Study On L-amino Acid Oxidase From Marine Pseudoateromanas Sp. B3

L-amino acid oxidase?LAAO;EC 1.4.3.2?is dimeric flavoprotein,its main biological property is antimicrobial activity and may act as defence or attack weapons via the H₂O₂.Most LAAOs from microorganisms appoint them as ideal molecular mechanism for the acquisition of nitrogen from diverse amino acid sources.Now,a novel LAAO with its substrate is applied to technology of hydrogen peroxide production in situ.Another field of application of LAAO is the use as biosensors and biotransformations.It has been reported to be used as anti-AIDS and-tumors medicines.In this dissertation,LAAO from marine microorganism had been studied.Measurement of a-keto acids,NH₄⁺ and H₂O₂ was carried out to show that marine bacterial B3 can produce L-amino acid oxidase.Fermentation condition researches showed that B3 can produce LAAO excellent at the condition with the agitation of 140 r/min,the temperature of 30?,the time of 72 h,the inoculation amount of 2%,the bacteria age of 24 h,the pH of 7.5,and in the 40 mL medium that include yeast extract 2.25 g/L,peptone 3.75 g/L,sea salt 30 g/L.The molecular identification of 16S rDNA was performed by constructing the phylogenetic tree combined physiological and biochemical analysis to show that B3 is proximal to Pseudoalteromonas spp.,the highest identity of 98.5%with Pseudoalteromonas rubra.Therefore it was designated as Pseudoalteromonas sp.B3.LAAO from Pseudoalteromonas sp.B3 was purified to homogeneity by a series of procedures including ammonium sulfate precipitation,desalting on Hiprep-26/10,chromatographies on DEAE-Sephacel,Phenyl hydrophobic chromatography,Sephacryl S-200 chromatography.LAAO exhibited maximal activity at 30?,and showed the optimal pH at 7 using L-Met as substrate.It was stable for 30 min at 50 ? and pH 4?8.The enzyme exhibited relatively broad range substrate specificity,oxidising a total of 11 L-amino acids and the L-Leu was the preferred substrate.The enzyme activity was promoted by metal ions Zn²⁺,Ca²⁺,Mg²⁺,K⁺ and Ge²⁺and inhibited by Mn²⁺,Cu²⁺,Co²⁺,Fe²⁺ and Ba²⁺.In the molecular cloning,we designed primers for cloning of LAAO of Pseudoalteromonas sp.B3 by Touch down-PCR and NEST-PCR methods.The DNA clones of LAAO showed that the open reading frame was 1608 bp in length encoding the protein of 535 amino acid residues,including flavin protein conservative district R-x-G-G.The deduced molecular mass of LAAO is 59571.46 Da,and theoretical isoelectric point is 6.11.The amino acid composition,cleavage sites,secondary structure and tertiary structure of LAAO were analyzed and predicted.Amino acid composition analysis shows that the highest levels of the amino acid is Ala?9.91%?,the second is Leu?9.53%?.The primary structure has no signal peptide.The secondary structure mainly contains 224 alpha helixs,72 extended strands and 239 bend regions.