Isolation, characterization, activity test and molecular identification of thermophilic bacteria producing proteases from Dolok Tinggi Raja Natural Hot Springs, North Sumatra, Indonesia (original) (raw)
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Screening of Thermostable Protease Producing Microorganisms Isolated from Indonesian Hotspring
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Although many proteases had been studied and characterized, only a few of them are commercially available. Protease thermostability is one of the crucial properties for industrialapplication. This research aimed to isolate and to screen the potential isolate which produce thermostable protease. There were 6 isolates (BII-1, BII-2, BII-3, BII-4, BII-6 and LII), isolated using solid Minimal Synthetic Medium (MSM) supplemented with 1.5% skim milk, that have, protease activity. Based on the 16S-rRNA gene sequencing analysis, isolates BII-1, BII-2 and BII- 6 were identified as Bacillus licheniformis, isolates BII-3 and BII-4 were identified as Bacillus subtilis, while isolate LII was identified as Brevibacillus thermoruber. Three isolates (BII-6, BII-4 and LII) were then further investigated for the second screening step using liquid MSM supplemented with 1% skim milk. The isolates (BII-6, BII-4 and LII) optimally produced protease when they were cultivated at 35, 30 and 50o C respectiv...
Protease Activity of Thermophilic Bacteria from Lejja Hot Springs in Soppeng South Sulawesi
JST (Jurnal Sains dan Teknologi)
This research aimed to observe quantitatively the effect of temperature and pH on protease activities from thermophilic bacteria collected from Lejja—Hot springs.  Spectrophotometry on the casein substrate was used to test the proteolytic activity of the crude protease.  Mixtures of enzyme and casein were incubated at various temperatures and pH for 20 minutes. The absorbance of tyrosine from protein hydrolysis was determined by spectrophotometry on λ 280 nm. Temperature and pH impacted on protease activity were determined at temperature (60; 65; 70; 75; 80; 85 and 90oC) and pH (6.0; 6.5; 7.0; 7.5; 8, 0; 8,5 and 9,0). Results showed that treatment of temperature variations and pH had a significant effect on protease activity. Crude extract of Bacillus licheniformis protease showed that the highest activity at 80oC and pH 7.5 was 0.1303 unit/ml/minute. Bacillus stearoformis showed the highest enzymatic activity of the protease at 85oC, and pH 7.5 was 0.1226 Unit/ ml/minut...
International Journal of Life Sciences, 2015
Article Information Thermophilic microorganisms have gained worldwide importance due to their remarkable potential to produce thermostable and thermoactive enzymes that have wide applications in pharmaceuticals and industries. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes. The aim of this research was therefore the isolation of protease producing thermophilic bacteria from Tashkooh or firing mount located in Ahvaz, Iran. 8 bacterial isolates were screened. These strains were examined for the existence of extracellular protease activity. All 8 isolated bacteria showed proteolytic activity on nutrient agar with skim-milk. 3 bacteria showed their optimum growth at alkaline pH and grew maximally at different temperature in the thermophilic range and had proteolytic activity at pH 11 in 70˚C and pH 9 in 55˚C.The best carbon source for proteolytic activity was starch. After performing some phenotypic tests determined that all the isolates were Gram positive, endospore forming rods, aerobic, capable to produce catalase, amylase and gelatinase enzymes and they were identified as Bacillus sp. The best isolated bacteria was identified molecularly with the aid of 16S rRNA sequencing and data revealed that the Bacillus subtilis strain G-13 (GenBank accession No. KJ139434.1).The investigation confirmed that the isolate to be a true thermophile and could be a source of thermostable protease which can be exploited for pharmaceutical and industrials applications.
Indonesian Journal of Biotechnology
Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.4...
2017
Thermophilic bacteria grow in relatively high temperature between 50 to 60°C. These organisms can produce specific enzymes that have been used in industrial and biotechnological applications. The basic method to derive a sequence for a uncultured bacteria is to use universal primers against the 16S rRNA gene region in a PCR step to increase the amount of DNA and then to sequence the amplicon. This study was carried out to screen the ability of thermophilic bacterial isolates for degrading protein by protease and to identify the most potent isolates using molecular techniques inferred by 16S rRNA gene. We are dependent on accurate sequences in databases, appropriate names associated with those sequences, and an accurate sequence for the isolate to be identified. Thirty seven bacterial isolates were cultured and characterized using nutrient casein plate method. Twenty out of thirty seven bacterial isolates have been recognized to be positive for casein test and selected for Gram staining method. Total DNA genomic were extracted by using I-genomic DNA Extraction Mini Kit, and polymerase chain reaction (PCR) operation assay was performed for appropriate target isolates. The phylogenetic analysis of these strains revealed that the genus of Bacillus is the most closely matched to the data from the GenBank. Bacillus subtilis, Bacillus sp, Bacillus amyloliquefaciens and Bacillus licheniformis were identified as species for the potential bacterial isolates. Two samples which are RSS6 and JF OUTER 2B have not been characterized through the phylogenetic tree. This might refer to either rare or new bacteria.
