Purifcaton and characterizaton of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia Sotharith Phon 1 , Andriat Ningrum 1 , Lucia Dhiantka Witasari 1,* Department of Food and Agricultural Products Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Jl. Flora No. 1, Yogyakarta 55281, Indonesia *Corresponding author: dhiantea_k@ugm.ac.id SUBMITTED 11 May 2021 REVISED 24 August 2021 ACCEPTED 27 January 2022 ABSTRACT Thermostable proteases that optmally withstand the high‐temperature conditons of thermophilic bacteria could be produced and purifed, which would be highly benefcial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditons. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytc index was measured in a solid medium. The expression of protease was optmized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purifed 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. Purifed thermostable protease was highly actvated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classifed as a thermostable alkaline serine protease. Its kinetc study revealed specifc actvity of 0.41 U/mg (Vmax) and 0.25 mg/mL (K M ). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high actvity at high temperatures and alkaline pH, which is vital for applicaton in industries such as leather processing and detergent formulaton. KEYWORDS Thermostable serine alkaline protease; Geobacillus sp. DS3; enzyme purifcaton, DEAE Sephadex A‐25 Indonesian Journal of Biotechnology VOLUME 27(2), 2022, 73‐79 | RESEARCH ARTICLE 1. Introducton Proteases, proteinases, or peptidases are several kinds of enzymes that have their specific function of degrading the protein molecules into shortchain peptides and amino acids (Sharma et al. 2017). Currently, proteases are re ported as one of the most widely used for the industry as well as industrial enzymes. The sales of industrial en zymes are around $4.2 billion in value worldwide (Singh et al. 2016; Suberu et al. 2019b), since the proteases play an important role as one of the largest groups of industrial enzymes. Proteases produced by microorganisms have be come a massive group of industrial enzymes, about more than 60% of the total global sale of enzymes (Souza et al. 2015). Hence, it has been utilized in several industrial ap plications and analytical processes, such as the production of leather, pharmaceuticals, protein processing and analy sis, foods biotechnology, cosmetic preparations, cleaning processes, diagnostic reagents, and peptide synthesis in dustries (Souza et al. 2015; Tavano et al. 2018; Zhou et al. 2018). Thermostable enzymes or thermozymes are com monly synthesized from thermophilic microorganisms at the thermophile condition, which optimal growth temper ature of more than 60 ºC and the hyperthermophiles with optimal growth temperatures of more than 80 ºC (Stetter 2006). Thermozymes are utilized for the enzymatic pro cess, which is conducted in hightemperature conditions. Generally, thermostable proteases are defined as proteases that optimally withstand hightemperature conditions and can exist with high catalytic efficiencies and provide resis tance from mesophilic microbial contamination (Hussein et al. 2015). The number of research involved in producing the thermostable enzyme from thermophilic bacteria is still limited in Indonesia. On the other hand, there are po tential geothermal sites since Indonesia is located in the ring of fire (volcano line) (Pambudi 2018), including crater Sikidang, Dieng which has the potential biodiver sity of thermophilic bacteria. In the previous study, it has been reported that the thermophilic bacterium Geobacillus sp. strain DS3 was isolated from Sikidang Crater, Dieng Plateau, Central Java (Witasari et al. 2010). Therefore, the potential protease from this isolated bacteria should be de termined and characterised. The purification of protease has a significant role since the purified enzyme will be utilized to determine the bio Indones J Biotechnol 27(2), 2022, 73‐79 | DOI 10.22146/ijbiotech.65822 www.jurnal.ugm.ac.id/ijbiotech Copyright © 2022 THE AUTHOR(S). This artcle is distributed under a Creatve Commons Atributon‐ShareAlike 4.0 Internatonal license.