Purification and Biochemical Characterization of a

Folia Microbiol. 52 (3), 231–236 (2007)

http://www.biomed.cas.cz/mbu/folia/

Purification and Biochemical Characterization of a Mycelial Alkaline Phosphatase without DNAase Activity Produced by Aspergillus caespitosus L.H.S. GUIMARÃESa, A.B. JÚNIORb, J.A. JORGEa, H.F. TERENZIa, M.L.T.M. POLIZELIa* aDepartamento de Biologia da Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, and bDepartamento de Bioquímica

e Imunologia da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto (São Paulo), Brazil Received 12 June 2006 Revised version 16 October 2006

ABSTRACT. Biochemical properties of a termostable alkaline phosphatase obtained from the mycelium extract of A. caespitosus were described. The enzyme was purified 42-fold with 32 % recovery by DEAEcellulose and concanavalin A-Sepharose chromatography. The molar mass estimated by Sephacryl S-200 or by 7 % SDS-PAGE was 138 kDa and 71 kDa, respectively, indicating a homodimer. Temperature and pH optima were 80 ºC and pH 9.0. This enzyme was highly glycosylated (≈74 % saccharide content). The activity was enhanced by Mg2+ (19–139 %), NH4+ (64 %), Na+ (51 %) and Mn2+ (38 %). 4-Nitrophenyl phosphate (4-NPP) was preferentially hydrolyzed, but glucose 1-phosphate (93 %), UTP (67 %) and O-phosphoamino acids also acted as substrates. νlim and Km were 3.78 nkat per mg protein and 270 μmol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 μmol/L in the presence of Mg2+, using 4-NPP as substrate. The purified alkaline phosphatase removed the 5´-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology.

Abbreviations 4-NPP Ampol AP(s) BAP ELISA

4-nitrophenyl phosphate 2-amino-2-methylpropan-1-ol alkaline phosphatase(s) bacterial alkaline phosphatase enzyme-linked immunosorbent assay

Glc-1-P PDA pUC SDS-PAGE

glucose 1-phosphate potato dextrose agar plasmid University of California sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Alkaline phosphatases (phosphoric monoester hydrolases) are ubiquitous in nature, occurring in prokaryotic and eukaryotic microorganisms, plants and animals. Phosphatases hydrolyze a wide variety of phosphate esters and anhydrides of phosphoric acid (Holander 1971). They have been classified as alkaline phosphatase (EC 3.1.3.1) with optimum activity at pH >8.0 and acid phosphatase (EC 3.1.3.2) with optimum pH < 6.0 (De Araújo et al. 1976). More recently, the phosphatases have been divided into five distinct families: alkaline phosphatases, protein phosphatases, purple acid phosphatases, higher-molar-mass acid phosphatases and low-molar-mass acid phosphatases (Vincent et al. 1992). The biotechnological potential of these enzymes has attracted much attention especially for recombinant DNA technology (Pereira et al. 1995) and ELISA (Dong and Zeikus 1997). Commercial preparations of AP are usually obtained from Escherichia coli (Seeburg et al. 1977) or calf intestine (Pozidis and Bouriotis 1995). In this context, enzymes obtained from filamentous fungi may be of interest, offering other advantageous properties for biotechnological applications (see, e.g., Gargova et al. 2006). AP activity was studied in several filamentous fungi, such as Scytalidium thermophilum (Guimarães et al. 2001), Humicola grisea var. thermoidea (Buainain et al. 1998) and Neurospora crassa (Morales et al. 2000; Bogo et al. 2006). Here we describe the purification and biochemical characterization of a termostable intracellular AP, without any accompanying DNAase activity, produced by the filamentous fungus Aspergillus caespitosus.

MATERIAL AND METHODS Microorganism. A. caespitosus (Raper and Fennel 1965) was identified and deposited in André Tosello Foundation (Campinas, São Paulo, Brazil) and maintained in our laboratory on slants of solid PDA *Corresponding author; fax +55 16 3602 4886, e-mail [email protected] .

