Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia

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 0.41 U/mg (V max ) and 0.25 mg/mL (K M ). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.


Introduction
Proteases, proteinases, or peptidases are several kinds of enzymes that have their specific function of degrading the protein molecules into shortchain 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 hightemperature conditions. Generally, thermostable proteases are defined as proteases that optimally withstand hightemperature 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 chemical function, such as the activity and its effect fac tors, including pH, temperature, activator, and inhibitor, to understand the character of the protease group. Based on the characteristic of active sites and mode of catalytic action, the proteases were grouped into aspartyl or car boxyl protease, cysteine or thiol protease, serine protease, and metalloproteases. Furthermore, regarding pH prefer ences, proteases could be subdivided into acidic, alkaline, and neutral proteases (Rawlings et al. 2018). Indeed, pro tease purification methods must be optimized in various strategies based on different purposes. However, the early purification methods could be started from ammonium sul fate precipitation and column chromatography (such as gel filtration and ion exchange).
In this present study, the purification and characteriza tion of protease are conducted to determine thermostable protease produced by Geobacillus sp. strain DS3 from Sikidang crater, Dieng plateau, Central Java, Indonesia.

Preliminary protease assay using solid medium
The main purpose of this step was to measure the prote olytic index. Expression of protease in Geobacillus sp. DS3 was determined using solid medium of Minimal Syn thetic Medium (MSM) (Zilda et al. 2013). The medium contained 0.1% (w/v) NaCl, 0.1% (w/v) K 2 HPO 4 , 0.01% (w/v) MgSO 4 ·7H 2 O, 0.05% (w/v) yeast extract and 2% (w/v) bacteriological agar was prepared and supplemented with 1% (w/v) skim milk. The incubation was carried out at 30, 40, 50, and 60 ºC for 24 h. The clear zone formed around the colony indicated the ability of the bacterium to produce protease and was designated as the Proteolytic Index (PI). The Proteolytic Index was determined by mea suring the diameter of the clear zone around the colony compared to the diameter of the colony.
The MSM medium without agar was used to optimize the expression of protease. Initially, the culture was incu bated for 24 h at 50 ºC for seed culture, then 0.5 mL of seed culture was subcultured into 5 mL liquid MSM medium. The growth culture was observed at various times of 0, 3, 6, 9, 15, 18, 21, and 24 h. In addition, the temperature optimum was conducted at 30, 40, 50, 60, 70, and 80 ºC.

Protease activity assay
Protease activity was performed using casein as a substrate by the modified Folin and Ciocalteu's method. Two hun dred microliter of the crude enzyme was mixed with 500 μL of 0.2 M GlycineNaOH buffer (pH 10.0) containing 1% (w/v) casein and incubated at 50 ºC for 10 min. The en zyme reaction was stopped by adding 2 mL of 10% (w/v) trichloroacetic acid. The mixture was incubated at room temperature for 15 min, followed by centrifugation at 3046 g at 20 ºC for 15 min. One milliliter of supernatant was mixed with 2.5 mL of 0.5 M Na 2 CO 3 and then 500 μL of 20% (v/v) 2 N Folin & Ciocalteu's reagent was added.
The mixture was incubated at 40 ºC for 10 min then the absorbance of the mixture was measured by GENESYS™ 150 Vis/UVVis Spectrophotometer at 660 nm. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per minute under the standard assay conditions.

Protein content
The protein concentration was determined using the Brad ford methods. A series of BSA standard solutions (0.25 1.4 mg/mL) was prepared for a standard curve. The as say was performed by pipetting a 10 µl sample and adding 1 mL of Bradford reagent. The mixture was incubated in a dark place for 5 min. The absorbance was read at 595 nm by GENESYS™ 150 Vis/UVVis Spectrophotometer.

