Proteases execute a large variety of functions and have important biotechnological applications. Proteases represent one of the three largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes. For an enzyme to be used as an detergent additive it should be stable and active in the presence of typical detergent ingredients, such as surfactants, builders, bleaching agents, bleach activators, fillers, fabric softeners and various other formulation aids.

An interesting application of alkaline protease was developed by Fujiwara and coworkers (Ishikawa et al. 1993). They reported the use of an alkaline protease to decompose the gelatinous coating of X-ray films, from which silver was recovered. A lthough protease s are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth.

We have isolated P. aeruginosa PD 100 which produces one protease (confirmed by Acidic-Native-PAGE and Native-PAGE). With respect to properties of this protease, it was considered of interest to investigate further the potential of the P. aeruginosa PD100 protease, in particular as an additive in various bio-formulations such as biological detergents, dehairing, amino acid production and contact-lens cleansing agents. The microorganism was grown for 24 hrs in the optimized medium and then the cells were harvested by centrifugation and the supernatant was used as the starting point for purification of the protease with ammonium sulphate precipitation follow with Sephadex G-50 chromatography and CM-Sephadex chromatography. The enzyme activity was performed spectrophotometrically according to Kunitz (Kunitz, 1947) with some modifications and protein concentration was determined by Lowry method. The enzyme was purified with these methods showed 49 fold of purification with an overall yield of 38% and a specific activity of 434 U/mg. The purified enzyme could be stored in 50 mM Tris-HCl buffer, pH 8, at -20ºC for 3 months without any apparent loss of activity.

The purified enzyme was incubated with different concentrations of SDS and Tween 80 for 1 hr and then assayed in the presence and absence of the detergents. With respect to the results from this experiment, it was found that the protease of P. aeruginosa PD100 enable to act in the presence of SDS and Tween 80. The enzyme showed 50% of maximal activity in 1% (w/v) SDS and 3 % (v/v) Tween 80. These results were confirmed by SDS-PAGE and zymograhy. This is one of the most interesting properties of the enzyme for different application such as usefulness in the clean up of DNA at high temperature due to its stability against SDS and temperature. Stability of the protease against different solvents (such as methanol, 1-propanol, 2-propanol, ethyleneglycol, ethylacetate, xylene, toluene, benzene) in reaction mixture in the presence of 10%, 20% and 50% of each solvent at 55ºC for 20 min revealed another property of the protease. This protease is very useful for fermentation and reactions in the presence of solvents. One of the most important advantages of this property is to reduce or abolish microbial contamination during degradation reaction.

Immobilization of the protease was performed by entrapment method using polyacrylamide gel. The immobilized enzyme retained 90% of total activity compared with the soluble enzyme. Also immobilized protease in comparison to the soluble one showed no critical change in pH optimum and stability. In contrast temperature stability of the immobilized protease compared to the soluble enzyme showed 15-20% increase as indicated by its inactivation pattern. Upon repeated use, the entrapped enzyme retained 83% of its initial activity after six cycles. The immobilized protease showed excellent storage stability and could be stored at 4ºC in 20 mM Tris-HCl buffer (pH 8) for more than 30 days without any loss in its initial activity. The immobilized enzyme can be recommended for amino acid production, clearing of juice and related applications.

The protease showed ability for digestion of different natural substrates with base of fibrin, albumin and collagen suggesting usefulness of this enzyme for different applications such as extraction of collagen from skin for collagen replacement therapy, waste treatment and other related applications. This protease showed high capability for removing proteins and stain from cloth and also it could be used as an alkaline protease in detergent powder or solution. Its ability to act in the presence of solvents and detergents can be exploited for this purpose.

The protease showed usefulness for dehairing of skin, since this protease can digest collagen, the process of dehairing must be controlled to avoid reducing the quality of the leather. As against traditional chemical methods, enzymatic processes yield products of improved quality and reduce the use of hazardous and polluting chemicals.

Properties of this protease such as alkaline pH, thermostability, solvent and detergent resistance, suggests the enzyme very useful for different applications. Since this protease has gelatinase activity, it can be recommended for an interesting application of alkaline protease to decompose the gelatinous coating of X-ray films, from which silver was recovered. The protease of P. aeruginosa PD100 with broad range of substrate specificity can be recommended for recovery of silver from photographic films, for animal food industry and clearing beverages, in leather industry for dehairing and bating skins, production of amino acid and peptides. Also it is useful for cleaning DNA during isolation of the DNA. Since this protease has high collagenolytic activity, it can be used for various applications such as: (a) cosmetics: for skin rejuvenation, wrinkle smoothing and dandruff removal, prophylactic on senile skin changes and also removal of the atrophied epithelium from the skin surface, (b) medicine: festering and wet wounds, (c) leather industry: for manufacturing of skin and leather and getting high quality suede, (d) biochemistry: for isolation of cells from many types of animal tissue. This study was fruitful in identifying one such enzyme, which can be exploited commercially.

References

ISHIKAWA, H.; ISHIMI, K.; SUGIURA, M.; SOWA, A. and FUJIWARA, N. Kinetics and mechanism of enzymatic hydrolysis of gelatin layers of X-ray film and release of silver particles. Journal of Fermentation and Bioengineering , 1993, vol. 76, no. 4, p. 300-305.

KUNITZ, M. Crystalline soybean Trypsin Inhibitor, II. General properties. Journal of General Physiology , March 1947, vol. 30, no. 4, p. 291-310.