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medium. For culture, a conidial suspension of spore concentration 100/μL (i.e. 105 spores per mL) was inoculated in Erlenmeyer flasks with 50 mL of SR liquid medium (initial pH 6; Rizzatti et al. 2001) supplemented with 1 % (W/V) xylan. The cultures were incubated for 3 d with agitation on an orbital shaker (1.67 Hz) at 40 ºC. Extraction of the intracellular enzyme. Three-d-old cultures were harvested by vacuum filtration. The filtrate was saved to measure the extracellular activity. Mycelial pads were blotted with filter paper, and ground in a porcelain mortar with sand at 4 ºC. AP activity was extracted with 100 mmol/L AMPOL (Merck) buffer (pH 8.5) at 4 ºC. The supernatant (23 700 g, 15 min, 4 ºC) was used as a source of crude intracellular AP. Determination of phosphatase activity and protein. AP activity was determined using 4-NPP (final concentration 2.7 mmol/L) as substrate in 100 mmol/L Ampol buffer (pH 8.5) at 70 ºC in a final volume of 600 μL. The reaction was stopped with 2 mL of saturated disodium tetraborate solution and 4-nitrophenol liberated quantified on 405 nm. Other phosphorylated substrates and racemic O-amino acids (O-phosphoserine, O-phosphotreonine and O-phosphotyrosine) were used and the Pi liberated was determined at 405 nm (Heinonen and Lahti 1981). Enzyme activity is given in nkat (the amount of enzyme that produces 1 nmol/s 4-nitrophenolate or of Pi under the assay conditions). Protein concentration was determined according to Lowry using bovine serum albumin as standard. Purification of the intracellular alkaline phosphatase. The crude extract was applied to a DEAEcellulose column (20 × 127 mm) equilibrated in 10 mmol/L Tris-HCl buffer (pH 7.5). The enzyme was eluted with a linear gradient of NaCl (0–500 mmol/L) in the same buffer. The fractions with AP activity were pooled, dialyzed and applied to a concanavalin A-Sepharose column (12 × 100 mm) equilibrated in 20 mmol/L Tris-HCl buffer (pH 7.5) plus 500 mmol/L NaCl and Mn2+ and Ca2+ (both 1 mmol/L). The peak of activity was eluted with a linear gradient of methyl α-D-mannopyranoside (0–500 mmol/L) in the same buffer. The fractions with activity were pooled and dialyzed against distilled water for 18 h at 4 ºC. Molar mass and saccharide content determination. The molar mass was determined by Sephacryl S-200 gel filtration in a column (18 × 1210 mm) equilibrated in 50 mmol/L Tris-HCl buffer (pH 7.5) plus 150 mmol/L NaCl. The molar-mass markers were: aldolase (158 kDa), bovine serum albumin (66 kDa) and carbonate dehydratase (29 kDa). Elution was done in the same buffer and 1.5 mL fractions were collected. The column was maintained at 4 ºC. The saccharide content was determined by phenol–sulfuric acid method (Dubois et al. 1956). Electrophoresis. Samples of intracellular AP were submitted to PAGE (6 %) electrophoresis according to Davis (1964) or 7 % SDS-PAGE (Laemmli 1970). Protein was stained with a silver solution (Blum et al. 1987). Myosin (205 kDa), β-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa) and carbonate dehydratase (29 kDa) were used as molar-mass markers. DNA assay. Plasmid DNA pUC 18 (1.13 μg/μL) (Amersham) was linearized with EcoRI and treated with commercial BAP (Invitrogen) or with intracellular AP of A. caespitosus. The treated samples and untreated controls were then incubated with T4 DNA ligase. All samples were applied to agarose gel with ethidium bromide and the bands were observed under UV light; standard of 1 kb was used. Kinetic parameters. νlim, Km and Hill coefficient (h) were determined with 4-NPP as substrate using Sigraf (Leone et al. 1992) and Enzyplot (Leone et al. 1995) softwares. Reproducibility of results. All results are the means of at least three independent experiments.

