Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications

Abstract

This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214 U/ml) under optimized conditions: carbon source, citric acid-1.5 % (w/w); inducer, soyabean meal-2 % (w/w); pH 11.0; shaking condition 37 °C for 48 h. The enzyme had pH and temperature optima of 9.0 and 60 °C, respectively. The enzyme displayed the molecular mass of 36 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0-11.0) and thermostability (20-70 °C). More than 70 % residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H(2)O(2) for 30 min. The protease activity was also enhanced by divalent cations such as Ba(2+), Ca(2+) and Mg(2+) and was strongly inhibited by Fe(2+), Zn(2+), Sr(2+), Hg(2+) and urea. The enzyme retained more than 50 % of its initial activity after pre-incubation for 1 h in the presence of 5 % (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1 % w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the eco-friendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.

Production of an alkaline protease using Bacillus pumilus D3 without inactivation by SDS, its characterization and purification

“…The highest levels of protease production was achieved in the culture grown at pH 11, thereby indicating the isolate can be classified as alkaliphilic microorganism (data shown in Figure 1). This result is in good agreement with the studies by some researchers, who reported maximum protease activity at pH 11 with B. pumilus [25][26][27] . Similarly, Tekin et al 28 also reported that B. cohnii produced maximum protease at pH 11.…”

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes

“…All the experiments and assays were performed in triplicate; and the standard deviation for each experiment was calculated using SPSS 14.0 and are indicated in the figures as error bars (JAYAKUMAR et al, 2012).…”

“…B001 where maximal protease production was detected at the mid stationary phase (PATEL et al, 2005;RAJA;PRABHAHAR, 2012). However, protease section by Bacillus pumilus MCAS8 was found to be at late stationary phase (48 h) (JAYAKUMAR et al, 2012). …”

“…The estimated cost of wheat bran was found to be 0.002 $ for one liter production medium (GOUDA, 2006). On the other hand, other carbon sources were reported for maximal protease production based on the source organism (JOSHI et al, 2008;SHIVANAND;JAYARAMAN, 2009;JAYAKUMAR et al, 2012). Less production of alkaline protease by B. agaradhaerens strain AK-R in the presence of simple sugar like glucose, galactose, sucrose, and maltose is mostly due to catabolite repression of protein biosynthesis (KANEKAR et al, 2002;DENG et al, 2010).…”

“…Alkaliphilic bacteria are group of microorganisms that grow better in alkaline environments, making them candidate strains for exploration of novel alkaline proteases for various biotechnological potential (JAYAKUMAR et al, 2012;GOHEL;SINGH, 2015). Soda lakes are one of the main natural habitats of alkaliphilic bacteria, representing the major types of naturally occurring highly alkaline environments with pH >11 (HORIKOSHI, 1999).…”