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β-Galactosidase from Coffea Arabica and its role in fruit ripening
Phytochemistry, Vol. 34, No. 2, pp. 355 360, 1993
0031-9422/93 $6.00+0.00 © 1993 Pergamon Press Ltd
Printed in Great Britain.
fl-GALACTOSIDASE FROM COFFEA A R A B I C A AND ITS ROLE IN FRUIT RIPENING KERITH D. GOLDEN,* MELFORD A. JOHN and ECCLESTONA. KEAN Department of Biochemistry, University of the West Indies, Mona, Kingston 7, Jamaica
(Received 23 November 1992)
Key Word Index--Coffea arabica; Rubiaceae; berry; ripening; arabinogalactan; galactan; fl-galactosidase; pectin; pectinase.
Abstract--fl-Galactosidase (EC 3.2.1.23) from ripe coffee berries was purified and characterized. The enzyme displayed activity against p-nitrophenyl-fl-D-galactopyranoside (PNPG) (Kin 0.33 mM), lactose (Kin 40 mM), arabinogalactan and galactan. Purification was carried out using (NH4)zSO4 precipitation, gel-filtration using Bio-Gel P-2 and P-200, ion-exchange chromatography on Cellex-CM and affinity chromatography on p-aminophenyl-fl-D-thiogalactopyranoside-agarose. Activity was highest within a pH range of 2.5-6, with an optimum at pH 4.4. The M, was estimated to be 2.9 x 104 by SDS-PAGE. The enzyme appeared to be a dimer of two identical subunits. It was inhibited by galactose (competitive, Ki 0.26 mM); p-chloromercuribenzoate (PCMB) at 0.29 mM completely abolished activity. The enzyme catalysed release of galactose from galactan and arabinogalactan; pectin yielded galactose only when the action of flgalactosidase was combined with that of an endopolygalacturonase from Aspergillus niger, fl-Galactosidase activity in coffee berries showed a progressive increase of more than four-fold as the fruit developed from the immature to ripe stage, with a slight decrease in fully ripe fruit. The same trend was observed with three other glycosidases, but not for Nacetyl glucosaminidase. It is suggested that fl-galactosidase plays a role in cell wall degradation such as occurs during fruit ripening.
INTRODUCTION fl-Galactosidase occurs widely in plants and has been studied in fruits [1-5], red kidney bean [6], seeds of Viona sinensis [7] and carrots [8]. Multiple forms of the enzyme have been identified in tomato [9], the seeds of-Vigna radiata [10] and mango [5]. Shadaksharaswamy and Ramachandra [11] reported on ~-galactosidase activity and changes in the oligosaccharide content of Coffea arabica and C. robusta. Carchon and DeBruyne [12] described the purification and characterization of ~tgalactosidase from C. canephora. However, very little work has been done on the activity of fl-galactosidase in coffee berries. The role of fl-galactosidase during the ripening of fruits is not clear. Bartley [1, 13] stated that (i) fl-galactosidase is able to hydrolyse galactan, (ii) galactose residues are lost from the walls of cortical ceils of apple as the fruit ripens and (iii) an increase in /~-galactosidase activity during ripening promotes the loss of galactose residues from the cell wail. On the other hand, Gross and Wallner 1-14] reported the inability of fl-galactosidase to hydrolyse a tomato cell wall polysaccharide which contained galactose and arabinose. This paper reports on the purification of fl-galactosidase from C. arabica, the ability
*Author to whom correspondence should be addressed. 355
of the enzyme to degrade pectic polysaccharides and its apparent increase in activity during the ripening of the coffee berry. RESULTSAND DISCUSSION
Purification of fl-galactosidase Data from a typical purification sequence is given in Table 1. The final product represents a 61-fold purification relative to the original homogenate. During ionexchange chromatography at pH 4 on Cellex-CM, all the enzyme remained bound to the resin. This contrasts with ca 36% of total carrot fl-galactosidase [8] which was not bound under similar conditions. This was one indication that coffee beans probably do not contain the isoenzyme forms reported for other plant sources [5, 11, 15]. Elution from the cation-exchange column was effected by 0.2 M Na acetate buffer; pH 6.0; of the two protein peaks emerging, only one contained fl-galactosidase activity (Fig. I). However, ~t-galactosidase was also present in this fraction; the heterogeneity was confirmed by SDSPAGE which revealed one major and one minor protein band (data not shown). In purifying fl-galactosidas¢ from plant sources, either affinity chromatography or isoelectric focusing [16, 17] has been required to obtain homogeneous preparations.
356
K. D. GOLDENet al. Table 1. Stages in the purification of fl-galactosidase from coffee berries Fraction
Protein (mg)
Specific activity (mg protein-l x 10 2)
Total units* Purification (#mol min-1)-1 (fold)
Recovery (%)
Supernatant of homogenate (NH4)2SO4p~,e, Bio-Gel P-2 filtrate Bio-Gel P-200 filtrate Cellulose CM eluate Affinity chromatography
37500 4489 1332 390 127 17
0.375 0.619 2.10 3.86 6.27 23
140 27.8 28.0 15.1 7.96 3.9
100 20 20 11 5.7 0.71
1 1.66 5.63 10.4 16.7 61
*One unit = amount of enzyme converting 1/~mol PNPG per min to nitrophenol.
~0 .~ ~1
1.6
%280 *Enzyme activity
1.2
•o
-
u
0
0.8
=t 0.4 &
,
The Km found for P N P G was 0.33 raM; for lactose, 40 mM. Activity (Vmax)for these respective substrates was in a ratio of ca 4: 1. With P N P G as substrate, the pH optimum was 4.4. Galactose was a competitive inhibitor with respect to P N P G hydrolysis. CuSO4 (K~, 1.17 mM), HgC12 (K~, 0.33 mM) and PCMB (K~, 1.50 #M) were noncompetitive inhibitors. At 0.2 mM, PCMB completely abolished enzyme activity. Evidently, free sulphydryl groups in the enzyme are essential for activity. All these properties are, in general, similar to those reported for flgalactosidase from other plant sources [4, 6, 7, 9, 16, 181 although the carrot enzyme seems to be exceptional in showing barely detectable activity on lactose. Action on pectic polysaccharides
0
40
120 160 Fraction no. (2 ml ) 80
Fig. 1. fl-Galactosidase purification at the stage of ionexchange chromatography on Cellex-CM.
