPI, indicating that trypsin-like protease may play a role in the decrease of the inhibitory activity during cultivation.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.