Acta Physiol Plant (2009) 31:81–87 DOI 10.1007/s11738-008-0203-1
ORIGINAL PAPER
Partial purification and characterization of pectinmethylesterase from ripening guava (Psidium guajava L.) fruits Koshik Mondal Æ Sarla P. Malhotra Æ Veena Jain Æ R. Singh
Received: 14 January 2008 / Revised: 27 June 2008 / Accepted: 7 July 2008 / Published online: 22 August 2008 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2008
Abstract Employing the techniques of (NH4)2SO4 fractionation, ion exchange chromatography on DEAE cellulose and gel filtration through Sephadex G-100, pectinmethylesterase (EC 3.1.1.11) was purified from guava (Psidium guajava L.) fruits var. Hisar Safeda harvested at turning stage of maturity to 129-fold with 28% recovery. Molecular weight as determined by gel filtration was found to be 51 kDa and the enzyme preparation exhibited the same molecular weight under native (NativePAGE) and denaturating conditions (SDS-PAGE) indicating that the enzyme was a monomer. With pectin as the substrate, it exhibited the Michaelis Menten kinetics with Km value of 3.1 g l-1. The enzyme was found to be stimulated by Ca?? and Na? and inhibited competitively by Dgalacturonic acid with Ki value of 1.97 mM. The enzyme was completely inactivated by iodine while with diethyl pyrocarbonate and N-acetylimidazole, the enzyme was inhibited up to the extent of 56 and 45%, respectively. However, DTNB had no inhibitory effect whatsoever precluding the participation of any –SH group in the active centre. It is tentatively proposed that the enzyme has tyrosine and histidine residues at its active centre. Keywords Fruit ripening Psidium guajava Pectinmethylesterase Purification
Communicated by G. Klobus. K. Mondal S. P. Malhotra (&) V. Jain R. Singh Plant Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, CCS Haryana Agricultural University, Hisar 125 004, Haryana, India e-mail:
[email protected];
[email protected]
Abbreviations SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis DTNB 5, 50 -Dithiobis-2-nitrobenzene PME Pectinmethylesterase PG Polygalacturonase
Introduction Pectinmethylesterase (PME) (EC 3.1.1.11) is known to catalyze demethoxylation of C-6 carboxylic groups of pectin compounds thereby providing the substrates to be depolymerized by polygalacturonase (PG) (Jansen and MacDonnell 1945) which preferentially degrades de-esterified (demethoxylated) rather than esterified (methylated) pectin (Koch and Nevins 1989). Thus, during fruit ripening, PME may play an important role in determining the extent to which pectin is accessible to degradation by PG and in reducing intercellular adhesiveness and tissue rigidity. Indeed, it has been suggested that the increased susceptibility of tomato fruit cell walls to PG during ripening is due to the action of PME. PME activity has been reported either to increase (Buescher and Furmanski 1978), or to remain constant (Missang et al. 2004) or to decrease (El-Zoghbi 1994) during ripening. It has been suggested that an increase in pectin solubility may result from an increase in methyl de-esterification of the polygalacturonates by PME (Awad 1985). PME also participates in cell wall pH regulation by generating protons and thus enhances the activity of cell wall hydrolases (Rexova-Benkova and Markovic 1976). It has also been proposed to be involved in cell extension (Moustacas et al. 1991) by inducing a partial autolysis of cell wall, which is successively reconstituted by
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glycosyltransferases. Based on the roles it plays in fruit softening during ripening and in cell wall extension during cell growth, PME is considered to be the enzyme of physiological relevance to plant metabolism. Because of its strategic role, PME has been purified from different sources (Lin et al. 1991; Javeri and Wicker 1991; Alonso et al. 1997) and its characteristics have been widely investigated. Guava (Psidium guajava L.) fruit belonging to family Myrtaceae, undergoes extensive softening during ripening and post-ripening. Changes in cell wall components including dissolution of pectin have been associated with softening of this fruit (Jain et al. 2001, 2003). Since no information, whatsoever, is available on the characteristics of guava PME, it prompted us to purify the enzyme from this fruit. We describe in this paper the isolation, partial purification and some physicochemical properties of PME from guava fruits.