Contamination by other proteins, notably ~-galactosidase, is likely otherwise [6, 8]. In the present case, thiogalactopyranoside coupled via a spacer arm of 12 atoms (10C) to beaded agarose proved to be effective. The enzyme that was bound and subsequently eluted gave a single band after SDS-PAGE. By reference to standards, a M, of 2.9 x 104 for flgalactosidase from coffee beans was indicated by SDSPAGE. After the enzyme was boiled with 2-mercaptoethanal, electrophoresis still showed only a single protein species, with an apparent M, of 1.4 × 104. This implied that the enzyme was comprised of two identical subunits, linked by one or more disulphide bridges. A homodimeric structure was reported by Konno et al. [8] for the enzyme from carrot cell cultures; however, the Mr of this enzyme was 1.05 x 105. Other plant sources have yielded flgalactosidase o f M r 5.8 x 104 (germinating seeds of Vigna sinensis [7]) and various other M,s ranging from 4.9 x 104 up to 3 x 10 5 have been reported by Dey and Campillo [15]. Is,enzymes of different Mr apparently co-exist in plants, although artifacts arising during isolation may confuse the picture [15]. The present case appears to be free of such complications and the M, seems to be the smallest yet reported for a plant fl-galactosidase.
Table 2 shows results for pectinase and fl-galactosidase acting separately and jointly. It should be noted that the release of free galactose only and not galacturonic acid or other sugar derivatives was monitored. The negative results for all three substrates incubated with pectinase (endopolygalacturonase) alone are as expected. This enzyme is reputed to produce random breakage of internal ~-(1--,4)-linked galacturonosyl bonds in the rhamnogalacturonan backbone of the pectin polymer [19, 20]. Hence, n o production of free galactose from the substrates by pectinase should occur. fl-Galactosidase acting alone released no galactose from pectin, but did so from arabinogalactan and galactan. In these two polysaccharides that constitute side chains on the pectin backbone, fl-(1--,4)-galactosidic bonds are prevalent and indeed predominate in the case of galactan. The enzyme evidently acts in exo-fashion on such linkages, cleavage occurring from the non-reducing ends of chains. The results with galactan are analogous to those for fl-galactosidase from apples .[1] and. carrot [8]. For arabinogalactan as substrate, our results show flgalactosidase action (Table 2), in contrast to those of others [14] who reported negative results for the enzyme from apple with this type of substrate. The detailed structure of apparently similar polysaccharides may vary with the source and also with the method used to isolate them. It was not directly shown by our work that arabinogalactan from coffee bean which contains fl-D-(1 ~3)-galactosidic bonds [21] is a substrate for coffee bean fl-galactosidase. However, the arabinogal-
fl-Galactosidase from Coffea arabica actan used in our experiments and found to be susceptible to the enzyme was from larch, and reportedly contains fl(1 ~3)- and fl-(l--*6)-galactopyranoside linkages [22]. Results from HPLC confirmed that fl-galactosidase, acting alone, released galactose from arabinogalactan. Arabinose and galactose were released from arabinogalactan by the joint action of fl-galactosidase and pectinase. Unidentified peaks with R,s ranging from 8 to 20 min were also observed and may indicate polysaccharides possessing structural complexity intermediate between arabinogalactan and free monosaccharide. It is noteworthy that the joint action of pectinase and fl-galactosidase released galactose from pectin, in contrast to negative results for the enzymes acting separately
357
(Table 2). This indicates that break-up of the rhamnogalacturonan backbone of pectin is necessary for fl-galactosidase action on segments of the heteropolymer. This is in apparent conflict with the findings of Pressey [9], who reported that one of three fl-galactosidases from tomato degraded a cell-wall polysaccharide containing 22% uronic acid, together with 58% galactose, 14.5% arabinose and lesser amounts of other sugars. Such a composition indicates considerable integrity of the rhamnogalacturonan backbone structure, and this suggests that the tomato enzyme (fl-galactosidase enzyme II) acts at an earlier stage of pectin degradation than coffee fl-galactosidase.
Activity of various #lycosidases durin# ripening Table 2. Galactose produced from peptic polysaccharides by the action of pectinase and fl-galactosidase Galactose formed (#g m1-1) from substrate* Enzyme added
Pectin
Arabinogalactan
Galactan
Pectinase fl-Galactosidase Pectinase and fl-galactosidase
n.d.'t n.d.
n.d. 18.7
n.d. 25.0
29.6
20.7
27.0
*Each value is the mean of three analyses. "j'n.d. = none detected. Enzyme as indicated was incubated in 700 #1, with each pectic polysaccharide for three days, then 200/d of each mixture was brought to a final volume of 1.4 ml containing 4.0 mM NAD ÷, 0.7 M glycine/0.3M hydrazine buffer, pH 8.6, and 0.45 units of fl-o-galaetose dehydrogenase. After 1 hr, A at 340 nm was determined. Galactose content per ml of initial incubation mixture is given.
Table 3 shows the changes occurring in activities of flgalactosidase and four other glycosidases, as the fruit matures and ripens. The extracts that were assayed were derived from entire berries. The same general trend is seen in all the enzymes except N-acetyl-fl-glucosaminidase. Activities tended to peak at the semi-ripe stage, having increased from lower values in the immature (green) fruit. fl-Galactosidase showed the greatest increase, more than six-fold per unit weight of berry, and more than four-fold per unit weight of protein in extracts. Expressed on the latter basis, the data suggest that the enzyme accounted for a progressively increasing proportion of total protein in the berries. However, the trend did not continue into the fully ripe stage. For N-acetyl glucosaminidase, activity per g berry (fr. wt) remained relatively constant and actually declined when expressed per unit weight of protein. This may reflect a very low turnover of the enzyme protein, as appears to be true for this enzyme during germination of peas [22]. It has long been known that increased activity
Table 3. Activity ofglycosidases at various stages of maturation of coffee berry [each value is 102 (mean + s.c.) of three replicate analyses] Stages* Enzyme assayed fl-Galactosidase 0t-Galactosidase ~t-Mannosidase N-Acetyl-fl-ogalactosiminidase N-Acetyl-fl-Dglucosiminidase
I
III
IV
13.06+0.98 15.62+1.48 8.45 + 0.20 10.10_+0.30 7.60+ 0.45 9.10+0.69
11.41+0.15 10.74_+0.21 7.77_ 0.24 7.32_+0.33 8.64_+0.30 8.14-+0.40
Activity# Specific activity:~ Activity Specific activity Activity Specific activity
2.09__+0.04 5.72+0.04 3.66_+0.10 8.88__+0.08 3.73 _+0.10 6.35 _+0.24 6.52+0.23 9.90+0.47 2.99 + 0.25 7.45 _+0.09 5.23+0.58 11.61+0.18
Activity Specific activity
5.02+0.35 6.81+0.04 8.09+0.81 8.77-+0.65 10.72+0.09 9.69+1.22
Activity Specific activity
8.13+0.40 7.38+0.13
13.01-+0.55 12.49__+0.52 13.55+0.55 11.47-+0.76 22.75 __+1.26 19.48_+1.0 16.21___0.08 10.79__+1.02
*Stages: I--immature; II-III--semi-ripe;IV--fully ripe. tActivity = units g-1 ft. wt. :~Specificactivity = activity mg-1 protein. PHYTO 34:2-D
II
K.D. GOLDENet al.
