Purification and Study of the Kinetic Properties of a-Galactosidase from Coconut Kernel

The low molecular weight a-galactosidaseisoenzyn~e of coconut kernel, was purified until hon~ogeneous as determined by polyacrylamide gel-electrophoresis. Its n~olecular weight ( by sodium dodecy! sulphate polyacrylamide gel-electrophoresis) was 22 300 daltons. The kinetic properties of the enzyme were studied using p-nitrophenyl-a-D-$alactopyranoside. The Knl and Vmax volues of the a-galactosidase were 2.5 :: 10-4M and 24 ,Lhnlo!es!mg protein!min respectively. The enzyme was inhibited by this substrate a t concentt.ations of 6 x 10-4M and. above. The product D-galactose inhibited the eilzyme competitively. Activation energy (as determined by the Arrhenius plot) wbs 10.1 l<i!ocalories. The a-gala-ctosidase is a glycoprotein with a carbohydrate content of 28%; it is stable upto 450C. 2-Galactosidase activity showed a broad pH optimum between 2.5 and 6.0 with nlaxinlum activity a t p H 5.5.


I. Introduction
Galactose containing oligosaccharides and polysaccharides are carbohydrate reserves ill hrgher plant tissues particularly in sseds.a -Galactosidase is an exo-carbohydrase that hydrolyses the terminal a -D-galactosidic linkage of these reserve carbohydrates.
Most l>lnnt seeds contain two a -galactosjdase isoenzymes of different n~olecular weighk8 Highest activity is found in mature seeds probably due to association of low molecular weight isoenzymes to form the higher molecular weight isoenzyme which has a higher specific activity.I5 However two major isoenzymes have been purified from 11uman plas,enta10 which.are not stlucturally related and do not share a precursor and product relationship.a -Galaclosidase is an important enzyme of the coconut seed as raffinose4 is the the predoniinant oligosaccharide and 61 % of the polysaccharides consist of galac-ton~aimans.~ a -Galactosidase from cocount kernel has been purified by Balasubramania et aI2 who separated two isoenzymes by Sephadex G-100 gel filtration in the presence of KC1.Their appa.rentnzolecular weights were 123 000 and ,21000 daltons.When chrmlatographed in the absence of KCl, an inactive protein peak cf high molecular weight and the low molec~~lar weight isoenzyme were obtained.
In this paper we report the purification of a -galactosidase from coconut kernel to apparent ho~nogeneity and also some kinetic studies carried out with the enzyme.
A unit of enzyme 'ctivity is defined as the aixount that hydrolyses 1 pmole of substrate!i~inimdx the conditions sptcified above.
The grotem determinations were made by the method of Lowry et a1 using crystalline botilne seritnl albumln as standard or by measuring the absorbmce at 280 nnl.

Purification of a -gn8astosidase
All oyeratioizs were carried out at approximately 4°C urjless specified otherwise.
Coconut kernel scraplings in McIlvaine buffer (pH 5.5) were homogenized using a Waring blender.The extract (2rnl/g) was psssed thro3gh a chcese clot11 and centrif~~ked at 25 000 g for 30 inin.The supzrnatant was retained.This supernatazlt was adjusted to pH 3.8 usirg 0.5 M citric acid.After standing for 30 min, the acidified solution was centrifuged at 25 000 g for 30 min.The pH of the acid supernatant was readjusted to pH 5.5 using a saturated solution of NazHPO,.
The acid supernatant (pH 5.5 ) was ammonium sulphate fractionated using solid a;mnloniu.isulphate.The fraction between 50% -75% saturation showed the lzighest specific activity.This precipitate was dissolved in 3 ml of McIlvaine buffer (pH 5.5) and dialysed against the same buffer.Frxtions (5 m!) were collected m a refrigerated fraction collector c?t a flow rate of 22 llll/h.

