Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

<p>Abstract</p> <p>Background</p> <p>Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus <it>Bacillus </it>are active producers of extra-cellular proteases, and the thermostability of enzyme production by <it>Bacillus </it>species has been well-studied by a number of researchers. In the present study, the <it>Bacillus subtilis </it>strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia.</p> <p>Results</p> <p>A thermostable organic solvent-tolerant protease producer had been identified as <it>Bacillus subtilis </it>strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v) of (AB₆₀₀= 0.5) inoculum size, in a culture medium (pH 7.0) and incubated for 24 h at 37°C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg). The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60°C, respectively.</p> <p>Conclusion</p> <p>Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic solvent. This unique property makes it attractive and useful to be used in industrial applications.</p

Optimisation of batch culture conditions for cell-envelope-associated proteinase production from lactobacillus delbrueckii subsp. lactis ATCC® 7830™

Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1 < X < 10 % vol/vol), agitation speed (0 < S < 200 rpm), varying incubation temperature (30 < T < 50 °C), starting pH (4.5 < pH < 7) and carbon/nitrogen ratio of production medium (0.2 < r < 5) had an individual effect on proteinase yield (p < 0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45 °C incubation temperature, 2 % (v/v) inoculum size of OD560 = 1, 150 rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8 h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions