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 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.

https://www.mdpi.com/1420-3049/25/11/2609/pdf

Effect of Cultural Conditions on Protease Production by a Thermophilic Geobacillus thermoglucosidasius SKF4 Isolated from Sungai Klah Hot Spring Park, Malaysia.

A large number of thermophilic representatives of the Geobacillus and Anoxybacillus genera have been isolated from geographically distant and physicochemically different environments, including high-, moderate-, and low-temperature habitats However, terrestrial hot springs are the main habitats for Geobacillus and Anoxybacillus species The members of these genera possess a variety of thermo-adaptive features that enable them to thrive at elevated temperatures Due to their ability to withstand harsh environmental conditions, geobacilli and anoxybacilli are a valuable source for provision of thermostable enzymes, such as amylases, lipases, proteases, etc, and other components Thermostable enzymes obtained from thermophilic bacilli have found a plethora of commercial applications due to their sturdiness and toughness in withstanding the heat generated in various biotechnological and industrial processes This chapter contains a review of studies of geobacilli and anoxybacilli from terrestrial geothermal springs worldwide with special emphasis on their distribution and diversity, ecological significance, adaptive mechanisms, enzymes, and biotechnological potential

Geobacillus and Anoxybacillus spp. from Terrestrial Geothermal Springs Worldwide: Diversity and Biotechnological Applications

In this study, it was aimed to determine the ability to produce protease enzyme of Thermomonas haemolytica isolated from geothermal Nenehatun hot spring in Turkey and utilization of this enzyme in the detergent industry to remove protein stains. The protease-producing strains were screened from hot springs, and a potential strain was identified as T. haemolytica according to morphological, physiological and biochemical characteristics and sequence of 16S rRNA gene. Maximum protease activity was observed at 55 °C and pH 9.0 at 72 h of incubation. Activity was very stable between 50 and 65 °C and pH 8.0–10.0, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA, EGTA, SDS, and urea. Some divalent metal ions such as Ca2+, Mg2+, and Mn2+ increased the enzyme activity, while Zn2+ and Cu2+ decreased. Michaelis–Menten constant (Km) and maximum velocity (Vmax) values were calculated by Lineweaver–Burk plot as 125 EU/ml and 1262 mg/ml, respectively. The biochemical characterization of the protease obtained from T. haemolytica was performed and applied on the blood and grass-stained fabrics with detergent to evaluate the stain removal performance of the enzyme. It was observed that the application of detergent with enzyme was more effective than the detergent without enzyme to clean up the stained fabrics. This is the first report of characterization of the protease of T. haemolytica. According to results obtained from this study, this new strain is a promising candidate for industrial applications in production of detergent.

Characterization and biotechnological application of protease from thermophilic Thermomonas haemolytica.

137 7.1 Background 137 7.2 Results 138 7.2.1 Isolation of acidophilic bacteria 138 7.2.2 Molecular characterization 138 7.2.3 Oxidation of iron by Acidithiobacillus ferrooxidans IO-2C in the presence of different iron substrates 139 7.2.4 Draft genome of Acidithiobacillus ferrooxidans IO-2C 144 7.3 Discussion 150 Chapter 8: Culture independent microbial diversity of extremophiles 152 Abstract 152 8.1 Background 152 8.2 Results 153 8.2.1 Microbial diversity of hot spring 153 8.2.2 Microbial diversity of saline soil 157 8.2.3 Microbial diversity of acid seep 162 8.3 Discussion 168 Chapter 9: Discussion 171 Conclusion 187 References 188

Ecology, diversity and metabolites analysis of extremophiles

http://www.cell.com/article/S2405844023001081/pdf

Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria

A thermophilic bacterium (TP-2) was isolated from the Tatta Pani hot spring in Azad Kashmir and was characterized using phenotypic and genotypic characters. The strain developed cream colored, round, smooth, flat and slimy colonies while the cells were Gram positive rods that ranged in size from about 2.1-3.6 μm to 0.2-0.3 μm in width. Sequence analysis of its 16S rRNA gene showed that isolate TP-2 had 89% homology with Geobacillus debilis. It grew within pH range of 5.5 to 8.5 with optimum growth at pH 7.0. The isolate showed optimum growth at 65oC and gave positive results for gelatin hydrolysis (GEL), ortho nitrophenyl-β-D-galactopyranosidase (ONPG), and nitrate production and produced acid from sucrose, glucose and maltose. It utilized glucose, fructose, maltose, lactose, sucrose, xylan, starch, filter paper and carboxymethylcellulose as sole carbon source. Isolate TP-2 produced significant amount of industrially important enzymes i.e. extracellular α-amylase, CMCase, FPase, Xylanase, Protease and Lipase and intracellular CMCase and FPase.

/pdf/characterization-of-a-novel-hydrolytic-enzyme-producing-3xdljghdes.pdf

Masroor Ellahi Babarl

Papers

Characterization Of A Novel Hydrolytic Enzyme Producing Thermophilic Bacterium Isolated From The Hot Spring Of Azad Kashmir-Pakistan