Isolation, Purification And Identification Of Bilirubin Oxidase From Myrothecium Roridum

We have known that Myrothecium roridum(MTR) 3.1967 is the one which produced the best bilirubin oxidase(BOD). We choose it as the research object.We have proved by the research that we can acquired more BOD if we use mushroom substrate and a single mycelium to active the MTR and add revulsant to the culture medium at the same time. We add 1.15×10⁵ spores to the 40% liquid potato culture medium. We cultivate MTR. spores at 26℃on 180r/min. We stop the cultivation after 96h. Thus we can acquired the best the original BOD liquid with 2.12u/mL activity.We have improved the method to measure BOD by using ascorbic acid as terminating agent. This new method can measure multi-samples at the same time with normal spectrophotometer and debase the errors in measurement. It can help BOD used in clinic.We identify this fungi which can produce bilirubin oxidase by agarose plate with bilirubin. We ascertain the BOD strips by bilirubin PAGE and retract BODs from PAGE which is dyed by imidazole-ZnCl₂. We know the MW (77kD&66kD) of the BODs which are retracted through SDS-PAGE and the isoelectric point (pH5.5) through IFE.In this research the purification is carried through metallic chelate chromatography and anion exchange chromatography. We have gained BOD with a 29.4u/mg activity after isolation and purification. We analyse the purified BODs by SDS-PAGE and IFE. The molecular weights are 65kD and 74kD and the isoelectric point is pH5.5. The best temperature of the enzyme activity is 40℃and the best pH is pH8.1. The BOD is more stabilizing at 4℃and should be deposited at a low temperature.