• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.353-360
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• 1998

A bacterial strain PN33 producing large amounts of extracellular pectin lyase (PNL, EC 4.2.2.10) was isolated from soil. The isolated bacterium was identified as a strain of Bacillus sp. Production of PNL by the strain was induced only by pectins, with a higher degree of esterification, which had been added to the culture medium as a sole carbon source. The optimal medium for PNL production was determined to consist of 10 g pectin, 2 g yeast extract, 4 g $K_2HPO_4{\cdot}3H_2O$, 0.6 g $MgSO_4$, and 0.11 g $CaCl_2$ per liter (pH 7.0). The PNL activity in the culture supernatant reached the highest level of 132 mU/ml after 32 h cultivation at $37^{\circ}C$ in the optimal medium. The PNL produced was purified to homogeneity by ammonium sulfate fractionation (50~80%), and cation exchange and size exclusion chromatographies. The molecular mass of the enzyme was estimated to be approximately 52 kDa by SDS-PAGE. Almost the same mass was determined by nondenaturing PAGE, indicating that the functional enzyme had a monomeric structure. As expected, the PNL exhibited higher activities on the highly esterified pectins whereas it gave no detectable activity on polygalacturonic acid. The enzyme showed the highest activity at the acidic pH of 6.0, exceptional for a bacterial PNL. Maximum activity was measured at $40^{\circ}C$, although the stability f the purified enzyme was poor at this temperature. alcium (1 mM) was found to activate the PNL activity by $50\%$, and also remarkably increased the thermal stability f the enzyme. Phenylmethylsulfonylfluoride (PMSF) and iethylpyrocarbonate (DEPC) inhibited the PNL activity lmost completely at the concentration of 5 mM. This result ndicates that some serine and histidine residues of the nzyme may play an essential role for catalytic function of he enzyme.

• 한국미생물·생명공학회지
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• 제30권4호
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• pp.325-331
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• 2002

아카시재목 버섯 균사체로부터 혈전용해효소를 생산하기 위한 최적 배양조건을 조사하였다. 복합배지 중 CVM이 가장 우수하였으며, 탄소원, 질소원, 인산원 및 무기질원으로 각각 2% galactose, 0.6% yeast extract와 0.1% $NaNO_3$, 0 0.1 % $K_2HPO_4$, 및 0.05% $MgSO_4$. $7H_2O$의 첨가에 의하여 효소의 활성이 가장 증가하였다. 따라서 F. fraxinea로부터 혈전용해효소를 생산하기 위한 최적 배지조건은 2% galactose, 0.6% yeast extract, 0.1 % $NaNO_3$, 0.1 % $K_2HPO_4$ 및 0.05% $MgSO_4$. $7H_2O$이다. 이상의 배지를 사용하여 배양온도 $25^{\circ}C$ 초기 pH 9에서 10일 동안 배양하였을 때 효소의 생산이 가장 높은 결과를 나타내었다. 배양액 중의 효소의 최적 온도 및 pH는 $40^{\circ}C$ 및 pH 10이었다. 본 효소의 활성이 PMSF와 aprotinin에 의하여 완전히 억제되는 것으로 보아 본 효소가 senne protease 계열의 효소임을 추정할 수 있었다.

• 사상체질의학회지
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• 제19권1호
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• pp.160-170
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• 2007