358
of many glycosidases accompanies fruit ripening. For example, Wallner and Walker [23] reported this for fl-l,3-glucanase and polygalacturonase in tomato, with the latter enzyme showing the most substantial change in the inner pericarp. These authors also cited reports of ripening-linked changes in polygalacturonase activity in various other fruits. The enzyme activities reported here for the different stages of ripening were derived from seeds and pericarp combined, but other data not reported suggest that the observed changes were similar in the two types of structures. Our findings are consistent with an active role for at least some glycosidases in the softening process and increasingly sweet taste that accompany ripening of coffee berries. The relevant enzymes would be those that participate in degradation of oligo- and polysaccharide components of primary cell walls, as well as the glycosyl components of glycoproteins and glycolipids [22, 24]. In this regard, Ahmad and Labavitch [25] argued against a role for a-galactosidase, ~t-mannosidase and polygalacturonase, even though these showed increasing activity in ripening pears. They cited the reputed absence of a-linked galactans and mannans in cell walls, and also their finding that the galactose and mannose content of the fruit did not increase with ripening. However, interconversion and utilization of free sugars could explain their findings. The present work has established that pectic polysaccharides are indeed potential substrates for fl-galactosidase of the coffee berry. This potential could be exploited in situ only if the enzyme has access to the substrates, by being located in or on cell walls. So far, only indirect evidence indicates such a location for fl-galactosidase of plants [9, 25, 26]. EXPERIMENTAL
Plant material. Coffee berries (Coffea arabica L. var. arabica) were collected from Hall's Farm in Mount Dakin, St Andrew, Jamaica. Enzymes and chemicals, fl-o-Galactose dehydrogenase (EC 1.1.1.48) from Pseudomonasfluorescens, glucose oxidase (EC 1.1.3.4) from Aspergillus niger and pectinase [endo-(1,4)-ct-o-polygalacturonase, EC 3.2.1.15] from A. niger were purchased from Sigma. p-Nitrophenyl-fl-Dgalactopyranoside (PNPG), NAD ÷, arabinogalactan from larchwood, pectin from citrus fruits and p-aminophenyl-fl-D-thiogalactopyranoside-agarose were also from Sigma. Bio-Gel P-2 and P-200, Cellex-CM (H + from) and a Fermentation Monitoring Column (with guard attached) came from Bio-Rad Laboratories. Galactan from gum arabic was purchased from Aldrich. Purification of fl-galactosidase. Seeds of ripe coffee berries were freed of pulp and washed with dist. H 2 0 , after which 300 g were then homogenized in 600 ml of 0.05 M NaOAc buffer pH 4.5, containing 0.1% mercaptoethanol (buffer A). After centrifugation, solid (NH4)2SO 4 was added to the supernatant and a fr. which pptd between 0 and 60% (w/v) was collected. The pellet was resuspended in and exhaustively dialysed against buffer A. Finally, the vol. was reduced to 25 ml by dialysis
against 10% polyethyleneglycol (M, 1450) soln containing 0.1% mercaptoethanol. Gel-filtration chromatography using Bio-Gel P-2 was carried out on a column (30 x 3.6 cm) equilibrated with buffer A. The void peak was collected (249 ml) and centrifuged to obtain a clear supernatant. The vol. was reduced to 50 ml by dialysing against 10% polyethyleneglycol and applied to a Bio-Gel P-200 column (30 cm x 3.6 cm) equilibrated with buffer A. Elution was carried out with this buffer and 2-ml frs collected. Frs that comprised a single peak displaying fl-galactosidase activity were pooled, and after adjustment to pH 4, the soln was applied to a Cellex-CM column (16 x 4.3 cm) equilibrated with 0.05 M NaOAc buffer pH 4 (buffer B). The column was washed with 110 ml of this buffer, and the bound enzyme was eluted with 400 ml of 0.2 M NaOAc buffer, pH 6 (buffer C). The frs of eluate containing flgalactosidase activity were pooled. At this stage, the vol. of enzyme soln from a typical purification was 89 ml; aliquots from this pool were subjected to affinity chromatography as the final purification step, as follows, p-Aminophenyl-fl-D-thiogalactopyranoside-agarose contained in a 10-ml syringe was equilibrated with 0.01 M NaOAc pH 5 (buffer D). Then an aliquot from the ion-exchange step (2.5 ml, containing ca 3.6 mg protein) which had been dialysed overnight against buffer D was applied to the column. Elution with the buffer and collection of ten 2-ml frs removed agalactosidase activity. The buffer was changed to 0.05 M NaOAc, pH 5, and this removed fl-galactosidase activity in ca 5 ml vol. Steps involving CC were performed at 18-22 ° with little effect on enzyme activity; however, 4-10 ° was maintained for all other operations such as dialysis and centrifugation. Assay of fl-galactosidase activity. Enzyme prepns (100 #1, containing 0.18-11 mg protein ml-1) were incubated at 37 ° in a final vol. of 400/zl, containing 34 mM NaOAc buffer, pH 4.5 and 2.0 mM PNPG. After 10 min, 1 ml of 0.3 M Na2CO 3 was added. Where incubations were carried out above pH 6, 2-(hydroxymethyl)-l,3propanedioi buffer (34 mM) was substituted for NaOAc. The resulting A was determined at 405 nm [5]. fl-Galactosidase activity with lactose. The incubation mixt. (350 #1) contained 29 mM NaOAc buffer, pH 4.5, 21 mM lactose and 0.4 mg enzyme protein. After 2 hr incubation at 40 °, aliquots were assayed for glucose using glucose oxidase [27]. Inhibition studies. P N P G at varied concns was used as substrate, with inhibitor concn fixed at 0.29 mM in the case of CuSO 4, HgCI 2 and galactose, or at 0.15/zM PCMB. The type of inhibition was determined from plots of 1/v vs 1/S. fl-Galactosidase action on pectin, arabinogalactan and galactan with or without pectinase. We aimed to determine whether or not fl-galactosidase acted on these substrates and the contribution of pectinase to any such action. This required the commercially available samples of all three substrates and also pectinase to be purified. For this, the substrates were gel-filtered on Bio-Gel P-2 and, in each
