Abstract The production of milk-clotting enzyme (MCE) by free and immobilized Bacillus circulans 25 cells was investigated. The investigation evaluates cell immobilization through entrapment and covalent binding to different carriers. B. circulans 25 cells immobilized covalently via glutaraldehyde (cross-linker) to natural wool exhibited higher effectiveness factor (0.76) compared to other carriers. Immobilized cells produce maximum level of MCE and highest ratio of milk-clotting activity/proteolytic activity (MCA/PA) after 24 h using 0.4g wet weight cells/g wool. In batch operation, decreasing biomass loading to 0.2 g enhanced the MCA/PA ratio and specific productivity by 5.94 and 59.88%, respectively. Immobilized cells on natural wool as an effective and suitable carrier were able to produce the same level of MCA, productivity and MCA/PA ratio for 9 repeated cycles (216 h). Under continuous operation immobilized cells in a packed-bed bioreactor were able to keep producing MCA with MCA/PA ratio for about 7 days at the same level. Moreover, continuous operation demonstrates a very good productivity of 0.385 KU/L/h, which is higher than the other production systems by 7.0-11.7 fold. Immobilized B. circulans 25 cells proved to be fully capable of continuous MCE production in a packed-bed bioreactor.

Aspergillus niger β-glucosidase was modified by covalent coupling to periodate activated polysaccharides (glycosylation). The conjugated enzyme to activated starch showed the highest specific activity (128.5 U/mg protein). Compared to the native enzyme, the conjugated form exhibited: a higher optimal reaction temperature, a lower Ea (activation energy), a higher Km (Michaelis constant) and Vmax (maximal reaction rate), and improved thermal stability. The calculated t1/2 (half-life) values of heat in-activation at 60 °C and 70 °C were 245.7 and 54.5 min respectively, whereas at these temperatures the native enzyme was less stable (t1/2of 200.0 and 49.5 min respectively). The conjugated enzyme retained 32.3 and 29.7%, respectively from its initial activity in presence of 5 mM Sodium Dodecyl Sulphate (SDS) and p-Chloro Mercuri Benzoate (p-CMB), while the native enzyme showed a remarkable loss of activity (retained activity 1.61 and 13.7%, respectively). The present work has established the potential of glycosylation to enhance the catalytic properties of β-glucosidase enzyme, making this enzyme potentially feasible for biotechnological applications.

β-Glucosidase from Aspergillus niger was immobilized on sponge by covalent binding through a spacer group (glutaraldehyde). Sponge-immobilized enzyme had the highest immobilization yield (95.67%) and retained 63.66% of the original activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme remains almost the same as for the free enzyme (pH 4.0). The optimum temperature for β-glucosidase activity was increased by 10 ºC after immobilization. The activation energy (Ea) of the immobilized β-glucosidase was lower than the free enzyme (3.34 and 4.55 kcal/mol), respectively. Immobilized β-glucosidase exhibited great thermal stability and retained all the initial activity after incubation at 55 ºC for 2 h; however, the free enzyme retained 89.25% under the same condition. The calculated half-life (t½) value of heat inactivation of immobilized enzyme at 60, 65 and 70 ºC was 213.62, 72.95 and 56.80 min, respectively, whereas at these temperatures the free enzyme was less stable (half-life of 200.0, 55.31 and 49.5 min, respectively). The deactivation rate constant at 65 ºC for the immobilized β-glucosidase is 9.5x10-3/ min, which was lower than that of the free form (12.53x10-3/ min). The immobilization process improved the pH stability of the enzyme (immobilized and free enzyme retained 69.35 and 39.86%, respectively, of their initial activity after 45 min at pH 7.5). The effect of some chemical substances on the activity of the immobilized and free β-glucosidase has been investigated. In the presence of sodium dodecyl sulfate (SDS) and p-chloromercuri benzoate (p-CMB) the immobilized enzyme retained 36.13 and 45.34%, respectively, of the initial activity, which is higher than that of free enzyme (13.71 and 1.61%, respectively). The Michaelis constant (Km) value of the free enzyme was 40.0 mM, while the apparent Km value for the immobilized enzyme was 46.51 mM. The maximum reaction rate (v max) of immobilized β-glucosidase was smaller than that of the free enzyme by 7.69%. Sponge-immobilized β-glucosidase was repeatedly used to hydrolyze cellobiose (5 and 8 cycles with retained activity of 67.32 and 51.04%, respectively).

