Partial Purification and Characterization of Invertase - Philippine ...

Philippine Journal of Science132 (2): 129-136, December 2003ISSN 0031 - 7683Partial Purification and Characterizationof Invertase from Flowers ofMadhuca longifolia (Mi)M.K.B Weerasooriya and H.P. YatawaraDepartment of ChemistryUniversity of Kelaniya, Sri LankaEnzyme Invertase (β-D-fructofuranosidase, EC 3.2.1.26), present in the flowers ofMadhuca longifolia plays an important role during the preparation of fermented Ayurvedicdrugs known as ‘Arishta’. This enzyme was partially purified with a yield of 11.6%, using(NH 4) 2SO 4fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulosechromatography at pH 6.5 and 4.2. The molecular mass of the enzyme as determined byelution through Sepharose 4B gel column was found to be about 210 kDa . The enzymeshowed a broad pH optimum between 4 -7 . Optimum assay temperature was 37ºC andabove 45ºC enzyme activity slowly declined and inactivated around 80ºC. ApproximateKm value for the enzyme for sucrose is 125 mM.Keywords: purification, characterization, invertase, Madhuca longifoliaIn ayurveda, fermentation processes have been usedfor ages to modify the potency of drugs and to extractand to preserve active constituents from medicinalplants. Therapeutic application of these fermenteddrugs are based on the properties of ingredientsand the method of preparation. In general, thesefermented ayurvedic preparations posses a moderatepotency and administered in the convalescent phaseof a disease (Namijoshi 1979, Caraka Samhita 1949).One such fermented preparations known as ‘Arishtas’are widely used in Sri Lanka as well as in otherSouth Asian countries. ‘Arishtas’ are weak alcoholicpreparations used for the treatment of a wide variety ofdiseases and specially, prescribed for diseases whichrequire long term treatments. Since, Arishtas are notperishable very often they are used as tonics (Kroeset al. 1992, Kroes 1990, Ayurvedic Pharmacopoeia1976 &1979).In the preparation of many Arishtas, dried flowersof Woodfordia fruticosa (L.) Kurz ( Malitta in Sinhalese,Dataki in Sanskrit; Family, Lythraceae), or Madhucalongifolia (Mi in Sinhalese; Family, Sapotaceae) areused to enhance the sucrose hydrolysis which leads toproduction of alcohol (Caraka Samhita 1949, AyurvedicPharmacopoeia 1976 &1979, Hand Book of DomesticMedicine 1978, Kroes 1990). Thus, produced alcoholcontributes preservation of drugs and also promotesthe extraction of biologically active components fromplant materials and absorption of active componentsfrom gastro-intestinal tract (Kroes 1990, AyurvedicPharmacopoeia 1976 &1979).So far, little is known about these enzymes. Hence,we aimed to purify and characterize the endogenousinvertases of the above two sources in view ofproviding better insight to rationalize, standardize andoptimize the quality and preparation procedures ofAyurvedic drugs used in Sri Lanka.In a previous work we reported the purification andproperties of invertase from the flowers of Woodfordiafruticosa (Weerasooriya and Yatawara 2002).Here, we present the kinetic properties of partiallypurified of invertase of Madhuca longifolia.129

