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: In this article , the fungal spore suspension was treated with different concentrations of ethyl methanesulfonate (EMS) (20-80 μg/mL) for different incubation periods (10-60 min).
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TL;DR: The two mutants were very effective in feather keratin-degrading in less than two days, UV- 18 was more efficient than UV-9, and the main objective was to enhance protease production of two bacterial strains, Bcillus sp.
Abstract: Microbial proteases contribute nearly 40% of the total worldwide enzyme market. Hence, with the view of this significance, the main objective of the present study was to enhance protease production of two bacterial strains, Bcillus sp. and Micrococcus varians using UV mutagenesis. Induction of mutation in both strains was carried out at different exposure times: 0, 3, 6, 9, 12, 15, 18 and 21 min at a distance of 10 between UV source and treated bacteria.Two best protease producer mutants for the two bacterial strains (UV-9 for Bcillus sp.and UV-18 forMicrococcus varians) were selected based on the clearance zone diameter of mutant colonies on 1% skimmed milk agar plates. UV-9 mutant showed 1.4 fold higher protease activity than the wild type in solid and liquid medium. However UV-18 mutant was found to produce 2.5 fold increases over the wild type on agar plates and 2.1 fold enhancement in liquid-medium assay.The two mutants were very effective in feather keratin-degrading in less than two days, UV- 18 was more efficient than UV-9.
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Cites background from "Isolation and characterization of a..."
...Shikha and Darmwal(2007) reported 1.44 fold increase in alkaline protease production over the wild strain of B. pantotheneticus while Dutta and Banerjee (2006) obtained 2.5 fold increase in protease production by UV-mutant Pseudomonas sp. Raoet al., (1998) reported that mutagenesis either by…...
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03 Sep 2016
TL;DR: Optimization of physical parameters by OVAT method revealed that mean generation time, 4% level inoculum, incubation time 72 hrs, pH 10, temperature 350C and agitation 150 rpm are ideal for enzyme production.
Abstract: The present investigations dealt with the optimization of the physical parameters for production of alkaline protease by alkaliphilic Bacillus subtilis SH-2 isolated from slaughter house soil of Warangal, Telangana State, India. Primary screening of four different samples revealed one potent isolate. Morphological and Biochemical characterization followed by Molecular signature of 16s rRNA homology confirmed that the isolate SH-2 belongs to Bacillus subtilis . Bacillus subtilis SH-2 was screened on four different reported mediums (M1213, M660, Horikoshi and Halophilic Bacillus medium) under shake culture conditions. Maximum alkaline protease production (500 EU/ml) obtained on M1213 and Horikoshi mediums. Further optimization of physical parameters by OVAT method revealed that mean generation time (41.18 min), 4% level inoculum, incubation time 72 hrs, pH 10, temperature 350C and agitation 150 rpm are ideal for enzyme production. OVAT method resulted in 2.2 fold increased production of alkaline protease production (1100 EU/ml).
1 citations
Cites background from "Isolation and characterization of a..."
...Microorganisms grow slowly at a temperature below or above the normal growth temperature because f a reduced rate of cellular production [27]....
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01 Jan 2020
TL;DR: UV irradiation was applied to develop a Pseudomonas aeruginosa mutant as a new source of protease overproduction, followed by cultural and nutritional optimizations to believe that the mutant PA-M25 could be a potential candidate to meet the growing protease demands.
Abstract: Over 60% of the global productions of industrial enzymes are proteolytic enzymes in which about 35% are alkaline proteases. The current microbial sources are unable to reach industrial demands of alkaline protease which led scientists to search new sources with enhanced enzyme activity. Therefore, we applied UV irradiation to develop a Pseudomonas aeruginosa mutant as a new source of protease overproduction, followed by cultural and nutritional optimizations. The mutagenesis was carried out by exposing parent strains to UV radiation (30w, 2537 Å) at 25 oC with a different time interval. The protease activity was estimated as relative protease activity and standard protease assay (OD660). Among all, mutant strain P. aeruginosa–M25 (PA-M25) exhibited 75.47% increased protease activity over the parent strain in submerged fermentation. It showed 612.84±2.50 U/ml of alkaline protease production compared to 349.26±2.57 U/ml by wild-type strain (significant at P≤0.005). Besides, the effects of nutritional factors on the protease production by PA-M25 were also studied. We found the optimal alkaline protease production in the medium (adjusted to pH 9.0) supplemented with 1% (w/v) glucose as carbon source, 0.5% (w/v) casein as nitrogen source and 2% (w/v) NaCl when incubated at 35 oC for 48 h without agitation. We believe that the mutant PA-M25 could be a potential candidate to meet the growing protease demands. However, further assessments regarding the characterization of the protease enzyme, as well as the industrial fitness of the mutant, are warranted.
1 citations
01 Jan 2019
TL;DR: All the bacterial species isolated had a better potentiality of degrading market fish wastes and Pseudomonas sp.
Abstract: Received: 27/Oct/2018, Accepted: 05/Dec/2018, Online: 31/Dec/2018 AbstractProteolytic enzyme, also called protease, proteinase, or peptidase, any of a group of enzymes that break the long chainlike molecules of protein into shorter fragments (peptides) and eventually into their components, amino acids proteolytic enzymes are present in bacteria. Protease are produced from various sources but only little information is available regarding the bacterial population in fish wastes of Chidambaram fish market and their efficiency in synthesizing protease enzyme. Isolated 25 bacterial strains and found that only 5 of them are capable of producing protease enzyme that are Pseudomonas sp. and Bacillus sp. was the best protease producer among the tested strains. In the present study, all the bacterial species isolated had a better potentiality of degrading market fish wastes.
1 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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