Screening Highly Efficient Caffeine Degrading Bacteria And Cloning Their Caffeine Metabolic Related Genes

China is a big country with mass caffeine production and consumption. Caffeine and other N-methylated xanthines are well known for applications in food and as pharmaceuticals. Microbial or enzymatic methods for producing and degrading these N-methylated xanthines have broader applications. Till now, few caffeine-degrading bacteria have been identified. Screening highly efficient caffeine degrading bacteria and cloning their caffeine metabolic related genes are very important to develop the bio-degradation method by microbe and solve related problems caused by the abuses of caffeine. The main content of this thesis is as follows:1. Caffeine-degrading microbes were obtained on selective culture media with caffeine as sole carbon and nitrogen source, two of which were identified and named as Pseudomonas putida ECUST5-2and Pseudomonas aeruginosa ECUST2-3(Novel strain). The degradation rate of ECUST5-2and ECUST2-3was0.039g h-1and0.024g h-1respectively. Both strains degraded caffeine by demethylation pathway.2. The circumstance of domestication was studied. Both ECUST5-2and ECUST2-3can tolerate to15-20g L-1caffeine, which was comparable to the international level. The biodegradation ability of ECUST5-2and ECUST2-3were optimized using response surface methodology. The key components for caffeine-degradation by ECUST5-2were caffeine,4, MgSO4and yeast extract. The significant factors by ECUST2-3were only and CaCl2. After optimization, caffeine degradation rate increased by3.28-fold and22.67-fold, respectively.3. The full length gene of ndmA and ndmB from Pseudomonas putida ECUST5-2was obtained by degenerate-PCR and thermal asymmetric interlaced PCR for the first time. Then ndmC and ndmD gene were successfully achieved. The expression plasmids of NdmA and NdmB were constructed. Then NdmA and NdmB were expressed in Escherichia coli, but they were mainly expressed as inclusion-body.