Conversion Condition And Expression Of L-amino Acid Oxidase From Marine Pseudoalteromanas Sp.R3

L-amino acid oxidase(LAAO;EC 1.4.3.2)is dimeric flavoprotein with various biological functions which are found to be probably related to the produced H2O2.LAAO can be applied to biosensors and biotransformations.Probably due to post translational modification of LAAO,only some heterologous expression systems have been reported hitherto.In this dissertation,LAAO from marine Pseudoalteromonas sp.R3 was used to convert L-amino acids to corresponding a-keto acids.After optimization of conversion condition,it was found that the optimum conversion condition was as follows:pH 8.31,temperature 36.21 ?,coenzyme FAD concentration 0.65 mM and Zn2+ concentration 10.14 mM.under the optimum condition,the productivity of a-keto acid can be increased to 0.847 mg/mL from 0.497 mg/mL,giving 70.42%higher.In the present study,a simple,rapid,sensitive,cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction was described.The pH value of detection solution suitable for Prussian blue agar assay was around 5 up to 12.This Prussian blue agar assay is sensitive to 0.5 mM<H2O2 ? 20 mM.Coupled with SDS-PAGE,this method can be directly used to determine the numbers and approximate molecular weights of LAAO in one step.The lao gene of Pseudoalteromonas sp.R3,encoding the L-amino acid oxidase,was amplified by PCR and cloned into the expression plasmids pET28b(+)and pET20b(+).The recombinant plasmids were then transformed into E.coli BL21(DE3)and separately screened on the resistant plate with 50 ?g/mL kanamycin and 50 ?g/mL ampicillin.LAAO can be efficiently expressed in engineered strains E.coli BL21(DE3)/pET28b(+)-lao and E.coi BL21(DE3)/pET20b(+)-lao by IPTG induction.But the activity of expressed LAAO is low,most probably due to the formation of insoluble protein.