FEMS Microbiology Letters 61 (1989) 319-322 319 Published by Elsevier

FEMSLE 03716

Purification and properties of urease from a cyanobacterium Anabaena doliolurn

Ashwani K u m a r Rai

Department of Botany, Banaras Hindu University, Varanasi 221 005, India

Received 8 May 1989 Accepted 5 June 1989

Key words: Cyanobacteria; Anabaena doliolum; Urease; Purification; Properties

1. SUMMARY

Urease was purified 39 fold from extracts of Anabaena doliolum. The enzyme had a MW of 228 000 as determined by gel filtration and con- sisted of six subunits of identical size with a MW of 36000. The enzyme activity was optimum at pH 7.0 and at 4 0 ° C with a K m of 115 /xM. Among the metal ions tested, Hg 2+, Ag 2÷ and Cu 2 + inhibited enzyme activity in decreasing order. p-hydroxymercuribenzoate (10/~M) and acetohy- droxyamic acid (100 ~tM) brought about a total inhibition of urease activity.

was purified from Bacillus pasteurii [3]. Thereafter many reports have emerged covering urease char- acterization from jackbean, soybean and many micro-organisms [4], leaving cyanobacteria little explored with the exception of a solitary report on the purification of urease from a halophilic non- heterocystous cyanobacterium Spirulina maxima [5].

The present study describes the purification and characterization of urease from a freshwater heterocystous cyanobacterium A. do#Mum.

3. MATERIALS A N D METHODS

2. INTRODUCTION

Our preliminary trials showed urea to be an excellent nitrogen source for A. doliolum. This observation prompted us to investigate the mecha- nism(s) by which the alga utilizes urea. Since, urea hydrolysis by crude extract of A. doliolum was unaffected by the addition of ATP or avidin and was almost abolished by hydroxyurea, we reported that urease (urea amidohydrolase [EC 3.5.1.5]) is the urea hydrolyzing enzyme [1].

Sumner [2] isolated the first crystalline urease from jackbean and subsequently bacterial urease

Anabaena dofiolum Bharadwaja (local isolate, pure and axenic population) was grown in Chu No. 10 medium as described before [1]. Exponen- tially growing algal cells were washed twice with sterile distilled water and finally with 50 mM K phosphate buffer, pH 7.0, containing 1 mM EDTA and 5 mM fl-mercaptoethanol (PEM buffer).

Urease activity was estimated by determining the amount of urea decomposed within a given period of time at 40°C. The reaction mixture contained 2/~mol of urea in a final volume of 500 /zl of 50 mM K phosphate buffer (pH 7.0). The reaction was initiated by the addition of enzyme

0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies

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solution (25-100 ~1) and terminated by the ad- dition of 4 ml of mixed reagent used for the colorimetric assay of urea [6]. Urea level was determined by diacetylmonoxime method [6] while protein was estimated as in [7], with bovine serum albumin (Sigma, U.S.A.) as the standard.

The unit of enzyme has been expressed as the amount that hydrolyzed 1 ffmol of urea per min at 40 °C (pH 7.0) and specific activity as units per mg of protein.

Unless otherwise stated, all extraction and purification procedures were carried out at or below 12°C in PEM buffer. The washed cells were homogenized in PEM buffer and the re- sultant mixture was centrifuged at 15 000 × g (30 min) to obtain a clear solution. The supernatant (crude extract), was treated with solid ammonium sulfphate to yield the protein precipitating at 55% saturation (calculated for 25 ° C). The precipitate was resuspended in a minimal volume of PEM buffer and dialyzed overnight against 2 litre of the same buffer giving 3 changes. Cold acetone ( - 2 0 ° C) was added to the dialyzed solution at 0 °C to give a final concentration of 70%. Precipi- tate collected by centrifugation at 15000 × g (15 rain) was dissolved in PEM buffer and dialyzed against the same buffer as earlier. After dialysis, the acetone fraction was loaded onto a 1.5 × 25 cm DEAE-Cellulose column equilibrated in PEM buffer. After passage of 1.5 1 of PEM buffer, linear gradient of 0-0.75 M NaC1 (PEM buffer) was passed through the column. The protein con- tent of column fractions was estimated at 280 nm and urease assays were performed on alternate

fraction. Select fractions containing the majority of urease activity were pooled, concentrated by Amicon ultrafiltration and dialyzed against PEM buffer. This dialyzed solution was subjected to gel filtration on a column of Sephadex G-200 equi- librated with PEM buffer and 0.25 M NaC1. Ac- tive fractions were combined, concentrated and dialyzed against PEM buffer containing 20% glycerol.

The molecular weight of the enzyme was determined by Sephadex G-200 gel filteration method [8]. The MW standards were carbonic anhydrase (29 kDa), albumin bovine serum (66 kDa), alcohol dehydrogenase (150 kDa),/~-amylase (200 kDa) and apoferritin (443 kDa), all from Sigma.

Polyacrylamide gel electrophoresis (7.5% w / v acrylamide) was performed at pH 8.3 (Tris-glycine) [9]. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed as in [10] with trypsin inhibitor (20.1 kDa), carbonic anhydrase (29 kDa), glyceraldehyde-3-phosphate dehydro- genase (36 kDa), albumin egg (45 kDa) and al- bumin bovine (66 kDa) as MW markers (all from Sigma, U.S.A.).