Weerasooriya & YatawaraMaterials and MethodsApparatusMicrobiological work was carried out under sterileconditions in a Labcare microbiological cabinet.Sterilization of the culture media, glass ware and otherconsumables prior and subsequent to use was carriedout in an autoclave at 15 p.s.i for 30 minutes.ChemicalsAll the bio-chemicals were purchased from FlukaChemical company, Sweden. All other chemicals usedwere of analytical grade.Plant materialsFlowers of Madhuca longifolia (Mi) were collectedfrom the garden of Pallkelle Ayurveda Hospital. Theywere identified by Mr. D.B.M.U. Dhanasekera andMrs.R.W. Edirisinghe by comparing with authenticsamples in the National Herbarium at PeradeniyaBotanical Gardens, Kandy. The collected flowers werecleaned and stored in the refrigerator.Enzyme purificationAll the purification steps were carried out at roomtemperature unless otherwise specified.Preparation of the crude extractMicroorganisms presence on the surface of theMadhuca longifolia flowers were removed by washingthe flowers with sterile distilled water. In each washing,the flowers were shaken for about 30 minutes and thisprocess was continued for nearly 55 times. The flowers(120 g) were next immersed in citrate buffer (pH 5.2,50 mM, 75.0 ml) containing 1 mM EDTA and 5 mM2-mercaptoethanol , ground with mortar and pestleand the crude extract thus obtained was centrifugedat 15,000 g for 30 minutes at 1°C. The supernatantsolution was filtered through a sterile filter paper, (0.22µm). A small aliquot which obtained from the filtrateswas cultured in nutrient Agar (NA) and potato dextroseagar (PDA) and incubated for 2 days at 37°C to checkfor the presence of any microflora.Since, no microflora was observed, above filtratewas used for the following ammonium sulphatefractionation.(NH 4)2 SO 4 fractionationInvertase in the crude extract was precipitatedat 1°C between 50-95% saturation of (NH 4)2 SO 4 ,centrifuged at 12,000g for 30 min and resuspended incitrate buffer (pH 5, 50mM) containing 1mM EDTA and5mM 2-mercaptoethanol. The enzyme preparationswere then stored in the freezer until required for furtherpurification and other analysis.Sepharose 4B gel filtration chromatographyEnzyme fraction was further purified by passingthrough a Sepharose-4B column (58x2cm) and elutedwith citrate buffer (pH 5.2, 50mM) containing 1mMEDTA, 5mM 2-mercaptoethanol and 50mM NaCl at aflow rate of 20ml/hr. Fractions (2 ml) were collected andthe enzyme activity was assayed.DEAE Cellulose chromatography at pH 6.5Active fractions eluted from sepharose-4B columnwere pooled and purified on a DEAE Cellulose column(12x 3cm) eluting with phosphate buffer (50mM, pH 6.5)containing 1mM EDTA and 5mM 2-mercaptoethanol.The enzyme was eluted with same buffer at a flow rateof 20ml/hr. Fractions (1.5 ml) were collected and theenzyme activity was assayed.DEAE Cellulose chromatography at pH 4.2Active fractions were pooled and chromatographedon second DEAE column (9 x 2.2 cm) eluting at a flowrate of 18ml/hr, with a linear gradient of 0-1M NaCl addedto citrate buffer (50mM citrate, pH 4.2) containing 1mMEDTA and 5mM 2-mercaptoethanol. Fractions (1.5 ml)were collected and the enzyme activity was assayed.The active fractions were pooled and concentrated byosmosis against solid polyethylene glycol.Sepharose 4B gel filtration chromatography(final step)Concentrated enzyme fraction was further purifiedby passing through a sepharose 4B column (12 x 1 cm)eluted with citrate buffer (pH 5.2, 50mM) containing1mM EDTA, 5mM 2-mercaptoethanol and 150mMNaCl. Fractions (1.0 ml) were collected and the enzymeactivity was assayed.Protein estimationQuantitative estimation of protein after each stepof purification was done by Bradford method (Scopes1982) using bovine serum albumin as the standardprotein.Invertase AssayActivity of invertase was assayed by Nelson’smethod as follows:The reaction mixture in a total volume of 2.5mlcontained citrate buffer (100mM, pH 5, 1.3ml)containing 1mM EDTA and 5mM 2-mercaptoethanoland sucrose (5%, 1.00 ml) . The reaction carried out at37 °C for 3 min, was initiated by the addition of enzyme(200µl , 1 nkat). Then Nelson’s reagent (1.00ml) was130

