Purification and Characterization of a Small Thermostable Protease from Streptomyces sp. CNXK100

Protease is a multifunctional enzyme that breaks down protein through the hydrolysis of peptide bonds, accounting for 60–65% of the world enzyme market (Mostafa et al. 2019; Mechri et al. 2022). It is widely used in many fields, such as detergents, diagnostics, tanning, therapeutics, silver recovery, waste treatment, and peptide synthesis. In recent years, microbial-derived proteases have been increasingly studied due to their specificity and friendly environment (Sharma et al. 2019; Šnajder et al. 2019; Parthasarathy and Gnanadoss 2020). Thermal stability is a crucial property of enzymes for industrial applications. Thermostable proteases are active in the range of 60–70°C without significant changes in their structure or properties. The ability to rapidly convert substrate at high temperatures is a significant factor in improving the efficiency of industrial operations (Zilda et al. 2012; Hussian and Leong 2023). Additionally, implementing high-temperature manufacturing processes offers other benefits, such as reducing the risk of microbial contamination, reducing fluid viscosity, and increasing heat and mass transfer (Synowiecki 2010; Hussian and Leong 2023).

Actinomycetes can biosynthesize a variety of efficient extracellular enzymes. In particular, Streptomyces species produce more than 80% of active natural products, which are essential in industrial fields (Spasic et al. 2018; Al-Dhabi et al. 2020a). Enzymes, including proteases derived from Streptomyces are receiving more and more attention due to their high substrate specificity (Spasic et al. 2018), ability to catalyze at high temperatures, wide pH range, resistance to organic solvents, surfactants, bleaching agents, proteolytic enzymes, and other denaturing agents (Abdullah Al-Dhabi et al. 2020b; Parthasarathy and Gnanadoss). Moreover, Streptomyces species such as S. clavuligerus, S. thermoviolaceus, S. rimouses and S. roseosporus have been shown to produce large quantities of proteases in different culture media (Abdullah Al-Dhabi et al. 2020b; Al-Dhabi et al. 2020a; Boughachiche et al. 2021). This study thus aimed to isolate and characterize a new thermostable extracellular protease from Streptomyces sp. CNXK100, thereby enhancing the database of actinomycete-derived proteases for potential industrial applications.

In the past few decades, there has been a growing interest in proteases that can function effectively at high temperatures and under harsh conditions, making them suitable for diverse applications. The CNXK100 thermostable protease was successfully purified in the present study, revealing a high specific activity. The purified CNXK100 protease, with a molecular weight of approximately 27 kDa, is similar to the protease from Streptomyces sp. D1 (28 kDa) (Mane et al. 2013), and these are among the smallest proteases in Streptomyces in comparison to those previously purified from S. griseorubens E44G (35 kDa) (Al-Askar et al. 2015) and S. koyangensis TN650 (45 kDa) (Ben Elhoul et al. 2015). The specific activity of this enzyme surpasses that of Streptomyces sp. AH.4 by 36 times (6.60 × 10⁴ U/mg) (Touioui et al. 2015) and exceeds that of S. clavuligerus Mit-1 by an impressive 350 times (6.80 × 10² U/mg) (Thumar and Singh 2007).

Significantly, the CNXK100 protease exhibited activity within a high-temperature range of up to 90°C, with an optimal temperature (75°C) surpassing those of specific proteases from Streptomyces sp. Al-Dhabi-82 (40°C) (Abdullah Al-Dhabi et al. 2020b), S. flavogriseus HS1 (50°C) (Ghorbel et al. 2014), and Streptomyces sp. AH4 (70°C) (Touioui et al. 2015).

The high-temperature activity of this protease may be attributed to the presence of numerous proline residues, intermolecular hydrogen bonds, disulfide, salt bridges, and hydrophobic interactions within its molecular structure (Zhou and Pang 2018; Xu et al. 2020). In addition, the CNXK100 protease demonstrated high activity in different buffers within a wide pH range of 5.0–10.0. This enzyme’s optimum pH range of 7.0–8.0 was lower than that of proteases from other strains, such as Streptomyces sp. GS-1 (pH 8.5) (Sarkar and Suthindhiran 2020), Streptomyces sp. M30 (pH 9.0) (Xin et al. 2015), and S. mutabilis (pH 10.0) (Mechri et al. 2022). This disparity in optimal pH values suggests that this protease can be characterized as a neutral enzyme (Singh and Bajaj 2017).