Purification of protease
The purification of protease was conducted by two steps purification systems; using ammonium sulfate precipita tion and DEAESephadex resin. The incubated culture at 50 ºC for 18 h was centrifuged at 3046 g. The supernatant was extracted with ammonium sulfate at various saturation as follows 020%, 2040%, 4060%, and 6080%. Each extract was separated by centrifuge at 4 ºC with 3046 g for 20 min. The obtained pellet was suspended in 0.2 M glycineNaOH buffer pH 10 and dialyzed against the same buffer overnight. After dialysis, the solution was added 85% glycerol with a ratio 1:1 to inhibit protein aggrega tion and stored at 20 ºC. The protease activity and protein concentration were determined.
The protein fraction of ammonium sulfate (4060%) was then subjected to further purification using DEAE Sephadex A25 prepacked column (15 cm × 10 cm). First, 0.5 mL dialysate was loaded into the prepared column that was equilibrated with 0.5 M GlycineNaOH buffer pH 10.0 (Buffer A). The unbound proteins were eluted by buffer A. Five milliliters of unbound fractions were col lected for 10 fractions. The bound proteins were eluted with 0.5 M NaCl dissolved in the same buffer. The 1.5 mL of bound fractions were collected and detected its pro tein content at a wavelength of 280 nm. The column was washed by the same buffer until the absorbance of the frac tion at 280 nm reached zero.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) assay
The molecular weights of purified protease were deter mined using SDSPAGE method. The gel consisted of 7% (w/v) and 12% (w/v) of stacking and separating gels, respectively. Samples were prepared by mixing purified enzymes in distilled water containing loading dye (No vagen). The prepared sample was incubated at 95 ºC for 5 min and then centrifuged at 1820 g for 5 s. Each sample and the protein marker (Spectra Multicolor Broad Range Protein Ladder, Thermoscientific) were loaded into the wells. Electrophoresis was performed at 120 V for 120 min. Protein bands were visualised by overnight staining with Coomassie dye. Protein bands were observed after destaining the gel the following day using the destaining solution.

The pH optimum, temperature optimum, and stability of enzyme
The pH optimum was determined using ammonium sul fate (4060%) extract containing 0.716 mg/mL of protein concentration. The optimal pH value of enzyme activity was performed from 5.8 -10.6 at 50 ºC, 1h. The standard buffers were used, including 0.2 M phosphate buffer (pH 5.8 -7.2), 50 mM TrisHCl buffer (pH 7.0 -9.0), and 50 mM GlycineNaOH buffer (pH 8.6 -10.6). Protease ac tivity assay were determined as described earlier.
The 4060% ammonium sulfate was precipitated in protein solution with a concentration of 0.716 mg/mL for the temperature and stability assays. The effect of temper ature on protease activity was conducted at optimum pH obtained. The range of temperatures was performed from 30, 40, 50, 60, 70, 75, and 80 ºC. The enzyme stability was measured by incubating the enzyme at 70 ºC for 3 h. Every hour, 50 μL of the enzyme was mixed with 500 μL of 50 mM GlycineNaOH buffer pH 9.6 containing 1% casein, and after 10 min the reaction was terminated by adding trichloroacetic acid (Rai and Mukherjee 2009). Protease activity was measured as described earlier.
Inhibitors of 1 mM phenylmethylsulphonyl fluoride (PMSF) for serine protease, 1 mM 2mercaptoethanol (2 ME) for cysteine protease, and 1 mM ethylenediaminete traacetic acid (EDTA) for metalloprotease were prepared. The ammonium sulfate extract (4060%) with a protein concentration of 0.716 mg/mL was premixed with the in hibitors and incubated at RT for 30 min. The residual ac tivity was measured using the protease activity assay. The result was expressed as the percentage of the protease ac tivity measured without an inhibitor (control).

Enzyme kinetic assay
The kinetic assay was conducted using pure enzyme after DEAESephadex A25 purification system. Various con centrations of casein (0.0025 -0.02 mg/mL) were used as substrate. Protease kinetic assay was performed at 50 ºC for 3 min. The kinetic rate constants, K M and V max were determined using the LineweaverBurke equation.

Statistical analysis
The proteolytic index, time, and temperature optimum for protease expression were statistically analyzed using SPSS (version 23) IBM oneway ANOVA with a probability level of p < 0.05. The other results are mostly represented as mean ±SD of at least three experiments.

