Catalytic, kinetic and thermodynamic properties of Bacillus pumilus FH9 keratinase conjugated with activated pectin.

Bacillus pumilus FH9 keratinase was covalently coupled to several oxidized polysaccharides. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. Among all preparations, the conjugated enzyme with oxidized pectin had the highest recovered activity (71.75%) and the highest thermal stability at 60°C (t1/2=333 min). Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher t1/2, and higher decimal reduction time values (D) within the temperature range of 50-80°C. The thermodynamic parameters (ΔH*, ΔG*, ΔS*) of irreversible thermal denaturation for the native and conjugated keratinase were also evaluated. The values of enthalpy of activation (ΔH*), free energy of activation (ΔG*), and free energy of transition state binding (ΔG*E-T) for keratin hydrolysis were lower for the conjugated enzyme. Moreover, there was highly significant impact on improving the values of Vmax/Km, kcat, kcat/Km, and ΔG*E-S for the modified enzyme. Both native and conjugated enzymes were slightly activated by CaCl2 and MgCl2. However, the inhibitory effects of EDTA, HgCl2 and ZnSO4 were more pronounced with the native enzyme.

Catalytic and thermodynamic properties of glycosylated Bacillus cereus cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) was covalently coupled to five oxidized polysaccharides differing in structure and chemical nature. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. The conjugated CGTase with oxidized dextran (MW 47000) had the highest retained specific activity (70.05%) and the highest half-life (T1/2) at 80°C. Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 60-80°C. The values of thermodynamic parameters for irreversible inactivation of native and conjugated CGTase indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy of deactivation (ΔH*). The results of thermodynamic analysis for cyclodextrin production from starch indicated that The enthalpy of activation (ΔH*) and free energy of activation (ΔG*), (free energy of transition state) ΔG*E-T and (free energy of substrate binding) ΔG*E-S values were lower for the conjugated CGTase. Similarly, there was significant impact on improvement of kcat, kcat/Km values. Both native and conjugated enzyme produce α-cyclodextrin from starch.

Biosynthesis of cyclodextrin glucosyltransferase by the free and immobilized cells of Bacillus cereus NRC7 in batch and continuous cultures.

AIMS: The objective of this study was to enhance the production of cyclodextrin glucanotransferase (CGTase) produced by a local isolate Bacillus cereus NRC7. METHODS AND RESULTS: In batch culture, maximal CGTase activity (69·0 U ml(-1)) was reached after 24-h incubation period. In continuous production of CGTase by the free cells of B. cereus NRC7, maximal reactor productivity (11·76 KU l(-1) h(-1)), with enzyme concentration of 49·0 U ml(-1) and specific productivity of 904·6 U per g wet cells per h, was attained at dilution rate of 0·24 h(-1), over a period of 640 h. Bacillus cereus NRC7 cells were immobilized on chitosan. The immobilization conditions with respect to matrix concentration and maximal cell loading were optimized for maximal CGTase production. In repeated batch operation, the activity of the immobilized cells was stable during ten cycles and the activity remained between 51 and 55 U ml(-1). In packed-bed reactor, the immobilized cells showed maximal productivity (27·18 KU l(-1) h(-1)) with enzyme concentration of 54·63 U ml(-1) and specific productivity of 151·89 U per g wet cells per h at dilution rate of 0·5 h(-1). The half-life of the immobilized cells was higher than 20 days. CONCLUSIONS: Continuous fermentation by the immobilized cells in packed-bed reactor is an appropriate potential technique for B. cereus NRC7 CGTase production that gave maximum productivity (27·18 KU l(-1) h(-1)), which was 9·47-, 2·31-, 12·24- and 12·94-fold higher than the free cells in batch, free cells in continuous, immobilized cells in batch and repeated batch cultures, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that evaluates CGTase productivity, in different fermentation modes, in terms of specific productivity (U per gram cells per h). In continuous fermentation by immobilized cells, maximal levels of CGTase productivity are higher than the previously reported values.

'Candida infection in the tongue and pharynx'.

60 patients with chronic pharyngitis were investigated. In all cases the tongue was coated, fissured or hairy. Scrapings from the tongue and swabs from the posteriors pharyngeal wall were examined for the presence of Candida albicans. In the tongue cultures were positive in all cases, and in the pharynx they were positive in 42 cases. After treatment with 'Canasten' solution, the condition was cured in 53 cases. This directs attention to the role of Candida albicans as a cause of chronic pharyngitis, and to the relationship between candidiasis of the tongue and that of the throat.