Screening and characterization of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM) strain, MSF 46

Abstract

Among the various bacterial isolates, the strain MSF 46 isolated from thorn forest soil samples, Tamil Nadu, India, was screened and characterized for its proteolytic activity. While the 16S rRNA sequencing and biochemical characterization revealed that the strain closely resembles Methylobacterium sp., methylotrophy of the strain was confirmed by the sequence homology of mxaF gene with other relative Methylobacterium sp. The alkaline protease was purified to homogeneity using DEAE cellulose ion exchange chromatography, with a 5.2-fold increase in specific activity and 34% recovery. The apparent molecular weight of the enzyme was determined as 40 kDa by SDS-PAGE study. The pH and temperature optima were 9.0 and 50°C respectively with maximum protease activity of 1164 U/ml. Protease of MSF 46 was active in a broad pH range 7.0-11.0 with a maximum at pH 8.5 and exhibited thermostability at 50°C. The enzyme activity was inhibited by PMSF but showed stability with Tween 20, Triton X-100 and hydrogen peroxide. Nearly 30% reduction in enzyme activity was observed in the presence of EDTA and DTT. The enzyme was effective in hydrolyzing gelatin, skimmed milk and blood clots and exhibited the potency for dehairing of goat skin and removing blood stain from cotton fabric. Significant morphological changes were observed under scanning electron microscope between cells grown in normal and casein amended medium. This first detailed report on the production of alkaline protease by a PPFM strain appears promising toward development of protocols for mass production, study of the molecular mechanism and other applications.ª 2014 Production and hosting by Elsevier B.V. on behalf

Purification and Characterization of a Detergent Compatible Alkaline Protease Produced by Bacillus ruris Isolated from Vegetable Oil Factory Effluent in Owo, Ondo State, Nigeria

“…Bacillus ruris protease had its highest activity at 50 ℃ (100%), while further temperature rise resulted in reduced protease activity decreasing to 77.042 % at 70 ℃. This result agrees with reports by (Adinarayana et al, 2003;Giri et al, 2011;Jayashree et al ., 2014;Niyonzima and More, 2014;Ramkumar et al, 2018) who reported protease stability at 50 ℃. This contrasts with Naidu (2011) who reported a reduction in protease activity beyond 35 ℃.…”

“…Findings from this study showed that surfactants such as tween 20, triton X, (SLS), (SDS), enhanced the protease activity of Bacillus ruris, while inhibitors such as 2-mercaptoethanol (BME), dithiothreitol (DTT), and ethylenediaminetetraacetic acid (EDTA) had varying inhibitory effects on Bacillus ruris protease as shown in Figure 7. This result is similar to reports obtained by Jayashree et al (2014) who reported improvement of protease activity by tween 20 and triton X and minimal effects of the oxidizing agents on protease enzyme. Further studies on enzyme inhibition could offer more insights regarding the nature of the proteases, its cofactor requirements and its active center (Jayashree et al, 2014).…”

“…The protease purification was found to decrease from 1 to 0.516 purification fold using Sephadex G-100 column chromatography. This is similar to reports by Josephine et al (2012) and Jayashree et al (2014) who reported a rise in the purification fold of Bacillus spp protease (Guleria et al, 2016). This disagrees with reports by Naidu (2011) and Bajaj and Jamwal (2013) who reported increased protein content when purification was carried out to obtain the pure protease enzyme.…”

Screening and Molecular Characterization of Actinomycetes from Mangrove Soil Producing Industrially Important Enzymes

“…The enzyme inactivated by TCA was used as the control. The amount of enzyme liberating 1µg of tyrosine per ml for 30 minutes at 37°C corresponds to one unit of protease activity (U) (Jayashree et al, 2014). Lowry's method was used in estimating protein concentration where Bovine serum albumin (BSA) was the standard (1951).…”

Antifungical Activity of AutochthonousBacillus subtilisIsolated fromProsopis julifloraagainst Phytopathogenic Fungi

“…After incubation, culture was harvested by centrifugation at 10,000 rpm for 10 min at 4℃. The cell free supernatant was used as the enzyme source for protease assay and the protease activity was determined as described by Jayashree et al [ 12 ]. One milliliter of enzyme solution was added to 1 mL 2% (w/v) casein solution (dissolved in 50mM phosphate buffer with pH 7) and incubated in water bath at 37℃ for 20 min.…”