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

Abstract

Background: 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 Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia.

Results: A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis 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 degrees 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 degrees C, respectively.

Conclusion: 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.

Figure 1

Phylogenetic position of strain Rand with other bacteria. The members of bacteria used include Bacillus malacitensis CECT 5687; Bacillus axarquiensis LMG 22476; Bacillus malacintesis LMG 22477; Brevibacterium halotolerans;Bacillus subtilis subsp. spizizenii BCRC 10447;Bacillus subtilis CCM 1999; Bacillus subtilis AU30; Bacillus subtilis isolate KCM-RG5; Bacillus subtilis isolate C10-1. Phylogenetic tree was inferred by using the neighbour-joining methods. The software package MEGA 4 was used for analysis.

Figure 2

Effect of Temperature on Protease Stability. The crude enzyme was incubated at different temperatures (37–70°C) for 30 min with shaking 150 rpm. Protease activity at 37°C was considered as 100%.

Figure 3

Effect of temperature on protease production. Culture media were incubated at 30, 37, 40, 45, 50, 55, 60 and 65°C with shaking at 150 rpm for 24 h. Protease activity at 37°C was considered as 100%.

Figure 4

Time course of protease activity and pH of the medium. Culture media were incubated at 37°C with shaking at 150 rpm for 48 h. Samples were taken at 4 h intervals to determine the protease activity and the pH level.

Figure 5

Effect of pH on protease production. Bacterial cultures were adjusted to pH 4, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0 and incubated at 37°C with 150 rpm for 24 h. Protease activity at pH 7 was considered as 100%.

Figure 6

Effect of agitation rate on protease production. Culture media were incubated at 37°C with different shaking rates (0, 50, 100, 150, 200 and 250 rpm) for 24 h. Protease activity at 200 rpm was considered as 100%.

Figure 7

Effect of inoculum size on protease production. Culture media were incubated with 1.0, 3.0, 5.0, 7.0, 9.0 and 11.0% (v/v) of inoculum and incubated at 37°C with shaking at 200 rpm for 24 h. Protease activity at with an inoculum size of 5% (v/v) was considered as 100%.