• The Plant Pathology Journal
• /
• v.31 no.3
• /
• pp.278-289
• /
• 2015

Indigenous strains of Trichoderma species isolated from rhizosphere soils of Tea gardens of Assam, north eastern state of India were assessed for in vitro antagonism against two important tea fungal pathogens namely Pestalotia theae and Fusarium solani. A potent antagonist against both tea pathogenic fungi, designated as SDRLIN1, was selected and identified as Trichoderma viride. The strain also showed substantial antifungal activity against five standard phytopathogenic fungi. Culture filtrate collected from stationary growth phase of the antagonist demonstrated a significantly higher degree of inhibitory activity against all the test fungi, demonstrating the presence of an optimal blend of extracellular antifungal metabolites. Moreover, quantitative enzyme assay of exponential and stationary culture filtrates revealed that the activity of cellulase, ${\beta}$-1,3-glucanase, pectinase, and amylase was highest in the exponential phase, whereas the activity of proteases and chitinase was noted highest in the stationary phase. Morphological changes such as hyphal swelling and distortion were also observed in the fungal pathogen grown on potato dextrose agar containing stationary phase culture filtrate. Moreover, the antifungal activity of the filtrate was significantly reduced but not entirely after heat or proteinase K treatment, demonstrating substantial role of certain unknown thermostable antifungal compound(s) in the inhibitory activity.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.6
• /
• pp.897-904
• /
• 2007

The aim of this paper is to discuss the methodology of our investigation of the dynamics of protein degradation and the total in situ protealytic activity in meso/eutrophic, eutrophic, and hypereutrophic freshwater environments. Analysis of the kinetics and rates of enzymatic release of amino acids in water samples preserved with sodium azide allows determination of the concentrations of labile proteins $(C_{LAB})$, and their half-life time $(T_{1/2})$. Moreover, it gives more realistic information on resultant activity in situ $(V_{T1/2})$ of ecto- and extracellular proteases that are responsible for the biological degradation of these compounds. Although the results provided by the proposed method are general y well correlated with those obtained by classical procedures, they better characterize the dynamics of protein degradation processes, especially in eutrophic or hypereutrophic lakes. In these environments, processes of protein decomposition occur mainly on the particles and depend primarily on a metabolic activity of seston-attached bacteria. The method was tested in three lakes. The different degree of eutrophication of these lakes was clearly demonstrated by the measured real proteolytic pattern and confirmed by conventional trophic state determinants.

• Applied Biological Chemistry
• /
• v.33 no.4
• /
• pp.325-329
• /
• 1990

This study was undertaken to determine thermodynamic characteristics of B. subtilis p-4 and B. licheniformis p-5 proteases isolated from fermented anchovy paste. $K_m$ values of two proteases for casein as a substrate were 0.38mM in p-4 protease and 0.18mM in p-5 protease, respectively. Denaturation constants($K_D$) of p-4 and p-5 proteases were $12.2{\times}10^{-5}/sec\;and\;19.0{\times}10^{-5}/sec\;at\;40^{\circ}C,\;and\;35.7{\times}10^{-5}/sec\;and\;46.3{\times}10^{-5}/sec\;at\;50^{\circ}C$, respectively. Activation energies($E_a$) of p-4 and p-S pmteases were 19.6 Kcal/mole and 15.2kcal/mole, respectively. Free energy of activation(${\Delta}G^*$), activation enthalpy(${\Delta}H^*$) and activation entropy(${\Delta}S^*$) at $40^{\circ}C$ were 23.21Kcal/mole, 18.98Kcal/mole and -13.50 eu, respectively for p-4 protease and 22.93Kcal/mo1e, 14.58Kcal/mole and -26.68 eu, respectively for p-5 protease. The major amino acids in p-4 protease(151 residues of amino acid) were Gly, Glu, Pro, Asp, Ser, Ala, Lys and Leu, while those in p-5 protease(247 residues of amino acid) were Gly, Glu, Asp, Ala and Leu. It may be concluded that heat denaturation of two proteases showed liner regression curve and p-5 protease was more sensitive to heat than p-4 protease.

• Journal of Microbiology
• /
• v.44 no.5
• /
• pp.537-547
• /
• 2006

This study was designed to determine whether or not Vibrio vulnificus metalloprotease VvpE can promote iron uptake via the proteolytic cleavage of human hemoglobin. We found that V. vulnificus utilized hemoglobin as an iron source more efficiently via the vulnibactin-mediated iron-uptake system than via the HupA-mediated iron-uptake system and, of the proteases produced by V. vulnificus, VvpE was found to be the only protease capable of destroying hemoglobin. However, VvpE expression, on both the transcriptional and protein levels, was suppressed in iron-limited media. However, vvpE transcription, but not extracellular VvpE production, was reactivated by the addition of hemoglobin or inorganic iron into iron-limited media. Moreover, vvpE transcription began only in the late growth phase when V. vulnificus had already consumed most of the iron for growth. In addition, neither vvpE mutation nor in trans vvpE complementation affected the ability of V. vulnificus to acquire iron or to grow in iron-limited media or in cirrhotic ascites containing hemoglobin. Hemoglobin added into iron-limited media was not destroyed, but gradually formed an insoluble aggregate during culture; this aggregation of hemoglobin occurred regardless of vvpE mutation or complementation. These results indicate that VvpE is not required for efficient iron uptake from hemoglobin. On the contrary, hemoglobin or iron is required for efficient vvpE transcription. In addition, a discrepancy exists between vvpE transcription and extracellular VvpE production in iron-limited media containing inorganic iron or hemoglobin, which suggests that additional unknown posttranscriptional events may be involved in the extracellular production of VvpE.