Partial Purification and Characterization of Invertase of M. longifolia flowersadded to the reaction mixture, boiled for 20 minutesand cooled to room temperature. Arseno-molybdatereagent (1.00 ml) was added and the mixture wasdiluted with water up to 10ml. Then absorbance wasmeasured at 510 nm.The standard curve was constructed for knownconcentration of product glucose against the absorbanceand the amount of glucose was determined. Theenzyme activity is expressed as as nmole of sucrosereacted min -1 under specified conditions.Determination of the molecular weightThe molecular mass of the purified enzymewas estimated by gel filtration through a column ofSepharose 4B, which had previously been calibratedwith Urease (340 kDa), Alcohol Dehydrogenase (141kDa), Bovine Serum Albumin (66 kDa), Egg albumin (45kDa), Pepsin (34 kDa) and Trypsin (24 kDa).For the characterisation of the purified enzyme,following experiments were carried out under thestandard assay conditions (1 nkat in assay). All theexperiments were performed three times in duplicatesunder the same conditions and mean standard deviationwere calculated.PH optimaThe optimum pH was determined by assaying theenzyme activity at different pH ranging from 2.6 to9.49 using the buffers glycine-HCl (pH 2.6), Citric acid-Na 2HPO 4(pH 3-7.5) and Tris-HCl (pH 8-8.9). Blankdeterminations were done simultaneously at all pH’swithout the enzyme.Temperature optimaThe optimum temperature was studied by assayingthe enzyme at different incubation temperaturesranging from 25°C to 65°C. Blank determinations weredone simultaneously at all temperatures without theenzyme.Thermal denaturationThermal stability of the purified enzyme was studiedby pre-incubating 1nkat of the enzyme at differenttemperatures ranging from 40 °C-100 °C for 30 min.Samples were cooled immediately in ice-water and theresidual enzyme activity was determined.Determination of KmKm value was obtained by assaying the invertaseactivity of purified preparation with sucrose as asubstrate at concentrations ranging from 10mM to250 mM.Results and DiscussionPreparation of the crude enzyme extractThe flowers were ground merely to disrupt onlythe plant tissues without harming the cellulolic wallsof micro-organisms, if any present in the flowers. Thisstep would avoid the release of microbial invertase intothe extracting solution.Subsequent filtering the above enzyme extractsthrough 0.22µm sterile filter under sterile conditions wasaimed at eliminating any micro-organisms, conidia orspores which could be present in the extract.PurificationThe enzyme was partially purified about 51.6fold by ammonium sulphate fractionation followed bygel filtration and anion exchange chromatography.The results of the enzyme purification are given inTable 1.During the purification by DEAE column at pH 6.5the enzyme didn’t bind to the column and was elutedbefore applying the gradient. However, at this DEAEstep enzyme showed purification of 15.95 fold with54.2% recovery. As the pH was changed to pH to 4.2.Table 1. Purification of invertase from Madhuca longifoliaStepTotal Protein (mg)Specific activity(nkat mg -1 protein)Recovery%Purfication fold(NH 4) 2SO 2Step585.2 0.42 100 1Gel filtration(1 st step)107.6 1.95 85.3 4.64Ion exchangeAt pH 6.519.9 6.7 54.2 15.95Ion exchangeAt pH 4. 25.48 11.95 26.6 22.6Gel filtration(2 st step) 1.32 21.7 11.6 51.6131

