Two distinct molecular forms of phytase from Klebsiella aerogenes: Evidence for unusually small active enzyme peptide

JOURNal. OF FERMENTATION AND BIOENGINEERING

Vol. 77, No. l, 23-27. 1994

Two Distinct Molecular Forms of Phytase from Klebsiella aerogenes: Evidence for Unusually Small Active Enzyme Peptide SUHAS M. TAMBE, 2 G A N G A D H A R S. KAKLIJ, I* SHASHIKANT M. KELKAR, 1 A~D LALIT J. P A R E K H 2 Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Bombay 4000851 and Department of Biochemistry, University of Baroda, Baroda 390002, 2 India Received 23 July 1993/Accepted 1 November 1993 The cell free extract from Klebsiella aerogenes could be resolved into two distinct fractions on DE-52, by isoelectrofocusing technique and on Sephadex G-200, one eluting in the void volume and the other at the end of the column run. The molecular weight of the second peak was 10-13 kDa and could possibly be a fragment of the native enzyme. The two species of the enzyme thus isolated differed in their Kin, pH optima, pI values and temperature sensitivity. Evidence is provided to show that low molecular weight enzyme is generated during isolation. This is one of the rare instances where such a small fragment of enzyme peptide retained a full complement of enzyme activity.

MgSO4 and 0.025 g yeast extract in a total volume of 100 ml. For induction of phytase the medium was supplemented with different carbon sources at a concentration of 0.25% w/v except the substrate phytate which was 2%. The culture was maintained on agar slants of the medium containing 2% phytate. The cells were grown overnight at 30°C in 20 ml aiiquots of medium containing carbon source. This was used as inoculum for induction of phytase in two liter flasks containing one liter medium at 30°C for 7 h. Preparation of cell free extract [CFE] The cells harvested by centrifugation (12,000 x g), were suspended in 10mM Tris-HCl buffer pH 8.0 containing 2 mM EDTA, 1 mM 2-mercaptoethanol, (buffer-A). The cells were then lysed by sonication at 20 kHz for 20 min. The lysate when centrifuged at 105,000xg for 1 h showed total phytase activity of extract in supernatant and therefore, routinely lysate was centrifused at 17,500xg and supernatant was termed as CFE. Phytase assay The assay reaction mixture contained 100 ~mol acetate pH4.5, 0.4pmol sodium phytate and enzyme in a final volume of 2.0 ml. After incubation at 60°C for 5 min the reaction was terminated by adding an equal volume of 10% TCA. The protein-free supernatant was estimated for liberated Pi by Fiske and Subba Row method (10) using potasium phosphate (monobasic) as standard. Soluble protein was measured by the method of Lowry et al. (1 l) using bovine serum albumin (BSA) as standard. A unit of enzyme activity is defined as the amount of enzyme that liberated 1 pmol of Pi min-X under the assay conditions. Isoelectrofocusing This was carried out in the pH range of 3.5 to l0 using 1 l0 ml LKB isoelectrofocusing column as described earlier (12). The pH gradient was developed by prefocusing at 300V for 18 h at 5°C with initial current of 6 mA which dropped to 0.5 mA at the end of the run. The enzyme preparation in 25% sucrose was applied onto the middle of the prefocused column and was again electrofocused for 18 h. The column was then emptied by collecting one ml fractions. The pHs of the fractions was determined at room temperature. The enzyme activity was estimated at 60°C as stated earlier.

Phytate or myo-inositol hexakis dihydrogen orthophosphate is one of the major phosphorus storage compounds in the seeds of higher plants, mostly cereals and legumes (1-5). The presence of phytate in dietary food may lead to deficiency of minerals such as Zn, Ca, Mg, Fe and inorganic phosphate (Pi), due to their chelation. The removal of phytate from food thus becomes of great importance. Germination (5) or fermentation are known to reduce the phytate content of cereals and pulses. Microbial phytases [myo-inositol hexakis dihydrogen phosphatase [EC 3.1.3.8] have been implicated in the breakdown of phytate to myo-inositol and Pi during fermentation (6-9). Various microorganisms such as fungi and bacteria are known to synthesize phytase. It is an inducible enzyme and its formation by various microorganisms is known to be influenced by the levels of inorganic phosphate (Pi), phytate and inositol. A strain of Kiebsiella aerogenes was isolated from the bajra grains, in our laboratory. This grew on phytate when provided as the sole source of carbon and phosphorus. This work was extended further since very little is known about the properties and regulation of cellular phytase from K. aerogenes. The present paper describes two distinct forms of active enzyme, one with an unusually low molecular weight. The formation, purification and differences in properties of the same have been brought out. MATERIALS AND METHODS