fl-Galactosidase from Coffea arabica case, the frs of the eluates saved for use were those eluted in well-defined bands containing carbohydrate as identified by the phenol-H2SO4 method [28]. To purify pectinase, 54 mg crude enzyme dissolved in buffer B was applied to a column (16 cm × 2 cm) of Dowex 50 (H +) that was equilibrated with the same buffer. After extensive washing, the buffer was changed to 0.20 M NaOAc, pH 6, and this eluted pectinase free of contaminating proteins such as fl-galactosidase, ~-galactosidase and ~-mannosidase that were detectable in the crude enzyme. To assay for pectinase, 0.25-1.0 mg enzyme protein was incubated with 0.25 mg purified pectin for 3.75 hr at 30 °. Reducing sugar formed was then measured by the method of ref. [29]. Each of the three substrates (1.5 mg in 300 #1) was incubated with pectinase alone (1.2 units), with fl-galactosidase alone (2.6 x 10 -2 units), and with both enzymes together. Controls containing substrate without enzyme were also incubated. In each case, the final vol. was 700 #1, to which 30 #1 toluene was added as bacteriostatic agent. After 3 days at 30°, the reaction mixt. was heated to 100° for 5 min and then centrifuged. The supernatant was assayed for free galactose using fl-D-galactose dehydrogenase [30]. Detection of ffalactose and arabinose by HPLC. Aliquots (10/d) of the supernatants from the above incubations were injd onto a Fermentation Monitoring Column (150 x 7.8mm) that was equilibrated with 0.01 M H2SO4. The column was eluted with 0.01 M H2SO 4 at a flow rate of 0.5 ml min- 1. Detection of each component was by UV absorption at 190 nm. As external standards to determine R,s, 10 #1 of a mixt. containing galactose and arabinose (each at 1.0-2 mg ml- ~) were injd.
Determination of enzyme purity and molecular size by SDS-PAGE. The method was essentially as described in ref. [31] using gel rods (10% polyacrylamide and 0.53% SDS) at pH 7.4. Samples were dissolved in buffer containing 1% (w/v) SDS, denatured at 100 ° for 5 rain and analysed as such, or were reduced by boiling in buffer containing 5% (w/v) 2-mercaptoethanol before analysis. Proteins used as standards (M, range 1.4--6.6 x 104) were electrophoresed simultaneously with and in rods adjacent to those containing samples of/~-galactosidase. Coomassie Brilliant Blue R was used for staining. Apparent relative mobility was calculated from the ratio of the distance migrated to that of total gel length. Protein determination. This was by the method of ref. [32] using BSA as standard.
Activity of various glycosidases in coffee berries at different stages of maturity. Coffee berries were harvested at different stages varying from immature to fully ripe, using external colour as a guide, viz., stage I--immature, green; stage II--mature, greenish-yellow; stage I I I - semi-ripe, yellow; stage IV--fully ripe, cherry red. Four 30-g batches of coffee berries each representing one stage were separately homogenized in 30 ml buffer A. After centrifuging, the supernatant from each batch was exhaustively dialysed against several changes of buffer A. Determinations of the following enzyme activities were done on the dialysed extracts: fl-galactosidase, ct-galac-
359
tosidase, ~-mannosidase, N-acetyl-fl-D-galactosaminidase and N-acetyl-fl-D-glucosaminidase. The procedure was in each case similar to that previously described for fl-galactosidase, substituting as substrate the p-nitrophenylesters of ~t-D-galactopyranoside, ~t-D-mannopyranoside, Nacetyl-fl-D-galactosaminide and N-acetyl-fl-D-glucosaminide as appropriate for each enzyme assay.
REFERENCES
1. Bartley, I. M. (1974) Phytochemistry 13, 2107. 2. Yamaki, S. and Matsuda, K. (1977) Plant Cell Physiol. 18, 81. 3. Etcheberrigaray, J. L., Vattmore, M. A. and Sampietro, A. R. (1981) Phytochemistry 20, 49. 4. Ohtani, K. and Misaki, A. (1983) Agric. Biol. Chem. 47, 2441. 5. John, M. A. (1984) Ph.D. Thesis. University of London. 6. Agrawal, K. M. L. and Bahl, O. P. (1968) J. Biol. Chem. 243, 103. 7. Biswas, T. M. (1986) Arch. Biochem. Biophys. 251,379. 8. Konno, H., Yamaseki, Y. and Katoh, K. (1986) Plant Physiol. 68, 46. 9. Pressey, R. (1983) Plant Physiol. 73, 132. 10. Kundu, R. K., DeKundu, P. and Banerjee, A. C. (1990) Phytochemistry 29, 2079. 11. Shadaksharaswamy, M. and Ramachandra, G. (1968)
Phytochemistry 7, 715. 12. Carchon, H. and De Bruyne, C. K. (1975) Carbohydr. Res. 41, 175. 13. Bartley, I. M. (1977) J. Exp. Botany 28, 943. 14. Gross, K. C. and WaNner, S. J. (1979) Plant Physiol. 63, 117. 15. Dey, P. M. and Campillo, E. D. (1984) Adv. Enzymol. 36, 91. 16. Li, S. C., Mazzotta, M. Y., Chien, S. F. and Li, Y. T. (1975) J. Biol. Chem. 250, 6786. 17. Simos, G., Kouras, T. G. and Geortgatsos, J. G. (1989) Phytochemistry 28, 2587. 18. Agrawal, K. M. L. and Bahl, O. P. (1972) Methods Enzymol. 28 B, 720. 19. Rexova-Benkova, L. and Markovic, O. (1976) Adv. Carbohydr. Chem. Biochem. 33, 323. 20. John, M. A. and Dey, P. H. (1986) Adv. Food. Res. 30, 139. 21. Dekker, R. F. H. and Richards, G. N. (1976) Adv. Carbohydr. Chem. Biochem. 32, 277. 22. Neely, R. S. and Beevers, L. (1980) J. Exp. Botany 31, 299. 23. Wallner, S. J. and Walker, J. E. (1975) Plant Physiol. 55, 94. 24. Flowers, H. M. and Sharon, H. M. (1975) Adv.
