Isolation and characterization of a newly isolated Pseudomonas mutant for protease production
TL;DR: In this paper, a potent bacterium for extracellular protease production was isolated from local soil and identified as Pseudomonas sp. RAJR 044, a mutant of this strain JNGR 242 with protease productivity 2.5 fold higher was obtained by ultraviolet irradiation under experimentally optimized conditions of pH 7.0, temperature of 34oC, inoculum volume of 1.0 mL and incubation time of 24 hours.
Abstract: A potent bacterium for extracellular protease production was isolated from local soil and identified as Pseudomonas sp. RAJR 044. A mutant of this strain JNGR 242 with protease productivity 2.5 fold higher was obtained by ultraviolet irradiation under experimentally optimized conditions of pH 7.0, temperature of 34oC, inoculum volume of 1.0 mL and incubation time of 24 hours. Comparative analysis of the chemical characteristics i.e. assimilation of carbon and nitrogen sources were also carried out. Maximum growth of the mutant strain in 2% gelatin agar plate was obtained in presence of dextrose (2%), maltose (2%), ammonium sulfate (2%) and potassium nitrate (2%) whereas, that of the parent strain was found in sucrose (2%) and ammonium nitrate (2%). The purified proteases from both the strains (parent and mutant) appeared as single homogeneous bands corresponding to 14.4 kDa molecular weight on SDS-PAGE. On studying the kinetic properties of both strains it was observed that the rate of casein hydrolysis was maximum at pH 8.0 and 7.0 and temperatures 45o C and 60o C for the parent and mutant strains respectively. It was also observed that both the extracellular proteases were inhibited by a serine protease inhibitor i.e. PMSF at 2mM concentration.
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TL;DR: The four Pseudomonas isolates of the present study were found to have potential in diesel degradation and can be recommended for bioremediation of sites that are contaminated with diesel.
Abstract: Background and objectives: Bacteria, most prevalently the Pseudomonas species possess high capacity to utilize and degrade petroleum hydrocarbons and are classified as the hydrocarbonoclastic microorganisms. Many species of the genus Pseudomonas are notorious for their aerobic degradation capacity, extracellular enzyme production and are metabolically versatile organisms capable of utilizing a wide range of hydrocarbons and other compounds. In this study, the ability of diesel utilization by some locally isolated Pseudomonas species was tested. Materials and Methods: From a local red laterite soil, four different Pseudomonas species were isolated on King’s B medium, characterized, identified and tested their potential in utilizing diesel, a petroleum hydrocarbon. At the same time, production of protease and urease enzymes during the utilization of diesel was also assayed following the standard procedures. Results: The isolates were grown well on diesel and subsequently produced the extracellular enzymes protease and urease at significant levels when compared to their production in the absence of diesel. Optimum temperature and pH for increased growth by four isolates was found to be 37 o C and pH 8.0 indicating the maximum utilization of diesel. All the isolates showed maximum growth in medium with 100% diesel than 100% glycerol as carbon source, when tested with different proportions of diesel and glycerol as carbon sources. Plasmid profile of the isolates revealed that, all four Pseudomonas isolates harbored two low molecular weight plasmids; one with 3 Kb size and the other with 10 kb to 12 Kb size. Conclusion: The four Pseudomonas isolates of the present study were found to have potential in diesel degradation and can be recommended for bioremediation of sites that are contaminated with diesel.
7 citations
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TL;DR: The results of the enzyme activity showed that the organisms; Aspergillus flavus, As pergillus niger, AsPergillus fumigatus and Penicillium italicum produced the protease enzyme maximally between day three and day five of incubation while the effect of temperature and thermal stability on the enzyme production showed temperature optimal for enzyme production was between 30 and 60 0 C.
Abstract: This study was undertaken to monitor the production of protease enzyme from soil fungal isolates obtained from Omo natural forest in Ogun State of Nigeria. The study also sought to determine the kinetic parameters of the enzyme with the aim of establishing the industrial and biotechnological importance of this microbial enzyme. The harvested mycelia of the fungi were separately homogenized in buffered culture medium for five days using shaker incubator in which the protease activity was monitored. The results of the enzyme activity showed that the organisms; Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Penicillium italicum produced the protease enzyme maximally between day three and day five of incubation while the effect of temperature and thermal stability on the enzyme production showed temperature optimal for enzyme production was between 30 and 60 0 C and the thermal stability on the enzyme activity was between 30 and 50 0 C. The optimal pH on the enzyme production was observed to be between pH 3.5 and 5.5 for the organisms. Keywords: Soil microorganism, fungal isolate, incubation period, microbial enzyme Nig J. Biotech . Vol. 23 (2011) 28 - 34
6 citations
TL;DR: P. vulgaris was identified as the major protease producer in optimized culture condition of 50 o C and pH6.5 and further research on optimization of other fermentation parameters using statistical tools with P. vulgarIS is needed to scale up the process.
