• Journal of Microbiology
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• 제33권4호
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• pp.315-321
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• 1995

The relationship between morphological differentiation and production of trypsin-like protease (TLP_ in streptomycetes was studied. All the Streptomyces spp.In this study produced TLP just before the onset of aerial mycelium formation. Addition of TLP inhibitor, TLCK, to the top surface of colonies inhibited aerial mycelium formation as well as TLP inhibitor, TLCK, to the top surface of colonies inhibited aerial mycelium formation as well as TLP activity. Addition of 2% glucose to the Bennett agar medium repressed both the aerial mycelium formation and TLP production in S. abuvaviensis, S. coelicolor A3(2), S exfoliatus, S. microflavus, S. roseus, s. lavendulae, and S. rochei. However the addition of glucose did not affect S. limosus, S. felleus, S. griseus, S. phaechromogenes, and S. rimosus. The glucose repression on aerial mycelium formation and production of TLP was relieved by the addition of glucose anti-metabolite (methyl .alpha.-glucopyranoside). Therefore, it was concluded that TLP production is coordinately regulated with morphological differentiation and TLP activity is essential for morphological differentiation in streptomycetes. The proposed role of TLP is that TLP participates in the degradation of substrate mycelium protein for providing nutrient for aerial mycelial growth.

• 한국식품영양과학회지
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• 제24권4호
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• pp.636-641
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• 1995

Proteolytic activities were compared using three species involving in squid jeotkal fermentation and showing positive reaction upon casein test : Pseudomonas D2, Flavovacterium odoratum and Acinetobacter calcoaceticus. Pseudomonas D2 produced highest activity of protease at 72h when incubated in our own modified medium(polypeptone, 0.5% ; tryptone, 0.5% ; NaCl, 3% ; pH, 7.5). Thus, this specie was selected for the further study. The growth pattern was coincided with the production of protease. Thus purification of protease was proceeded by ethanol precipitation, sephadex G-100 gel filtration, and DEAE sepharose ion exchange chromatography. The purified protease showed highest activity at pH 7.0 and 5$0^{\circ}C$. The enzyme was very stable over the wide ragnes of the temperature ; even with one hour heat treatment at 7$0^{\circ}C$, the enzyme showed substantial amount of the activity toward casein. In addition, the enzyme was stable over the wide range of pH. Molecular weight of the protease was determined to be 17.4 kD by SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• 제19권1호
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• pp.99-107
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• 2009

Strain PUPC1 produces an antifungal protease as well as plant growth promoting enzymes such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase and phosphatase. Morphological, cultural, and physiological characteristics as well as 16S rRNA gene-sequence-based phylogenetic analysis confirmed the taxonomic affiliation of PUPC1 as Chryseobacterium aquaticum. The optimum growth of PUPC1 was observed at pH 6.0 and $30^{\circ}C$, and maximum protease production was observed in medium B amended with 1% tryptone, 0.5% sucrose, and 0.005% $MnCl_2$. The protease was purified by ammonium sulfate precipitation, Sephadex G-75 gel filtration chromatography, and electroelution from preparative SDS-PAGE. The protease had a molecular mass of 18.5 kDa. The optimum pH and temperature stability of the protease were pH 5.0-10.0 and temperature $40-70^{\circ}C$. Chryseobacterium aquaticum PUPC1 and its protease showed a broad-spectrum antifungal activity against phytopathogenic fungi. Strain PUPC1 also exhibited plant growth promoting traits. The objective of the present investigation was to isolate a strain for agricultural application for plant growth promotion and biocontrol of fungal diseases.

• 한국미생물·생명공학회지
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• 제34권1호
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• pp.7-14
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• 2006

가금류의 feather은 전 세계적으로 발생되지 않는 지역이 없을 만큼 광범위하게 발생되어지는 폐기물로써, 현재까지 많은 연구자들에 의하여 그 이용성에 대한 연구가 활발하게 이루지고 있다. 그러나 disulfide 결합, 수소결합 및 이온결합에 의하여 매우 강한 결합력을 가지는 feather 구성 단백질의 화학적인 성질 때문에 분해하여 사용하기에 많은 어려움이 있다. 가금류 폐기물을 이용한 사료의 제조과정에서 가열 및 가압 등의 과정을 거치면서 아미노산의 손실이 발생하고 낮은 소화율 등이 문제로 야기되어지고 있다. 이러한 문제를 해결하기 위한 노력으로 미생물이 생산하는 keratinolytic protease를 이용하고자 산업적으로 이용 가능성을 가지는 균주를 분리하였다. 토양 시료로부터 Bacillus sp. SMMJ-2, FL-3, NO-4 및 RM-12 4종의 keratinolytic protease 생성균을 분리하였다. 이 균주들은 5.0% skim milk agar plate에서 높은 protease 활성을 나타내었으며, 2.0% whole chicken feather agar plate에서도 많은 점질성의 물질을 생산하였다. Keratinolytic protease 생산을 위하여 배지4($0.7%\;K_{2}HPO_{4},\;0.2%\;KH_{2}PO_{4},\;0.1%$ fructose, 1.2% soybean meal, $0.01%\;Na_{2}CO_3$, pH 7.0)에서 Bacillus sp. SMMJ-2에 의한 keratinolytic protease의 생산을 조사한 결과, 배양 3일에 가장 높은 keratinolytic protease 활성을 나타내었다. 그러나, Basal medium(0.05% NaCl, $0.03%\;Na_{2}HPO_{4},\;0.04%\;NaH_{2}PO_{4},\;0.5%$ whole chicken feather, pH 7.0)에 유일한 탄소 및 질소원으로 whole chicken feather를 첨가하고 배양한 결과, $30^{\circ}C$에서 일주일 이상 배양하였을 때 feather은 모두 분해되었으나 깃대는 분리하지 못하였고 배지 4에 질소원으로 1.2% whole chicken feather를 첨가하였을 때 40시간 이내에 깃대를 포함한 feather를 모두 분해하였다. Bacillus sp. SMMJ-2에 의한 keratinolytic protease의 생산은 접종 후 9시간이 지나면서 생산되기 시작하여 24시간 동안 배양하였을 때 최대 활성의 86%를 나타내는 것으로 조사되었다. Bacillus sp. SMMJ-2에 의하여 생산되어지는 keratinolytic protease 활성은 $30^{\circ}C$, 180 rpm으로 3일간 배양했을 때 106 units/ml/min 이였으며, protease 활성은 540 units/ml/min을 나타내었다. 온도와 pH에 대한 효소의 안정성은 $50\%$ acetone을 이용하여 분리한 효소로 조사한 결과, $30{\sim}50^{\circ}C$까지는 80% 이상의 잔존효소활성을 나타내었고, $60^{\circ}C$ 이상에는 20분간 열처리로 효소가 거의 실활 되었다. 효소의 pH 안정성은 pH $6.0{\sim}12.0$에서는 비교적 안정한 것으로 조사되었다.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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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.