Enzymol. 48, 29. 25. Ahmad, A. E. and Labavitch, J. M. (1980) Plant Physiol. 65, 1014. 26. Dopico, B., Nicolas, G. and Labrador, E. (1990) Physiol. Plant. 80, 629.
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27. Keston, A. S. (1956) Abstracts of Paper, 129th Meeting, A.C.S. 28. Dubois, M., Gilles, K. A., Hamilton, J. K., Roberts, P. A. and Smith, F. (1956) Analyt. Chem. 28, 350. 29. Nelson, N. (1944) J. Biol. Chem. 153, 373.
30. Wallenfels, K. and Kurz, G. (1966) Methods Enzymol. 9, 112. 31. Kamicker, B. J. and Prill, J. W. (1986) A E M 51,487. 32. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265.
0031-9422/93 $6.00+0.00 © 1993 Pergamon Press Ltd
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fl-GALACTOSIDASE FROM COFFEA A R A B I C A AND ITS ROLE IN FRUIT RIPENING KERITH D. GOLDEN,* MELFORD A. JOHN and ECCLESTONA. KEAN Department of Biochemistry, University of the West Indies, Mona, Kingston 7, Jamaica
(Received 23 November 1992)
Key Word Index--Coffea arabica; Rubiaceae; berry; ripening; arabinogalactan; galactan; fl-galactosidase; pectin; pectinase.
Abstract--fl-Galactosidase (EC 3.2.1.23) from ripe coffee berries was purified and characterized. The enzyme displayed activity against p-nitrophenyl-fl-D-galactopyranoside (PNPG) (Kin 0.33 mM), lactose (Kin 40 mM), arabinogalactan and galactan. Purification was carried out using (NH4)zSO4 precipitation, gel-filtration using Bio-Gel P-2 and P-200, ion-exchange chromatography on Cellex-CM and affinity chromatography on p-aminophenyl-fl-D-thiogalactopyranoside-agarose. Activity was highest within a pH range of 2.5-6, with an optimum at pH 4.4. The M, was estimated to be 2.9 x 104 by SDS-PAGE. The enzyme appeared to be a dimer of two identical subunits. It was inhibited by galactose (competitive, Ki 0.26 mM); p-chloromercuribenzoate (PCMB) at 0.29 mM completely abolished activity. The enzyme catalysed release of galactose from galactan and arabinogalactan; pectin yielded galactose only when the action of flgalactosidase was combined with that of an endopolygalacturonase from Aspergillus niger, fl-Galactosidase activity in coffee berries showed a progressive increase of more than four-fold as the fruit developed from the immature to ripe stage, with a slight decrease in fully ripe fruit. The same trend was observed with three other glycosidases, but not for Nacetyl glucosaminidase. It is suggested that fl-galactosidase plays a role in cell wall degradation such as occurs during fruit ripening.
INTRODUCTION fl-Galactosidase occurs widely in plants and has been studied in fruits [1-5], red kidney bean [6], seeds of Viona sinensis [7] and carrots [8]. Multiple forms of the enzyme have been identified in tomato [9], the seeds of-Vigna radiata [10] and mango [5]. Shadaksharaswamy and Ramachandra [11] reported on ~-galactosidase activity and changes in the oligosaccharide content of Coffea arabica and C. robusta. Carchon and DeBruyne [12] described the purification and characterization of ~tgalactosidase from C. canephora. However, very little work has been done on the activity of fl-galactosidase in coffee berries. The role of fl-galactosidase during the ripening of fruits is not clear. Bartley [1, 13] stated that (i) fl-galactosidase is able to hydrolyse galactan, (ii) galactose residues are lost from the walls of cortical ceils of apple as the fruit ripens and (iii) an increase in /~-galactosidase activity during ripening promotes the loss of galactose residues from the cell wail. On the other hand, Gross and Wallner 1-14] reported the inability of fl-galactosidase to hydrolyse a tomato cell wall polysaccharide which contained galactose and arabinose. This paper reports on the purification of fl-galactosidase from C. arabica, the ability
*Author to whom correspondence should be addressed. 355
of the enzyme to degrade pectic polysaccharides and its apparent increase in activity during the ripening of the coffee berry. RESULTSAND DISCUSSION
Purification of fl-galactosidase Data from a typical purification sequence is given in Table 1. The final product represents a 61-fold purification relative to the original homogenate. During ionexchange chromatography at pH 4 on Cellex-CM, all the enzyme remained bound to the resin. This contrasts with ca 36% of total carrot fl-galactosidase [8] which was not bound under similar conditions. This was one indication that coffee beans probably do not contain the isoenzyme forms reported for other plant sources [5, 11, 15]. Elution from the cation-exchange column was effected by 0.2 M Na acetate buffer; pH 6.0; of the two protein peaks emerging, only one contained fl-galactosidase activity (Fig. I). However, ~t-galactosidase was also present in this fraction; the heterogeneity was confirmed by SDSPAGE which revealed one major and one minor protein band (data not shown). In purifying fl-galactosidas¢ from plant sources, either affinity chromatography or isoelectric focusing [16, 17] has been required to obtain homogeneous preparations.