Abstract: Aims: The present study was investigated to optimize and partially purify the proteases produced by the food borne bacterial strains. Methodology and Results: Four bacterial strains such as Bacillus cereus, Proteus vulgaris, P. mirabilis and Enterobacter aerogenes were isolated from food wastes. These strains were individually inoculated in to the formulated culture media supplied with three different concentrations (1:1 to 1:3) of raw milk as major substrate. Among the concentrations, 1:2 ratio of substrate supplied medium showed maximum (0.133 to 8.000 IU/mL) protease production by all the tested organisms. After optimization, the organisms were tested for protease production at various pH (3 to 9), and temperature (30 to 80 °C). The result showed that all the organisms were capable of producing maximum protease at pH 6 (8.533 to 10.133 IU/mL) and at 50 °C (8.666 to 10.666 IU/mL). The crude enzymes produced by the tested organisms were individually purified by two different methods viz sodium alginate and ammonium sulphate-butanol methods. The purity of the protease determined in these two methods was ranged between 3.24 to 5.44 I and 3.13 to 5.55 IU/mL respectively. The partially purified enzymes were further analysed through SDS-PAGE; accordingly the molecular weight of protein produced by the test organisms was determined in between 49.44 and 50.98 kDa. Conclusion, significance and impact of study: Among the tested strains P. vulgaris was identified as the major protease producer in optimized culture condition of 50 o C and pH6. The molecular mass of the partially purified protease of P. vulgaris was 50.32 KDa. Further research on optimization of other fermentation parameters using statistical tools with P. vulgaris is needed to scale up the process.
6 citations
Cites background from "Isolation and characterization of a..."
...Banerjee and Dutta (2006) pointed out that the protease recovery by acetone precipitation of mutant and parent Pseudomonas sp. observed by SDS-PAGE....
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01 Jan 2010
TL;DR: Some fundamental properties of partially purified enzyme like effects of different temperatures and pH values on protease activity were studied to indicate its potential application in detergent formulation and leather processing.
Abstract: The aim of the study was to enhance the production alkaline protease by employing combined mutagenic treatments. The parent strain Bacillus pumilus was exposed to UV irradiation followed by EMS (Ethyl methane sulfonate) and MMS (Methyl methane sulfonate) to improve the yield of alkaline protease. After each treatment, the mutants were screened on skim milk agar plates for the selection of best proteolytic mutant. Among various mutants, the mutant Bacillus pumilus M15 was selected as best positive mutant produced 1.95 fold more enzyme over the parent strain in similar culture conditions. The enzyme was partially purified by precipitating with (NH4)2SO4 at different saturation level (40-80 %). Approximately 2.1 fold increase in enzyme activity was achieved from the initial culture broth at 70 % saturation level with 83.6 % enzyme recovery. Some fundamental properties of partially purified enzyme like effects of different temperatures and pH values on protease activity were also studied. Maximum protease activity was obtained at pH 10 at 60 o C. These properties indicate its potential application in detergent formulation and leather processing.
5 citations
Cites background from "Isolation and characterization of a..."
...A 2.5 fold increase in alkaline protease production by UV mutant Pseudomonas sp. JNGR242 was also observed in another investigation (Dutta and Banerjee 2006)....
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01 Jan 1975
TL;DR: In this paper, the effect of pH of medium (3.0-8.0), temp. of incubation (20-45°C), N source (inorganic or organic), and glucose concn. in the medium (0.05-3. 0%) on acid protease production by Asp. niger in a medium containing 4% wheat flour was made.
Abstract: A study was made of the effect of pH of medium (3.0-8.0), temp. of incubation (20-45°C), N source (inorganic or organic), and glucose concn. in the medium (0.05-3.0%) on acid protease production by Asp. niger in a medium containing 4% wheat flour. A study was also made of protease production on different media (wheat, moong, mash, gram, masur + jowar, soybean, barley, bajra), wheat giving the best results. Max. enzyme production was achieved under the following conditions: initial p H of 7, incubation temp. of 35°C, and nitrate as N source. Addition of glucose to the medium enhanced mycelial growth but had an adverse effect on enzyme production.
4 citations
References
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
Journal Article•
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
TL;DR: Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions and will enable researchers to tailor new lipases for biotechnological applications.
Abstract: ▪ Abstract Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond–forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an α/β hydrolase fold, a catalytic ...
1,072 citations
"Isolation and characterization of a..." refers background in this paper
...…J. R. and Banerjee, R. Brazilian Archives of Biology and Technology 38 Takami et al., 1990; Mabrouk et al., 1999) as well as on mutation of Pseudomonas sp. for lipase production (Xiu-Gong Gao et al., 2000; Jaeger et al., 1999) and polysaccharide production (West, 2002) have been reported....
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TL;DR: Alkalophilic Bacillus sp.
Abstract: An alkalophilic Bacillus sp. no. AH-101, which produced extremely thermostable alkaline protease, was isolated among 200 soil samples. The enzyme production reached its maximum level of 1500 units/ml after about 24 h in alkaline medium (pH 9.5). The enzyme was most active toward casein at pH 12–13 and stable to 10 min incubation at 60° C from pH 5–13. Calcium ions were effective in stabilizing the enzyme especially at higher temperatures. The optimum and stable temperatures were about 80° C and below about 70° C respectively in the presence of 5 mM calcium ions. The enzyme was completely inactivated by phenylmethane sulphonyl fluoride, but little affected by ethylenediaminetetraacetic acid, urea, sodium dodecylbenzenesulphonate and sodium dodecyl sulphate. The molecular weight and sedimentation constant were approximately 30 000 and 3.0S respectively, and the isoelectric point was at pH 9.2. These results indicte that no. AH-101 alkaline protease is more stable against both temperature and highly alkaline conditions than any other protease so far reported.
191 citations
"Isolation and characterization of a..." refers background in this paper
...…inhibitor, enzyme concentration, pH, redox potential, temperature, etc. Studies on protease production from Bacillus sp. (Manachini et al., 1988; Takami et al., 1989; Dutta, J. R. and Banerjee, R. Brazilian Archives of Biology and Technology 38 Takami et al., 1990; Mabrouk et al., 1999) as well…...
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