Bacillus subtilis NRC33a levansucrase was immobilised on different carriers using different immobilisation methods including physical adsorption, covalent binding, ionic binding and entrapment. The immobilised enzyme prepared by covalent binding on chitosan through 3% gluteraldehyde had the highest immobilization yield (81.51%). Therefore, it was used as a typical example for Bacillus subtilis NRC33a immobilised levansucrase and its properties were investigated. The time of the reaction and substrate concentration revealed that the activity of the immobilised enzyme was relatively lower than the free enzyme. The immobilised levansucrase showed a slight increase in activity compared with the free enzyme above 35°C. The activation energies were 6.62 and 9.27 kcal mol-1 for free and immobilised enzyme respectively. Although the thermal stability of the immobilised levansucrase was significantly improved in comparison to the free form, the deactivation energy of the immobilised enzyme was lower than that of the free enzyme. The half life of the immobilised and free levansucrase was also determined. The effect of different pH values reported that at acidic pH the activity of the immobilised levansucrase was higher than that of the free enzyme. The study of pH stability of free and immobilised levansucrase showed that the immobilisation process protected the enzyme from alkaline and severe acidic media. The effect of various metal ions showed that the free levansucrase was more sensitive to the inhibitory effect of the investigated substances. Immobilised levansucrase retained 51.13% after 14 repeated uses.

Oxidative stress and proteases have been implicated in several diseases and extensive evidence indicates that antioxidants and protease inhibitors help prevent organ functional damage. Leiurus quinquestriatus (LQQ) scorpion venom causes cellular injuries that may lead to multiple organ failure. Thus, the capability of the antioxidant "natural standardized extract of Gingko biloba leaves (Gin, EGb 761)" and the non-selective protease inhibitor, aprotinin, in ameliorating venom-induced biochemical alterations indicative of cellular injury and oxidative stress was studied to determine their effectiveness in protecting rats from venom-evoked cellular damages. Thus, in this study, rats were treated with LQQ venom (0.3mg.kg-1, subcutaneously) alone or after Gin (150mg.kg-1, orally, daily for 2 weeks before venom) and/or aprotinin (Apr, 46000 KIU.kg-1, intraperitoneally, 30 min before venom). Control groups were injected with saline or treatment modalities. Lungs and hearts were excised after decapitating rats (n=8/group) 60 min after venom injection and the following activities were measured: reduced glutathione (GSH), malondialdehyde (MDA) - an index of lipid peroxidation, glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH). Our findings demonstrate that LQQ venomsignificantly elevated GSH (p<0.05 vs. control), MDA (p<0.05), G6PD (p<0.05), and LDH activities (p<0.001) in hearts of envenomed rats. The venom also elevated MDA (p<0.05 vs. control) and reduced GSH and GPx (p<0.05) in the lungs of envenomed rats. In general, pretreatment with EGb761 attenuated LQQ venom-evoked increases in GSH (p<0.05 vs. venom), MDA in rat hearts and lungs (p<0.05 vs. venom), plus LDH in the heart (p<0.01). Aprotinin alone significantly reduced the venom-elicited increase in G6PD and LDH activities and the decrease in GPx levels (p<0.05). In general, these protective effects of EGb761 on GSH, MDA (p<0.01 vs. venom) and LDH (p<0.001) in the heart and/or lung were potentiated when combined with aprotinin. We concluded that the effectiveness of EGb761 and Apr in ameliorating venom-evoked biochemical changes indicative of necrosis and free radical generation point out the involvement of oxidative stress and proteases in venom-evoked cellular damages seen in this study in isolated rat hearts and lungs.