356
K. D. GOLDENet al. Table 1. Stages in the purification of fl-galactosidase from coffee berries Fraction
Protein (mg)
Specific activity (mg protein-l x 10 2)
Total units* Purification (#mol min-1)-1 (fold)
Recovery (%)
Supernatant of homogenate (NH4)2SO4p~,e, Bio-Gel P-2 filtrate Bio-Gel P-200 filtrate Cellulose CM eluate Affinity chromatography
37500 4489 1332 390 127 17
0.375 0.619 2.10 3.86 6.27 23
140 27.8 28.0 15.1 7.96 3.9
100 20 20 11 5.7 0.71
1 1.66 5.63 10.4 16.7 61
*One unit = amount of enzyme converting 1/~mol PNPG per min to nitrophenol.
~0 .~ ~1
1.6
%280 *Enzyme activity
1.2
•o
-
u
0
0.8
=t 0.4 &
,
The Km found for P N P G was 0.33 raM; for lactose, 40 mM. Activity (Vmax)for these respective substrates was in a ratio of ca 4: 1. With P N P G as substrate, the pH optimum was 4.4. Galactose was a competitive inhibitor with respect to P N P G hydrolysis. CuSO4 (K~, 1.17 mM), HgC12 (K~, 0.33 mM) and PCMB (K~, 1.50 #M) were noncompetitive inhibitors. At 0.2 mM, PCMB completely abolished enzyme activity. Evidently, free sulphydryl groups in the enzyme are essential for activity. All these properties are, in general, similar to those reported for flgalactosidase from other plant sources [4, 6, 7, 9, 16, 181 although the carrot enzyme seems to be exceptional in showing barely detectable activity on lactose. Action on pectic polysaccharides
0
40
120 160 Fraction no. (2 ml ) 80
Fig. 1. fl-Galactosidase purification at the stage of ionexchange chromatography on Cellex-CM.
Contamination by other proteins, notably ~-galactosidase, is likely otherwise [6, 8]. In the present case, thiogalactopyranoside coupled via a spacer arm of 12 atoms (10C) to beaded agarose proved to be effective. The enzyme that was bound and subsequently eluted gave a single band after SDS-PAGE. By reference to standards, a M, of 2.9 x 104 for flgalactosidase from coffee beans was indicated by SDSPAGE. After the enzyme was boiled with 2-mercaptoethanal, electrophoresis still showed only a single protein species, with an apparent M, of 1.4 × 104. This implied that the enzyme was comprised of two identical subunits, linked by one or more disulphide bridges. A homodimeric structure was reported by Konno et al. [8] for the enzyme from carrot cell cultures; however, the Mr of this enzyme was 1.05 x 105. Other plant sources have yielded flgalactosidase o f M r 5.8 x 104 (germinating seeds of Vigna sinensis [7]) and various other M,s ranging from 4.9 x 104 up to 3 x 10 5 have been reported by Dey and Campillo [15]. Is,enzymes of different Mr apparently co-exist in plants, although artifacts arising during isolation may confuse the picture [15]. The present case appears to be free of such complications and the M, seems to be the smallest yet reported for a plant fl-galactosidase.
Table 2 shows results for pectinase and fl-galactosidase acting separately and jointly. It should be noted that the release of free galactose only and not galacturonic acid or other sugar derivatives was monitored. The negative results for all three substrates incubated with pectinase (endopolygalacturonase) alone are as expected. This enzyme is reputed to produce random breakage of internal ~-(1--,4)-linked galacturonosyl bonds in the rhamnogalacturonan backbone of the pectin polymer [19, 20]. Hence, n o production of free galactose from the substrates by pectinase should occur. fl-Galactosidase acting alone released no galactose from pectin, but did so from arabinogalactan and galactan. In these two polysaccharides that constitute side chains on the pectin backbone, fl-(1--,4)-galactosidic bonds are prevalent and indeed predominate in the case of galactan. The enzyme evidently acts in exo-fashion on such linkages, cleavage occurring from the non-reducing ends of chains. The results with galactan are analogous to those for fl-galactosidase from apples .[1] and. carrot [8]. For arabinogalactan as substrate, our results show flgalactosidase action (Table 2), in contrast to those of others [14] who reported negative results for the enzyme from apple with this type of substrate. The detailed structure of apparently similar polysaccharides may vary with the source and also with the method used to isolate them. It was not directly shown by our work that arabinogalactan from coffee bean which contains fl-D-(1 ~3)-galactosidic bonds [21] is a substrate for coffee bean fl-galactosidase. However, the arabinogal-
fl-Galactosidase from Coffea arabica actan used in our experiments and found to be susceptible to the enzyme was from larch, and reportedly contains fl(1 ~3)- and fl-(l--*6)-galactopyranoside linkages [22]. Results from HPLC confirmed that fl-galactosidase, acting alone, released galactose from arabinogalactan. Arabinose and galactose were released from arabinogalactan by the joint action of fl-galactosidase and pectinase. Unidentified peaks with R,s ranging from 8 to 20 min were also observed and may indicate polysaccharides possessing structural complexity intermediate between arabinogalactan and free monosaccharide. It is noteworthy that the joint action of pectinase and fl-galactosidase released galactose from pectin, in contrast to negative results for the enzymes acting separately
357
(Table 2). This indicates that break-up of the rhamnogalacturonan backbone of pectin is necessary for fl-galactosidase action on segments of the heteropolymer. This is in apparent conflict with the findings of Pressey [9], who reported that one of three fl-galactosidases from tomato degraded a cell-wall polysaccharide containing 22% uronic acid, together with 58% galactose, 14.5% arabinose and lesser amounts of other sugars. Such a composition indicates considerable integrity of the rhamnogalacturonan backbone structure, and this suggests that the tomato enzyme (fl-galactosidase enzyme II) acts at an earlier stage of pectin degradation than coffee fl-galactosidase.
Activity of various #lycosidases durin# ripening Table 2. Galactose produced from peptic polysaccharides by the action of pectinase and fl-galactosidase Galactose formed (#g m1-1) from substrate* Enzyme added
Pectin
Arabinogalactan
Galactan
Pectinase fl-Galactosidase Pectinase and fl-galactosidase
n.d.'t n.d.
n.d. 18.7
n.d. 25.0
29.6
20.7
27.0
*Each value is the mean of three analyses. "j'n.d. = none detected. Enzyme as indicated was incubated in 700 #1, with each pectic polysaccharide for three days, then 200/d of each mixture was brought to a final volume of 1.4 ml containing 4.0 mM NAD ÷, 0.7 M glycine/0.3M hydrazine buffer, pH 8.6, and 0.45 units of fl-o-galaetose dehydrogenase. After 1 hr, A at 340 nm was determined. Galactose content per ml of initial incubation mixture is given.