Preliminary protease assay using solid medium
To identify the protease expression in Geobacillus sp., the preliminary assay using MSM agar containing 1% skim milk at various temperatures was conducted. The clear zone surrounding Geobacillus sp. DS3 colonies after 24 h incubation indicated the protease activity was the highest, followed by a higher proteolytic index. Incubation at 50 ºC showed the highest proteolytic index (Table 1). Unfortu nately, the colonies and clear zone did not appear in higher temperature incubation at 60 ºC . In a previous study, it was reported that 6 isolates BII1, BII2, and BII6 (Bacil lus licheniformis); BII3 and BII4 (Bacillus subtilis); and LII (Brevibacillus thermoruber) isolated from Indonesian hot spring formed clear zone on MSM plate agar after in cubating at 55 ºC for 30 h (Zilda et al. 2013). Thus, the preliminary result revealed that Geobacillus sp. DS3 from Sikidang crater, Central Java has the highest protease ac tivity at 50 ºC in solid MSM medium.

Optimizing the expression of protease
We performed incubation at various times and tempera tures to determine optimum conditions for protease ex pression in Geobacillus sp. DS3. The protease activity substantially increased from 0 h until 18 h and then grad ually decreased after 18 h incubation (Figure 1a). The highest protease activity was observed at 18 h incubation. Therefore, it was selected for further protease expression conditions in Geobacillus sp. DS3 at a different level of temperature conditions. The protease activity was dramat ically raised from 40 ºC and suddenly dropped after 50 ºC. Thus, Geobacillus sp. DS3 produced protease with the highest enzyme activity at 50 ºC for 18 h of incuba tion. Protease produced from isolate LII of Brevibacillus thermoruber, from Padang Cermin, Lampung, Indonesia, expresses the highest activity at 50 ºC for 22 h incubation (Zilda et al. 2013).

Purification of protease
The highest protease activity at 4060% saturation in am monium sulfate purification system. Therefore, this am monium sulfate extract was utilized for the following pu rification step using DEAESephadex A25. The bound fraction of DEAESephadex A25 purification showed two protein peaks at fractions 19 and 26. Protease activity and protein content of several peaks were measured, but only the highest peak of fraction 19 provided detectable protease activity ( Table 2).
The SDSPAGE assay of ammonium sulfate extract (4060%) in lane 3 contained several slightly visible bands around 30, 32, and 60 kDa. Lane 5 (ammonium sulfate extract (020%) showed a thick band at 30 kDa but did not exhibit any protease activity. Unfortunately, lane 6 (fraction 19 of DEAESephadex A25 purification) was invisible. In general, the molecular weight of alkaline serine proteases from Bacillus species has been reported to be 30-45 kDa. The molecular weight of alkaline ser ine proteases from B. subtilis RD7, and Bacillus lehensis JO26 were reported to be 43 kDa (Suberu et al. 2019a), and 34.6 kDa (Bhatt and Singh 2020), respectively. The purified thermostable alkaline serine protease gene of Geobacillus stearothermophilus B1172 has a molecular weight of 39 kDa (Iqbal et al. 2015). The molecular weight of alkaline serine protease from Bacillus cereus strain S8 was 71 kDa (nonreducing) and 35 kDa and 22 kDa (re ducing), indicating the enzyme existence as a dimer in its native state. Furthermore, after gel filtration (Sephadex G200) chromatography, its purified enzyme showed a molecular weight of 22 kDa (Lakshmi et al. 2018). Based on this information, we predicted our protease has a molec ular weight of around 32 kDa.

