• Korean Journal of Food Science and Technology
• /
• v.23 no.4
• /
• pp.452-458
• /
• 1991

Psychrotrophs producing protease were isolated during ripening periods of Cheddar cheese and one of them containing the highest protease activity was identified as Pseudomonas fluorescens 65. The extracelluar proteolytic enzyme was partially purified from P. fluorescens 65 through the Sephadex G-100 gel filtration. The protease was eluted between 190 ml and 230 ml of elution volume of sodium phosphate buffer. The purified protease showed a single band in SDS-PAGE and its molecular weight was 47,000. The composition of amino acid for the protease was determined and the most abundant amino acids were glutamic acid (14.96%) and serine (13.86%). The optimum temperature and pH for the activity was $45{\sim}50^{\circ}C$ and 6.0, respectively.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.2
• /
• pp.264-271
• /
• 2006

Two fibrinolytic enzymes were purified from the culture supernatant of Fomitella fraxinea mycelia by ion-exchange and gel filtration chromatographies, and were designated as F. fraxenia proteases 1 and 2 (FFP1 and FFP2). The apparent molecular masses of the enzymes were estimated to be 32 kDa and 42 kDa, respectively, by SDS-PAGE and gel filtration chromatography. Both enzymes had the same optimal temperature ($40^{\circ}C$), but different pH optima (10.0 and 5.0 for FFP1 and FFP2, respectively). FFP1 was relatively stable at pH 7.0-9.0 and temperature below $30^{\circ}C$, whereas FFP2 was very stable in the pH range of 4-11 and temperature below $40^{\circ}C$. FFPI activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and aprotinin, indicating that this enzyme is a serine protease. The activity of FFP2 was enhanced by the addition of $CO^{2+}$ and $Zn^{2+}$ and inhibited by $Cu^{2+},\;Ni^{2+}$, and $Hg^{2+}$. Furthermore, FFP2 activity was strongly inhibited by EDTA and 1,10-phenanthroline, implying that the enzyme is a metalloprotease. Both enzymes readily hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$- and $B{\beta}$-chains of fibrinogen over ${\gamma}$-chain. FFP1 showed broad substrate specificity for synthetic substrates, but FFP2 did not. $K_{m}$ and $V_{max}$ values of FFP1 for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA, were 0.213 mM and 39.68 units/ml, respectively. The first 15 amino acids of the N-terminal sequences of both enzymes were APXXPXGPWGPQRIS and ARPP(G)VDGQ(R,I)SK(L)ETLPE, respectively.

• Korean Journal of Microbiology
• /
• v.32 no.4
• /
• pp.291-296
• /
• 1994

A bacterial strain NO. AM-28, showing proteolytic activity against defatted soybean was isolated from domestic soil. The isolated strain was identified as Aeromonas hydrophila by both the biochemical tests using API kit and the analysis of cellular fatty acid profile with MIDI system. The protease production from A. hydrophila AM-28 was highly enhanced when it was cultivated in the medium containing glycerol as a carbon source, tryptone or $(NH_4)_2HPO_4$ as a nitrogen source, and $CaCl_2$ as a mineral source. The optimal pH and temperature for the enzyme was 8.0 and $65^{\circ}C$, respectively. The enzyme was stable up to $55^{\circ}C$ and at pH values ranging from 7.0 to 13.0. The enzyme activity was inhibited by phenylmethylsulfonyl fluoride and EDTA, indicating that serine residue and metal ions be involved in enzyme activity.

• Journal of Microbiology and Biotechnology
• /
• v.34 no.2
• /
• pp.436-456
• /
• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

• Korean Journal of Microbiology
• /
• v.45 no.4
• /
• pp.291-299
• /
• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• Microbiology and Biotechnology Letters
• /
• v.17 no.6
• /
• pp.545-551
• /
• 1989

Thermostable alkaline protease of alkalophilic Bacillus sp. No. 8-16 has been purified, and the properties of the enzyme investigated. The characteristic point of the organism used is especially good growth in alkaline and thermal condition. The alkaline protease of the strain No. 8-16 was purified from crude enzyme by acetone precipitation, CM-cellulose ion exchange chromatography, Sephadex G-100 and Sephadex G-75 gel filtration. Through the series of chromatograpies, the enzyme was purified to homogeneity with specific activity of 37 fold higher than that of the crude broth. Characteristics of the purified enzyme were as follow; $K_m$ value for the enzyme was 1.3 mg/ml, the alkaline protease showed a maximal activity at 7$0^{\circ}C$ and from the pH 6.0 through 12.0, and stable for 1 hr. at 6$0^{\circ}C$. The moleclar weight of the enzyme was estimated to be 33,000 by Sephadex G-100 gel filtration. The activity of the alkaline protease was inhibited by iodoacetic acid and Ag$^+$, Hg$^+$, PMSF (phenylmethylsulfonyl fluoride), and activated by $Ca^{2+}$ and Mn$^{2+}$.

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

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

• BMB Reports
• /
• v.35 no.2
• /
• pp.206-212
• /
• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• Journal of Life Science
• /
• v.23 no.12
• /
• pp.1486-1494
• /
• 2013

A high protease-producing strain was isolated and identified from the digestive tract of octopus vulgaris by detecting a hydrolysis circle of protease and its activity. The strain was identified by morphology observation, biochemical experiments, and 16S rRNA sequence analysis. The protease obtained from the strain was purified by a three-step process involving ammonium sulfate precipitation, carboxy methyl-cellulose (CM-52) cation-exchange chromatography, and DEAE-Sephadex A50 anion-exchange chromatography. The properties of protease were characterized as well. The strain Bacillus sp. QDV-3, which produced the highest activity of protease, was isolated. On the basis of the phenotypic and biochemical characterization and 16S rRNA gene-sequencing studies, the isolate was identified as follows: domain: Bacteria; phylum: Firmicutes; class: Bacilli; order: Bacillales; family: Bacillaceae; and genus: Bacillus. The isolate was shown to have a 99.2% similarity with Bacillus flexus. A high active protease designated as QDV-E, with a molecular weight of 61.6 kDa, was obtained. The enzyme was found to be active in the pH range of 9.0-9.5 and its optimum temperature was $40^{\circ}C$. The protease activity retained more than 96% at the temperature of $50^{\circ}C$ for 60 min. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity, thus confirming that this protease isolated from Bacillus sp. QDV-3 is an alkaline serine protease. Metal ions, $Mn^{2+}$ and $Mg^{2+}$, were determined to enhance the protease activity, whereas $Ba^{2+}$, $Zn^{2+}$, and $Cu^{2+}$ were found to inactivate the enzyme.