In vitro and in silico characterization of alkaline serine protease from Bacillus subtilis D9 recovered from Saudi Arabia

Amal Mahmoud,Essam Kotb,Amany I Alqosaibi,Ahmed A Al-Karmalawy,Ibtesam S Al-Dhuayan,Hameedah Alabkari

doi:10.1016/j.heliyon.2021.e08148

Amal Mahmoud, Essam Kotb + Show 4 more

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https://doi.org/10.1016/j.heliyon.2021.e08148

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Abstract

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.

Highlights

As one of the main industrial enzymes, proteases are responsible for around 60% of the world enzyme market (Clarridge, 2004; Raveendran et al, 2018; Razzaq et al, 2019)
Of 93 soil bacterial isolates collected from the Eastern Province (Dhahran) and Riyadh (Shaqra) of Saudi Arabia, 12 bacterial isolates have showed the maximum alkaline protease activity upon preliminary screening
The 12 high alkaline protease-producing bacterial isolates were identified through amplification and sequence analysis of the 16S rRNA gene

Summary

Introduction

As one of the main industrial enzymes, proteases are responsible for around 60% of the world enzyme market (Clarridge, 2004; Raveendran et al, 2018; Razzaq et al, 2019). Serine proteases represent one-third of the share in the enzyme market (Page and Di Cera, 2008). Microbial proteases are preferred over animal and plant proteases due to their easy genetic manipulation and wide biochemical diversity (Chanalia et al, 2011). A wide variety of organisms produce alkaline proteases. Are mostly the microbial source to generate the proteases. Bacillus-derived alkaline proteases have numerous applications in industry, including organic synthesis, food, pharmacology, leather, and bioremediation (Tarhriz et al, 2014; Al-Dhuayan et al, 2021)

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