Isolation and characterization of alkaline phosphatase of Saccharopolyspora erythraea from fermentation broth of erythromycin production

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1 Indian Journal of Biotechnology Vol 3, October 24, pp Isolation and characterization of alkaline phosphatase of Saccharopolyspora erythraea from fermentation broth of erythromycin production Subhasree Bhattacharjee, Ananta K Das and Sunil K Mandal* Department of Pharmaceutical Technology, Division of Microbiology, Jadavpur University, Kolkata 7 32, India Received 18 July 23; accepted 14 November 23 Alkaline phosphatase having two ph optima (8.4 and 9.2) was excreted in substantial amount by Saccharopolyspora erythraea during erythromycin production and was precipitated from the broth with 6-8 % final saturation of ammonium sulphate. PAS and silver nitrate staining of SDS-gel electrophoresis depicted eight distinct bands of glycoproteins in the precipitate. Buffer A (ph 8.4) eluted the glycoproteins from native gels and showed four bands of phosphatase with optimum activity at ph 8.4 and four at ph 9.2. After three successive native gel electrophoreses and elutions, four isomers of ph 8.4 were isolated with Buffer A and four of ph 9.2 with Buffer B (ph 9.2). The eight isoenzymes of alkaline phosphatase were purified more than 2-folds and were characterized as glycoproteins of different molecular weights, turnover numbers and extensive sugar percentages in their molecules. Keywords: alkaline phosphatase, glycoprotein, native gel electrophoresis, PAS-staining, SDS-PAGE, Saccharopolyspora erythraea IPC Code: Int. Cl. 7 A 1 N 63/4 Introduction Alkaline phosphatase dephosphorylates inactive derivatives of many antibiotics in the final step of biosynthesis, and a direct relationship between intracellular enzyme level and antibiotic formation were well established 1-5. One non-specific alkaline phosphatase (optimum ph 9) increased sharply in the broth during stationary phase when chlortetracycline was produced at limiting concentration of inorganic phosphate 6. Alkaline phosphatase of Saccharopolyspora erythraea, having two ph optima (8.4 and 9.2) was found related to formation and excretion of erythromycin from inside to outside of the cells 7. The enzyme, therefore, might be two separate substratespecific enzymes or a single non-specific phosphatase giving peak activity at two phs. The role of alkaline phosphatase would not be well defined unless the enzyme is isolated in pure form. Present paper reports isolation and characterization of alkaline phosphatase of S. erythraea by gel electrophoreis. Materials and Methods Precipitation of Alkaline Phosphatase by Ammonium Sulphate After separation of cell mass, 12 hrs clear fermentation broth of glucose-sodium nitrate medium 7 *Author for correspondence: Tel: ; Fax: sunil_microbiologist@yahoo.co.in was treated with ammonium sulphate of 5, 6, 8 and 1% final saturations. The chilled precipitates from different saturations of ammonium sulphate were collected by cold centrifugations (39, g) for 2 min. Individual precipitate was dialysed for 3 hrs at 4ºC against two changes of Buffer A (ph 8.4), and was assayed for phosphatase activity and protein content. Four types of buffers [A & A 1 (ph 8.4), B & B 1 (ph 9.2)] were used for elution of the enzymes. Buffer A (.5 M Tris-HCl, ph 8.4) and Buffer B (.5 M glycine NaOH, ph 9.2) contained.2 mm PMSF (phenylmethyl sulphonate fluoride),.2 mm dithiothreitol and 1mM CaCl 2.2H 2 O. Without CaCl 2.2H 2 O, Buffers A and B were named as Buffers A 1 and B 1, respectively. Gel Electrophoresis The precipitate having two peaks of phosphatase activity at ph 8.4 and ph 9.2 was subjected to SDS- PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis), using 6 ma current. On completion of electrophoresis, the gel was stained by silver nitrate 8,9. The same precipitate was used for native PAGE at 4 to 6ºC. The native gel was cut into several pieces at 2 mm intervals, crushed and treated with different buffers. All the chemicals for electrophoresis and dyes were purchased from SRL Pvt. Ltd.