The substrate and other biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The gel filtration media were obtained from Pharmacia Fine Chemicals (Uppsaia, Sweden). DE-52 was a product obtained from Whatman (Whatman Ltd., England). All other chemicals used were of analytical grade and were obtained from standard sources. Growth and culture conditions K. aerogenes isolated from bajara grains at Department of Biochemistry, University of Baroda, was grown in a medium containing 0.7g K2HPO4; 0.2g KH2PO4; 0.05 g NH4NO3; 0.02g * Corresponding author. 23

24

TAMBE ET AL.

J. FERMENT.Bzom~o., RESULTS

Induction of phytase F r o m the different carbon sources tried, only p h y t a t e and inositol could significantly induce phytase (Table 1). Highest activity was observed with phytate and decreased with inositol, acetate and nutrient b r o t h in decreasing order. However, D-glucose or citrate were ineffective. The induction o f the enzyme by nutrient b r o t h is not unusual since Aerobactor aerogenes (13), now called as K. aerogenes also showed induction. In the case o f Klebsiella sp. 2, only phytate was f o u n d to induce phytase activity, while inositol could not induce any enzyme activity (14). In o r d e r to o b t a i n sufficient cell growth a n d easy availability, inositol was used as the inducer in the studies further described here. Evidence for two distinct active forms of enzyme Ion exchange chromatography on DE-52 The elution profile o f C F E on DE-52 shown in Fig. 1, indicated that phytase activity could be resolved into two forms. One was u n b o u n d a n d a p p e a r e d in washings with bufferA. A m o n g the several protein peaks eluted b y the 0.0-0.5 salt gradient, only retained protein that eluted at 0.26 M NaCI had phytase activity.

Gel permeation chromatography and molecular weights of the two forms The gel filtration o f the C F E on Sephadex G-200 also indicated presence o f two enzyme peaks as shown in Fig. 2A, one eluting in the void volume TABLE 1. Effect of different carbon sources on growth and phytase activity Growth

Phytase activity (units O.D. -1)

Carbon source

(O.D-~0nm)

D-Glucose Citrate Acetate Inositol Sodium phytate Nutrient broth

0.27_+0.015 0.09_+0.010 0.15_+0.015 0.27 _+0.005 0.03 _+0.011 0.56_+0.017

and the other eluting in the total volume o f the column. The DE-52 u n b o u n d fraction concentrated by a m m o nium sulphate precipitation when subjected to gel filtration on the same Sephadex G-200 column eluted at the position o f the second peak. The molecular weight o f the second peak as determined b y calibrating the column (Fig. 2B), was very low and therefore eluted at the end o f run, just after cytochrome c, indicating a molecular weight less t h a n 12.8 kDa. W h e n this fraction was subjected to A m i c o n ultrafiltration using a YM-10 m e m b r a n e having an exclusion limit o f 1 0 k D a , the enzyme remained on the m e m b r a n e . Thus, these studies indicated that the molecular weight o f this fraction was greater than 10 kDa, and less than 13 kDa. The desalted b o u n d fraction eluted in the void volume o f G-200 suggesting the molecular weight to be greater t h a n 600 kDa. This was then fractionated on Sepharose 6B (Fig. 3A). The molecular weight o f the protein in the present studies, calculated b y calibrating the Sepharose 6B column, was a p p r o x i m a t e l y 700 k D a (Fig. 3B). IsoelectricpHofthe two forms The C F E , when subjected to isoelectrofocusing, showed presence o f two different fractions corresponding to pI values a p p r o x i m a t e l y o f 3.7 and 10.5 as shown in Fig. 4. The gel filtration o f the two fractions obtained f r o m electrofocusing column on Sephadex G-200 indicated that the fraction with pI 3.7 was the high molecular species (700 kDa), while one with a pI o f 10.5 corresponded to the low molecular species (10-13 kDa) [data not presented]. Mechanism of generation of low molecular weight species The high molecular weight species obtained in void volume on Sephadex G-200 was treated with 0.15 M