RESULTS Purification of intracellular alkaline phosphatase. A crude cell extract was the source of intracellular AP. The enzyme was eluted on DEAE-cellulose as a single peak with 345 mmol/L NaCl in 10 mmol/L Tris-HCl (pH 7.5). Fractions with activity were pooled, dialyzed and applied to Con A-Sepharose. A single peak of activity was eluted with 123 mmol/L methyl α-D-mannopyranoside. The enzyme was purified 42-fold with ≈32 % recovery (Table I). The intracellular AP was purified until apparent electrophoretic homogeneity Table I. Purification of intracellular alkaline phosphatase from A. caespitosus Purification step

Total activity, nkat

Crude extract DEAE-cellulose Con A-Sepharosea aConcanavalin A-Sepharose.

1490 488 476

Total protein, mg 75.8 5.51 0.59

Specific activity, nkat/mg 19.7 88.6 807.0

Recovery, % 100.0 32.8 32.0

Purification, fold 1 4.68 42.6

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was reached, showing a single band on 7 % SDS-PAGE (Fig. 1). Also a single-protein band which was positively stained for AP activity was observed on 6 % PAGE. Biochemical properties. The molar mass of the intracellular AP was 71 kDa (7 % SDS-PAGE), or 138 kDa (Sephacryl S-200 gel filtration) which indicates that AP can be a homodimer. The temperature optimum for the purified enzyme was 80 ºC (Fig. 2A); it was stable at 60–70 ºC with t50 of 1 h at 70 ºC (Fig. 2B) and exhibited pH optimum of 9.0 in Ampol buffer (Fig. 2C). The purified enzyme was a glycoprotein with 74 % saccharidee content. Kinetic parameters νlim and Km, varied in the absence or presence of Mg2+ with 4-NPP as substrate. The values for νlim and Km were 3.78 nkat per mg protein and 270 μmol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 μmol/L in the presence of Mg2+. The enzyme activity was enhanced by Mg2+ (19– 139 %), NH4+ (64 %), Na+ (51 %) and Mn2+ (38 %), to a lesser extent also by Ba2+ and Zn2+ (Table II). The enzyme was inhibited by Ca2+, Hg2+ and Cu2+; EDTA inhibited 70–75 % of its activity. These results showed that the enzyme is a metalloenzyme dependent on divalent ions, especially Mg2+. The enzyme hydrolyzed preferably 4-NPP, Glu-1-P (93 %) and UTP (67.5 %) (Table III). The rate of hydrolysis for ATP, glycerol 2-phosphate and ITP was ≈51–58 %. A lower rate of hydrolysis was observed for others substrates, including O-phosphotyrosine (23.7 %), O-phosphothreonine (22.1 %) Fig. 1. SDS-PAGE of a purified fraction after and O-phosphoserine (9.27 %). concanavalin A-Sepharose elution; 1 – 7.0 % SDS-PAGE stained with silver, 2 – 6.0 % DNA assay. The enzyme (Fig. 3) removed the PAGE stained for alkaline phosphatase activity, 5´-phosphate group of linearized plasmid DNA, impeding the 3 – 6.0 % PAGE stained with silver; for molarrecircularization of plasmid DNA (as observed with a commass markers see Materials and Methods. mercial BAP). DNAase activity was not observed.

Fig. 2. Intracellular alkaline phosphatase activity; A: effect of temperature ( °C), B: thermal stability (for 0–60 min) at 60 (open circles) and 70 ºC (closed circles), C: effect of pH.

DISCUSSION A. caespitosus produces two APs, one of these being an extracellular enzyme previously characterized (Guimarães et al. 2003b), the other an intracellular form described in the present study. The intracel-

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lular AP is shown to be a homodimeric glycoprotein with two identical subunits of 71 kDa, different from the extracellular AP (which shows two subunits of 57 kDa; Guimarães et al. 2003b). This difference could be attributed to the different saccharide content (74 vs. 56 %) of the two enzymes. Dimeric structures of AP Table II. Influence of selected compounds (concentration of 1 and 10 mmol/L) on intracellular alkaline phosphatase activity from A. caespitosus