Acta Physiol Plant (2009) 31:81–87
fruit tissue (10 g) was blended in a pre-chilled pestle and mortar using acid washed quartz sand with 50 ml of chilled 0.1 M Tris-HCl buffer (pH 7.5), containing 10% NaCl. The resulting homogenate was passed through four layers of cheese cloth and the filtrate centrifuged at 10,0009g for 30 min. The supernatant so obtained was referred to as the crude extract. (NH4)2SO4 fractionation PME in the crude extract was precipitated between 30 and 70% saturation of (NH4)2SO4. The precipitates obtained were collected by centrifugation at 10,0009g for 30 min and suspended in 0.1 M Tris-HCl buffer (pH 7.5) and dialyzed overnight against the same buffer (diluted 1:10) for 24 h with repeated changes of the buffer. Following steps of enzyme purification were carried out in cold lab (LKB LPLC system for enzyme purification).
Materials and methods
Ion exchange chromatography
Fruit samples
The dialyzed enzyme was loaded onto a DEAE cellulose column (effective bed dimensions 34 9 3 cm) previously equilibrated with 0.1 M phosphate buffer (pH 7.5). The column was first eluted with 0.1 M Tris-HCl buffer (pH 7.5) and then with a linear gradient of 0–0.4 M KCl in the same buffer at a flow rate of 30 ml h-1. The fractions of 3 ml each were collected and analyzed for protein content (A280) and enzyme activity. Active fractions were pooled together, concentrated against solid sucrose and subjected to gel filtration chromatography.
Guava fruits were harvested at different stages of maturity viz. immature green (IG), mature green (MG), colour turning (T), ripe (R) and over-ripe (OR) from the 10-year old trees from the Horticultural Farms at CCS Haryana Agricultural University, Hisar, for the purpose of studying the PME profile. However, fruits harvested at colour turning stage were used for enzyme purification. Chemicals
Gel filtration All the chemicals and biochemicals used during the present course of investigations were purchased either from Sigma Chemical Co. (St. Louis, MO, USA), Hi-Media or SRL (SISCO Research Laboratories Pvt. Ltd., India) and were of high purity. Pectin was purchased from SISCO Research Laboratories Pvt. Ltd, India and it had 7% methoxy content. Enzyme purification Unless otherwise stated, all steps of enzyme purification were carried out at 0–4°C.
The concentrated enzyme was then loaded onto a Sephadex G-100 column (effective bed dimensions 51 9 1.5 cm) previously equilibrated with Tris-HCl buffer (pH 7.5). The enzyme was eluted with this buffer at a flow rate of 12 ml h-1. The active fractions of 3 ml each eluted as a single peak were pooled together, concentrated again by osmosis against solid sucrose and stored at 4°C for further studies. Fold purification was calculated as the ratio of the specific activity at each step to that in crude extract while per cent yield was calculated as % of total activity in crude extract remaining at each step of purification.
Preparation of crude extract Enzyme assay The extraction conditions were standardized with respect to type, pH, molarity and ionic strength of the buffer, concentration of stabilizing agents and the other constituents of the extraction medium to achieve maximum extraction of the enzyme from guava (Psidium guajava L.) fruits. Fresh
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Preliminary experiments were conducted to ensure linearity with respect to enzyme concentration and time of incubation. Enzyme was assayed using continuous spectrophotometric method of Hagerman and Austin (1986).
Acta Physiol Plant (2009) 31:81–87
The standard reaction mixture in a total volume of 3.0 ml prepared in a glass cuvette, consisted of 2.5 ml of 0.5% (w/v) apple pectin in buffer (2 mM Tris-HCl, pH 7.5), 0.4 ml of 0.01% (w/v) solution of bromothymol blue prepared in same buffer and 0.1 ml of the enzyme preparartion. The cuvette was shaken quickly and absorbance at 620 (A620) was monitored at room temperature. This initial reading was taken as zero time of incubation. After 30 min, the change in absorbance at 620 nm was determined. The assay was calibrated with galacturonic acid (50–500 lg). One enzyme unit of enzyme activity is defined as the amount of enzyme which liberates 1 lg galacturonic acid/ 30 min. Specific activity was calculated as enzymes units/ mg protein. Protein estimation The protein content of the individual fractions obtained after column chromatographic steps was monitored by measuring the extinction at 280 nm. Quantitative estimation of protein after each step of purification was done by the method of Lowry et al. (1951). Determination of purity and molecular weight The purity of the enzyme preparation obtained after gel filtration chromatography on Sephadex G-100 was judged by native-PAGE at 4°C in 10% gels, using Tris-glycine buffer (pH 8.3) following the method of Davis (1964). The molecular mass of the purified enzyme was estimated by gel filtration through a column of Sephadex G-100, which had previously been calibrated with cytochrome c (12.4 kDa), lysozyme (14.3 kDa), b-lactoglobulin (18.0 kDa), pepsin (34.7 kDa), egg albumin (45 kDa), bovine serum albumin (66 kDa) and alcohol dehydrogenase (150 kDa). The subunit molecular mass was determined by SDS-PAGE according to the procedure followed by Laemmili (1970).
