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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01 Jan 2013
TL;DR: These four Pseudomonas isolates exhibited high potential in utilizing kerosenes and therefore useful for bioremediation of sites that contaminated with kerosene.
Abstract: The present work relates to a simple, safe, and efficient process for the complete utilization of kerosene using fluorescent pseudomonads. Fluorescent pseudomonads used in this study were isolated from local red soil collected at Acharya Nagarjuna University Campus, Guntur Dt., (AP) India. The isolates were identified as Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas putida and Pseudomonas fluorescens on the basis of biochemical characteristics. The isolates were screened for their ability to grow on nutrient broth medium in presence of petroleum hydrocarbon (kerosene) and also utilization of kerosene as their sole source of carbon and energy. All the isolates showed maximum growth in medium with 6% kerosene concentration when tested with different concentrations of kerosene viz., 0%, 2%, 4%, 6%, 8% and 10%. The isolates were able to grow well on kerosene and subsequently produced the extracellular enzymes protease and urease at significant level compared with control (without kerosene). Plasmid profile of the isolates revealed that, all the four Pseudomonas isolates harbored two low molecular weight plasmids one with 3Kb size and the other with 10Kb to 12Kb size. Thus, these four Pseudomonas isolates exhibited high potential in utilizing kerosene and therefore useful for bioremediation of sites that contaminated with kerosene.
3 citations
Cites background from "Isolation and characterization of a..."
...species isolated from local soil (Dutta and Banerjee, 2006)....
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...…B-Kerosene; Values are average of triplicates with standard deviation; Growth – t-cal value = + 4.95; p-value = 0.0001 **; Urease – t-cal value = +7.73; p-value = 0.0000 **; ** Values are < 0.01 (1%), so results are highly significant species isolated from local soil (Dutta and Banerjee, 2006)....
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01 Jan 2014
TL;DR: Strain improvement studies showed that mutation was the best compared to immobilization technique showing 108% higher than the parent strain using UV light as a mutagenic agent.
Abstract: The improvement of microbial strains offers the greatest opportunity for the cost reduction without significant capital investment. The purpose of the present investigation is to enhance the production by subjecting the indigenous lipase producing strain Pseudomonas aeruginosa RAS-4 to strain improvement by using UV light as a physical mutagen and immobilization. Strain improvement studies showed that mutation was the best compared to immobilization technique showing 108% higher than the parent strain using UV light as a mutagenic agent. The molecular weight of lipase was determined to be 42 kDa. Localization of the lipase specific gene was carried out by plasmid curing and acridine orange proved to be the best curing agent revealing that the lipase gene is associated with the genomic DNA.
2 citations
01 Jan 2009
TL;DR: Among the twenty different fungal members isolated from the rhizosphere of wild yam, three fungal isolates were found to be the best protease producers and identified as Aspergillus sp.
Abstract: Among the twenty different fungal members isolated from the rhizosphere of wild yam, Dioscorea wallichii, three fungal isolates were found to be the best protease producers and identified as Aspergillus sp., Penicillium sp. and Trichoderma sp. The protease production studies of these three isolates were conducted up to 12 days. After nine days there was no significant increase in the enzyme production. Maximum protease activity was observed in the range of pH 6 and temperature 30˚C. Protease from all the three isolates was stable up to 2 h in dettol, tween 20, tween 80 and soap solution. Among the different immobilization techniques used agar block was the most effective. The molecular weight, zymogram analysis and destaining activity of the protease enzyme were also studied.
2 citations
Cites methods from "Isolation and characterization of a..."
...wallichii were grown in gelatin agar media and compared for the protease production by adding mercuric chloride test reagent [12]....
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Dissertation•
07 Jan 2010
2 citations
Cites background or methods from "Isolation and characterization of a..."
...By manipulating the parameters like change of temperature, pH, inoculum volume, substrate volume and incubation period that govern the metabolic processes of the microorganisms the production rate can be increased to industrial level [145-147]....
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...For mutation by UV radiation, all the bacterial single cultures like Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum and co-cultures like Lactococcus lactis + Lactobacillus brevis, Lactococcus lactis + Lactobacillus plantarum and Lactobacillus brevis + Lactobacillus plantarum of 1 ml each were taken in tubes and one control for each was kept aside and the rest of the tubes were exposed to UV at an intensity of 254 nm irradiation [145,202] for about 30, 60, 90 and 120 min....
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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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