Compound

None BaCl2 CaCl2 CuCl2 CuSO4 EDTA HgCl2

Residual activity, % 1

10

100.0

100.0

91.5 68.3 86.6 64.6 25.6 35.4

107.3 73.2 75.6 29.3 29.3 25.6

Compound

MgCl2 MgSO4 MnCl2 NaCl NH4Cl ZnCl2

Residual activity, % 1

10

132.7 120.5 75.6 96.3 106.2 106.1

227.8 239.0 138.8 151.2 165.6 41.5

Table III. Effect of several substrates on the hydrolysis of intracellular alkaline phosphatase of A. caespitosus Substrate 4-Nitrophenyl phosphate Glucose 1-phosphate Uridine 5´-triphosphate Adenosine 5´-triphosphate Glycerol 2-phosphate Inosine 5´-triphosphate

Relative activity, % 100.0 93.3 67.5 57.8 53.7 51.5

Substrate

Relative activity, %

Adenosine 5´-(P β-glucosyl)diphosphate O-Phosphotyrosine O-Phosphothreonine Glucose 6-phosphate O-Phosphoserine Fructose 6-phosphate

29.8 23.7 22.1 19.4 9.3 5.2

were observed also for the conidial (63 kDa) and mycelial (58.5 kDa) AP of S. thermophilum (Guimarães et al. 2001) but, in contrast, the extracellular AP of Penicillium chrysogenum is a monomeric glycoprotein with 58 kDa (Politino et al. 1996), confirming the structural diversity of these biomolecules. Perhaps the higher saccharide content of the intracellular enzyme also contributes to the higher temperature optimum and higher

Fig. 3. Electrophoresis on agarose gel of plasmid DNA treated with extracellular alkaline phosphatase of A. caespitosus; 1 – markers of 1 kDa; 2 – intact plasmid DNA, 3 – linearized plasmid DNA; 4 – linearized plasmid DNA treated with T4 DNA ligase, 5 – linearized plasmid DNA treated with BAP, 6 – linearized plasmid DNA treated with extracellular alkaline phosphatase of A. caespitosus, 7 – linearized plasmid DNA treated with BAP and T4 DNA ligase, 8 – linearized plasmid DNA treated with extracellular alkaline phosphatase of A. caespitosus and T4 DNA.

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thermostability observed for this enzyme than for the extracellular one (Guimarães et al. 2003b). The temperature optimum of the intracellular AP of A. caespitosus was higher than that produced by N. crassa (Say et al. 1996), P. chrysogenum (Politino et al. 1996) and H. grisea (Buainain et al. 1998). Elevated temperature optimum is favorable for biotechnological applications, because it reduces contamination and the reaction time. Other phosphatases with elevated temperature optimum have been reported also for S. thermophilum (Guimarães et al. 2001). The activity of the intracellular AP from A. caespitosus was dependent, similarly to the extracellular form (Guimarães et al. 2001), on divalent ions. Nearly the same was reported for the enzymes from P. chrysogenum (Politino et al. 1996), H. grisea (Buainain et al. 1998), S. thermophilum (Guimarães et al. 2001). Divalent ions are considered to be essential for both the catalytic activity and stability of various APs (Genge et al. 1998), Mg2+ being a potent AP activator and Zn2+ an inhibitor (Nakazato et al. 1997). EDTA promoted inhibition of activity, confirming that intracellular AP is a metalloenzyme. Both the extracellular and the intracellular forms of AP from A. caespitosus hydrolyzed rapidly the synthetic substrate 4-NPP; however, differences were observed for both enzymes regarding the hydrolysis of other substrates, such as Glc-1-P, UTP, ATP, glycerol 2-phosphate, ITP and O-phosphoamino acids. In addition, the intracellular AP produced by A. caespitosus was effective in removing 5´-phosphate of plasmid DNA without hydrolyzing the DNA molecule which was not observed for the extracelullar enzyme (data not shown). This can indicate the prospective use of this enzyme in recombinant DNA protocols as suggested for the AP activity of N. crassa by Pereira et al. (1995). 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