Organic solvent tolerant metallo protease of novel isolate Serratia marcescens PPB-26: production and characterization (original) (raw)
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Alkaline proteases hold a great potential for application in the food, detergent and leather industries due to the increasing trend to develop environmental friendly technologies. In this study, an attempt is made to isolate and characterise the bacteria from the protein rich soil sample as well as to optimise the culture conditions for the production of protease enzyme. The molecular sequencing of the bacterium and the phylogenetic tree formulated revealed the presence of Serratia marcescens. In the subsequent phase effect of temperature, pH, activator and inhibitor was also studied on the activity of protease enzyme by using casein and hair as substrates. The enzyme further was shown to have the ability to remove the blood from the blood stained surgical instruments as well as to recover silver from the X ray film. The optimum production of protease by Serratia marcescens was observed at pH 10 and temperature 37°C. The presence of CaCl 2 (activator) increased the enzyme activity and the presence of HgCl 2 (inhibitor) reduced the protease activity. The protease enzyme was able to act on casein and hair as well as able to remove the blood from the surgical instruments. Treatment of X-Ray films with protease resulted in the silver bound with gelatin being stripped off in to the reaction mixture at pH 10 and temperature 40 o C. The alkaline protease enzyme obtained in the present study by using Serratia marcescens is found to be stable and active in alkaline condition, so could be of significant use for detergent and leather processing technology as well as for dehairing and silver recovery.
PRODUCTION AND CHARACTERIZATION OF THERMO TOLERANT ALKALINE PROTEASE FROM SERRATIA MARCESCENS
Abstract - Alkaline proteases hold a great potential for application in the food, detergent and leather industries due to the increasing trend to develop environmental friendly technologies. In this study, an attempt is made to isolate and characterise the bacteria from the protein rich soil sample as well as to optimise the culture conditions for the production of protease enzyme. The molecular sequencing of the bacterium and the phylogenetic tree formulated revealed the presence of Serratia marcescens. In the subsequent phase effect of temperature, pH, activator and inhibitor was also studied on the activity of protease enzyme by using casein and hair as substrates. The enzyme further was shown to have the ability to remove the blood from the blood stained surgical instruments as well as to recover silver from the X ray film. The optimum production of protease by Serratia marcescens was observed at pH 10 and temperature 37°C. The presence of CaCl2 (activator) increased the enzyme activity and the presence of HgCl2 (inhibitor) reduced the protease activity. The protease enzyme was able to act on casein and hair as well as able to remove the blood from the surgical instruments. Treatment of X-Ray films with protease resulted in the silver bound with gelatin being stripped off in to the reaction mixture at pH 10 and temperature 40oC. The alkaline protease enzyme obtained in the present study by using Serratia marcescens is found to be stable and active in alkaline condition, so could be of significant use for detergent and leather processing technology as well as for dehairing and silver recovery.
Production and Characterization of Protease from Serratia marcescens
2014
Extracellular protease produced by Serratia marcescens isolated from the gut of termite (Centrocestus formosanus) was investigated in this study. The effect of temperature, pH, carbon and nitrogen sources on protease production was examined. The extracellular protease was separated from the culture supernatant of the organism through precipitation with ammonium sulfate. The protease was purified by Sephadex G-150 gel filtration and diethylaminoethanol (DEAE) - Sephadex A-50 ion exchange chromatography. The effect of temperature, pH, and salts on the proteolytic activity of the enzyme was also investigated. Optimal protease production was obtained after about 12-hour incubation. The optimum temperature and pH for protease production was found to be 40°C and pH 7, respectively. Protease production was highest when rice bran and casein were used as the carbon and nitrogen sources, respectively. The native molecular weight of the protease was estimated to be 56 kDA. The optimal temperat...
PRODUCTION, CHARACTERIZATION & OPTIMIZATION OF POTENT PROTEASE (SERRATIOPEPTIDASE) FROM SERRATIA MARCESCENS E 15 *Chaudhari Snehal Anil, Mali Anil Kashinath. Department of Microbiology, Yashwantrao Chavan College of Science, Karad (MH) - 415103 Corresponding Author: Chaudhari Snehal Anil, Email: snehal.chaudhari90@gmail.com Abstract: Production, characterization & optimization of serratiopeptidase from Serratia marcescens E 15 was the base for this study. Serratia marcescens E 15 was allowed to grow in trypticase soy broth. The serratiopeptidase(STP) enzyme activity was determined according to the gelatin clearing zone. Enzyme assay was carried out &protein content was determined. A standard serratiopeptidase tablet “Serric-5” (5mg) containing 10000 Units of STP enzyme was used for the assay. Protease assay was carried out to determine the Units of protease in STP. Optimum conditions for STP production were 37oC temperature, 25hrs. Incubation period & pH=7.3. The STP enzyme was partially purified by ammonium sulphate precipitation & dialysis. STP enzyme activity increased with the increase in enzyme concentration. Keywords: Production, Characterization, Serratiopeptidase, Serratia marcescens E 15
Purification and Characterization of 56 kDa cold active Protease from Serratia marcescens
African Journal of Microbiology Research, 2011
The extracellular cold active protease produced from Serratia marcescens TS1. The protease was purified to homogeneity from the production medium by ammonium sulphateation then followed by acetone precipitation with 80% saturation. The cold active protease was fractionized by diethylaminoethyl (DEAE) cellulose column chromotography. The molecular weight of protease was approximately 56 KDa. The isoelectric point was close to 6.4. The maximal activity towards casein was found at 40°C and its pH activity was at 8.0. The protease was strongly inactivated by HgCl 2 metal ion and reactivated by FeSO 4 thus indicated as metalloprotease. The protease was inhibited by Na 2 ethylenediaminetetraacetic acid (EDTA). The protease of S. marcescens TS1 showed a potential application in the laundry industry by removeing the blood, chocolate and egg youlk stains from the white cotton cloths in a short period without changing texture of cloths.
