Four major enzymes commonly used in the market are lipases, proteases, amylases, and cellulases. For instance, in both academic and industrial levels, microbial lipases have been well studied for industrial and biotechnological applications compared to others. Immobilization is done to minimize the cost. The improvement of enzyme properties enables the reusability of enzymes and facilitates enzymes used in a continuous process. Immobilized enzymes are enzymes physically confined in a particularly defined region with retention to their catalytic activities. Immobilized enzymes can be used repeatedly compared to free enzymes, which are unable to catalyze reactions continuously in the system. Immobilization also provides a higher pH value and thermal stability for enzymes toward synthesis. The main parameter influencing the immobilization is the support used to immobilize the enzyme. The support should have a large surface area, high rigidity, suitable shape and particle size, reusability, and resistance to microbial attachment, which will enhance the stability of the enzyme. The diffusion of the substrate in the carrier is more favorable on hydrophobic supports instead of hydrophilic supports. The methods used for enzyme immobilization also play a crucial role in immobilization performance. The combination of immobilization methods will increase the binding force between enzymes and the support, thus reducing the leakage of the enzymes from the support. The adsorption of lipase on a hydrophobic support causes the interfacial activation of lipase during immobilization. The adsorption method also causes less or no change in enzyme conformation, especially on the active site of the enzyme. Thus, this method is the most used in the immobilization process for industrial applications.

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°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. 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.