1. Objectives This study was performed to find the activities and characteristics of purified thrombolytic enzymes from Holotrichia extracts. 2. Methods In the first time, a coarse enzyme fluid was made by using the freedried Holotrichia extracts. After manufacturing total soluble proteins and purifing enzymes, it was evauluated the activities and characteristics of this enzyme's dissolving capability to fibrin and thrombus. This study was taken using azocasein assay, fibrin-plate method, native-PAGE and fibrin zymography. 3. Results A soluble proteins were efficiently extracted form freezedried Holotrichia extracts. And, this purified enzyme had a ten times fibrinolytic capability compare with ustulation Holotrichia sample. In native PAGE and fibrin zymography, Holotrichia extracts showed the respectable fibrinolytic activity. Also, It had higher thrombolytic activities compared with general thrombolytic enzyme 'plasmin'. In experiment of various protease inhibitors of the purified enzyme from Holotrichia extracts on the azocaseinolytic activity, the enzyme was strongly inhibited by EDTA ${\cdot}$ EGTA, and weakly by APMSF ${\cdot}$ PMSF ${\cdot}$ TPCK. 4. Conclusion Holotrichia extracts has the thrombolytic activities, and it will operate directly th fibrin-clot and thrombus.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• 대한약침학회지
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• 제17권1호
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• pp.44-50
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• 2014

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.624-631
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• 2007

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by tractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63kDa protease (PRO63) at pH5.0. The optimum temperature for the two proteases was $65^{\circ}C$. The PRO33 had a $K_m$ value of 6.6mM and a $V_{max}$ value of $10.31{\mu}mol/l/min$, and PRO63 l7.6mM and $9.08{\mu}mol/l/min$, with casein as substrate. They were thermostable at $60^{\circ}C$. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at $70^{\circ}C$ for 1h. The thermal stability of the two enzymes was significantly enhanced by $Ca^{2+}$. The residual activity of PRO33 and PRO63 at $70^{\circ}C$ after 60min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing $Ca^{2+}$. These properties make them applicable for many biotechnological purposes.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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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.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.829-835
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• 2004

A fibrinolytic enzyme has been found in several bacteria isolated from fermented food. This study was carried out to investigate the purification and characteristics of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 originated from Chungkookjang. The fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate fractionation and chromatographies on DEAE-cellulose and on Sephadex G-100. The final specific activity of the purified enzyme increased 11.0-fold, and the protein amount in the purified enzyme was about 16% of that in the culture supernatant. The molecular weight of the purified enzyme was estimated to be about 45,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 7.0 and $60^{\circ}C$, respectively. The enzyme activity was relatively stable up to $60^{\circ}C$ over the pH range of 7.0-10.0. The fibrinolytic enzyme activity increased by $Ca^{2+}$ and $Cu^{2+}$, whereas it was inhibited by $Hg^{2+}$ and $Ba^{2+}$. In addition, it was severely inhibited by PMSF and DFT. It is suggested that the purified enzyme was a serine protease for the fibrinolysis. The purified enzyme could completely hydrolyze fibrin in vitro within 8 h. Hence, it is suggested that the purified enzyme can be put into practice as an effective thrombolytic agent.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.264-271
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• 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.

• Fisheries and Aquatic Sciences
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• 제1권2호
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• pp.201-208
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• 1998

A protease, which had no tryptic and chymotryptic activity, was purified from the hepatopancreas of shrimp, P. japonicus, through ammonium sulfate fractionation, Q­Sepharose ionic exchange, benzamidine Sepharose 6B affinity, and Sephacryl S-100 gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 24 kDa by gel filtration and showed a single peptide band by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protease had a low ratio of acidic to basic amino acids, which is different with pro teases from marine animals. The enzyme was partially inhibited by benzamidine, tosyl-L-lysine chioromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), and pepstatin. The enzyme did not have any activity against benzoyl-D,L-arginine p-nitroanilide (BAPNA) or benzoyl-L-tyrosine ethyl ester (BTEE) which is a specific substrate of trypsin and chymotrypsin, respectively. However, the enzyme showed activity forward N-CBZ-L-tyrosine p-nitrophenyl ester (CBZ-Tyr-pNE), N­CBZ-L-tryptophan p-nitrophenyl ester (CBZ-Trp-pNE), and N-CBZ-L-proline p-nitrophenyl ester (CBZ-Pro-pNE). The protease did not showed tryptic and chymotryptic activity, which was not reported in shrimp hepatopancreas.