pH optimum, temperature optimum, and stability of enzyme
Different standard buffers (phosphate buffer, trisHCl buffer and glycineNaOH buffer) with various pH ranges from 5.8 10.6 were applied to determine the optimum pH for protease. The result showed that the crude en zyme has pH optimum in glycineNaOH buffer at 9.6 for protease activity. At pH below 7, its protease activity gradually decreased from 50% of the maximum activity. The protease activity was maximal at 70 ºC. At a tem perature below 40 ºC revealed, protease activity reduc tion was approximately 60% of the maximum activity. At 80 ºC, it only exhibited 25% of the maximum activity. Furthermore, the result of enzyme stability at 70 ºC in dicated that the enzyme was highly stable for 1 h incu bation and gradually decreased after 2 h incubation. It still showed protease activity around 63% of its maximal stability at 3 h incubation. These results suggested that the enzyme belongs to the thermostable alkaline protease. An alkaline serine protease from G. stearothermophilus (GsProS8) showed optimal activity at pH 8.5 and 50 °C (Chang et al. 2021). Whereas a serine protease from ther mophilic Geobacillus sp. GS53 optimally worked at 55°C and pH 8 (Baykara et al. 2021). Iqbal et al. (2015) also reported that a thermostable alkaline serine protease from G. stearothermophilus B1172 was stable at 90 °C and pH 9. The alkaline protease from B. cereus strain S8 showed optimum temperature and pH of 70 ºC and 10, respectively (Lakshmi et al. 2018). Besides, Shrinivas and Naik (2011) reported thermostable alkaline protease from Bacillus sp. JB 99 showed an optimal temperature at 70 ºC and pH op timum at 11. Thermostable alkaline protease in this study which is stable at 70°C and pH 9.6 could be applied in the detergent industry. Typically, a detergent protease needs to be active, stable, and compatible with the alkaline envi ronment encountered under harsh washing conditions: pH 9 11, the temperature of 20 60°C, as well as high con centrations of salt, bleach, and surfactant.

FIGURE 2
Effect of pH and temperature on protease activity

Effect of protease inhibitors
Protease inhibitors were subjected to identify groups at the active site of the enzyme. 2ME (cysteine protease inhibitor) and EDTA (metalloprotease inhibitor) slightly affected the protease activity ( Figure 3). The result indi cated that SHgroup residue did not work in the protease activity of this enzyme. In addition, ions or metals may not be essential for the stability and activity of this enzyme. In contrast, the protease activity is inhibited by PMSF (serine protease inhibitor) due to its lowest residual activity (50%) (Figure 3). Through this finding, the enzyme could be classified as a serine protease. Similarly, some proteases such as GsProS8 from G. stearothermophilus (Chang et al. 2021), alkaline protease from B. cereus strain S8 (Lakshmi et al. 2018), thermostable alkaline protease from Bacillus sp. JB 99 (Shrinivas and Naik 2011) were strongly inhib ited by PMSF.
Keratinases are a group of mostly extracellular serine proteases that are able to degrade keratins into amino acids. Keratin is a group of fibrous structural proteins of hair, wool, nails, hooves, horns and feather quills, and the epithelial cells in the outermost layers of the skin. There fore, thermostable serine protease in this study could be potentially used for leather processing applications.

Enzyme kinetic
The LineweaverBurk curve (Figure 4) was obtained by plotting 1/V versus 1/S with a casein substrate concentra tion of 0.0025 -0.02 mg/mL. The enzyme has the value V max of 0.41 U/mg, while K M was 0.25 mg/mL. GsProS8 exhibited Vmax of 231.50 U/mg and KM of 7.37 mg/mL (Chang et al. 2021). A serine protease of thermophilic Geobacillus sp. GS53 showed 137.8 U/mg of specific ac tivity (Baykara et al. 2021). Lakshmi et al. (2018) reported that alkaline protease from B. cereus strain S8 mentioned K M and V max of 3.3 mg/mL and 15 U/mg, respectively. Lower K M value of protease reflects a stronger binding affinity of this enzyme to substrate. Thus, the thermostable alkaline protease from Geobacillus sp. DS3 in this study exhibited a higher affinity than the other alkaline serine proteases.

Conclusions
In conclusion, thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, In donesia, had an optimal condition for enzyme activity at 70 ºC with pH 9.6. However, further studies related to the effect of surfactant, metal ion, organic solvents, and bleaching agents that are most crucial for industries are required to be performed.