Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia (original) (raw)
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Molecules, 2020
Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to ...
Thermophilic bacteria are biotechnologically important group of microorganisms due to their capability to produce a variety of thermostable enzymes, such as proteases. The aim of the present study was the optimization of protease production by thermophilic bacteria isolated from hot springs (54°C-55.5°C) in Maha Oya, Sri Lanka. Four isolates of thermophilic bacteria that belong to the genera Geobacillus, which were previously been isolated and identified, were used in the present investigation to study their protease production. Among the four isolates, Geobacillus toebii (DMBUK 107191) reached a maximum protein concentration with a level of 858 µg/mL and Geobacillus kaustophilus (DMBUK 107161) showed maximum protease activity of 2.232 units/mL (55°C, pH 7). Studies on the result of temperature and pH on protease activity discovered that the extreme protease activity was detected at 60°C over a pH range of 6-8. The isolates utilized several carbon and nitrogen sources for the production of proteases. Among the various carbon sources used, bacteria showed maximum protease activity in the presence of sucrose and fructose. Furthermore, bacteria showed a maximum yield of protease activity when gelatin was used as the nitrogen source.
Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus
Journal of Basic Microbiology, 2014
Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(þ), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg À1 , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90°C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg À1. The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.
2021
This research aims to produce thermostable alkaline protease from Bacillus subtilis isolated from La Hot Spring, Terengganu, Malaysia. The study was also conducted to determine the optimum conditions for protease production and stability by considering several parameters including pH, temperature and salt concentration. All seven bacteria were screened on skim milk agar overnight at 37 °C. Three strains with the highest proteolytic activity were identified in protease specific medium. The thermostable alkaline protease had an optimum temperature of 60 °C which achieved 85.73, 82.90 and 83.05 U/mL of protease activity for the three strains respectively. Furthermore, the strains exhibited significant activity of more than 90% from their original activity. Meanwhile, the optimum pH for protease production was pH 9 with the protease activity of 76.76, 79.71 and 88.39 U/mL for TB4, TB6 and TB9 strains, respectively. Proteases were found stable at pH 9 where the loss did not exceed 30% of...
Waste and Biomass Valorization, 2020
Current study was planned keeping in mind the importance of proteases and their role in industry. In the present study protease was produced using Luria Bertani medium. The LB medium was supplemented with various carbon and nitrogen sources separately for the enhancement of growth of Geobacillus SBS-4S and for protease production. The optimization studies demonstrated the increase in protease production from 10.6 to 24.4 U/mL or 34.5 U/mL when the medium was supplemented with additional 2% yeast extract or 5% wheat bran respectively. Under the optimal conditions we could produce 46.2 U/mL of protease. The protein was purified by column chromatography and the purified protein was utilized for characterization studies. SDS-PAGE analysis confirmed the molecular weight of protease as 37 kDa. The enzyme exhibited its maximal activity at 60 °C and pH 9.0. Presence of Ca 2+ and Mn 2+ at a final concentration of I mM in the activity assay mixture enhanced protease activity from 100% to 133 and 150% respectively. Protease activity was slightly reduced (92%) in the presence of SDS whereas the presence of non-ionic detergents Triton X-100, Tween-20 and Tween-80 reduced the enzyme activity to 87, 82 and 69% respectively. Thermostability studies demonstrated that the protein was stable with 50% residual activity after an incubation of 2 h and 25 min at 60 °C in the presence of 1 mM Mn 2+. The kinetic studies demonstrated the K m and V max values of 16.67 mg/mL and 143 U/mL respectively. The stability of protease at wide range of pH and temperature makes this enzyme suitable for its utilization in detergent and poultry feed industry.
This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214 U/ml) under optimized conditions: carbon source, citric acid-1.5 % (w/w); inducer, soyabean meal-2 % (w/w); pH 11.0; shaking condition 37°C for 48 h. The enzyme had pH and temperature optima of 9.0 and 60°C, respectively. The enzyme displayed the molecular mass of 36 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0-11.0) and thermostability (20-70°C). More than 70 % residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H 2 O 2 for 30 min. The protease activity was also enhanced by divalent cations such as Ba 2+ , Ca 2+ and Mg 2+ and was strongly inhibited by Fe 2+ , Zn 2+ , Sr 2+ , Hg 2+ and urea. The enzyme retained more than 50 % of its initial activity after pre-incubation for 1 h in the presence of 5 % (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1 % w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the ecofriendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.
2007
A new s train I RDO alkalophilic Geobacillus ca ldoxylosilyticus was isolated f rom a T ikehau pond i n the Atoll, Polynesian France. It was identified o n the basis of 16s rRNA gene homology, GC, where it was 95% related to t his strain. This strain is unique in that it could g row easily and produced alkaline protease in presence of o ne gram of wh eat b ran or ric e stalk w ithout an y su pplements. Growth of Geobacillus co ldoxylosilyticus under solid s tate f ermentation ( SSF) using wheat bran or rice stalk a s the sole carbon and n itrogen sources produced a ppreciable levels of alkaline protease (1890 U/gm, 300 U/gm) respectively. The optimized parameters for e nzyme product ion wer e s tudied. The neem ext ract played a si gnificant role in activation and stability of the p artially purified alkaline p rotease. The hydrolytic activity of the alkaline protease towards different natural substrates improved in presence of 0.05 % neem extract. This enzyme sho wed keratinaceous ...
2013
A thermophile alkaline protease producing Bacillus sp. strains isolated from soil samples. Enzyme synthesis occurred at temperatures between 20oC and 60oC with an optimum of 55°C and between the pH range of 6.0-13.0 with an optimum pH of 10.0 on skimmilk agar plates. Analyses of molecular mass of the partially purified enzyme was carried out by SDS-PAGE which revealed two bands as 112 and 97 kDa. The enzyme was active in a broad temperature range between 30oC and 100oC, with an optimum at 70oC, and maximum activity was at pH 11.0. The enzyme also presented the alkaline-stable properties with a remaining activity around 94%, at pH 6.0-13.0 for 24 h, at 55°C. The enzyme activity was highly stable between 30-100°C with a remaining activity 91%, after pre-incubation for 1 h. Enzyme was exposured to various NaCl concentrations (3-30%), and the highest residual activity was determined in the presence of 3% NaCl (87%). While after being exposed with β-mercaptoethanol (1%), PMSF (3mM), Twee...