Results and discussion Extraction and purification Figure 1 shows the profile of PME activity during ripening and post ripening of guava fruit cv. L-49 and Hisar Safeda. There was continuous increase in the PME activity reaching the maximum during the turning stage after which the activity decreased until over ripe stage. Since Hisar Safeda had much higher PME activity than L-49, the guava fruits of cv. Hisar Safeda harvested at turning stage were used for the purification purposes. PME associates with other cell components including cell walls (Jansen et al. 1960). As a consequence, extraction was incomplete in buffers of low
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ionic strength. In preliminary experiments, the enzyme was found to be incompletely extracted by buffers of low ionic salt content. However, the 0.1 N Tris-HCl buffer (pH 7.5) containing 10% NaCl effectively released PME from the tissue and the residue gave no evidence of further bound enzyme. It is likely that guava fruits at the turning stage have high content of organic acids like malic and citric acid which requires a high cation addition for extraction at pH 7.5 and this raises the ionic strength of buffer used for extraction. The summary of results showing stepwise purification of PME has been presented in Table 1. The enzyme was purified about 129-fold with 28% recovery by conventional methods of purification viz., ammonium sulphate precipitation, ion exchange chromatography and gel filtration. Enzyme from the crude extract with total activity of 326 units and specific activity of 1.45 was precipitated between 30 and 70% ammonium sulphate at 4°C. During this step, about 34% activity was lost. The enzyme fractionated at 70% (NH4)2SO4 saturation was dissolved in 0.1 M Tris-HCl buffer (pH 7.5), dialyzed and submitted to ion exchange chromatography on DEAE-cellulose. This step resulted in 77-fold purification with specific activity of 112. The pooled fractions contained about 60% of the applied activity and 39% of the original activity. The dialyzed enzyme did not adsorb to DEAE-cellulose and got eluted as a single peak before applying the KCl gradient (Fig. 2) indicating that PME from guava fruit carried either a net positive charge or did not carry any charge under the experimental conditions. In this respect, the guava PME seems to resemble that of banana (Brady 1976), flax cell wall (Gafe et al. 1992) and lemon (MacDonald et al. 1993). Fractions were pooled on the basis of specific activity, concentrated against solid sucrose and applied to a column of sephadex G-100. Most of the PME activity eluted as a single peak (Fig. 3) which was 129-fold purified with specific activity of 187 and recovery of 27.5% activity of the original crude preparation. The final enzyme preparation gave one major band on native-PAGE (Plate 1) alongwith a few very minor and faint bands indicating that the enzyme could not be purified to homogeneity. It was a partially purified preparation. Since this is the first report of purification of guava PME, the results could not be compared with the literature values. Characterization of PME The enzyme was found to be thermostable as it could retain 95–100% activity when incubated at 50°C for 45 min. Preincubation of enzyme at 60, 70 and 80°C for the same period resulted in 15, 40 and 64% loss in activity, respectively. However, at 90°C, the enzyme was completely inactive after 45 min of pre-incubation. Thermostable forms of the enzyme have been reported in persimmon
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Acta Physiol Plant (2009) 31:81–87 Protein Enz. Act.