Metal ions can exhibit varying effects on different thermostable proteases from Streptomyces strains. In the presence of the heavy metal Cu²⁺, the activity of the studied enzyme remained unaffected, whereas Cu²⁺ induced a loss of protease activity from Brachystreptospora xinjiangensis OM-6 (Gohel and Singh 2013) and Streptomyces sp. P3 (Cheng et al. 2015). Besides, Mn²⁺ and Fe²⁺ induced a partial reduction in the activity of the CNXK100 protease whereas both were observed to completely abolish the protease activity from S. olivochromogenes (Simkhada et al. 2010). This observation underscores the potential applicability of the enzyme under conditions characterized by the presence of these heavy metals.

Among the five tested substrates, the CNXK100 protease displayed the highest hydrolysis activity toward casein. It exhibited similarities to proteases from other Streptomyces strains, including S. koyangensis TN650 (Ben Elhoul et al. 2015), Streptomyces sp. LCJ12A (Parthasarathy and Gnanadoss 2020), and S. speibonae (Tran et al. 2022). Consequently, this protease could be employed to produce peptones and peptides from casein (Rao et al. 2020).

The K_M value serves as an indicator of the enzyme’s affinity for its substrate. The K_M of the CNXK100 protease was 1.3–4.2 times higher than that of the metalloprotease from S. olivochromogenes (0.74 mg/ml) (Simkhada et al. 2010) and the alkaline serine protease from S. griseus (2.50 mg/ml) (El-Khonezy et al. 2015). However, the V_max value of the CNXK100 protease, representing the maximum reaction rate of substrate conversion, was remarkably higher (846–17200 times) than those of the protease from S. olivochromogenes (3876 U/mg) (Simkhada et al. 2010) and S. griseus (190 U/mg) (El-Khonezy et al. 2015).

The CNXK100 protease has been assessed for its thermal stability, exhibiting resilience up to 65°C for 60 minutes, with its activity surpassing that of the protease derived from Streptomyces sp. P3 (Cheng et al. 2015). However, it is imperative to note that this protease underwent heat treatment at 70°C for 30 minutes during the purification process. Consequently, this protease is considered a relatively thermostable enzyme, and its activity is anticipated to remain stable for at least 30 minutes at 70°C.

In addition, the CNXK100 protease demonstrated notable substrate hydrolysis activity across a wide range of pH levels following incubation in three distinct buffers: citrate-phosphate, phosphate, and Tris. Mainly, slightly enhanced activity was observed when the enzyme was subjected to processing at pH 6.0 in citratephosphate buffer or within the pH range of 6.0–8.0 in phosphate buffer, surpassing the corresponding control. Possessing inherent pH robustness and compatibility with diverse buffer systems, the CNXK100 protease holds promise for applications in the medical field (pH 5.0–6.0) (Singh et al. 2016), as well as in the domains of bakery and brewing (pH 6.0–8.0) (Gurumallesh et al. 2019), and various formulations within the industrial area of detergent production (pH 8.0–10.0) (Ben Elhoul et al. 2015). The pH stability of CNXK100 protease aligns with that of proteases from S. flavogriseus (pH 4.5–9.5) (Mostafa et al. 2019), Streptomyces sp. XE-1 (pH 5.0–9.0) (El-Khonezy et al. 2015), and S. koyangensis TN650 (pH 5.0–11.0) (Ben Elhoul et al. 2015).

Furthermore, ethanol, isopropanol, chloroform, and methanol were found to only marginally decrease the enzymatic activity of CNXK100 protease, with a maximum reduction of 20%. In contrast, ethanol resulted in a 40% reduction in the activity of protease derived from S. koyangensis TN650 (Ben Elhoul et al. 2015) and Streptomyces sp. M30 (Xin et al. 2015). On the other hand, chloroform resulted in 40% and 90% reductions in the activity of Streptomyces sp. GS-1 (Sarkar and Suthindhiran 2020) and S. koyangensis TN650 protease, respectively (Ben Elhoul et al. 2015), while isopropanol caused reductions of 50% and 70% in the activity of S. koyangensis TN650 protease (Ben Elhoul et al. 2015) and Streptomyces sp. M30 protease, respectively (Xin et al. 2015). The stability of the CNXK100 protease was further exemplified through its resistance to Triton X-100, Tween, and SDS. On the contrary, Triton X-100 induced a 40% reduction in the activity of a new S. flavogriseus HS1 protease (Ghorbel et al. 2014). In comparison, Tween caused a reduction exceeding 40% in the activity of serine proteases from Thermoactinomyces spp. (Aksoy et al. 2012). Additionally, a 0.5% SDS solution resulted in approximately 75% reduction in the activity of antifungal protease from Streptomyces sp. A6 (Singh and Chhatpar 2011) and even completely abolishes the activity of S. flavogriseus HS1 protease (Ghorbel et al. 2014).

