PJB-2024-625
MOLECULAR AND IN-SILICO CHARACTERIZATION OF ALKALINE PROTEASES FROM DIFFERENT BACILLUS ISOLATES
Hussain Ahmad
Abstract
MOLECULAR AND IN-SILICO CHARACTERIZATION OF ALKALINE PROTEASES FROM DIFFERENT BACILLUS ISOLATES
Hussain Ahmad and Noreen Asim
Institute of Biotechnology and Genetic Engineering,
Faculty of Crop Production Sciences
The University of Agriculture, Peshawar-Pakistan
December, 2024
ABSTRACT
Alkaline proteases possess high commercial value because of their multiple applications in the industrial sector. The present study was designed for alkaline protease gene isolation from different Bacillus isolates, sequencing, and in-silico characterization. Further, the alkaline protease enzyme from these isolates were purified through SDS- PAGE and gelatin zymography. All the isolates showed good protease activity on gelatin agar media, the zone of hydrolysis by Bacillus paramycoids (D1) was 3mm, Bacillus zhangzhouensis (D2) 3mm, Alcaligenes faecalis (D3) 3mm, Bacillus cereus (D4) 2mm and Bacillus altitudes (D5) 2mm respectively. Gene-specific primers were used for alkaline protease gene isolation and sequencing, Blast results confirm the presence of this gene in these isolates. The gene sequence was translated to protein its 3D structure prediction was carried out. 3D structure of these proteins showed the presence of almost similar proteolytic domain. These structures were further validated by ProCheck resulting in 91.5%, 91.6%, 90.0%, 92.3%, and 85.9% residues in most favoured regions for D1, D2, D3, D4, and D5 respectively. The energy graph produced by ProSA showed z-scores -7.54, -6.33, -8.48, -9.36, 6.54 for D1, D2, D3, D4, and D5 respectively, indicating the stability of the predicted models. The ERRAT database provides the quantitative measurement for different Bacillus species, Bacillus ceruse(96.928), Bacillus paramycoids (96.198), and Bacillus zhangzhouensis (96.154), indicating higher resolution of protein structures. The SDS-PAGE showed a mixture of proteases on the base of molecular weight, while the gelatin zymography analysis revealed 10, 15, 20, 25, 50, and 75 KDa bands on gelatin zymgram. The study concludes the presence and effectiveness of alkaline proteases present in these isolates which may play a significant role in industrial applications due to their stability, hydrophilicity, and enhanced thermostability.
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