Purification and characterization of haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: A potential additive for laundry detergents
Highlights
► An extracellular protease is characterized from polyextremotolerant strain. ► The reaction has been catalyzed at pH (10), temperature (60 °C), and salinity (15%). ► Enzyme has functional activity in various organic solvents (50% v/v). ► Enzyme has stability with surfactants and commercial detergents. ► Study showed its substantial role in laundry industry and prospects for peptide synthesis.
Introduction
Global concern for the environment has attracted the researchers to investigate enzymes as replacement of chemical catalysts in various biochemical processes. The continual exploration of enzymes and their utilities have expanded their industrial market with the growth of 7.6% per year (David et al., 2009). Of the various industrial enzymes, proteases alone contribute approximately 60% of the total sales in the world (Banik and Prakash, 2004) and bacteria belonging to genus Bacillus produce most commercial proteases used today (Maurer, 2004). Proteases are the most versatile enzyme with a long history of catalytic applications in food and pharmaceutical industries. Recently, their roles in synthesis of bioactive peptides and as additive in commercial detergents are gaining attention. The composition and nature of the surfactants in detergents greatly manifest the stability of the enzyme. The other prerequisites for the utilizing proteolytic enzymes in detergent formulations include their functional ability and stability at alkaline pH in presence of various surfactants and detergents. The natural proteases in general are not stable under these conditions (Gupta et al., 2005). However several physiochemical methods such as chemical modification, immobilization, entrapment, protein engineering and directed evolution have been employed for their stabilization at various extreme conditions (Ogino and Ishikawa, 2001). Even the gene-shuffling techniques have not resulted in successful products. There have been extensive studies on screening of microbes to obtain proteases with functional ability under the required extreme conditions (Maurer, 2004) so that the overall process becomes environment friendly, feasible and economic. In this context, alkaline proteases of microbial origin have been extensively studied however detergent stable proteases were characterized from a few bacterial species (Deng et al., 2010). Since it is known that extremophiles could be the potent producers of enzymes with functional ability at extreme physico-chemical conditions, Shivanand and Jayaraman (2009) characterized salt stable protease from halotolerant bacteria. Similarly organic solvent tolerant bacteria were reported to produce solvent stable proteases (Xu et al., 2010, Shah et al., 2010). In this way it would be further interesting to investigate polyextremotolerant microbes and their extracellular enzymes with an assumption that they would have wider adaptability in various environmental conditions (Jain et al., 2010). However, the information on polyextremotolerants has been less explored in comparison to extremotolerant microbes.
In our previous study, a strain belonging to genus Bacillus was isolated from RO reject that showed characteristic features of sustaining the three extremities such as pH (10), temperature (60 °C) and pressure tension of 450 psi under absolute oxygen atmosphere (Jain et al., 2010). The present study has been designed to extrapolate the potential of this strain as a source of enzyme protease with distinct functional properties. Central to our study is the investigation of functional ability of protease under different ranges of pH, temperature, and salinity along with their combined effect. This study also investigated the possible utility of the enzyme as an additive in detergent matrices.
Section snippets
Microorganism
The bacterial strain employed in this study was isolated from the reverse osmosis reject obtained after desalination of brackish water. The 16S rDNA sequence of the isolated strain showed 94% similarity with Bacillus licheniformis and was characterized as a thermoalkalitolerant strain (Jain et al., 2010).
Screening for protease enzyme
The screening for secretion of extracellular protease enzyme was performed by streaking the bacterial strain on milk agar plates containing (g/l): yeast extract (10); agar (20); sodium chloride
Characteristics of the microorganism
The isolated bacterium Bacillus SM2014 was a thermo-alkali-tolerant strain (Jain et al., 2010). This strain further showed tolerance to broad range of salt concentration from 0% to 15% representing its haloduric physiology (Supplementary Fig. 1). This is another unique property of the bacterium together with already reported distinct characteristics.
Enzyme production medium and culture condition
The time course study on protease production in basal media showed an increment in protease activity with the growth of bacterium and maximum
Conclusion
This study extrapolate the potential of a polyextremotolerant strain after characterization of alkaline, halothermoactive, surfactant stable, SDS induced serine type protease as its extracellular secretion which have functional activity at broad pH ranges (8–12), temperatures (20–80 °C) and salinity up to 20%. Moreover the stability of the enzyme in the presence of various surfactants, bleach, oxidizing agents, solvents and in saline conditions together with wash performance analysis confirmed
Acknowledgements
We gratefully acknowledge Dr. P. K. Ghosh (Director, CSMCRI) for his valuable suggestions. Deepti Jain and Sanjiv K. Mishra wish to acknowledge CSIR for awarding Senior Research Fellowship. Imran Pancha and Anupama Shrivastav acknowledge CSIR for the financial support. We would like to thanks Analytical Section for using FT-IR and HPLC.
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