2 BHATTACHARJEE et al: ISOLATlON AND CHARACTERIZATION OF ALKALINE PHOSPHATASE OF S. ERYTHRAEA 559 Periodic Acid-SchifT (PAS) and Silver Nitrate Staining A staining technique with Periodic Acid-Schiffs reagent and immediately followed by silver nitrate lo,ll were used for staining of the alkaline phosphatase. After SDS-electrophoresis, gels were treated with 2% periodate and then gently agitated with concentrated Schiff's reagent (a mixture of 4 g pararosaniline and 6.8 g potassium metabisulphite dissolved in 2 rnl of.25 N HCl) until bands turned magenta. The magenta colour was reduced with 2% sodium metabisulphite and the decolourised gel was immediately stained with silver nitrate solutions. Enzyme Assay The activity of alkaline phosphatase at ph 8.4 and ph 9.2 was determined in a reaction mixture of a final volume of 2 ml containing.1 rn1 of enzyme source, 2.5 umole of disodium-p-nitrophenyl phosphate, 19 umole of a buffer and 4 umole of MgCh Tris-HCI buffer of ph 8.4 and glycine-naoh buffer of ph 9.2. The reaction mixtures were incubated at 37 C. After incubation for 3 min, 3 m1 of 1% trichloroacetic acid (TCA) was added and centrifuged at 3 rpm for 1 min. The level of released inorganic phosphate (P;) in the filtrate was determined by King's method'f in Beckman DU-64/UV spectrophotometer. Estimation content of the enzyme was estimated 13, using bovine serum albumin. Estimation of Sugar in Glycoprotein Neutral hexoses in glycoprotein were estimated by anthrone reagent method'" and n-glucose was used as standard. The absorbance was measured at 62 nm in Beckman DU-64/UV spectrophotometer. Determination of Molecular Weight and Turnover Number A mixture of myosin (2,), f3-galactosidase (116,25), phosphorylase (97,4), bovine serum albumin (66,2), ovalbumin (45,), carbonic anhydrase (31,), soybean trypsin inhibitor (21,5) and lysozyme (14,4) were used as marker proteins and purchased from Sigma. After SDS-gel electrophoresis, the bands of the marker proteins and glycoprotein bands of the enzyme were stained by Coomasie blue and PAS-silver stain, respectively. The mol wt of the unknown was calculated by its Rr value from the best-fit curve of Rj values versus log of mol wt of the marker proteins. Turnover number (molecular activity) of the enzymes was determined from mol wt of the enzymes and their specific activity at optimum ph, and was expressed as moles of inorganic phosphate (P;) per mole of the enzyme. Results and Discussion Alkaline phosphatase with two ph optima (8.4 and 9.2) increased more than 3-folds in precipitation from 6-8% final saturation of ammonium sulphate. The precipitate after SDS-gel electrophoresis and silver nitrate staining, showed a lot of protein bands including major and minor (Fig.I-E ). 1 To identify the active bands of alkaline phosphatase, native gel electrophoresis was performed and stained with fast-blue stain, which showed phosphatase to be diffused throughout the gel. The enzyme was then eluted from fresh native gels with different buffers (A, A), B and B(). Buffer A eluted maximum enzyme protein and resulted in 1 times increase of specific activity at ph 8.4, whereas Buffer B eluted less protein but increased enzyme activity at ph 9.2 (Table 1). The diffused activity of phosphatase led us to presume that the alkaline phosphatase might be glycoproteins. Therefore, eluates with Buffer A and B were concentrated by dehydration with the help of polyglycol bags. The concentrated enzyme after SDS- PAGE was stained by silver nitrate (Fig.l-E ) 2 and by PAS technique, which showed bands of glycoproteins within four PAS-positive zones (PAS-I, PAS-D, PASill & PAS-IV) (Fig.I-E ). 3 The positive zones were.m. M,... 2OO,OOO M~t16,25 Ml"'97,4 Fig.I-Pholographs of marker proteins (M), silver stain of broth proteins (E I ), silver stain of enriched phosphatases (Ej), PASzones (E3) and distinct glycoprotein bands in PAS-zones (Ea) after staining of SDS-gel electrophoresis

