Short Communication: Isolation and characterization of thermophilic actinobacteria as proteolytic enzyme producer from Ie Seuum Hot Spring, Aceh Besar, Indonesia (original) (raw)
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Biodiversitas Journal of Biological Diversity
Protease is an enzyme that catalyzes the hydrolysis of peptide bonds in protein. Actinobacteria are one of bacterial groups that is able to produce protease. Actinobacteria are Gram-positive bacteria and mostly aerobic. This study aimed to identify protease-producing actinobacteria ATIS61 isolate using 16S rRNA gene and to characterize the protease activity. This study was experimental research, consisted of amplification and sequencing of the 16S rRNA gene, and protease activity test. The 16S rRNA gene analysis showed that ATIS61 isolate was closely related to Nocardia sp. strain 335427 with a 99.88% similarity and the result of phylogenetic tree construction was related to Nocardia farcinica strain ARS8 with Bootstrap 94%. Protease activity test showed the highest activity was on the eighth day of incubation at 0.115 U/mL. Protease activity based on temperature showed the highest activity at 40°C of 0.156 U/mL and the stability of protease towards temperature was stable at 40°C and 50°C. Protease activity showed that the highest protease activity was at pH 8 of 0.096 U/mL and the highest protease stability was also at pH 8. The addition of HgCl2 showed that it could inhibit protease activity with a value of 0.059 U/mL.
Isolation of thermophilic actinomycetes producers of thermostable proteases
In this study we isolated and screened 58 thermophilic actinomycetes strains for proteolytic activity. The 11A strain originally isolated from a soil sample collected from Antarctic areas that showed highest proteolytic activity was chosen for further analyses. The accomplished observations on the microbial growth in continuous shake flasks confirmed (he strain's thermophilic nature and identified its maximal protease synthesis at 45°C for 48h. The pH profile for enzyme production was investigated as well. The observed pH range was from 6.5 to 8.5 and the optimal value for the maximal enzyme synthesis was determined at pH 8.0. The influence of different temperatures on the enzyme activity was observed for period of 2 days. The enzyme retained more than 90% from its initial activity during incubation at 50°C for 48h. It was also found that the enzyme is relatively stable up to 80°C. The influence of different concentrations of carbon and nitrogen nutrient sources on the level of enzyme production was also investigated.
Screening of Thermostable Protease Producing Microorganisms Isolated from Indonesian Hotspring
Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology, 2013
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...
Comparative study of proteolytic activity of protease-producing bacteria isolated from thua nao
Thua nao is a Thai traditional fermented soya bean product with distinct flavour and aroma. This study was carried out to screen the potential protease-producing bacteria during the fermentation process. Of 171 strains, there were 169 bacterial isolates (98.8%) exhibiting proteolytic activity. Two potential isolates namely TN51 and TN69 showing highest activity as indicated by the widest clear zones (2.73 and 2.65 cm, respectively) were selected for further study. Using five different protein-based media, we found that the medium composition had an influence on the enzyme activity.
2021
Abstract. Fachrial E, Krisdianilo V, Harmileni, Lister INE, Nugroho TT, Saryono. 2021. Isolation, characterization, activity test and molecular identification of thermophilic bacteria producing proteases from Dolok Tinggi Raja Natural Hot Springs, North Sumatra, Indonesia. Biodiversitas 22: 1725-1732. Proteases are complex enzymes that can be applied in both commercial products and physiological fields. This enzyme accelerates the breakdown of peptide bonds in polypeptides and proteins using hydrolysis reactions, transforming them into simpler bonds. In recent years, the demand for protease enzymes in Indonesia is still dependent on imported products, and there has also been an increase in their use. This is because there are many advantages in terms of efficiency, environmental friendliness, and its ability to catalyze a reaction without by-products. Proteases dominate the enzyme market with a total of 70% sales. Furthermore, they are manufactured from thermophilic bacteria as a so...
STUDY OF THE EFFECT OF TEMPERATURE AND TIME OF CULTIVATION OF THERMOPHILIC ACTINOMYCETE ISOLATE 11A
The effect of temperature and time of cultivation of thermophilic actinomycete isolate 11A on proteolitic activity was investigated and modeled by using a central composite design (CCD). The two independent variables (temperature and time of cultivation) were studied at three levels with 4 repetitions at the central point totaling of 48 experimental runs. From the analysis of the results was determined that the temperature of cultivation has the greatest effect on the protease production of the isolate. The most favorable conditions for the enzyme production were determined from the obtained regression model. The results were compared with a previous study in which a systematic investigation of the operating parameters was carried out.
2018
Protease is an enzyme that is economical and very widely used in various fields of industry. This study aims to isolate and screen the bacteria producing acid, neutral and alkaline protease enzymes from the dadih. Isolation of bacteria from the dadih using the method plate count while screening bacteria producing protease enzymes by using Skim Milk Agar selection medium at various levels of acidity. The results showed that 111 bacterial isolates from dadih, 27 isolates of bacteria produce protease, where 5 bacterial isolates have the potential to produce acid protease, 6 isolate of protease neutral and 16 isolates as alkaline protease producer.
Isolation of Thermostable Protease Producing Bacteria from the Microbial Mats
Journal of University of Shanghai for Science and Technology, 2021
As thermostable protease has more commercial value in different industries, the aim of this study was to search for such an enzyme producing bacteria from the microbial mats. Investigation was continued on the isolate for its ability to produce mass amount of enzyme and its activity under suitable optimized conditions. Different parameters including cheap carbon and nitrogen substrates, inoculum size and temperature was selected to optimize the enzyme production conditions. Initially five different isolates from two microbial mats collected from different sources were analyzed for its ability to produce thermostable protease after exposing to higher temperature incubation conditions. Test culture tentatively named as 1F from microbial mat-1 was selected as more enzyme producer among the ten isolates. The organism was selected based on the zone of clearance on skim milk agar by the isolate, that indicating more protease production. Under each optimization parameter, each type of carb...
International Journal of Research Studies in Biosciences, 2016
With the increasing demand for industrial enzymes with novel properties, the production of enzymes tolerant to extreme conditions has also increased. Due to their unique macromolecular properties, thermophiles possess high metabolic rates, physically and chemically stable enzymes and higher end product yields than similar mesophilic species. Actinomycetes are bacteria which require minimal nutritional requirement for their growth and hence are preferred over thermophilic bacteria as source of industrial enzymes. In this study, thermophilic actinomycetes were isolated from hot water springs situated at Vajreshwari near Mumbai, Maharashtra and screened for the production of cellulase and amylase. Five isolates of thermophilic actinomyces obtained, tested positive for both amylolytic and cellulolytic activity. Two out of the five isolates were chosen for large scale production of the enzyme. The enzyme activity of the crude extract was determined using DNSA assay for reducing sugar. The optimum conditions for activity of amylase from isolate no. 4 were 55 o C at pH 4.8 and for cellulase were 55 o C at pH 9. Amylase and cellulase with high stability and good activity at elevated temperature and acidic /alkaline conditions may find commercial applications in various fields such as such starch processing, food industries and detergents industries.