Characterization of partially purified alkaline protease secreted by halophilic bacterium Citricoccus sp. isolated from agricultural soil of northern India

Abstract

The present study reports the isolation of protease producing bacteria Bact2 from agricultural soil of Regional Centre of Soil Salinity Research Institute, Lucknow, India. It was identified as Citricoccus sp. (KC522120.1) by 16S rRNA gene sequencing. The optimum pH and temperature for maximum enzyme production was observed to be at 10 and 40 °C respectively. The enzyme protease was partially purified and recovery of enzyme was 77.16%. A 1.66 fold increase in enzyme activity was also observed in comparison to original. After purification, the partially purified enzyme (PPE) was characterized. The optimum pH and temperature of partially purified enzyme (PPE) activity was observed to be at 10 and 40 °C respectively. The activity of PPE was enhanced in presence of Ca²⁺ and Mg²⁺ while it was inhibited by Cu⁺⁺ and Na⁺. The PPE was completely inhibited by Phenyl methyl sulphonyl fluoride (PMSF) indicating that it belongs to serine protease group. Finally, enzyme was found to be compatible with detergents and effective in removing stains of tea and ink indicating it's application in detergent industry. Besides, to our knowledge till date production of alkaline protease from Citricoccus sp. has been reported first time in the present manuscript.

Introduction

The chemical biocatalysts used in industrial processes could be replaced by enzymes to increase efficiency and ensuring ecological and economic sustainability of the process. Proteases are the most dominant group of enzyme constituting sixty percent of the entire enzyme industry (Sharma et al., 2017) and hydrolyze peptide bond between protein with paramount application in industrial as well as pharmaceutical sector (Lakshmi et al., 2018). They have wide applications across various industries such as detergent, food, bakery, leather, infant formulas etc. due to their attractive features like ease in production, thermo tolerance and ability to perform at varied pH range (Genckal and Tari, 2006). The commercial proteases available in market are mostly extracted from Bacillus strains due to their stability at high temperature and pH (Ibrahim et al., 2015, Al-Hakim et al., 2018).

A large percentage of these enzymes are used widely in many industries especially in detergent industry which constitute single largest market for enzyme at twenty to thirty percent of total sales (Haddar et al., 2009). Enzymes used in detergent should be able to resist harsh situations like surfactants, variation in pH and temperature (Verma et al., 2011a, Verma et al., 2011b, Waghmare et al., 2015). A large number of microorganisms have been reported to produce alkaline proteases under various physiochemical and nutritional conditions e.g Bacillus, Micrococcus, Pseudomonas and Streptomyces etc in last twenty years (Hashem et al., 2015, Khajuria et al., 2015). Still, there is always a need for novel microbe producing proteases with characteristics matching the need of industries. The proteases produced by microbes present in natural environment are usually unstable under extreme conditions, despite several attempts of physicochemical treatments, protein engineering and gene-shuffling methods. In this context, main focus has been given to extremophiles known for producing enzymes steady at extreme physicochemical conditions leading to denaturation of enzymes produced by their natural counterparts. Actually, limited reports are available describing the alkaline proteases isolated from halophilic and halotolerant microorganisms (Thebti et al., 2016, Ahmed et al., 2016) and only few of them have been reported further for their potential use in detergent industry. From last two decades, alkaline protease was purified and characterized by several microorganisms viz., Bacillus (Ibrahim et al., 2015), Micrococcus, Staphylococcus (Kumari, 2014), Streptomyces (Al-Askar et al., 2015). Therefore, there is a continuous need to search for novel and better strains of microorganisms that produce alkaline protease in higher titers with better characteristics. Although the genus Citricoccus was reported for the first time by Alterburger et al. (2002) viz., Citrococcus murelis followed by another species of Citricoccus alkalitolerant by Li et al. (2005). Both species were found positive for lipase and glucosidase activities, there is no report available to our knowledge describing protease production any Citricoccus sp. Hence, we launched a screening programme for halotolerant bacteria isolated from saline agricultural soil for production of extracellular proteases. The bacterium Bact2 showing highest production of proteases was selected for further studies. The present work describes the identification of the isolate and characterization of the interesting biochemical properties of its alkaline protease making it a very potential candidate for it's application in detergent industry. To our knowledge, this is the first report stating the production of alkaline proteases from Citricoccus species.