Purification and characterization of two novel extra cellular proteases from Serratia rubidaea
Process Biochemistry, 2007
A protease, producing bacterial culture (isolate ‘C’) was obtained from slaughterhouse waste samples, Hyderabad, India. It was related to Serratia rubidaea on the basis of 16S r RNA gene sequencing and biochemical properties. Cultural characters of S. rubidaea identified it as a psychrophile secreting protease at 10–30 °C. Single step purification of culture supernatant on sephacryl S-100 column revealed two proteases CP-1 and CP-2. The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 97 and 45 kDa, respectively. N-terminal sequencing of CP-1 revealed a novel surface protein of S. rubidaea and CP-2 protease has shown 100% homology with protease of Serratia sp. A fold purification of 1.5 with 54% recovery was achieved in CP1 and purification of CP-2 resulted in 88% yield with a fold purification of 2. The optimum pH values of CP-1 and CP-2 were shown to be 10 and 8, respectively. The maximum activities for the enzymes were at 40 °C and 30 °C. Both the proteases are inhibited by EDTA indicating that they are metallo proteases. The activity of CP-1 was enhanced with Cu2+ that of CP-2 was enhanced with Zn2+ and Ca2+. These proteases have stability in presence of detergents, surfactants and solvents. These properties make these proteases an ideal choice for application in detergent formulations, food, leather industries, vaccine and enzyme peptide synthesis.
Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marcescens A3 and Pseudomonas putida A2 based on 16S rDNA sequencing. Protease produced through fermentation of organic MSW by these bacteria under some optimized physicochemical parameters was partially purified and characterized. The estimated molecular mass of the partially purified protease from S. marcescens and P. putida was approximately 25 and 38 kDa, respectively. Protease from both sources showed low K m 0.3 and 0.5 mg ml À1 and high V max 333 and 500 mmole min À1 at 40 °C, and thermodynamics analysis suggested formation of ordered enzyme-substrate (E-S) complexes. The activation energy (E a) and temperature quotient (Q 10) of protease from S. marcescens and P. putida were 16.2 and 19.9 kJ/mol, and 1.4 and 1.3 at temperature range from 20 to 40 °C, respectively. Protease of the both bacterial isolates was serine and cysteine type. The protease retained approximately 97% of activity in the presence of sodium dodecyl sulphate. It was observed that the purified protease of S. marcescens could remove blood stains from white cotton cloth and degrade chicken flesh remarkably. Our study revealed that organic MSW can be used as raw materials for bacterial protease production and the protease produced by S. marcescens A3 might be potential for applications.
Fermentation of individual proteins for protease production by Serratia marcescens
Biochemical Engineering Journal, 2004
The major whey proteins -lactoglobulin, ␣-lactalbumin, bovine serum albumin (BSA) and immunoglobulins were tested as the sole source of carbon and nitrogen for producing proteases by fermentation with Serratia marcescens. Cell growth, protein consumption and protease production were monitored during these fermentations. The maximum level of protease activity was achieved with BSA. Several substances described as important factors in protease synthesis were then added as supplements to a medium prepared with BSA to ascertain their influence on protease production. Likewise, in order to study the possibility of a synergic effect of whey proteins on protease production, a protein mixture simulating the protein composition of whey was also tested. However, low levels of activity were obtained, similar to those achieved with -lactoglobulin. These results were also compared with those obtained when real whey is employed as substrate. A complete turnaround in the tendency of the kind of protease produced was found when the culture medium was solely based on bovine proteins and salts.
ANADOLU UNIVERSITY JOURNAL OF SCIENCE AND TECHNOLOGY –C Life Sciences and Biotechnology
The protease producing bacteria were screened from Indonesian traditional fermented food, tauco and terasi, and 4 halophilic protease producers were isolated. Among these isolates, halophilic bacterial isolate TANN 4 was recorded as the best protease producer. Extracellular protease from isolate TANN 4 was partially purified using ammonium sulfate precipitation. The protease was partially purified with final yield of 72.87 % and 25.41 fold purity. This moderate thermoactive and alkaliphilic protease showed a pH optimum of 8.0 and temperature optimum was 50 °C. The enzyme was also active at salt concentrations ranging from 1 to 15 % (w/v), with optimum activity at 1 % NaCl (w/v). Ethylenediaminetetraacetic acid (EDTA) completely inhibited the enzyme activity suggesting that it was a metalloprotease. Among metal ions, the Ca 2+ , K + and Mg 2+ ions enhanced the activity of enzyme. The KM and Vmax values exhibited by partially purified protease were 0.0649 mM and 216.45 U mg −1 using casein as substrate. The molecular weight was estimated to be 19.8 kDa on SDS PAGE. The enzyme was also fairly stable in Triton X-100, SDS, 1 % commercial detergents (OMO and Ariel) and 25 % methanol and it was capable of hydrolyzing casein, hemoglobin and bovine serum albumin (BSA). These characteristics make this halophilic bacterial extracellular metalloprotease seem to be potentially useful for biotechnological and industrial applications. Automated ribotyping analysis revealed that 3 isolates (TANN 4, TR 2 and TR 4) resembled Halobacillus trueperi that exhibited 71, 68 and 69 % similarity respectively, and isolate (TR 1) resembled Virgibacillus pantothenticus with 64 % similarity.