Abstract
Endophytes are largely recognised as treasures of novel bioactive compounds with medicinal and therapeutic applications. However, their potential as sources of industrially valuable protease enzymes is under explored. Proteases are enzymes which catalyse the cleavage of peptides and have various industrial applications. Microbial proteases play a significant role in the enzyme industry and are used in the leather industry, food and beverage, detergents, silk degumming and silver recovery from X-rays. The use of microbial proteases for degradation of proteins offers an environmentally friendly, cost effective, and sustainable alternative to processes which rely on the use of harmful chemicals for protein degradation. Therefore, a continuum in research for novel sources of protease enzymes which meet industrial standards and improved efficacy in protein degradation is pertinent. The present study investigated the production of industrially applicable proteases from two bacterial endophytes, Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 which have been previously isolated from Dicoma Anomala medicinal plant, and the potential of their proteases for application as detergent additives and feather degrading proteases. Furthermore, through whole genome sequencing, families of relevant and industrially valuable proteins were identified from Bacillus sp. strain MHSD17 genome. Results obtained from this study showed that both Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 endophytes exhibited extracellular protease production. Through optimisation of cultural parameters, it was shown that Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 grew optimally when cultured in protease production media at pH 9 and had optimal protease production on days 1 and 4, respectively. Characterisation of biochemical properties revealed that crude enzyme extracts of both Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17, had an optimal protease activity at a temperature of 50°C, at pH 10. The endophytes exhibited varying thermostability and pH stability. Activity in the presence of surfactants revealed that Bacillus sp. strain MHSD16 exhibited 64% and 79% residual activity after pre-incubation in 1% Sodium dodecyl sulphate (SDS) and Triton X-100, respectively. For Bacillus sp. strain MHSD17, 79% and 85% residual activity was observed after pre-incubation in 1% SDS and Triton X-100, respectively. Based on these and various other biochemical findings, the potential applications of Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 crude enzyme extracts in laundry detergent additives and chicken feather degradation were investigated. Bacillus sp. strain MHSD17 exhibited optimal characteristics for use as a detergent bio-additive while also achieving 80.45% of chicken feather degradation. Whole genome sequencing of Bacillus sp. strain MHSD17 revealed the presence of industrially valuable proteases such as neutral metalloprotease (NprB), S1 and S8 family of serine peptidases as well as M4 family of metalloproteases. These findings not only advance our understanding of microbial proteases but also highlight the potential for bioengineering and industrial exploitation of protease enzymes.
Keywords: Protease, endophytes, whole genome sequencing, industrial protease, serine peptidase, metallopeptidase.