Table 3 shows the changes occurring in activities of flgalactosidase and four other glycosidases, as the fruit matures and ripens. The extracts that were assayed were derived from entire berries. The same general trend is seen in all the enzymes except N-acetyl-fl-glucosaminidase. Activities tended to peak at the semi-ripe stage, having increased from lower values in the immature (green) fruit. fl-Galactosidase showed the greatest increase, more than six-fold per unit weight of berry, and more than four-fold per unit weight of protein in extracts. Expressed on the latter basis, the data suggest that the enzyme accounted for a progressively increasing proportion of total protein in the berries. However, the trend did not continue into the fully ripe stage. For N-acetyl glucosaminidase, activity per g berry (fr. wt) remained relatively constant and actually declined when expressed per unit weight of protein. This may reflect a very low turnover of the enzyme protein, as appears to be true for this enzyme during germination of peas [22]. It has long been known that increased activity
Table 3. Activity ofglycosidases at various stages of maturation of coffee berry [each value is 102 (mean + s.c.) of three replicate analyses] Stages* Enzyme assayed fl-Galactosidase 0t-Galactosidase ~t-Mannosidase N-Acetyl-fl-ogalactosiminidase N-Acetyl-fl-Dglucosiminidase
I
III
IV
13.06+0.98 15.62+1.48 8.45 + 0.20 10.10_+0.30 7.60+ 0.45 9.10+0.69
11.41+0.15 10.74_+0.21 7.77_ 0.24 7.32_+0.33 8.64_+0.30 8.14-+0.40
Activity# Specific activity:~ Activity Specific activity Activity Specific activity
2.09__+0.04 5.72+0.04 3.66_+0.10 8.88__+0.08 3.73 _+0.10 6.35 _+0.24 6.52+0.23 9.90+0.47 2.99 + 0.25 7.45 _+0.09 5.23+0.58 11.61+0.18
Activity Specific activity
5.02+0.35 6.81+0.04 8.09+0.81 8.77-+0.65 10.72+0.09 9.69+1.22
Activity Specific activity
8.13+0.40 7.38+0.13
13.01-+0.55 12.49__+0.52 13.55+0.55 11.47-+0.76 22.75 __+1.26 19.48_+1.0 16.21___0.08 10.79__+1.02
*Stages: I--immature; II-III--semi-ripe;IV--fully ripe. tActivity = units g-1 ft. wt. :~Specificactivity = activity mg-1 protein. PHYTO 34:2-D
II
K.D. GOLDENet al.
358
of many glycosidases accompanies fruit ripening. For example, Wallner and Walker [23] reported this for fl-l,3-glucanase and polygalacturonase in tomato, with the latter enzyme showing the most substantial change in the inner pericarp. These authors also cited reports of ripening-linked changes in polygalacturonase activity in various other fruits. The enzyme activities reported here for the different stages of ripening were derived from seeds and pericarp combined, but other data not reported suggest that the observed changes were similar in the two types of structures. Our findings are consistent with an active role for at least some glycosidases in the softening process and increasingly sweet taste that accompany ripening of coffee berries. The relevant enzymes would be those that participate in degradation of oligo- and polysaccharide components of primary cell walls, as well as the glycosyl components of glycoproteins and glycolipids [22, 24]. In this regard, Ahmad and Labavitch [25] argued against a role for a-galactosidase, ~t-mannosidase and polygalacturonase, even though these showed increasing activity in ripening pears. They cited the reputed absence of a-linked galactans and mannans in cell walls, and also their finding that the galactose and mannose content of the fruit did not increase with ripening. However, interconversion and utilization of free sugars could explain their findings. The present work has established that pectic polysaccharides are indeed potential substrates for fl-galactosidase of the coffee berry. This potential could be exploited in situ only if the enzyme has access to the substrates, by being located in or on cell walls. So far, only indirect evidence indicates such a location for fl-galactosidase of plants [9, 25, 26]. EXPERIMENTAL
Plant material. Coffee berries (Coffea arabica L. var. arabica) were collected from Hall's Farm in Mount Dakin, St Andrew, Jamaica. Enzymes and chemicals, fl-o-Galactose dehydrogenase (EC 1.1.1.48) from Pseudomonasfluorescens, glucose oxidase (EC 1.1.3.4) from Aspergillus niger and pectinase [endo-(1,4)-ct-o-polygalacturonase, EC 3.2.1.15] from A. niger were purchased from Sigma. p-Nitrophenyl-fl-Dgalactopyranoside (PNPG), NAD ÷, arabinogalactan from larchwood, pectin from citrus fruits and p-aminophenyl-fl-D-thiogalactopyranoside-agarose were also from Sigma. Bio-Gel P-2 and P-200, Cellex-CM (H + from) and a Fermentation Monitoring Column (with guard attached) came from Bio-Rad Laboratories. Galactan from gum arabic was purchased from Aldrich. Purification of fl-galactosidase. Seeds of ripe coffee berries were freed of pulp and washed with dist. H 2 0 , after which 300 g were then homogenized in 600 ml of 0.05 M NaOAc buffer pH 4.5, containing 0.1% mercaptoethanol (buffer A). After centrifugation, solid (NH4)2SO 4 was added to the supernatant and a fr. which pptd between 0 and 60% (w/v) was collected. The pellet was resuspended in and exhaustively dialysed against buffer A. Finally, the vol. was reduced to 25 ml by dialysis
