[The measurement of intra-cellular AOX in recombinant Pichia pastoris]

The activities of intracellular alcohol oxidase(AOX) in recombinant P. pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment. The enzyme vitality and specific enzyme vitality were defined nd the condition for detecting the enzyme vitality was also established. The experimental results showed that with a certain quantity of biomass in a phosphate buffer containing methanol, the consuming rate of dissolved-oxygen reflected the enzyme vitality of intracellular AOX. It was also found that the pH of the buffer could be very freely between 4.7 and 7.4 and the suitable optical dersity of cell concentration at 600 nm was between 0.5 and 2.0. Furthermore, the values of qo2max and Km of AOX versus oxygen consumption, which were 0.409 s-1 and 0.16 respectively, were calculated. It is a simple and sensitive and feasible method for quick measuring of AOX.