Weerasooriya & YatawaraTable 2. Properties of Invertase from Madhuca longifoliaPropertyMolecular mass (kDa) 210Optimum pH 4- 7Optimum temperature 37°CKm (37°C, pH 5)Thermal stability125 mMEnzyme is stable up to 45°C.Beyond this point activity is slowlydeclined and inactivated around80° Cthe enzyme retained in DEAE column. Upon gradientelution, the enzyme showed activity peak at 0.42MNaCl.In the final purification step, reloading the activeenzyme fractions (obtained from above DEAE step)on Sepharose 4B gave a slightly broader peak.Native PAGE of this enzyme fraction (obtainedafter fractionation through final Sepharose 4Bcolumn) showed that enzyme has been partiallypurified.Molecular weightNative molecular mass of the enzyme as determinedby elution through Sepharose 4B gel filtration ~210kDa(Fig. 1). Interestingly this value falls in the molecularmass range that reported invertases from Woodfordiafruticosa, another invertase source used for thepreparation of arishta (Weerasooriya and Yatawara2002), banana fruit (Sum et al. 1980) , Iraqui date fruit(Mahrouf & Zerki 1982), rice grains (Chang et al. 1994)and sprouting soya bean (Chen & Black 1992) .Optimum pHThe purified invertase of Madhuca was quite stableand showed broad pH optimum between 4.0 - 7.0(Fig. 2) which was quite comparable to that reportedfor invertases from Woodfordia (Weerasooriya andYatawara 2002), Iraqui date fruit (Mahrouf & Zerki1982), Allocasia leaves (Nakasone & Yasui 1982),Ricinus communis (Prado et al. 1985 ) and sugarcaneleaf (Minouru 1942).Optimum temperatureEnzyme showed a higher enzyme activity in therange of 30ºC to 45ºC with a maximum of 37ºC (Fig.3)which was very close to that reported for invertases5.85.65.4Log Molecular Weight5.254.84.64.44.240 4 8 12 16 20 24 28 32 36 40 44 48Elution volume (ml)Figure 1. Determination of the molecular weight of Invertase of Madhuca longifolia (Urease 340kDa, Alcohol dehydrogenase141kDa, Bovine serum albumin 66kDa, Egg albumin 45kDa, pepsin 34kDa and Trypsin 24kDa were used as the standards).132

Partial Purification and Characterization of Invertase of M. longifolia flowers4035Invertase activity (nmole/min)3025201510502.07 3.07 4.03 5.11 6.12 7.04 8.06 8.97pH valueFigure 2. Invertase activity of Madhuca longifolia as a function of pH (pH 2.6 glycine HCl 100mM; pH 3-7.5 Citric acid-Na 2HPO 4100mM; pH 8-8.9 Tris-HCl 100mM; 37°C, 3min, Mean and standard deviation were determined from six replicates.)3530Invertase activity (nmole/min)252015105020 30 35 37 40 50 55 60 65 70 75Incubation temperature 0 CFigure 3. Invertase activity of Madhuca longifolia as a function of incubation temperature 0.1M citrate pH 5, temeperaturerange 25-65°C, 3 min, Mean and standard deviation were determined from six replicates).133

Weerasooriya & Yatawaraof Woodfordia (Weerasooriya and Yatawara 2002),Allocasia leaves (Nakasone & Yusai 1982), sugarcaneleaf (Minouru 1942) and sugar beet (Bohuslavska etal. 1984 ) .Thermal inactivationBeyond 45ºC, invertase activity of Madhucaslowly declined and became almost nil by 80ºC (Fig.4)similar to that reported for invertases of Woodfordia(Weerasooriya and Yatawara 2002), Allocasia leaves(Chen & Black 1992) and potato tubers (Isla et al. 1998). However, invertase of Madhuca seems to be moreheat sensitive than invertases of muscat bailey A grapes(Porntaveewat et al. 1994), semillon grapes (Nakanishiet al. 1991) and yeast particularly, Saccharomycesecerevisae (Raheja et al. 1998) and Saccharomycesefragilis (Kulikora 1986) which were reported to be stableup to 80ºC.Kinetic constants (km)The Km values of purified Madhuca invertase forsucrose was found to be ~ 125 mM which was very closeto the values that reported for invertases of Woodfordia,(Weerasooriya and Yatawara 2002). invertases fromSolanum tuberosum tubers (Isla et al. 1999) and higherthan Km values that reported for invertases of ricegrain (Km ~ 70mM) (Chang et al. 1994), and potatotubers (Km ~ 100mM) (Isla et al. 1998).In a