3 56 INDIAN J BIOTECHNOL, OCTOBER 24 immediately followed by silver nitrate staining and depicted eight distinct bands of glycoproteins, indicating three in PAS-I, two in PAS-II, two in PAS- III and a single band in PAS-IV (Fig.1-E 4 ). The identified different PAS-positive areas on fresh native gels were treated with Buffer A and Buffer B, and the respective eluates were analysed for protein, phosphatase activity and sugar content. From the results (Table 2) it appeared that Buffer A eluted more enzyme proteins and both the enzymes with optimum activity at ph 8.4 and 9.2; whereas Buffer B eluted less protein and only the enzyme of optimum activity at ph 9.2. Substantial amounts of sugars were measurable from all PAS-positive zones due to the glycoprotein structure of alkaline phosphatases. Subsequently, after native gel electrophoresis, each PAS-positive zone was treated with respective buffer and several aliquotes of each zone area were pooled together and dehydrated with polyglycol bags. The concentrated eluates were again subjected to native gel electrophoresis and each phosphatase +ve zone was cut into pieces according to the number of enzyme bands. Each band of the enzyme was isolated with buffer. In this procedure, after native gel electrophoresis and elution with respective Buffer A and Buffer B, each band of the enzyme was isolated in more purified form (Table 3). Buffer A (ph 8.4) isolated eight active isomers of the enzyme and identified both the bands of PAS-II and PAS-III as isoenzymes of ph 8.4 phosphatase, and three bands of PAS-I and the single band of PAS-IV as isoenzymes of ph 9.2 phosphatase. Buffer B (ph 9.2) eluted much less protein as compared to Buffer A (ph 8.4) and isolated only the three bands (PAS-I) and the single band (PAS-IV) in active form, which showed phosphatase activity optimum at ph 9.2 (Table 3). It is realized from the results that the isoenzymes of ph 8.4 phosphatase were unstable and have been inactivated at alkaline ph of Buffer B (ph 9.2). Electrophoretic analysis finally led us to isolate eight isoenzymes of alkaline phosphatase and purify more than 2-folds (Table 4). Substantial amounts of carbohydrate were found in their molecules and distinct molecular weight of each isoenzyme was determined by its R f value on SDS-PAGE from the best fitted curve of R f values versus log of mol wt of markers. The distinct molecular weights, turnover numbers and estimated percentages of sugar characterized alkaline phosphatase of S. erythraea to be comprised of two phosphatases with different ph optima(ph 8.4 and ph 9.2), each having four isoenzymes of different mol wt and glycoproteins in structure (Table 5). Table 1 Effect of different buffers on elution of alkaline phosphatase from native gels after electrophoresis of precipitate (from 4 ml broth with ammonium sulphate of 6-8% final saturation) Buffer Enzyme activity(pi) (μmole) Specific activity (μmole/mg) Broth(4 ml) Enzyme precipitate Buffer A(pH 8.4) Buffer A 1 (ph 8.4) Buffer B(pH 9.2) Buffer B 1 (ph 9.2) Table 2 Estimation of phosphatase activity, protein and sugar content of alkaline phosphatase of S. erythraea in four PAS-positive zones after two successive native gel electrophoresis and elution with buffer Eluant Buffer A (ph 8.4) Buffer B (ph 9.2) PAS +ve zone PAS I PAS II PAS III PAS-IV PAS I PAS II PAS III PAS-IV Enzyme precipitate (mg ) Phosphatase activity (P) (μmole) Specific activity (μmole/mg) Sugar in glycoprotein

4 BHATTACHARJEE et al: ISOLATION AND CHARACTERIZATION OF ALKALINE PHOSPHATASE OF S. ERYTHRAEA 561 Table 3 Phosphatase activity, protein and sugar contents of each eight isomers of alkaline phosphatase after three successive native gel electrophoresis and elution with buffer Eluant PAS +ve zone Glycoprotein band Sugar Phosphatase activity (P i ) (μmole) Specific activity (μmole/mg) Enzyme precipitate Buffer A (ph 8.4) Ia PAS-I Ib Ic PAS-II IIa Iib PAS-III IIIa IIIb PAS-IV IV Buffer B (ph 9.2) Ia PAS-I Ib Ic PAS-II IIa Iib PAS-III IIIa.1.12 IIIb.9.2 PAS-IV IV Table 4 Purification of alkaline phosphatase of S. erythraea in successive gel electrophoresis and elution with buffer Successive gel electrophoresis & elution with buffer Broth (4ml) Enzyme precipitate 1 st gel electrophoresis: Buffer A Buffer B 2 nd gel electrophoresis: Buffer A Buffer B 3 rd gel electrophoresis: Buffer ApH 8.4 phosphatase (PAS-II &III) ph 9.2 phosphatase (PAS-I &IV) Buffer BpH 8.4 phosphatase (PAS-II &III)pH 9.2 phosphatase (PAS-I &IV) Yield of protein(%) Specific activity (μmole/mg) Increase of sp. activity (folds) Table 5 Molecular weights, turnover numbers (K cat ), percentages of sugar in molecules and R f values of eight isomers of alkaline phosphatase of