Asian Journal of Pharmaceutical and Clinical Research
Objective: The main objective of this study is to investigate the industrial applications of a thermophillic alkaline protease from a hot water spring bacterial isolate “A” and to study its production, optimization, and purification.Methods: The alkaline protease was produced using shake flask studies maintaining a pH of 9.0 and a temperature of 50°C. Optimization studies of the enzyme were carried out using variable pH, temperature, organic carbon, and nitrogen sources followed by purification of the enzyme using DEAE-cellulose ion exchange chromatography technique. Stability of the enzyme was analyzed in the presence of organic solvents and surfactants. The efficiency of the enzyme in the removal of proteinaceous stains in the presence of strong detergents under extreme conditions was assessed. The fibrinolytic activity of the enzyme in dissolving the blood clot was confirmed.Results: The isolated alkaline protease was purified to homogeneity with a 16-fold increase. Media optim...
Exploration of a Hot Spring for Thermostable Protease Producers
Journal of Microbiology, Biotechnology and Food Sciences, 2017
Proteases from thermophilic sources have been reported to have optimum catalytic activities at the high temperatures. Therefore thermostable proteases are receiving considerable attention for their usefulness in various enzymatic industrial processes. Proteases have found applications in laundry detergents as additives, leather finishing, silk industry, feeds modification, food processing, brewing, pharmaceuticals, diagnostic reagents, peptide synthesis, and silver recovery from X ray/photographic film. Some thermostable protease producers have been reported from the genera Pyrococcus, Thermococcus, Staphylothermus, Desulfurococcus, Pyrobaculum and Sulfolobus. In archaea, the hyperthermophilic Desulfurococcus strain was reported to produce the extremely thermostable serine protease (Hanzawa et al., 1996). Thermophilic fungi from the genera Achaetomium, Chaetomium, Penicillium, Rhizopus, Sporotrichum, Torula and Rhizomucor have also been reported to produce novel thermostable proteases (Emi et al., 1976). Among bacteria, the genus Bacillus is the dominant source of thermostable proteases. Here we have selected the terrestrial Unkeshwar hot spring (19°51'21.3"N and 78°15'00.9"E) from Nanded district of Maharashtra state of, India and explored it for the isolation of various thermophiles. Further we have identified the industrially important thermostable protease producers. Production and characterization of thermostable proteases from the selected isolates were also assessed. MATERIALS AND METHODS Isolation and identification of thermophiles Water samples from Unkeshwar hot spring were collected in March, August and December 2011 and March 2012. Temperature and pH of the water samples were recorded individually at the time of collection of samples (Pathak et al., 2014; Jadhav and Pathak, 2015). Abiotic characterization of Unkeshwar hot spring water samples was performed and published (Pathak and Rathod, 2014). Composite water samples were spread on nutrient agar, tryptone-yeast extract agar, tryptone-yeast glucose agar, Vogel Johnson Agar, glucose sodium azide glycerol agar, thiosulphate agar, J agar, brain heart infusion agar, Gram negative agar and Bacillus agar plates and these plates were incubated at 50 o C to isolate different thermophiles (Pathak and Rathod, 2015). Typical morphological characters viz. colony shape, size, margin, elevation, consistency, opacity and pigmentation of selected isolates were recorded. Microscopic characters viz. cell shape, cell size, sporulation and motility of selected isolates were observed (Pathak and Rathod, 2015). Optimization of physical parameters for maximum growth of selected isolates was thoroughly assessed (Polkade et al., 2015). Sugar utilization pattern of selected isolates was determined by using dextrose,
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 ...
African Journal of Microbiology Research, 2012
A moderately thermophilic bacterium designated EP1001 isolated from an alkaline hot spring (conditions in the hot spring: temperature 53-54°C and pH 9.3) in Zimbabwe was investigated for protease production. Studies to determine the growth and protease enzyme production by the novel bacterium were carried out in ordinary batch, regulated batch and fed-batch modes of fermentation using a BioFlow III 3 L fermentor. Biomass production and protease production were dependent on fermentation mode. Higher biomass formation and protease production were obtained in fed-batch fermentations compared with that in ordinary and regulated batch cultivations. The protease enzyme was found to be a thermostable alkaline serine protease with optima activity at 75°C and pH 10. The enzyme had a half life of 45 min at 80°C and 12 h at 70°C. It was stable over the pH range of 5.0 to 11.0. The enzyme was inhibited by phenylmethane-sulfonyl fluoride and EDTA but not by N-Tosyl-L phenanylalanine chloromethyl, iodoacetamide and O-phenathroline. The ions Ca 2+ and Fe 2+ at 0.5 and 2.5 mM concentration were stimulatory, while Mg 2+ and Mn 2+ had little effect on the enzyme activity. The enzyme produced by bacterium (EP1001) was concluded to be an alkaline protease that requires calcium and iron ions for its activity.