summary, Invertase from Madhuca longifoliafits many characteristics described for invertaseisolated from Woodfordia fruticosa (another invertasesource which used for the preparation of Arishta)such as optimum pH, optimum temeperature, thermalstability, molecular mass and Km value. Km ofWoodfordia invertase for sucrose is reported to be~160 mM whereas that of Madhuca invertase foundto be ~125mM. This indicates possibly substratesucrose shows higher affinity towards Madhucainvertase rather than that of Woodfordia. All arishtapreparations in Sri Lanka are carried out at roomtemperature which is around 35-37ºC . This is inagreement with the temperature 37ºC which showsthe optimum activity of the enzyme. pH of the solutionis also very importance during the preparation ofArishta. As described in Kroes 1990, all arishtapreparations favour the weak acidic condition.Alkaline conditions are unfavourable for the reaction.This is in agreement with optimum pH of the enzyme.As reported by Kroes 1992, high content of gallicacid, (a weak acid present in preparation medium)presumably maintain the pH of the solution around 4-60Invertase activity (nmole/min)5040302010050 60 70 80 90 100Temperature 0 CFigure 4. Thermal stability of invertase of Madhuca longifolia (Incubation of 1nkat of enzyme in 0.1M citrate buffer pH 5 for30 min at different temperature 40-100°C and then measured for activity under standard conditions; 0.1M citrate pH 5, 37°C,3min, Mean and standard deviation were determined from six replicates).134

Partial Purification and Characterization of Invertase of M. longifolia flowers201/rate x 10 -2 (nmole/min) -1151050-25 -5 15 35 55 75 95 115-51/[Substrate](M) -1Figure 5. Determination of Km of Madhuca invertase - (Line-Weaver Burk plot; 0.1M citrate pH 5, 37°C, 3min, substrateconcentration ranging from 10-250mM; Mean and standard deviation were determined from six replicates.)7 in order to provide optimum activity of the enzyme.Apart from these data further information particularlyon the inhibitors of two enzymes are to be obtained.All these data will enable to upgrade the quality andpreparation procedures of all arishtas in Sri Lankaas well as in India.AcknowledgementsNational Science Foundation, Sri Lanka isacknowledged for the financial support.ReferencesAyurvedic Pharmacopoeia Vol. I, part I (1976), part2 (1979). Department of Ayurveda, Colombo, SriLanka.Bohuslavska M, Zahradnicek J, MichaljanicovaD & Kotyk A. 1984. Effects of chemical andphysiochemical factors on sugar beat invertaseactivity. Biol. Plant, 26 (4): 303-8.Caraka Samhita (Engl. Transl.) 1949. ShreeGulabkunverba Ayurvedic Society, Jamagun,India.Chang YY, Yjuang RH, Su JC & Sung HY. 1994.Partial purification and characterization of invertaseisozymes from rice grains. Biochem. Mol. Biol.33(3): 607-15.Chen JQ & Black CC. 1992. Biochemical andImmunological properties of alkaline invertase fromsoya bean hypocotyls. Arch. Biochem. Biophysics.15: 295 (1), 61-9.Hand Book of Domestic Medicine and CommonAyurvedic Remedies (1978). Central Council forResearch in Indian Medicine and Homeopathy,Ministry of Health and Family Welfare, New Delhi,pp. 334-335.Isla MI, Vattuone MA & Sampietro AR. 1998.Hydrolysis of sucrose within isolated vacuolesfrom Solanum tuberosum L.tubers. Planta.205(4): 601-5.Isla MI, Vattuone MA, Ordonez RM & Sampietro AR.1999. Invertase activity associated with the wallsof Solanum tuberose tubers. Phytochemistry.50(4): 525.Kroes BH. 1990. “Nimba Arishta; impact of thepreparation process on chemical parameters andimmnunomodulatory activity’’ Ph.D. Thesis Universityof Uretch. Netherland.135

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