0.0 0.0 1.20_+0.115 3.74 _ 0.150 11.66_+0.524 1.01 _+0.066

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carbon sources at 0.25% w/v, except phytate which was used at 2% w/v, with identical inoculum and phytase activity was estimated. The values are mean_+S.E. of three experiments. 1.5

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FIG. 1. DE-52 ion exchange chromatography. Pre-swollen DE52 was suspended in buffer-A. The column (49 × 2.5 cm) was packed and equilibrated with the same buffer. The CFE in the same buffer was passed through the column and washed with a bed-volumes of the same buffer. The adsorbed proteins were eluted by a linear gradient of 0 to 0.5 M NaCI in equilibrating buffer. Fractions of 6 ml were collected, at flow rate of one ml rain -1 and were assayed for protein (---9 and phytase activity (--). Active fractions were pooled and used in further experiments.

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FIG. 2. (A) Gel filtration on Sephadcx G-200. The enzyme prcparation (10.0 rag) was loaded on the gel column (100 x 1.5 cm) pre-equilibrated with buffer A and was washed with three bed-volumes of the same buffer. Fractions of 5.0 ml each were collected at flow rate of 0.3 ml rain -1. (B) Calibration curve. Sephadex G-200 column described above was calibrated using aldolase (140 kDa), hexokinase (96 kDa), bovine serum albumin (68 kDa), ovalbumin (46 kDa) and cytochrome C (12.4kDa). The arrow indicates the position for phytase.

VOL 77, 1994

PHYTASE FROM K. AEROGENES

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KCI and/or 0.005% Triton X-100. This treated preparation, when passed through the same column, resulted in an additional peak eluting at the position of the low molecular weight fraction (Fig. 5). The high molecular weight fraction obtained from Sephadex G-200 column having a pI 3.7, when stored at 5°C for 7.0d and electrofocused as described earlier, resulted in a single peak as shown in Fig. 6. It appears at a position corresponding to pI 10.5, clearly pointing to the conversion of high molecular weight species of enzyme into low molecular weight fraction. Properties of the two e n z y m e species

Kinetic properties

The two species differed in their substrate saturation kinetics and had Km values 0.062 and 3,5

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loss o f their activity after 680C were overlapping. The

inset shows the profile between 52-80°C, for 10 rain incubation, at every 2°C rise of temperature, for low molecular weight form. pH optima The optimum pH for the activity of low molecular weight enzyme was found to be 5.2 while that of the high molecular weight enzyme species, was 4.5 (Fig. 8). The crude preparation showed activity in broad pH range with optima at 4.5. Both were active over a pH range of 3.6 to 6.0. Specificity The substrate specificity of the low molecular weight enzyme was examined after DE-52 chromatography since it was sufficiently pure at this stage. Among the various substrates including glycolytic intermediates, nucleotide phosphates tried at concentra-

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0.114mM for high and low molecular weight species, respectively. Thermo-stability The stability of the two enzyme species was checked by keeping aliquots of the enzyme at different temperatures (30-70°C) for 5 min or 10 min and were assayed for activity. The activity thus estimated was compared with that of an aliquot which was kept at 0°C. The two species gave inactivation patterns as shown in Fig. 7. The high molecular weight species was relatively stable, retaining 85% and 75% activity after exposure for 5 and 10 min, respectively, over a wide range of temperatures. On the other hand, the low molecular weight enzyme was labile and lost 70% activity after exposure to 30°C which was constant upto 600C. However, it showed activation at 63°C yielding 60% and 70% activity after exposure of 10 and 5 min, respectively. The profiles of

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FIG. 5. Sephadex G-200 gel filtration of KC1 and detergent treated void volume peak from previous Sephadex G-200.

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26

TAMBEET AL.

