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The strategy to prepare a novel biocatalyst by the immobilization of chitosanase onto liposome (ICL) was carried out based on the direct interaction of liposomes with cell membrane of Streptomyces griseus cell. The ICL was characterized in relation to the molecular weight of protein, the chitosanase activity, the effect of the surface hydration of various liposomes on hydrolysis activity of immobilized chitosanase and the stability of ICL under various extreme conditions. The SDS-PAGE analysis of the purified ICL sample shows the existence of a protein with approximately 39kDa that corresponded to the sum of weight of the mature chitosanase and its signal peptide (38.8kDa). The above protein of ICL also expresses the chitosanase activity that is significantly higher than that of the conventional chitosanase. Furthermore, the surface hydration of liposomes used to prepare ICL that affected the activity of immobilized chitosanase verified the importance of liposome surfaces. Indeed, the stability of ICL assayed by measuring the chitosanase activity is significantly higher than that of conventional chitosanase under various temperatures and pH conditions. These characteristics of ICL show the possible preparation of the biocatalysts that can be prepared by immobilizing enzymes onto liposome vesicles properly.

The effective production and secretion of chitosanase from S. griseus cells, in the presence of hydrogen peroxide, were studied by the treatment of the cells with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes under heat condition. The variation of the conformation and activity of the chitosanase caused by the interaction of liposomes with target chitosanase under stress condition was systematically investigated by using circular dichroism (CD) spectra and dielectric dispersion analysis (DDA). The effect of the oxidative stress (hydrogen peroxide) on the lipid and protein peroxide of cell membrane of S. griseus pretreated with and without liposomes under heat stress at 41 degrees C was further carried out. The possible utilization of membrane-membrane interaction between liposomes and cell membrane induced by the heat treatment was further investigated to enhance the production of chitosanase from S. griseus under oxidative stress condition.

A direct interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes with membrane of Streptomyces griseus cell under the heat stress at 41 degrees C increased the chitosanase production and release to 2.2 times higher than that at 37 degrees C without the POPC liposomes. Amount of chitosanase released across the lipid mimicking cell membrane (LMCM) liposome under a heat at 41 degrees C in the presence of POPC liposomes was 17% of initially-entrapped chitosanase while it was only 1% in the absence of POPC liposomes, even under a heat stress at 41 degrees C, clearly showing the importance of the direct interaction between membrane and membrane.

The effects of heat stress and liposome treatment on the growth of Streptomyces griseus cells and chitosanase production were investigated on the basis of using the designed strategy of a stress-mediated bioprocess. The effective conditions for increasing the interaction between chitosanase and the 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) liposome under heat stress condition were determined on the basis of the results of circular dichroism (CD) and dielectric dispersion analysis (DDA). Under these effective conditions, S. griseus cells were cultivated for the effective production of chitosanase. The results obtained from both CD spectra and DDA showed that heat stress enhances the interaction of the POPC liposomes and chitosanase. The strongest interaction between them could be obtained in the specific temperature range of 40-45 degrees C. The enhancement of the target chitosanase production was conducted under heat stress at 41 degrees C in the presence and absence of the POPC liposomes. The growth rates of S. griseus cells in the cases of heat (41 degrees C) and heat (41 degrees C)/POPC treatments were respectively 1.2 and 1.4 times higher than that obtained under the control condition. In the heat (41 degrees C) and heat (41 degrees C)/POPC treatments, chitosanase activity increased to 1.8 and 2 times, respectively, higher than that obtained under the control condition. Heat stress and the addition of the POPC liposomes could therefore be utilized to induce the potential functions of bacterial cells for the enhancement of the final target production.