Study On The Utilization Of D-Amino Acids By Marine Microbes And Activity Analyses Of Amino Acid Racemases

Marine dissolved organic carbon(DOC)pool is the largest of marine organic carbon storage pool,which is the fundamental part of marine carbon cycles.D-amino acids(D-AAs)is the major constituent of marine DOC pool.However,the bioavailability and the microbial metabolism mechanism of D-AAs are not clear yet,and the study on bioavailability and metabolic mechanism of D-AAs is related to cognize the source and fate of D-AAs in marine environment.This dissertation investigated and studied utilization and metabolic mechanism of D-AAs of marine bacteria and Thaumarchaeota.The main experiments and results can be concluded as the following:1.Ecological survery on bioavailability of D-AAs in South China Sea.The natural seawater was coated on 19 kinds of D-AAs plates that D-AAs were used as carbon and nitrogen sources,and the utilization of D-AAs by marine bacteria were analyzed.The results showed that:(1)No colony growed on the 66.7%of plates that D-AAs were used as sole carbon and nitrogen source,suggesting that D-AAs were difficult to use by marine bacteria.(2)D-AAs-utilizing isolates were classified as Gammaproteobacteria,Actinobacteria,Alphaproteobacteria,Bacilli and Flavobacteria,and the majority(72%)of them are Gammaproteobacteria.(3)Bacteria in the pelagic surface waters of South China Sea showed higher capabilities to utilize D-AAs.2.Study on utilization of D-AAs by Thaumarchaeota Nitrosopumilus maritimus SCM1.The culture experiment of Thaumarchaeota N.maritimus SCM1 in which D-AAs were used as sole carbon source suggested that:10 out of the 19 tested D-AAs(D-Met,D-Asp,D-Trp,D-Glu,D-Val,D-Pro,D-Ile,D-Phe,D-Leu and D-Asn)could promote the growth of N.maritimus SGM1.The other 9 tested D-AAs(D-Ser,D-His,D-Cys,D-Gln,D-Lys,D-Ala,D-Arg,D-Tyr and D-Thr)could inhibite the growth of N.maritimus SCM1.The NH3 coming from partial D-AAs degrading could be involved in the ammonia-oxidizing process of N.maritimus SCM1 and serve asnitrogen and energy source for N.maritimus SCM1 growth.3.Preliminary study on metabolic mechanism of D-AAs.The amino acid racemase genes were cloned from Alteromonas macleodii DSM17117 and N.maritimus SCM1,and expressed in vitro using Escherichia coli(E.coli)expression system.The study on biological activity and substrate specificity of the racemases found that:the alanine racemase(AlaR)of A.macleodii DSM17117 showed a high substrate specificity with Ala;the aspartate racemase(AspR)and Asp/Glu/hydantoin racemase(AGHR)of A.macleodii DSM17117 showed similar biological characteristics and both of them showed biological activity to asparagine and glutamine.The glutamate racemase(GluR)of N.maritimus SCM1 expressed successfully in the E.coli expression system,but biological activity of GluR didn't be detected.