The CNXK100 protease also demonstrated higher stability than other microbial proteases in the presence of two oxidative agents, NaClO and H₂O₂. While 1.0% NaClO has been reported to reduce the activity of Bacillus siamensis protease by 20% (Tarek et al. 2023), 0.5–3.0% NaClO concentrations resulted in an enhancement of 5.0–20% in the activity of CNXK100 protease. Additionally, 0.5% H₂O₂ reduced the activity of S. flavogriseus HS1 protease by 40% (Ghorbel et al. 2014), whereas this effect on CNXK100 protease was only observed at a 3.0% H₂O₂ concentration.

Limited research exists on the tolerance of proteases from the genus Streptomyces towards common proteolytic enzymes. Pepsin primarily cleaves peptide bonds at the N-terminal positions of aromatic amino acids, including tyrosine, tryptophan, and phenylalanine (Ahn et al. 2013), while chymotrypsin cleaves peptide bonds at the carboxyl positions of tyrosine and phenylalanine, as well as at the N-terminal position of tryptophan (Zhou et al. 2011). Neither of these enzymes exhibited a significant effect on the CNXK100 protease, suggesting that these amino acids do not play a crucial role in the protease’s active site. Trypsin, known for its ability to hydrolyze peptide bonds on the carboxyl side of arginine or lysine at the C-terminal, excluding those followed by proline, only causes a marginal reduction of approximately 30% in the activity of the CNXK100 protease (Vorob’ev 2019). Meanwhile, proteinase K, a nonspecific serine endopeptidase, can cleave peptide bonds of aromatic, aliphatic, and hydrophobic amino acids at the C-terminal (Mótyán et al. 2013), thereby exerting a substantial influence on CNXK100 protease structure and its catalytic function. The CNXK100 protease has been found to possess fibrinolytic activity, a characteristic documented in a few other studies involving proteases derived from S. radiopugnans VITSD8 (Verma et al. 2018), S. rubiginosus VITPSS1 (Dhamodharan et al. 2019), and Streptomyces sp. 214L–11 (He et al. 2023). Due to its stability towards pepsin, trypsin, chymotrypsin, and fibrinolytic activity, the CNXK100 protease holds potential applications in medications (Bose et al. 2019).

The enduring characteristics of CNXK100 protease with surfactants, oxidizing agents, and diverse proteases were also demonstrated via its sustained functionality and even enhanced blood stain removal capability when used in conjunction with commercial detergents. This indicates its promising potential for utilization in the detergent industry.

Last but not least, to our knowledge, limited research has been conducted on the storage stability of purified proteases from Streptomyces. Nevertheless, the CNXK100 protease storage stability mirrors a few proteases derived from Bacillus, which retained 80–90% activity after 2 months at 4°C (Kotb et al. 2023; Wang et al. 2018).

In this study, one of the smallest thermostable proteases from Streptomyces sp. CNXK100 was successfully purified and characterized. The protease exhibited optimal activity at high temperatures and demonstrated activity in diverse buffers across a wide pH range. Additionally, metal ions were found to be important for enzyme activity and exhibited significant fibrinolytic activity. Notably, the protease displayed a preference for casein, with a V_max value thousands of times higher than other Streptomyces proteases. The enzyme demonstrated its stability at high temperatures in a wide pH range and resistance to polar and non-polar organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Although the protease exhibited partially decreased activity in commercial detergents, their combination increased the destaining effect, particularly on blood stains. Moreover, the enzyme remained stable for at least 70 days at 4°C. In conclusion, the thermostable protease originating from Streptomyces sp. CNXK100 is a versatile candidate for various applications in different fields.