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Single step purification of D-amino acid oxidase and mPEG-conjugated-D-amino acid oxidase from Trignopsis variabilis by HPLC.


D-amino-acid oxidase ( is a flavoprotein that 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 [1] T. variabilis (TvDAAO) is characterized by higher turnover number, good stability under a wide range of reaction conditions [2]. D-amino-acid oxidase is present in wide variety of microorganisms,birds, amphibians, reptiles and fishes (Krebs, 1951). The yeast enzymes are widely used in biotechnological applications, e.g. for oxidation of cephalosporin C in the two-step formation of 7-aminocephalosporanic acid [3] and for a human gene therapy paradigm that involves oxidant-mediated tumor cell death [4]. The purification of D-Amino acid oxidase has been described by using Sephadex G75 [5]. Purification of D-Amino acid oxidase from Trignopsis variabilis has been carried out by sonication, then precipitation of protein has been carried out by salts and solvens extraction methodology and finally adsorption column (charcoal) and anion exchange chromatography [6]. Purification of D-Amino acid oxidase from Trignopsis variabilis has been carried out by sequential liquid liquid extraction method [7]. Size exclusion chromatography of native DAO and PEGDAO from porcine kidney has been performed by using Superose 6 HR column[8]. Tumor targeted delivery of mPEG-conjugated enzymes have been studied by In vitro cytotoxicity MTT assay [9] and TBARS assay[10]. In the present study, D-amino acid oxidase from T. variabilis has been purified by HPLC. The retention time, enzyme activity and protein content of purified D-Amino acid oxidase and mPEGConjugatedDAmino acid Oxidase has been investigated. mPEG-Conjugated-DAmino acid Oxidase has also been purified by HPLC and retention time, enzyme activity and protein content of mPEG-Conjugated-D-Amino acid Oxidase has also been calculated.

Materials and Methods

Sodium pyrophosphate (98 % purity, lot no 1567 )Acetone ( lot no SC4F540157) and used were purchased from Loba chemicals. Trignopsis Variabilis MTCC 1354 was procured from IMTECH, Chandigarh, India. The buffer used was prepared in HPLC water purchased from Milipore, USA. Fifty milimolar Sodium pyrophosphate buffer was freshly prepared and pH was adjusted with phosphoric acid using pH meter (Century CP901) with pH electrode.


A Hitachi liquid chromatograph (model L-6000) has been used with UV-VIS detector (model L 4200). A high pressure pump, a 100 [micro]l syringe, degasser has been used. A RP-18 (5 [micro]M) has purchased from Merck, Germany. The flow rates have been optimized from 0.5ml/min to 1.5 ml/min.The mobile phase consists of 50mM Sodium pyrophosphate buffer pH 7.2. As, the Enzyme is to be detected, the detector wavelength had been set at 280 nm. The injection Volume was 10 [micro]l.

Sample preparation

Microorganism and Culture conditions

Trignopsis Variabilis MTCC1354 was grown in a medium (50 ml) containing malt extract (3g/l), yeast extract (3g/l), peptone (5g/l) and glucose (10g/l) with shaking at 200 rpm and 30oC for 48 hours. This media was sterilized at 121[degrees]C, 15 psi pressure for 15minutes.

Cell disintegration

50 ml of Biomass had been harvested.Then contrifuged at 5000 rpm for 20 min at 4[degrees]C. The pellet had been dissolved with acetone and mixed gently with glass rod to form precipitates.Centifuged at 2000 rpm for 20 min and collected the contents to beaker and subjected to shaking at 4oC overnight.The acetone powder had been prepared. The acetone powder had been dissolved in sample buffer Sodium tetrapyrophosphate buffer pH 7.2 in a ratio of 2:1. Microcentrifuged the mixture at 5000 rpm for 5min and supernatant had collected.

Preparation of purified enzyme by HPLC.