• 대한미생물학회지
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• 제16권1호
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• pp.19-28
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• 1981

The Pseudomonas infection has been increased in incidence and suspected as a cause of opportunistic pathogen. Protease and elastase produced by Pseudomonas aeruginosa are reported to be closely associated with pathogenicity of Pseudomonas aeruginosa. We examined, in this work, the relationship between production of exoenzyme of Pseudomonas aeruginosa and susceptibility to antimicrobial agents in view of possible application to the management of Pseudomonas infection. 1. In 295 Pseudomonas aeruginosa isolated from clinical specimens, 34.6% were from pus, 20.7% from sputum, 15.6% from wound including burn sites and 12.9% from urine. 2. Distribution of protease and elastase production by clinically isolated Pseudomonas aeruginosa, showed that protease and elastase producing strains were 83.1%, protease producing strains were 7.5%, elastase producing strains were 2.0%, and non producing strains were 7.5%. 3. MIC(minimum inhibitory concentration) peak for tetracycline and chloramphenicol were observed at 25mcg/ml and 200mcg/ml respectively, but there were no Pseudomonas aeruginosa which correspond to MIC peak, 6.25mcg/ml. Gentamicin of aminoglycosides was highly susceptible to Pseudomonas aeruginosa clinically isolated from pus, sputum and wound sites, but susceptible to isolates from nasal discharge and urine. Regarding MIC peak of carbenicillin, 100mcg/ml, 81.8% of Pseudomonas aeruginosa were from urine, 54.8% from wound including burn sites, 52.7% from pus, and 50.8% from sputum. 4. Enzyme producing strains showed no susceptibility to kanamycine and carbenicillin at low concentration, but protease producing strains tend to resistant to antimicrobial agents.

• 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.

• 한국미생물·생명공학회지
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• 제5권3호
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• pp.153-158
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• 1977

Rhizopus japonicus S-62에 의한 acid protease의 생산조건 및 효소의 내열성을 검사하여 다음과 같은 결과를 얻었다. 1) 산성 protease의 생산에 있어 wheat bran medium에 관한 sucrose, yeast, ammonium chloride, sodium phosphate monobasic의 최적 첨가 농도는 각각 0.5%, 2.0%, 0.4%이었다. 2) 내열제로서KH$_2$PO$_4$와 NaH$_2$PO$_4$는 가장 효과적이었다. 3) 효소액에 KH$_2$PO$_4$와 NaH$_2$PO$_4$를 각각 2%씩 첨가하고 5$0^{\circ}C$에서 10분간 가열처리하였을 때 잔존청성은 다 같이 100%를 나타내었다. 4. 55$^{\circ}C$이상에서는 KH$_2$PO$_4$와 NaH$_2$PO$_4$의 과열변과는 거의 없었다.

• 한국미생물·생명공학회지
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• 제49권2호
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• pp.181-191
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• 2021

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

• 한국식품영양학회지
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• 제6권2호
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• pp.89-95
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• 1993

A Nuluk, a Korean traditional Koji for brewing, was made with wheat flour and Aspergillus oryzae L2 which had a good aroma and strong abilities In producing saccharogenic and dextrogenic enzymes. The cultural conditions for the production of saccharogenic and proteolytic enzymes were tested. The productivity of dextrogenic enzyme was improved when Nuluk was made with unsteamed wheat flour as compared with steamed one, but that of proteolytic enzyme was reduced. The addition of water containing 0.5% hydrochloric acid was unfavorable for the production of those two enzymes. The optimum ratio of water added to wheat flour for the production of those two enzymes was 28$^{\circ}C$ on the basis of wheat flour, The productivity of saccharogenic enzyme was enhanced when the Nuluk was molded after 20 hours of precultivation, but that of proteolytic enzyme was reduced as compared with no molding. The optimum temperatures for the production of saccharogenic enzyme and proteolytic enzyme were 36$^{\circ}C$ and 28$^{\circ}C$, respectively.