4. RESULTS AND DISCUSSION

A summary of the purification of urease from A. doliolum is given in Table 1. Gel filtration through Sephadex G-200 was effective in increas- ing the purity of the enzyme. Urease could be purified 39 fold from crude extract with a yield of

Table 1

Purification of urease from A. doliolum

Steps Total protein Total activity Specific activity Fold Yield (mg) (units) (units/rag) (%)

Crude extract 176.31 42.28 0.24 1 100 Ammonium sulphate

fractionation 98.73 34.97 0.35 1.47 82.71 Acetone fractionation 78.08 33.10 0.42 1.76 78.28 DEAE-cellulose

chromatography 21.57 25.34 1.17 4.89 59.93 Sephadex G-200

chromatography 1.90 17.85 9.39 39.14 42.21

(A)

tB)

Fig. 1. Polyacrylamide gel electrophoresis of A. doliolum urease. (A) Polyacrylamide gel electrophoresis, (B) SDS-polyacryla- mide gel electrophoresis. The enzyme protein was 20 /zg (A)

and 10/~g (B).

42%. The enzyme eluted from Sephadex G-200 was homogenous with a single protein band observed on polyacrylamide gel electrophoresis with and without SDS (Fig. 1). The MW of the native enzyme corresponded to 228000 when passed through a calibrated Sephadex G-200 col- umn. SDS-polyacrylamide gel electrophoresis gave a subunit band of MW of 36000. Thus, urease f rom A. doliolum appeared to consist of six sub- units of identical size. Although the MW of native jackbean urease obtained by equilibrium ultra- centrifugation is reported to be in the range of 590000_+ 30000 and the subunit as 95000 [11], there have also been reports of very small urease subunits (30000-32000) [12,13]. The MW of A. doliolum urease obtained in the present investiga- tion is similar to the values reported for urease from Spirulina maxima [5].

The enzyme was less stable when stored at 4 ° C in 50 mM K phosphate buffer, pH 7.0 containing l mM EDTA and 5 mM /3-mercaptoethanol, as the activity declined by 70% over a 2 week period with the precipitation of some amount. The en- zyme was quite stable when stored at - 2 0 ° C in 50 mM K phosphate buffer (pH 7.0) containing 1 mM EDTA, 5 mM /3-mercaptoethanol and 20% glycerol with no loss of activity over 2 months.

The enzyme activity was maximum at 40 °C and was half at 22 as well as 58°C. The opt imum p H for the activity of the purified enzyme was 7.0 in 50 mM K phosphate buffer and half maximum activity at p H 5.9 and 10.2. On the other hand, urease from cyanobacterium Spirulina maxima showed a sharp opt imum at p H 8.7 in tris-maleate buffer [5]. Different p H optima depending upon the type of buffer, have been reported for the enzyme [4,14,15].

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The K m values of enzyme have been found to vary depending upon the source, pH and type of buffer [15]. Urease from A. doliolum followed Michaelis-Menten kinetics. The plot of S / V against S gave an apparent K m of 115 /~M. The affinity of A. doliolum urease for urea is thus significantly higher than that from jackbean (3-5 mM) [4], Klebsiella aerogenea (0.7 raM) [16], Brevibacterium ammoniagenes (18-72 mM) [15] or from the cyanobacterium Anabaena cylindrica (1.3 mM) [17], but similar to the urease from Spirulina maxima (0.12 raM) [5]. Because of this property, urease from A. doliolum and Spirulina maxima may be considered most suited for detecting urea even in traces by the reaction rate assay compared to the conventional methods employing ureases from other sources.

The purified enzyme preparat ion when prein- cubated (1 h) with various concentrations of metals at room temperature in 50 mM K phosphate buffer (pH 7.0), lost the activity completely at 10 /~M of either Hg 2+, Ag 2+ or Cu 2+ (50% activity was inhibited at 0.6, 0.7 and 1 /~M, respectively). Co 2+, Ni 2+ or Zn 2+ however, even at much higher concentration (20 #M), had no effect on the en- zyme activity.

Sulfhydryl reagent p-hydroxymercuribenzoate (10/~M) and the chelating agent acetohydroxamic acid (100/~M) inhibited the activity of the purified enzyme completely in similar fashion as reported earlier for the crude extract of A. doliolum [1].

R E F E R E N C E S

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[2] Sumner, J.B. (1926) J. Biol. Chem. 69, 435-441. [3] Larson, A.D. and Kallio, R.E. (1954) J. Bacteriol. 68,

67-73. [4] Reithel, F.J. (1971) in The Enzymes (Boyer, P.D., ed.),

Vol. 4, pp. 1-21. [5] Carvajal, N., Fernandez, M., Rodriguez, J.P. and Donoso,

M. (1982) Phytochemistry 21, 2821-2823. [6] Wootton, I.D.P. (1974) Microanalysis in Medical

Biochemistry, 5th ed., pp. 73-99. J. and A. Churchill Ltd., London.

[7] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.

[8] Andrews, J. (1964) Biochem. J. 91,222-223.

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[9] Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427. [10] Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244,

4406-4412. [11] Dixon, N.E., Hinds, J.A., Fihelly, A.K., Gallola, C.,

Winzor, D.J., Blakeley, R.L. and Zerner, B. (1980) Can. J. Biochem. 58, 1323-1334.

[12] Contaxis, C.C. and Reithel, F.J. (1972) Can. J. Biochem. 50, 461-473.

[13] Staples, S.J. and Reithel, F.J. (1976) Arch. Biochem. Bio- phys. 174, 651-657.

[14] Sumner, J.B. (1951) in The Enzymes (Sumner, J.B. and Myrback, K. eds.), Vol. 1, pp. 873-892, Academic Press, New York.

[15] Nakano, H., Takenishi, S. and Watanabe, Y. (1984) Agric. Biol. Chem. 48, 1495-1502.

[16] Friedrich, B. and Magasanik, B. (1977) J. Bacteriol. 131, 446-452.

[17] Mackerras, A.H. and Smith, G.D. (1986) J. Gen. Micro- biol. 132, 2749-2752.