The Study Of The Enzymatic Properties About Protocatechuate 3,4-dioxygenase And Its Immobilization

The protocatechuate branches metabolic pathway is important for the the biodegradation of aromatic compounds.The protocatechuate 3,4-dioxygenase?P34O?catalyzes the ring-opening step in the catabolism of aromatic compounds through the protocatechuate branch of the ?-ketoadipate pathway.In this study,the sizes of the two gene fragments encoding P34O which are amplified by using the genomes Rhizobium sp.LMB-1 and Acinetobacter sp,LMB-5 are approximately 1300 bp.In addition,the sizes of recombinant P34O expressed by recombinant strains LMB-1/proP34O and LMB-5/proP34O are close to the predicted size.In order to analyze the properties of recombinant P34O,the differences in the enzymatic properties of the two recombinant P34O are studied.The optimum pH of two recombinant enzymes both are 7.5,and the optimal temperature of the recombinase expressed from the LMB-1/proP34O is 60 ?.However,the recombinant P34O from the LMB-5/proP34O shows the highest activity at 50 ?.The relative activity of the P34O from LMB-1/proP34O decrease slowly after incubating 450 min at 60 ?.However,the activity of the enzyme from LMB-5/proP34O is reduced to 20%,after only incubating 15 min at 60 ?.It is observed that the activity of P34O from LMB-1/proP34O only can be activate by Fe3+,but Fe3+,Al3+ and Mg2+ all can increase the activity of one from LMB-5/proP34O.Meanwhile,the transcriptional level of pcaG and pcaH gene in two wild strains both is increased by the induction of the aromatic compounds such as PAEs.In order to further improve the properties of P34O,the enzyme from LMB-1/proP34O is immobilized onto 3-APTES-modified Fe₃O₄ nanoparticles?NPs?by the glutaraldehyde method.The results show that the optimum temperature of the immobilized enzyme is increased 10 ?,compared to the free enzyme,and the thermal stability of immobilized P34O is better than that of the free enzyme.However,the optimum pH was unaffected.The Km,Vmax and Kcat values of immobilized enzyme are 159.29?M,19.16 ?M/min and 958/min,respectively,and the corresponding values of free enzyme are 65.34 ?.M,12.29 ?gM/min and 615/min,respectively.The immobilized P34O on Fe₃O₄ NPs be reused 10 times to retain 64%relative activity.