S. erythraea after final purification by gel electrophoresis & elution with buffer A PAS +ve zone Isomers (ph optima) PAS-I Ia (ph 9.2) Ib (ph 9.2) Ic (ph 9.2) PAS-II IIa (ph 8.4) Iib (ph 8.4) PAS-III IIIa (ph 8.4) IIIb (ph 8.4) % of Sugar in glycoprotein Turnover number (K cat ) (min 1 ) Isomers of alkaline phosphatase Molecular weight (Dalton) , , , , , , ,258.5 R f Marker protein Mol. Wt. (Dalton) 2, a 116,25 b 97,4 c 66,2 d R f.39 a.29 b.255 c 45, e.67 f.49 e.412 d 31, f 21,5 g.849 g PAS-IV IV (ph 9.2) , ,4 h.98 h Superscripts a, b, c, d, e, f, g & h denote Myosin, β-galactosidase, Phosphorylase B, Bovine serum albumin, Ovalbumin, Carbonic anhydrase, Soybean trypsin inhibitor and Lysozyme respectively. Authors reported 7 two alkaline phosphatases of S. erythraea with different ph optima (8.4 and 9.2) and a direct relationship with erythromycin production. The enzymes were Ca ++ -dependent, and activated and

5 562 INDIAN J BIOTECHNOL, OCTOBER 24 modulated by Ca ++, which in turn did serve as a messenger and drived the enzymes to move along with erythromycin from inside to outside of the cells. Another biosynthetic role of intracellular alkaline phosphatase was elucidated when D-glucose was taken up inside the cells as glucose-6-phosphate. Above certain concentration, glucose-6-phosphate repressed the alkaline phosphatases. But one isoenzyme of optimum ph 8.4 hydrolysed glucose-6-phosphate and regulated its limited concentration, providing D-glucose for the synthesis of neutral sugars in the molecule of erythromycin 7. The synthesis of neutral sugars from D- glucose was described earlier 15. The isoenzyme of optimum ph 8.4 regulated the required concentration of D-glucose for neutral sugar synthesis and thus relieved the repressive effect of glucose-6-phosphate on other isoenzymes of alkaline phosphatase involved in erythromycin biosynthesis. The biosynthetic roles were evaluated before isolation and characterization of the enzyme. After the present study, where alkaline phosphatase comprised eight isoenzymes of different mol wts, it is realized that each of the isoenzymes would have played a role in the pathway to erythromycin biosynthesis. There is no doubt that present work will be helpful in future to evaluate the individual role of the isoenzymes in erythromycin biosynthesis. Acknowledgement Financial help from UGC, New Delhi is acknowledged. References 1 Majumdar M K & Majumdar S K, Relationship between alkaline phosphatase and neomycin formation by S. fradiae, Biochem J, 122 (1971) Miller A L & Walker J B, Accumulation of streptomycin in cultures of streptomycin producers grown in a high phosphate medium, J Bacteriol, 14 (197) Pass L & Raczynska-Bojanowska K, Inhibition mechanisms of viomycin synthesis by inorganic phosphate, Acta Biochim Pol, 15 (1968) Mertz F P & Doolin L E, Effect of inorganic phosphate on the biosynthesis of vancomycin, Can J Microbiol, 19 (1973) Martin J F et al, Proc 3 rd Int Symp Genetics of industrial microorganisms, edited by O K Sobek & A L Laskin (American Society for Microbiology, Washington DC) 1979, Malik V S, Genetics and biochemistry of secondary metabolism, Adv Appl Microbiol, 28 (1982) Bhattacharjee S et al, Alkaline phosphatase and erythromycin production by Saccharopolyspora erythraea, Indian J Microbiol, 42 (22) Blum S, Beier H & Gross H J Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8 (1987) Switzer R C 3 rd, Merril C R & Shilfrin S A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels, Anal Biochem, 98 (1997) Zacharius R M, Zell T E, Morrison J H & Woodlock J J, Glycoprotein staining following electrophoresis on acrylamide gels, Anal Biochem, 3 (1969) Jay G D, Culp D J & Jahnke M R, Silver staining of extensively glycosylated proteins on sodium dodecyl sulphate polyacrylamide gels: Enhancement by carbohydrate-binding dyes, Anal Biochem, 185 (199) King E J, The colorimetric determination of phosphorus, Biochem J, 26 (1932) Lowry D H Rosenbrongh N J, Faar A L & Randal R J, measurement with Folin Phenol reagent, J Biol Chem, 193 (1951) Robert G S, Analysis of sugars found in glycoproteins, in Methods in enzymology, Vol III, edited by E F Neufield & V Ginsburg (Academic Press, New York) 1996, Butte J C & Corcoran J W, The biogenesis of desosamine, an aminotrideoxy sugar in erythromycin, Fed Proc, 21 (1962) 89.