Continuous ATP regeneration process with stable acetate kinase.

Heat-stable acetate kinase (AK) from Bacillus stearothermophilus was successfully immobilized covalently to Sepharose resin by several conventional methods including carbodiimide, hydroxysuccinimide, cyanogen bromide, and glutaraldehyde and also by a new method which utilizes a bifunctional ADP derivative as a spacer. The latter method gave a higher yield in terms of enzyme activity than the conventional methods. The properties and kinetics of the immobilized AK were studied batchwise and in a column. The Michaelis-Menten equation could be applied to the immobilized AK column. The apparent Km values of ADP and acetyl phosphate for immobilized AK were not significantly different from those for free AK. The pH-activity profile of immobilized AK was similar to that of free AK. The heat stability of immobilized AK was markedly improved as compared with free AK. The immobilized AK retained more than 80% of the initial activity after continuous operation at 30 degrees C for 1 month. It was shown that the immobilized AK from B. stearothermophilus could be utilized as an ATP regeneration system in the bioreactor.