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Journal ArticleDOI

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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Citations
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Journal Article
TL;DR: This review attempts to focus on some of the difficulties observed in the earlier work and tried to find out possible solutions and bridging up this gap by introducing recent information regarding proteinases.

65 citations

Journal ArticleDOI
TL;DR: In this paper, the authors improved the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments.
Abstract: The purpose of the present investigations was to improve the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments viz. UV irradiations, NTG (N-methyl-N-nitro-N-nitrosoguinidine) and MMS (methyl methane sulfonate). After screening on skim milk agar plates, a total of nine positive mutants were selected for shake flask experiments. Among these, the best proteolytic mutant designated as UV-9 showed 1.4 fold higher alkaline protease activity in preoptimized growth medium than the parent strain. The fermentation profile and kinetic parameters such u(h-1), Yp/s, Yp/x, Yx/s, qs, Qs, qp and Qp also indicated the superiority of the selected mutant UV-9 for alkaline protease production over the parent strain and rest of the mutants. The dehairing capability of mutant UV-9 alkaline protease was analyzed by soaking goat skin pieces for different time intervals (3-15 h) at 40 o C. A complete dehairing without degradation of collagen was achieved after 12 h, indicating its commercial exploitation in leather industry.

30 citations


Cites background from "Isolation and characterization of a..."

  • ...Dutta and Banerjee (2006) observed 2.5 fold increased in alkaline protease production by UV mutant Pseudomonas sp. JNGR242....

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Book ChapterDOI
01 Jan 2019
TL;DR: This chapter includes microbial enzymes used in food processing and the food industry, their physicochemical and biological properties, recent developments, and future prospects.
Abstract: Enzymes, the natural catalysts for chemical reactions, are produced by all living cells Their role in food processing has also been recognized for many centuries Even before this knowledge about enzymes, they have been used in a number of processes such as the tenderization of meat using papaya leaves, soy sauce preparation, curd or cheese making, baking, brewing, etc From animals to plants to microbial sources, enzymes may be extracted from any living organisms Of the hundred or so enzymes being used in industries, more than half are of microbial origin In the food industry, microbial enzymes have been extensively used to increase the diversity, variety, and quality of food Microorganisms as an enzyme source are always preferred over other sources as large amounts of enzymes can be produced from them in a controlled manner that is also faster and cheaper Moreover, the minimum of potentially harmful content is present in microbial enzymes in comparison to those of plants and animals This chapter includes microbial enzymes used in food processing and the food industry, their physicochemical and biological properties, recent developments, and future prospects

17 citations

Journal ArticleDOI
TL;DR: In this paper, a review aimed at discussing the recent research activities on milk fermentative enzymes, with focus on the broad spectrum of enzyme origins and current aspects of genetic engineering is presented.
Abstract: Aside from bacteria, yeasts and moulds, enzymes from animal, vegetal and microbial sources are increasingly utilized for milk fermentation providing a broad spectrum of innovative product conceptions. In order to alter texture and flavour or to improve the nutritional value of milk-based products from different animal milks, microbial and enzymatic fermentation procedures are traditionally established worldwide. To date, genomic and proteomic approaches enable new selection strategies for precise enzymes for modern product applications. Hereby, generating beneficial health ingredients from milk is a main aspect. New insights into the biochemical mechanisms of enzymatic digestion and genetic engineering lead to enzymes with exact defined functions for explicit ripening flavour development or the improvement of texture of fermented milk products. The ability to synthesize complex exo-polysaccharides or to release bioactive peptides by accurate proteolytic activities of enzymes or to enzymatically cross-link the protein matrix in order to modify the texture characteristics of fermented milk products is a raising facet, especially for specific pharma- or nutraceutical applications. This review aimed at discussing the recent research activities on milk fermentative enzymes, with focus on the broad spectrum of enzyme origins and current aspects of genetic engineering. New approaches on proteolytic, lipolytic, glycolytic as well as milk clotting and protein cross-linking enzymatic activities are examined and associated with possible product applications. From technical prospective, advantages and disadvantages of immobilized enzymes within milk fermentation processes are critically discussed.

16 citations


Cites background from "Isolation and characterization of a..."

  • ...Industrial processes prefer enzymes predominantly derived from microbial sources with strong metabolic activity, assessable for genetic modifications and suitable for submerse cultivation in forced aerated continuous stirred reactors admitting short generation times and simple nutrient requirements for a reliable production [4, 5]....

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Journal ArticleDOI
X C Wang1, Hong-Yan Zhao1, Gang Liu1, Xiaojie Cheng1, Hong Feng1 
TL;DR: Strain improvement for protease production can serve as an alternative strategy to promote feather degradation and could be potentially used in various industries.
Abstract: The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries.

15 citations


Cites background from "Isolation and characterization of a..."

  • ...However, the protease activity in Pseudomonas sp RAJR044 was enhanced 2.5-fold by UV mutagenesis (Dutta and Banerjee, 2006)....

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References
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Journal ArticleDOI
15 Aug 1970-Nature
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
01 Jan 1970-Nature
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

Journal ArticleDOI
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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Journal ArticleDOI
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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01 Jan 1980

179 citations