10 [micro]l of the crude enzyme D-Amino acid oxidase had injected in the injection valve of HPLC having Lichrospher(R) WR 300 RP-18 (5[micro]m) column using Sodium pyrophosphate buffer 50mM. pH 7.2 as mobile phase at UV detection 280 nm. The flow rate had been optimized by adjusting 0.5-1.5ml/min. Under these conditions, protein had eluted 1 ml fraction each. The enzyme activity had been calculated by the standard phenyl hydrazine methad [11]. The protein content (mg/ml)of each fraction was calculated by using 1.55A260-0.76A280 [12].Then 10 [micro]l of the purified D-AAO had been injected using similar conditions to know the retention time of purified D-AAO.

Purification of mPEG-propionaldehyde -conjugated D-Amino acid oxidase

Purified D-amino acid oxidase obtained by HPLC has been covalently conjugated with mPEG-Propionaldehyde. The molecular weight of D-Amino acid oxidase is39 kDa and mPEG-propionaldehyde is 2 kDa.The conjugated mPEG-propionaldehydeD-Amino acid oxidase has molecular weight 41kDa. 10[micro]l of mPEG- propionaldehyde conjugated D-amino acid oxidase had been injected into HPLC column to know the change in retention time using same conditions as above. The enzyme activity of D-Amino acid oxidase and protein content (mg/ml)of each fraction had calculated.

Results and Discussion

Cell disintegration

D-Amino acid Oxidase is intracellular enzyme. The acetone disrupts the cell membranes and the enzyme extudes out. The supernatant was used to calculate the enzyme activity by the standard phenylhydrazine method.

HPLC assay development of crude and purified enzyme.

As the flow rate had been optimized by adjusting 0.5 ml/min-1.5ml/min. At flow rate 0.5 ml/min, there had been spreading of D-AAO units in different fractions from 3rd fraction to 6th fractions, showing the peaks from 2.98 to 6.97 min as shown in fig 1.


When the flow rate was adjusted to 1.5 ml/min, the units of Enzyme were spreading from 2nd to 7th fraction as shown in the peaks from 1.18 to 6.18 in fig. 2.


But in comparision at the flow rate of 1 ml/min, the maximum D-AAO units were found only in two fractions i.e. fraction no 3rd and 4th.The crude Enzyme shows the peaks from 2.33 to 3.90 giving maximum units in fraction no 3rd with specific activity 72 I.U/mg. As shown in fig 3 peaks 2.78, 3.69 and 3.9 were longer, the elution starts at 2.78 min and ends at 3.90.So the retention time of crude enzyme is 2.78,3.69 and 3.90. There may be present some other proteins in the crude soup of the enzyme, which showed a huge peaks at 4.51, 8.70 and 11.43 but the units of D-Amino Acid Oxidase were maximum in the 3rd fraction So we considered the retention time of crude enzyme is 2.78 min as shown in fig 3.


The purified enzyme showed the same retention time showing peak at 2.74 with specific activity 153.3 I.U./mg. but the purification fold was improved.


HPLC assay development of mPEG-propionaldehyde -conjugated D-AAO After close inspection of chromatogram of conjugated D-AAO reveals that the retention time of D-Amino acid oxidase was changed as the peaks shift from 2.7 to 2.03 when mPEG-propionaldehyde was conjugated with D-Amino acid oxidase, molecular weight had been increased by conjugation with mPEG-propionaldehyde.


Purification Fold

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.


Crude D-Amino Acid Oxidase had 186 units with sp. acitivity 28 I.U./mg. Out of 186 units of crude D-Amino Acid Oxidase, 114 units had been recovered in fractions no 3 (80 units) and fraction no 4 (34 units). The specific activities of the fractions no 3 and 4 were 72.7 I.U/mg and 42.5 I.U./mg. The percentage recovery was of crude D-AAO is 61 %. As 10[micro]l of mPEG- propionaldehyde conjugated D-amino acid oxidase was injected, Out of 0.8 units of purified D-Amino acid oxidase, 0.46 units had been recovered showing 57% recovery. Then purified 3rd fraction was conjugated, out of 0.8 units of purified PEG-Conjugated-D-Amino acid oxidase, 0.43 units have been recovered showing 54% recovery.


