In this study, we have isolated and characterized proteolytic soil bacteria and their alkaline protease. Based on 16S rRNA sequence analysis, 12 isolates with the highest protease activity were classified as B. subtilis and B. cereus groups. B. subtilis D9 isolate showing the highest protease activity was selected for in vitro and in silico analysis for its ِِAKD9 protease. The enzyme has a molecular mass of 48 kDa, exhibiting optimal activity at 50 °C pH 9.5, and showed high stability till 65 °C and pH 8–11 for 1 h. Fe³⁺‏ stimulated, but Zn²⁺ and Hg²⁺ strongly inhibited the protease activity. Also, the maximum inhibition with PMSF indicated serine protease-type of AKD9 protease. AkD9 alkaline serine protease gene showed high sequence similarity and close phylogenetic relationship with AprX serine protease of B. subtilis isolates. Functional prediction of AKD9 resulted in the detection of subtilase domain, peptidase_S8 family, and subtilase active sites. Moreover, prediction of physicochemical properties indicated that AKD9 serine protease is hydrophilic, thermostable, and alkali-halo stable. Secondary structure prediction revealed the dominance of the coils enhances AKD9 activity and stability under saline and alkaline conditions. Based on molecular docking, AKD9 showed very promising binding affinities towards casein substrate with expected intrinsic proteolytic activities matching our obtained in vitro results. In conclusion, AKD9 alkaline serine protease seems to be a significant candidate for industrial applications because of its stability, hydrophilicity, enhanced thermostability, and alkali-halo stability.

Forty-six promising chitinolytic isolates were recovered during a screening for chitinolytic bacteria in the environment of Saudi Arabia. The top three isolates belonged to the genus Streptomyces. Streptomyces variabilis Am1 was able to excrete the highest amount of chitinases, reaching the maximum at 84 h with 0.5% yeast extract and nitrogen source and 2% galactose as a carbon source. Purification of chitinase by DEAE-Cellulose and Sephadex G₇₅ improved the specific activity to 18.6-fold and the recovery to 23.8% and showed a mass at 56 kDa. The optimal catalysis of the purified chitinase was at 40 °C and pH 8 with high thermostability and pH stability as reflected by a midpoint temperature value of 66.6 °C and stability at pH 4–9. The protein reagents SDS, EDTA, and EGTA significantly inhibited the enzyme and the EDTA-chelated chitinase restored its activity after the addition of Fe²⁺ ions suggesting a metallo-chitinase type with ferric ions as cofactors. Chitinase exerted high antifungal activity against some phytopathogenic fungi. Interestingly, the tested Streptomyces were able to produce chitosan nanocubes along with chitosan from chitin degradation which may be an additional power in their antifungal activity in nature. This work also reveals the importance of unexplored environments as a pool of promising microorganisms with biotechnological applications.

In this study, thirty-seven alkaline protease-producing bacteria were recovered from different regions of Saudi Arabia. The proteolytic strain with the highest productivity was identified as Bacillus sp. Mar64. Maximum productivity of Mar64P alkaline protease was reached at 60 h, pH 9.0, and 45 °C using 1% tyrosine and 0.5% maltose as nitrogen and carbon supplies, respectively. Specific activity was intensified to 8.5-fold with a recovery of 12.4% and SDS—PAGE revealed one band at 28 kDa after enzyme purification. Mar64P was maximally active at 55 °C and pH 11.0 with thermal stability up to 70 °C and pH stability at 7.0–12.0 for 1 h. It was inhibited by EDTA and unaffected by PMSF, therefore tentatively classified as metalloprotease-type. Storage efficacy was effective for up to eight weeks and it was durable in presence of organic solvents (20%, v/v) such as acetonitrile, acetone, and isopropanol upto to 15 days. The enzyme was compatible with dry detergents at both low and high temperature, in addition, was successful in removing various stains such as blood, egg yolk, chocolate, tea, coffee, and sweat. Furthermore, it was successful in removing skin hairs and hydrolyzing gelatin of waste X-ray films. Collectively, due to these unique properties, Mar64P could be considered an environmentally friendly candidate in both detergent and leather industries.

Ninety-one elastase-producing bacterial isolates were recovered from different localities of the Eastern Province of Saudi Arabia. Elastase from the best isolate Priestia megaterium gasm32, from luncheon samples was purified to electrophoretic homogeneity using DEAE-Sepharose CL-6B and Sephadex G-100 chromatographic techniques. The recovery was 17.7%, the purification fold was 11.7x, and the molecular mass was 30 kDa. Enzymatic activity was highly repressed by Ba²⁺ and almost completely lost by EDTA, but it was greatly stimulated by Cu²⁺ ions, suggesting a metalloprotease type. The enzyme was stable at 45°C and pH 6.0–10.0 for 2 hours. Ca²⁺ ions considerably enhanced the stability of the heat-treated enzyme. The V_max and K_m against the synthetic substrate elastin–Congo red were 6.03 mg/mL, and 8.82 U/mg, respectively. Interestingly, the enzyme showed potent antibacterial activity against many bacterial pathogens. Under SEM, most bacterial cells showed loss of integrity, damage, and perforation. SEM micrographs also showed a time-dependent gradual breakdown of elastin fibers exposed to elastase. After 3 hours, intact elastin fibers disappeared, leaving irregular pieces. Given these good features, this elastase may be a promising candidate for treating damaged skin fibers with the inhibition of contaminating bacteria.