International Journal of Biotechnology & Biochemistry

D-amino-acid oxidase (1.4.3.3) is a flavoprotein. It catalyses oxidative deamination of D-amino acids to 2-Oxo acids producing NH₃ and H₂O₂. In yeast, the enzyme is involved in the catabolic utilization of D-amino acids. T. variabilis (TvDAAO) is characterized by higher turnover number, good stability under a wide range of reaction conditions. Enzymes such as the D-amino acid oxidase, which is almost ubiquitous to organisms of many kingdoms, is missing in all plants investigated. D-Amino acid oxidase has been purified using Lichrospher (R) WR 300 RP-18 (5μm) column by HPLC from Trignopsis variabilis and the Retention time, enzyme activity and protein content of purified D-Amino acid oxidase and mPEG-Conjugated-D-Amino acid Oxidase have been investigated. D-Amino Acid Oxidase units are maximum in the 3rd fraction having the specific activity 72 I.U/mg. The retention time of crude enzyme is 2.33 to 3.90 minute. The retention time of purified D-Amino Acid Oxidase is 2.74 min having the specific activity 153.3 I.U./mg. But the retention time of mPEG-conjugated D-Amino Acid Oxidase is 2^nd minute as the molecular weight of D-amino acid oxidase has been increased by conjugation with mPEG propionaldehyde, so the retention time of conjugated D-amino acid oxidase has decreased. The purification fold of purified fractions of free D-AAO and conjugated D-AAO is almost same as 5.5. There is almost no change in the enzyme activity of D-AAO after conjugation.