The Retention time, enzyme activity and protein content of purified D-Amino acid oxidase and mPEG-Conjugated-D-Amino acid Oxidase had calculated. As the enzyme D-AAO was covalently conjugated with mPEG, there was shift in fraction from higher to lower based on the same principle as molecular sieving applied with high pressure. D-AAO shows no antitumor activity alone nor with D-Amino acid but when it is conjugated with mPEG-propionaldehyde, then it may show antitumor activity due to enhanced permeability and retention effect (8).This method followed in the present study is helpful for In vitro MTT cytotoxicity assay to know the antitumor effects of conjugates.


[1] Pollegioni, L., Porrini, D., Molla, G. and Pilone, M.S., Eur. J. Biochem. 267 (2000) 6624- 6632

[2] Pollegioni, L., Langkau, B., Tischer, W., Ghisla, S. & Pilone, M.S. J. Biol. Chem. 268 (1993) 13850-13857.

[3] Pilone, M.S., Buto, S. & Pollegioni, L. Biotechnol. Lett. 17(1995) 199-204.

[4] Stegman, L.D., Zheng, H., Neal, E.R., Ben-Yoseph, O., Pollegioni, L., Pilone, M.S. & Ross, B. D. Hum. Gene Ther. 9 (1998) 185-193.

[5] Yagi K, Nishikimi M, Oishi N, Takai A. Biochim Biophys Acta. 212(2) (1970) 243- 247.

[6] Berg C.P. and Rodden. F.A. Anal. Biochem. 71(1) (1976) 214-222.

[7] Singh, P., Verma, N. and Marwaha, S.S. Biotechnol Letts. 23(18) (2001) 1479-1483.

[8] Fang, J. Sawa, T., Akaike, T. and Maeda, H.Cancer Research. 62 (2002) 31383143.

[9] Mueller, H., Kassack, M.U. and Wiese, M. Biomolecular Screening J.9 (6) (2004) 506-515.

[10] Yagi, K. Free Radical and Antioxidants Protocols. 108 (1998) 101-106.

[11] Case E.M. Biochem. J. 26 (1932) 753-758.

[12] Schleif, R.F. and P.C. Wensink. Practical methods in Molecular Biology New York, NY, Springer-verlag, 1981, p74.

(1) * Neelam Verma and (2) Shipra Kalra

(1) Department of Biotechnology, Punjabi University, Patiala, Punjab, India e-mail :

(2) Department of Biotechnology, Punjabi University, Patiala, Punjab, India e-mail :
Table 1

0.5ml/min     Enzyme      Protein    Specific
              Activity    Conc.      Activity
              (I.U)       (mg/ml)    (I.U/mg)

1.            0.8         0.10       8
2.            7.0         0.50       14
3.            30          1.90       15.7
4.            26          1.5        17.33
5.            26          1.6        16.25
6.            30          0.55       54.5
7.            36          0.65       55.3
8.            12          0.40       30
1.0 ml/min
1.            0.08        0.238      0.33
2.            21.6        0.77       28.0
3.            48.3        0.59       81.8
4.            35.3        0.36       98.8
5.            28.6        0.20       143
6.            18.6        0.30       62
7.            11.0        0.20       55
8.            1.6         0.11       14.5
1.            3.8         0.3        12.66
2.            4.5         0.4        11.25
3.            80          1.1        72.7
4.            34          0.8        42.5
5.            3.5         0.5        7.00
6.            3.6         0.30       12.0
7.            0.20        0.20       0.0005
8.            0.34        0.21       1.61

Table 2

Flow Rate             Crude Enzyme          Purified     mPEG-
(1ml/min)                                   D-AAO        Conjugated

Retention Time        2.78 min and 3.60     2.74 min     2.03

Specific Activity     28                    153.3        153.5

Fraction No.          3rd and 4th           3rd          3rd

Peaks                 2.33 to 3.99 min      2.74 min     2.03

Peak Area             40%                   71%          78%

Percent Recovery      61%                   57.5%        54%

Enzyme Units          186 units             0.46 units   0.43 units

Protein Content       6.6                   0.0030       0.0028

Purification Fold     --                    5.5          5.5
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Author:Verma, Neelam; Kalra, Shipra
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Date:May 1, 2008
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