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FIG. 7. Thermo-stability. The aliquot of the enzyme adjusted to 1 mg/ml with BSA was incubated at indicated temperatures in buffer-A for 5 or 10 rain and was assayed for enzyme activity. High molecular weight fraction (©, • ), low molecular weight fraction (o, • ) for 5 or 10 min respectively. Inset shows the data for every 2°C rise, between 50-80°C, for 13 kDa fraction incubated for 10 min only. tion of 25 raM, only p-nitrophenyl phosphate (p-NPP) and phytate could serve as substrates for this enzyme. The activity towards p N P P was 45% that of phytate. The content of free phosphate in each substrate was determined by running 0 time controls. DISCUSSION The foregoing results, while describing some of the properties of the phytase from K. aerogenes have brought out the presence of two different forms of phytase from K. aerogenes one possibly native, of 700 kDa and the other, a fraction of the native enzyme which has full complement of activity with an exceedingly low molecular weight between 10 and 13 kDa. This is presumably the first instance where such a very small fraction of the enzyme is shown to exhibit the enzyme activity, suggesting intact active site, The conversion of high molecular weight species into small molecular weight active enzyme species after salt/ detergent treatment or storage supports the notion that this fraction originated during the isolation procedure, possibly due to cleavage of some sensitive peptide linkage. Despite the cleavage and size of the peptide, it is note-worthy that it is enzymatically active. The significant differences observed in Kin, p H optima, thermo-

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FIG. 8. pH activity profiles of high and low molecular weight enzyme fractions. In the range of pH 2.2-3.6, a 50 mM glycine.HCI and in the range of 3.8-6.0 a 50 mM succinate.NaOH buffers were used. High molecular weight fraction (----) and low molecular weight fraction (--). Vertical bars represent the ---S.E.

stability and pI values are consistent with the sizes of the proteins and their isoelectric pHs. It is quite clear from the data that the 700 kDa protein has a pI of 3.7 and therefore, was retained on DE-52 at pH 8.0. It forms floculant turbidity at pH 4.5 in the absence of substrate with significant loss of activity. However, this loss of activity could be prevented in the presence of the substrate (data not shown) and it was also fairly stable to heat inactivation. On the contrary, the small enzyme peptide fraction (10--13 kDa), having a pI 10.5, was not retained by DE-52 and was not sensitive to lower pH. Phytases from fungi are known to be extracellular (15, 16), while those from bacteria are intracellular with the exception of Bacillis subtilis (13, 14, 17, 18). The CFE, on ultracentrifugation at 105,000 × g for 1 h, showed presence of total phytase activity in the supernatant suggesting that the phytase from K. aerogenes was cytoplasmic (data not shown). Phytases from other microbial sources which have been purified show wide range of differences in their pH optima (pH2.5 to 7.6), molecular weights (37--490 kDa), thermo-stability (60--75°C) and substrate specificity (14, 17-24). The pI of 3.7 for 700 kDa fraction is close to the pI of 4.6 for Aspergillus ficuum NRRL 3135 phytase having molecular weight of 85-100 kDa (21). The K. aerogenes phytase, similar to that of Pseudomonas did not show activity against /%glycerophosphate and other phosphorylated glycolytic intermediates, nor nucleotide phosphates except paranitrophenyl phosphate (18). However, the phytase from Schwanniomyces castellii, even though preferentially hydrolysed phytate, also showed activity with other glycolytic intermediates and nitro phenol phosphates (19). The pH optima and Km values for K. aerogenes phytase are well within the reported range. However, the molecular weight of the native enzyme was significantly higher than that of Schwanniomyces castellii (19), and further, there is no report so far suggesting the presence of either isozymes of enzyme or such a low molecular weight forms of phytase. The possibility that 700 kDa fraction could be formed by non-covalent association of 13 kDa enzyme with other larger protein can not be ruled out. This possibility remains to be ascertained, since 700 kDa phytase fraction posed problem of its convertion to 13 kDa fraction during further processing to get the homogeneous preparation.

Voz. 77, 1994

PHYTASE FROM K. AEROGENES ACKNOWLEDGMENT

Research fellowship to Mr. Suhas Tambe, for conducting this work as part of his Ph.D. degree was from CSIR. REFERENCES 1. Lott, J. N.A.: Accumulation of seed reserves of phosphorus and other minerals, p. 139-166. In Murray, D. R. (ed.), Seed physiology, vol. 1. Acad. Press, Sydney (1984).

13. 14. 15.

2. Gopalan, C., Ramasastri, B., and Balasubramaniam, S.C.: 3. 4.

5. 6.

