Production and partial purification of protease by selected bacterial strains using raw milk as substrate

Abstract

Aims: The present study was investigated to optimize and partially purify the proteases produced by the food borne bacterial strains. Methodology and Results: Four bacterial strains such as Bacillus cereus, Proteus vulgaris, P. mirabilis and Enterobacter aerogenes were isolated from food wastes. These strains were individually inoculated in to the formulated culture media supplied with three different concentrations (1:1 to 1:3) of raw milk as major substrate. Among the concentrations, 1:2 ratio of substrate supplied medium showed maximum (0.133 to 8.000 IU/mL) protease production by all the tested organisms. After optimization, the organisms were tested for protease production at various pH (3 to 9), and temperature (30 to 80 °C). The result showed that all the organisms were capable of producing maximum protease at pH 6 (8.533 to 10.133 IU/mL) and at 50 °C (8.666 to 10.666 IU/mL). The crude enzymes produced by the tested organisms were individually purified by two different methods viz sodium alginate and ammonium sulphate-butanol methods. The purity of the protease determined in these two methods was ranged between 3.24 to 5.44 I and 3.13 to 5.55 IU/mL respectively. The partially purified enzymes were further analysed through SDS-PAGE; accordingly the molecular weight of protein produced by the test organisms was determined in between 49.44 and 50.98 kDa. Conclusion, significance and impact of study: Among the tested strains P. vulgaris was identified as the major protease producer in optimized culture condition of 50 o C and pH6. The molecular mass of the partially purified protease of P. vulgaris was 50.32 KDa. Further research on optimization of other fermentation parameters using statistical tools with P. vulgaris is needed to scale up the process.

Optimization of protease production from Bacillus halodurans under solid state fermentation using agrowastes

“… Bajaj et al (2013) stated that the secretion of protease varies in microorganisms and it is dependent on environmental factors. Prakash et al (2011) reported that these factors are most probably inducing the production of enzyme by microbial isolate. In this work, the Bacillus halodurans was experimented for the maximizing the enzyme yield by optimizing pH as well as incubation temperature.…”

Optimization and partial purification of a protease produced by selected bacterial strains grown on trash fish meal substrate and its antagonistic property against bacterial pathogens

“…The important environmental factors such as pH and temperature are mostly influencing the enzyme production (Prakash et al, 2011). In the present study, the test organisms were tried for their maximum ability on protease production in different pH and temperature, accordingly, it was observed that maximum protease production was recorded at pH 6 by all the tested organisms.…”

“…There were many substrates used for protease production, which include skimmed milk, peptone, casein, raw milk, etc. Some of the agricultural wastes and animal wastes were also used as substrates for the production of protease, because they are readily available, economically very cheap and have high protein content (Prakash et al, 2011). Bacillus subtilis Y-108 produced 20.2 U ml À 1 protease, when grown in crustacean shell waste and this protease is highly active at pH 8 and 50 1C temperature (Yang et al, 1999).…”

“…Traditionally, much of this material has been converted to powdered fish meal by a combined process of cooking and separation of soluble from insoluble (Ferreira and Hultin, 1994). In view of their high protein content of high quality, fish represents a potential source of industrial peptones for a wide range of applications (Prakash et al, 2011). The fish-processing industry generates considerable quantities of byproducts as waste that includes viscera, shell (from the crustacean and molluscan processing), scales, fins, bone frames and mantles of squids.…”

Development of a NS2B/NS3 protease inhibition assay using AlphaScreen® beads for screening of anti-dengue activities

“…Briefly, NS2B/NS3 expression plasmid was propagated in Escherichia coli strain XL 1-Blue MRF (Agilent Technologies, Santa Clara, CA).The enzyme was isolated and purified using Ni 2+ -NTA (nickel-nitrilotriacetic acid) resin-based column. The specific proteolytic activity of NS2B/NS3 protease was determined using similar formula as published for other proteases [29, 30]. Protease assay using fluorogenic peptide susbtrate (BOC-Gly-Arg-Arg-AMC) was also used to determine the NS2B/NS3 inhibition activities of the controls and derivatives [22].…”