• Journal of agriculture & life science
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• v.45 no.6
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• pp.201-212
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• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• Applied Chemistry for Engineering
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• v.16 no.5
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• pp.705-711
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• 2005

The reaction of lauric acid vinyl ester with n-butanol was established to give lauric acid butyl ester by subtilisin A-catalyzed transesterification in organic media containing nonionic surfactants. Pyridine has been used as an organic solvent since it provides higher yields than other organic solvents. However, because of stench of pyridine, compounds enzymatically synthesized in pyridine may be unsuitable as ingredients for foods and drugs. Hence, in order to select an organic solvent to replace pyridine, sugar esters were synthesized with the protease in various organic media. However, no solvent paralleled pyridine in yields. On the basis of this result, pyridine-based W/O microemulsions containing nonionic surfactants and water were used in enzymatic synthesis of the sugar ester, and when Tween 60, Brij 56, or 1-O-octyl-${\beta}$-D-glucopyranose was used for the W/O microemulsions, the yield was higher.

• Journal of Life Science
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• v.23 no.1
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• pp.15-23
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• 2013

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. AprE51-6 showing increased catalytic activity was produced previously. To enhance the thermostability of AprE51-6, 2 residues, Gly-166 and Asn-218 based on B. subtilis subtilisin E were mutated by site-directed mutagenesis. The results of the mutational analysis showed that substitution of arginine for Gly-166 (AprE51-7) increased the fibrinolytic activity 1.8-fold. An N218S mutant (AprE51-8) also increased the fibrinolytic activity up to 4.5-fold in a fibrin plate assay. Purified AprE51-7 and AprE51-8 mutants had a 1.9- and a 2.5-fold higher $k_{cat}$, respectively, and a 2.1-1.9-fold lower $K_m$, respectively. This resulted in a 3.8- and a 4.7-fold increase in catalytic efficiency ($k_{cat}/K_m$), respectively, relative to that of wild-type AprE51. AprE51-8 had a broader pH range than AprE51-6 and nattokinase, especially at an alkaline pH value. In addition, AprE51-8 showed higher thermostability than AprE51-6 at $60^{\circ}C$. The half-lives of AprE51-7 and AprE51-8 at $50^{\circ}C$ were 21.5 and 27.3 min, respectively, which are 2.0 and 2.6 times longer, respectively, than that of the wild-type AprE51.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.36 no.5 s.108
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• pp.53-61
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• 2004

[ $Poly(_{D}-ldactide)\;(_{D}-PLA)$ ] was synthesized to have low molecular weight for miscible blends with a high molecular $poly(_{L}-lactide)\;(_{L} -PLA)$. The blends were prepared by dissolving the two components of $_{L}-PLA\;and\;_{D}-PLA\;(w/w)$ in chloroform (l00/0, 90/10, 70/30, 50/50, 30/70, 0/100). The miscibility of these miscible blends was characterized by gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and the selective degradability by enzymes (proteinase K, subtilisin and $\alpha$-chymotrypsin). The coating efficiency of PLA blends onto paper was determined and the degrading activity cellulases by on these blends. The miscibility, coating efficiency and enzymatic degradability of these blends were decreased according to increasing of $_{D}-PLA$ blending part. Such results were attributed to the extent of coating application of PLA, with better miscibility (compatibility), coating efficiency and degradability due to a higher $_{L}-PLA$ content.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.65-70
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• 1990

The objective of the current study is to elucidate the biological roles of proteinase inhibitor in microorganisms. As the first step, a strain of Streptomyces fradiae was selected as a producer of extracellular proteinase inhibitor. The proteinase inhibitor was purified from culture broth through ultrafiltration, gel-filtration and ion-exchange chromatography. Molecular weight of the proteinase inhibitor was estimated to be 16, 800 by SDS polyacrylamide gel electrophoresis. It was found that the proteinase inhibitor inhibited only alkaline serine proteinases such as subtilisin, $\alpha$-chymotrypsin and Promase E but not trypsin and other proteinases. The mode of inhibition against Pronase E with succinyl-phenylalanine-p-nitroanilide as a substrate was competitive.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.327-331
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• 2000

The application of LFH-PCR(long flanking homology region-PCR) for Bacillus subtilis gene disruption is presented. Without plasmid- or phage-vector construction, only by PCR, based on a DNA sequence retrieved from B. subtilis genome data base, kanamycin resistance gene was inserted into two genes of B. subtilis involved in sporulation, spoIIIE and spoIIIG. The effect of gene disruption on subtilisin expression was examined and the sporulation frequency of two mutants was compared to that of the host strain. For this purpose, only 2 or 3 rounds of PCR were required with 4 primers. We first demonstrated the possibility of LFH-PCR for rapid gene disruption to characterize an unknown functional gene of B. subtilis or other prokaryote in the genomic era.

• YAKHAK HOEJI
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• v.39 no.1
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• pp.55-60
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• 1995

The acetone precipitates of the hot water extract of Torilis ftuctus showed strong hemostatic activity which was not inhibited by aspirin. This activity was not through platelet activation but possibly through activating some coagulation factors in plasma. The hemostatic action of the precipitates was not active by oral adminitration and no behavioral toxicity was appeared in treated mice. However, mice treated with the acetone precipitates through tail vein showed serious tremor and then were killed probably by the thrombus produced in the body. The hemostatic activity was still remained after treatment with $\beta$-glucosidase, $\beta$-alactosidase, $\alpha$-amylase, subtilisin BPN', or trypsin but completely lost by acid hydrolysis. The active components seemed to be a complex of unidentified macromolecules to which some phenolic compounds were strongly bound.

• Parasites, Hosts and Diseases
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• v.45 no.4
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• pp.283-285
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• 2007

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed up regulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.

• Journal of Microbiology
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• v.44 no.6
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.80-85
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• 2010

Two different ${\alpha}$-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.