Nutritive value of Indian food, p. 125-131. National Institute of Nutrition, ICMR, Hyderabad (1978). Nagal, Y. and Funahaskl, S.: Phytase activity from bran: purification and substrate specificity. Agric. Biol. Chem., 26, 794-803 (1962). Adams, C.A. and Novellie, L.: Acid hydrolases and autolytic properties of bodies and spherosomes isolated from ungermihated seeds of Sorghum bicolor (finn.). Moench. Plant Physiol., 55, 7-11 (1975). Mandal, N. C. and Blawas, B. B.: Metabolism of inositol phosphates. II. Phytase synthesis during germination in cotyledons of mung beans (Phaseolus aureus). Plant Physiol., 45, 4-7 (1970). Naevert, B., Sandstrom, B., and Cederblad, A.: Reduction of phytate content of bran by leavening in bread and its effect on Zn absorption in man. Br. J. Nutr., 53, 47-53 (1985).

16. 17. 18.

19. 20.

7. Ramakrlshnan, C.V., Parekh, L.J., Akolkar, P.N., Rao, 8. 9.

10. 11. 12.

G.S., and Bhandari, S.D.: Studies on soy idli fermentation. Plant Foods Man., 12, 15-33 (1976). Sutardi and Buckle, K.A.: Reduction in phytic acid levels in soybeans during tempeh production, storage and frying. J. Food Sci., 50, 260-263 (1985). Dhankher, N. and Chauhan, B.M.: Effect of temperature and fermentation time on phytic acid and polyphenol content of rabadi, a fermented pearl millet food. J. Food Sci., 52, 828829 (1987). Flake, C. M. and SubbaRow, Y. T.: The colorimetric determination of phosphorus. J. Biol. Chem., 66, 375-400 (1925). Lowry, O.H., Rosebrongh, N.J., Farr, A.L., and Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). Menezes, L., Kelkar, S.M., and Kakllj, G. S.: Glucose-6-phos-

21. 22.

27

phate dehydrogenase and 6-phosphogiuconate dehydrogenase from Lactobacillus casei: responses with different modulators. Ind. J. Biochem. Biophys., 26, 329-333 (1989). Greaves, M. P., Anderson, G., and Wehley, D. M.: The hydrolysis of inositol phosphates by Aerobacter aerogenes. Biochim. Biophys. Acta, 132, 412-418 (1967). Shah, V. and Parekh, L. J.: Purification and properties: phytase from Klebsiella sp. no-2. Ind. J. Biochem. Biophys., 27, 98-102 (1990). Shleb, T. R., Wodzlnskl, R. J., and Ware, J. H.: Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillusficuum. J. Bacteriol., 100, 1161-1165 (1969). Howson, S. G. and Darts, R. P.: Production of phytate hydrolysing enzyme by some fungi. Enzyme Microb. Technol., 5, 377382 (1983). Powar, V. g. and Jagannathan, V.: Purification and properties of phytate-specific phosphatase from Bacillus subtilis. J. Bacteriol., 151, 1102-1108 (1982). Irving, G. C. ,L and Cosgrove, D. J.: Inositol phosphate phosphatases of microbiological origin: some properties of a partially purified bacterial (Pseudomonas sp.) phytase. Aust. J. Biol. Sci., 24, 544-557 (1971). Lanrent, S., Christel, L., Helene, B., Guy, M., and Pierre, G.: Purification and properties of the phytase from Schwanniomyces castellii. J. Ferment. Bioeng., 74, 7-11 (1992). Irving, G. C.J. and Cosgrove, D.J.: Inositol phosphatases of microbiological origin: some properties of the partially purified phosphatases of Aspergillusficuum NRRL 3135. Aust. J. Biol. Sci., 27, 361-368 (1974). Ullah, A. H. J. and Gibson, D.M.: Extracellular phytase (EC 3.1.3.8) from Aspergillusficuum NNRL 3135. Purific. Characterizat. Prep. Biochem., 17, 63-91 (1987). Yamada, K., Mlnoda, Y., and Yamamoto, S.: Phytase from Aspergillus terreus. I. Production, purification and some general properties of the enzyme. Agric. Biol. Chem., 32, 1275-1282

(1968). 23. Skowronaskl, T.: Some properties of partially purified phytase from Aspergillus niger. Acta Micro. Pol., 27, 41--48 (1978). 24. Yamamoto, S., Minoda, Y., and Yamada, K.: Chemical and physicochemical properties of phytase from Aspergillus terreus. Agric. Biol. Chem., 36, 2097-2103 (1972).