Expression And Catalytic Characterization Of A New Bacterial Pyranose Oxidase

Rice straw resource is tremendous in China and its effective utilization remains significant for solving problems of environmental pollution and resource waste.Therefore,the development of efficient straw degradation microbial resources has become a recent research focus.Pantoea ananatis Sd-1 is an endophytic bacterium isolated from rice seeds and has a strong degradation ability of rice straw.The complete genome sequence of Sd-1 was obtained and the preliminary study of its degradation mechanism has been completed.In order to further explore the degradation mechanisms and essential protein in P.ananatis Sd-1,a new bacterial pyranose oxidase(P20)was identified and characterized from Sd-1.This is the second reported bacterial P20 by far and it might be potential to replace fungal P20x in biotechnical applications.The research content in this paper mainly includes the following three parts.(1)Prokaryotic expression and purification of P20 from Sd-1.The putative GMC family oxidordeuctase gene in Sd-1 was analyzed by phylogenetic sequence comparison,conserved domains prediction and homology-based structural modeling and the result indicated that it is a gene encoding for a bacterial P20.Then it was cloned and prokaryotic expressed in E.coli.After condition optimization,the recombinant P20 was purified successfully.(2)Characterization of P20 from Sd-1.Native-PAGE and western blot analysis showed that the recombinant P20 has the similar spatial structure as fungal P20,the biochemical characters also indicated that it is a new bacterial P20.Effects of pH and temperature on the recombinant P20 enzyme activity and stability and the catalytic efficiency of different saccharides by the recombinant P20 were proved.Effects of the recombinant P20 on lignin degradation by laccase revealed that P20 had promoted the degradation ability of bacterial laccase.(3)Homologous expression of P20 gene from Sd-1.Homologous expression of P20 gene was tested to investigate its effects on Sd-1 growth and the ability of lignocellulose degradation.However,Sd-1 was almost devitalized at the end of cultivation period due to the high level of H2O2 accumulation induced by lignocellulose.