Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere

Abstract

A thermostable extracellular alkaline protease producing Bacillus amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth promoting activities. Strain SP1 was purified to 6.48-fold using four-step purification protocol and characterized in detail for its robustness and ecofriendly application in leather and detergent industries. Structural analysis revealed that the protease was monomeric and had a molecular weight of 43 kDa. It exhibited optimum activity at 60°C in alkaline environment (pH 8.0) and stable in the presence of surfactants and oxidizing agents. Enzyme was thermostable at 50°C and retained more than 70% activity after 30 min incubation. It has shown stain removal property and dehairing of goat skin without chemical assistance and hydrolyzing fibrous proteins. This protease showed Km of 0.125 mg ml(-1) and V(max) of 12820 μg ml(-1) indicating its excellent affinity and catalytic role. Thermal inactivation of the pure enzyme followed first-order kinetics. The half life of the pure enzyme at 50, 60, and 65°C was 77, 19.80, and 13.33 min, respectively. The activation energy was 37.19 KJ mol(-1). The results suggest that the B. amyloliquefaciens SP1 has a potential application in different industries.

Potential application spectrum of microbial proteases for clean and green industrial production

“…are well characterized for usage as detergent additives, e.g. B. subtilis, B. cohnii, B. clausii, B. licheniformis and B. brevis (Guleria et al 2016). Alcalase was the first detergent protease developed by Novozyme during 1960s.…”

“…B. altitudinis GVC11 , B. cereus MCM B-326 (Nilegaonkar et al 2007), B. cereus VITSN04 (Sundararajan et al, 2011), B. halodurans JB 99 (Shrinivas et al 2012), B. pumilus MCAS8 (Jayakumar et al 2012), B. subtilis (Sathiya 2013) and B. subtilis KT004404 (Rehman et al 2017). A thermostable alkaline protease from Bacillus amyloliquefaciens SP1 exhibited excellent dehairing (goat skin) potential (Guleria et al 2016).…”

“…Therefore, discovery of sturdy enzymes that are capable of functioning under industrial processes has gained considerable research impetus in recent years (Sharma and Bajaj 2017). There is continuous and intense research focus on targeting processapt robust enzymes either by exploiting the enormous natural microbial diversity or by bioengineering (Singh and Bajaj 2015;Guleria et al 2016). Microbial enzymes may potentially be utilized for numerous industrial applications and may help replacing toxic and hazardous chemical catalysts (Nigam et al 2012).…”

“…For biotechnological processes involving protein production, several species of Bacillus, viz. B. subtilis, B. amyloliquefaciens and B. licheniformis and others, have become the most popular due to their excellent fermentation properties, high product yields and the complete lack of toxic by-products (Dijl and Hecker 2013;Singh and Bajaj 2016;Guleria et al 2016). The enzymes being marketed by Novo Industry, Denmark, are produced from Bacillus spp.…”

“…There are several reports of thermostable and wide range pH stable proteases from Bacillus spp. which have excellent compatibility for industrial processes (Baweja et al 2016;Guleria et al 2016). The genus Bacillus is represented by Gram-positive, spore-forming, rod-shape bacteria that are obligate aerobes or facultative anaerobes (Dijl and Hecker 2013).…”