• Mycobiology
• /
• v.30 no.3
• /
• pp.160-165
• /
• 2002

Aspergillus niger has been used as a host to express many heterologous proteins. It has been known that the presence of an- abundant protease is a limiting factor to express a heterologous protein. The protease deficient mutant of A. niger was obtained using UV-irradiation. A total of $1{\times}10^5$ spores were irradiated with $10{\sim}20%$ survival dose of UV, 600 $J/m^2$ at 280 nm, and the resulting spores were screened on the casein-gelatin plates. Ten putative protease deficient mutants showing the reduced halo area around colonies were further analyzed to differentiate the protease deficient mutant from other mutant types. Among ten putative mutants, seven mutants showed significant growth defect on nutrient rich medium and two mutants appeared to be the secretory mutants, which resulted in the impaired secretion of extracellular proteins including proteases. A mutant $pro^--20$ showed reduced halo zone without any notable changes in growth rate. In addition, the starchdegrading and glucose oxidase activities in the culture filtrate of $pro^--20$ mutant showed the similar range as that of the parental strain, which suggested that the $pro^--20$ mutant ought to be the protease deficient mutant rather than a secretory mutant. The reduced proteolytic activity of the $pro^--20$ was demonstrated using SDS-fibrin zymography gel. The reduced extracellular proteolysis was quantified by casein degradation assay and, comparing with the parental strain, less than 30% residual extracellular protease activity was detected in the culture filtrate of the $pro^--20$ mutant. The bio-activity of an exogenously supplemented hGM-CSF(human Granulocyte-Macrophage Colony Stimulating Factor) in the culture filtrate of $pro^--20$ mutant was detected until eight times more diluted preparations than that of the parental strain.

• Korean Journal of Microbiology
• /
• v.45 no.1
• /
• pp.69-73
• /
• 2009

Proteases are degradative enzymes which hydrolyze a peptide bond between amino acids and they are abundantly applied to commercial field. In order to investigate optimal medium compositions of carbon and nitrogen source for enzyme production, modified STY medium containing 0.15% yeast extract were used as basal medium. When galactose was used as carbon source, enzyme activity showed 1.3 higher than that of glucose. For nitrogen source addition of casamino acids to basal medium in place of tryptone showed lowest activity, whereas addition of malt extract showed maximal activity. The optimum temperature and pH of extracellular protease were found to be $35^{\circ}C$ an pH 8.5.

• Korean Journal of Microbiology
• /
• v.43 no.3
• /
• pp.222-226
• /
• 2007

Proteases are degradative enzymes which hydrolyze a peptide bond between amino acids and they are abundantly applied to commercial field. In order to screen new source of pretense, bacteria secreting extracellular pretense were isolated by enrichment culture from deep sea water samples of East Sea, Korea. A bacterium, named as HJ19, showed the best growth and the largest clear zone in plates supplemented skim milk at $30^{\circ}C$. The partial DNA sequence analysis of the 16S rRNA gene, phenotypic tests and morphology identified that this strain was In genus Micrococcus. The strain HJ19 could not grow at $10^{\circ}C$ but it started growth and showed pretense activity at $20^{\circ}C$. The optimal growth was at $37^{\circ}C$ and the maximal protease activity at $30^{\circ}C$ was about 480unit/ml.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.6
• /
• pp.753-758
• /
• 2000

Human leptin is identified as a 16kDa (146 amino acids) protein secreted from adipocytes which influences body weight homeostasis. In order to produce active leptin, the human obese gene coding for leptin was expressed in Bacillus subtilis WB600 strain which is deficient in six extracellular proteases. The recombinant leptin was produced in a culture supernatant, and in a culture supernatant, it was contained as high as 48% for total proteins. After simple purification steps, which consisted of ammonium sulfate precipitation and anion-exchange column chromatography, 2.3 mg of leptin with a purity greater than 95% was obtained from the 0.51 culture with the recovery yield of 38.3%. The purified leptin showed the correct folding structure with one disulfied bond.

• Microbiology and Biotechnology Letters
• /
• v.23 no.3
• /
• pp.322-328
• /
• 1995

A thermophilic bacteria showing proteolytic activity against defatted soybean was isolated from soil. It was identified as Bacillus amyloliquefaciens based on its morphological and physiological characteristics. The Bacillus amyloliquefaciens NS 15-4 was cultivated at 50$\circ$C by rotary shaking in a medium containing defatted soybean. An extracellular protease from this strain was purified to homogeneity by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The molecular weight of the enzyme was estimated to be approximately 30,000 by SDS-PAGE and the N-terminal amino acid sequence of the enzyme was turned out to be AQSVPYGISQIKAPA. The optimum temperature and pH for the enzyme reaction were 60$\circ$C and 11, respectively, and its thermostability was increased by the addition of calcium ion. The enzyme was inactivated by phenylmethylsulfonylfluoride, suggesting it be a serine protease. Comparing with other commercial proteases, the enzyme showed relatively high proteolytic activity against defatted soybean, a water-insoluble protein substrate.

• Proceedings of the PSK Conference
• /
• 2002.10a
• /
• pp.331.2-331.2
• /
• 2002

Actinomycetes CS0707 has been isolated in soil sample from location in the Jeju province. Korea, and produces thermostable extracellular proteases. Actinomycetes CS0703 showed the highest protease activity at late exponential phase when grown in OSYM medium (oatmeal 2.0%, soybean meal 1 %, dried yeast 1 %, mannitol 1 %) at $48^{\circ}C$. Three forms of protease(Ta-1, TA-2 and Ta-3) were fractionated by Ultrogel AcA 54 column chromatography, and further purified through ammonium sulfate fractionation, ultramembrane filtration, and DEAE-sepharose CL-6B column chromatography. (omitted)