Concenti~ntion
Selected fraction from Sephadex gel filtration were pooled and concentrated to about 4 ml uslng an Amicon ultrafilt~etion cell with an UM 2 membrane under nitrogen (2.5 atm).The concentrated enzylne solution was cerltrifuged at 7009 g for 10 min and the supernatant was retained.

2.5,. Polyacrylamide gel electrophoresis
Electrophoresjs was carried out by the method of Davis6 using a Shandon apparatus.
Polyacrylamide gels (5 ;/,I were prepared and loaded with 200 p l (2.5 p g protein) of the purified enzyme (diluted 2 : 1 with glycerol).McIlvaine buffer pH 6.5 was used in the reservoir.Gels were stained for proteins using Coomsssie blue.Their a-galactosidase activities were tested by incubatins with 4 -methyl umbelleferyla -Dgalactoside (2.5 m M ) at room temperature for 30 min.T!le reaction was te~minated with.0.5 M NaOH and the fluorescent bands observed under UV light.

Determinatioil of molecular weight
The molecular weight of the purified enzyme was determined using SDS (Sodium Dodecyl Sulphate ) polyacrylarnide gel (10%) electrophoresis by the method of Weber et al.ls Standard proteins (10 , L L ~ each.)were in a total volume of 200 pl.a -Galactosidase (3 pg) was taken separately in.200 id.T o all solutioas 100 p l of glycerol, 5 , L L ~ of n~ercaptoetl~anol and 5 ,LLI of Bromopl~enol blue were added.Electrop.horesiswas conducted for 3 h at 8 m A per tube constant current.

Purification of agalactosidase
The purification of agalactosidase of coconut kernel is summarised in Table I.
The multistep purification gave an overall yield of 11 P/, and the agalactosidase was purified 510 fold.

Test for purity
Polyacrylamide gel electrophoresis of purified enzyme (2.5 p g ) gave single and coincident band for protein and csgalactosidase activity.

Determination of molecular weight
The molecular weight of the purified enzyine determined by SDS polyac~ylamide gel electrophoresis appeared to be 22 300 daltons (Figurc I).The effect of Hg2+ ions on s-galactosidase activity was studied by incubating a -galactosidase at rooin temper~ture (2g°C) ir, McIlvaine buffer (pH 5.5) containing mercuric chloride (lpM) for 15 min.It was then assayed for enzyme activity.

The inhititor concenttation was kept constant
Pqf) while tlze subst-rate concentration in the assay medium was varied from M to 5 x M. In case of Dgalactose it was added to the assay medium a t a constant level (10 mM) while the substrate concentration was varied as before.The Lineweaver -Burk plot shows that Hg2+ ions inhibit a -galactosidase noncompetitively.The inhibition by Dgalactose is competitive (Figure 2).

Stability of a-galactosidase at various temperatures
The enzyme was very stable upto 45OC beyond ~vllich the activity declined very sharply.The enzyme lost 92% of its activity when incubated at 60°C for 15 min.
3.8.Gllycoprotein nature of a-galactosidase Purified a-galactosidase was tested for the Gresence of ~artohydrates by the method of Dubois et a19 using glucose as the standard.The carbohydrate content of purified a-galactosidase was 28 %.