against 10% polyethyleneglycol (M, 1450) soln containing 0.1% mercaptoethanol. Gel-filtration chromatography using Bio-Gel P-2 was carried out on a column (30 x 3.6 cm) equilibrated with buffer A. The void peak was collected (249 ml) and centrifuged to obtain a clear supernatant. The vol. was reduced to 50 ml by dialysing against 10% polyethyleneglycol and applied to a Bio-Gel P-200 column (30 cm x 3.6 cm) equilibrated with buffer A. Elution was carried out with this buffer and 2-ml frs collected. Frs that comprised a single peak displaying fl-galactosidase activity were pooled, and after adjustment to pH 4, the soln was applied to a Cellex-CM column (16 x 4.3 cm) equilibrated with 0.05 M NaOAc buffer pH 4 (buffer B). The column was washed with 110 ml of this buffer, and the bound enzyme was eluted with 400 ml of 0.2 M NaOAc buffer, pH 6 (buffer C). The frs of eluate containing flgalactosidase activity were pooled. At this stage, the vol. of enzyme soln from a typical purification was 89 ml; aliquots from this pool were subjected to affinity chromatography as the final purification step, as follows, p-Aminophenyl-fl-D-thiogalactopyranoside-agarose contained in a 10-ml syringe was equilibrated with 0.01 M NaOAc pH 5 (buffer D). Then an aliquot from the ion-exchange step (2.5 ml, containing ca 3.6 mg protein) which had been dialysed overnight against buffer D was applied to the column. Elution with the buffer and collection of ten 2-ml frs removed agalactosidase activity. The buffer was changed to 0.05 M NaOAc, pH 5, and this removed fl-galactosidase activity in ca 5 ml vol. Steps involving CC were performed at 18-22 ° with little effect on enzyme activity; however, 4-10 ° was maintained for all other operations such as dialysis and centrifugation. Assay of fl-galactosidase activity. Enzyme prepns (100 #1, containing 0.18-11 mg protein ml-1) were incubated at 37 ° in a final vol. of 400/zl, containing 34 mM NaOAc buffer, pH 4.5 and 2.0 mM PNPG. After 10 min, 1 ml of 0.3 M Na2CO 3 was added. Where incubations were carried out above pH 6, 2-(hydroxymethyl)-l,3propanedioi buffer (34 mM) was substituted for NaOAc. The resulting A was determined at 405 nm [5]. fl-Galactosidase activity with lactose. The incubation mixt. (350 #1) contained 29 mM NaOAc buffer, pH 4.5, 21 mM lactose and 0.4 mg enzyme protein. After 2 hr incubation at 40 °, aliquots were assayed for glucose using glucose oxidase [27]. Inhibition studies. P N P G at varied concns was used as substrate, with inhibitor concn fixed at 0.29 mM in the case of CuSO 4, HgCI 2 and galactose, or at 0.15/zM PCMB. The type of inhibition was determined from plots of 1/v vs 1/S. fl-Galactosidase action on pectin, arabinogalactan and galactan with or without pectinase. We aimed to determine whether or not fl-galactosidase acted on these substrates and the contribution of pectinase to any such action. This required the commercially available samples of all three substrates and also pectinase to be purified. For this, the substrates were gel-filtered on Bio-Gel P-2 and, in each
fl-Galactosidase from Coffea arabica case, the frs of the eluates saved for use were those eluted in well-defined bands containing carbohydrate as identified by the phenol-H2SO4 method [28]. To purify pectinase, 54 mg crude enzyme dissolved in buffer B was applied to a column (16 cm × 2 cm) of Dowex 50 (H +) that was equilibrated with the same buffer. After extensive washing, the buffer was changed to 0.20 M NaOAc, pH 6, and this eluted pectinase free of contaminating proteins such as fl-galactosidase, ~-galactosidase and ~-mannosidase that were detectable in the crude enzyme. To assay for pectinase, 0.25-1.0 mg enzyme protein was incubated with 0.25 mg purified pectin for 3.75 hr at 30 °. Reducing sugar formed was then measured by the method of ref. [29]. Each of the three substrates (1.5 mg in 300 #1) was incubated with pectinase alone (1.2 units), with fl-galactosidase alone (2.6 x 10 -2 units), and with both enzymes together. Controls containing substrate without enzyme were also incubated. In each case, the final vol. was 700 #1, to which 30 #1 toluene was added as bacteriostatic agent. After 3 days at 30°, the reaction mixt. was heated to 100° for 5 min and then centrifuged. The supernatant was assayed for free galactose using fl-D-galactose dehydrogenase [30]. Detection of ffalactose and arabinose by HPLC. Aliquots (10/d) of the supernatants from the above incubations were injd onto a Fermentation Monitoring Column (150 x 7.8mm) that was equilibrated with 0.01 M H2SO4. The column was eluted with 0.01 M H2SO 4 at a flow rate of 0.5 ml min- 1. Detection of each component was by UV absorption at 190 nm. As external standards to determine R,s, 10 #1 of a mixt. containing galactose and arabinose (each at 1.0-2 mg ml- ~) were injd.
Determination of enzyme purity and molecular size by SDS-PAGE. The method was essentially as described in ref. [31] using gel rods (10% polyacrylamide and 0.53% SDS) at pH 7.4. Samples were dissolved in buffer containing 1% (w/v) SDS, denatured at 100 ° for 5 rain and analysed as such, or were reduced by boiling in buffer containing 5% (w/v) 2-mercaptoethanol before analysis. Proteins used as standards (M, range 1.4--6.6 x 104) were electrophoresed simultaneously with and in rods adjacent to those containing samples of/~-galactosidase. Coomassie Brilliant Blue R was used for staining. Apparent relative mobility was calculated from the ratio of the distance migrated to that of total gel length. Protein determination. This was by the method of ref. [32] using BSA as standard.
Activity of various glycosidases in coffee berries at different stages of maturity. Coffee berries were harvested at different stages varying from immature to fully ripe, using external colour as a guide, viz., stage I--immature, green; stage II--mature, greenish-yellow; stage I I I - semi-ripe, yellow; stage IV--fully ripe, cherry red. Four 30-g batches of coffee berries each representing one stage were separately homogenized in 30 ml buffer A. After centrifuging, the supernatant from each batch was exhaustively dialysed against several changes of buffer A. Determinations of the following enzyme activities were done on the dialysed extracts: fl-galactosidase, ct-galac-
359
tosidase, ~-mannosidase, N-acetyl-fl-D-galactosaminidase and N-acetyl-fl-D-glucosaminidase. The procedure was in each case similar to that previously described for fl-galactosidase, substituting as substrate the p-nitrophenylesters of ~t-D-galactopyranoside, ~t-D-mannopyranoside, Nacetyl-fl-D-galactosaminide and N-acetyl-fl-D-glucosaminide as appropriate for each enzyme assay.
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30. Wallenfels, K. and Kurz, G. (1966) Methods Enzymol. 9, 112. 31. Kamicker, B. J. and Prill, J. W. (1986) A E M 51,487. 32. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265.