Discussion
Homogeneous a-galactosidase has been obtained from seeds of Vicia faba7, Vicia sativa.14sweet aln~ond,~' white clover19 and fungus Mortierellu vina;eal7 and also from the lzun~an placenta.1°The a -galactosidase of coconut kernel obtained by our method was purified 510 fold and the yjeld was 11 %.The yield and p~lrity are collzparable to the values obtained for Viciafaba?Vicis sativa,I4 sweet almond,12 white cloverI9 and MortiereIla v i ~a c e a .~~ The specific activity of the purifieci coconut a -galactosidase is equal to the specific actixfities of the enzymes obtained from V. sativa14 and sweet a1mond.l'Molecular weight of the purified z-%alactosidase isoenzyme of coconut was deterinined by SDS polyacrvlamide gel electrophoresis to be 22 300 daltons.The cr. galactosidase being a glycoprotein could impair the binding of SDS. 18This would result in lowering the electrophoretic mobi!ity.The molecular weight obtained for the same enzyme by Balasubramaniam et a12 from gel filtration was 21 400 daltons.I-1ydra.tion of glycoproteins and its affinity for dextran could affect the mobility of the en,zyme in gel filtration.However the molecular weights of a -galactosidase Frum various seeds have been determined by gel filtration and the values obtained for their !owmolecular weight isoenzymes varied between 25,000 daltons and 40,000 d a l t o n ~. ~ High concentrations of p-nitroplzenyl -a -Dgalactopyranoside inhibited the agalactosidase of coconut.The inhibition by this substrate has also been observed nit11 the agalactosidase of V. faba7 and sweet almond.12Km and Vmax val~les obtained in this study are lowel than the values obtained using the same substrate in sweet almond,12 V. sativa,14 and white c l o v e ~l ~ but similar to that of the l~tgh molecular weight isoenzyme F~om V. jaha.7 Like the a -galactosidase from coconut kernel agalactosidase from M , vinacea17 is inhibited nonconlpetitively by Hg2+ ions while that from V. faba7 and P. ur7zygd~,lzcs~ are competitively inhibited by Hg2+ ions.D-Galactose competitively inhibits coconut agalactosidase.It also iiilzibits competitively agalactosidase from V. faba7 but the enzyme from M. vir7ace~'~ shows a mixed type inhibition.
The pH optima of a-galactosidase varied to a considerable extent when p-nitro-pl~enyla-:D-galactopyranoside was used as substrate.Many a -galactosidases showed two pH optima7,s an(! the agalactosidase of coconut also appears to have two peaks with.~~zaximnum activity at pH 5.5.The sharp decline in activity between

Pur fication and Study of the Kinetic Properties of r-Galucto~idase
203 pH 6 and 8 obselvec!with coconut a: -galactosidase indicates the presence of a catalytic group with a pRa of 7 at tlze active site which cotlld be an imidazole group of histidine.A silllilar decline in activity is observed in the a -galactosidase of sweet almond uslng p-nitrophenyl-a-D gdactopyranoside as substrate and studies on the effect of pH on its Km and Vmax indicate the presence of imida,zole and casboxyl groups at the active site.6Preliminary fluorescence spectrophotometric studies indicate the involvelnent of a tryptophan at or near the active site of a-gdzctosidase from coconut.
The energy of activation of a: -galactosidase of coconut is lower than tlze reported values from other plaats sources but similar to that of M. vi~~rrcea'~ and Prunus aniygd~lzo.~The thermal stability that was observed in a -galactosidase of cocor~~it kernel is colnmon to a -galactosidase from V. faba7 and M. 11ir~area17 that are glycoproteins.The carbohydrate content is highel than of o: -galactosidase from M .~~i n a c e a ' ~ but is sillzilar to the high molec~.ilarweight a -galactosidase isoenzynle from V. f a b ~~.~

3. 5 .
Effect of pH on enzyme activity.a -Galactosidase in Mcllvaine b -~f f z ~showzd optimum activity at pH 5.5.Below pH 5.5 the activity was co~lstant and high.While above pH 5.5 the activity declined very steeply (Figure3).

Figure 3 .
Figure 3.Effect of pH on a-galactosidase activity.Bars indicate standard deviations obtained from four experiments.

3. 6 .
Effect of temperaturea -Galactosidase activity increased with increase in temperature fiom 10CC to 50°C.Arrhe~iius plot for agaiactosidase (Figure4gave a straight line graph showing that the energy of activation did not change with the chang2 in tempzrature.The energy of activation was 13.1 Kcal.

Figure 4 .
Figure 4. Arrhenius plot of a-gatactosidase for the substrate p-nitrophenyl-a-D galactopyranoside.

TABLE 1 .
Purification of a-galactosidase from coconut !ternel Volume Total enzyme Total Specific activity Recovery Purification