Absorbance (280 nm)
L-49
18
HS
16 14 12
0.4
0.035
0.35
0.03
0.3
0.025
0.25
0.02
0.2
0.015
0.15
0.01
0.1
0.005
10
-1
0.04
0.05 0
5 9 13 17 21 25 29 32 36 40 44 48 51 54 57 61 64 68 71 75 79 83 87 95 11 0
0
8
Fraction Number
6
Fig. 2 Elution profile of PME on DEAE-cellulose
0 IG
MG
T
R
OR
Stages Fig. 1 Pectinmethylesterase activity in guava fruit at different stages of ripening
0.4
0.03
0.35
0.025
0.3 0.25
0.02
0.2 0.015
0.15
0.01
0.1
0.005
0.05
95 11 0
85
88
80
60
70
55
39
47
34
28
18
23
5
0
10
0
(Alonso et al. 1997), sweet cherry (Alonso et al. 1996), lemon endocarp (MacDonald et al. 1993), orange (Amaral et al. 2005), plums (Nunes et al. 2006) and bergamot fruit (Laratta et al. 2008). Compared to alcohol dehydrogenase, bovine albumin, ovalbumin, pepsin, carbonic anhydrase, b-lactoglobulin and cytochrome-c, and assuming a globular configuration, PME activity appeared to be associated with a protein of molecular weight 51 kDa (Fig. 4). SDS-PAGE (Plate 2) yielded a single protein band of Mr 51 kDa (Fig. 5) indicating the monomeric nature of guava PME. The present value of Mr falls within the range of reported values of 23– 110 kDa for PME from different fruits (Giovane et al. 1990; Gafe et al. 1992; Bordenave and Goldberg 1993; Nunes et al. 2006). The detailed studies of PME from germinating Vigna sinensis seeds (Nighojkar et al. 1994) and bergamot fruit (Laratta et al. 2008) have described the enzyme as a protein of Mr. 54 and 45 kDa. This similarity in the Mr from such very distinct botanical material
-1
Protein Enz. Act.
0.035
2
Enzyme activity (mg gal ml )
4
Absorbance (280 nm)
mg galacturonic acid released/30 min g-1 fresh wt.
20
Enzyme activity (mg gal ml )
84
Fraction Number
Fig. 3 Elution profile of PME on Sephadex G-100
suggests that this property of the enzyme may be a quite general one. The Km of PME was measured with aqueous solutions of apple pectin in the range of 1–10 mg ml-1 without adding any salt to the substrate. The enzyme exhibited a typical hyperbolic velocity saturation curve (Fig. 6) with Km value of 3.1 mg ml-1 of pectin revealing that PME from guava follows Michaelis Menten kinetics. The Km value is high compared to that of enzymes from different sources (0.4– 2.4 g l-1) (Gafe et al. 1992; Hagerman and Austin 1986; Komae et al. 1990; Laratta et al. 2008) indicating a low affinity of guava PME for apple pectin. In all these studies, citrus pectin having 70% methylation at C6 carboxylic group was used as the substrate. It is quite likely that the extent of methoxylation in pectin from different sources
Table 1 Summary of purification of PME from guava (Psidium guajava L.) fruits Sp. activity (mg gal min-1 mg-1 protein)
Fold purification
Yield (%)
S. no.
Purification step
Volume (ml)
Total protein (mg)
Total activity (mg galacturonic acid liberated min-1)
1.
Crude extract
350
224.62
326.42
1.45
1.00
100
2.
(NH4)2 SO4 fraction (30–70%)
55
65.46
215.56
3.29
2.26
66.03
3. 4.
DEAE-cellulose Sephadex G-100
26 12
1.15 0.48
128.39 89.87
111.64 187.22
76.99 129.11
39.33 27.53
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Acta Physiol Plant (2009) 31:81–87
Plate 1 Native-PAGE pattern of purified PME from guava (Psidium guajava L.) fruit. Lane 0: standard markers, Lane 1: crude extract, Lane 2: (30–70%) (NH4)2 SO4 fraction, Lane 3: DEAE cellulose column fraction, Lane 4: purified enzyme
Fig. 4 Determination of molecular weight of partially purified PME by gel filtration through Sephadex G-100. Alcohol dehydrogenase = 150 kDa, albumin bovine = 66 kDa, ovalbumin = 45 kDa, pepsin = 34.7 kDa, b-lactoglobulin = 18 kDa, lysozyme = 14.3 kDa, cytochrome c = 12.4 kDa
may be responsible for determining the affinity of the enzyme for the substrate. The bivalent cation Ca?? activated the enzyme to a greater extent than the monovalent cation Na?. Ca?? as CaCl2 activated PME about 150% at 5 mM final
85
Plate 2 SDS-PAGE patteren of purified PME from guava (Psidium guajava L.) fruit. Lane 0: standard markers, Lane 1: purified enzyme
Fig. 5 Determination of molecular weight of partially purified PME by SDS-PAGE. Alcohol dehydrogenase = 150 kDa, albumin bovine = 66 kDa, ovalbumin = 45 kDa, pepsin = 34.7 kDa, b-lactoglobulin = 18 kDa, lysozyme = 14.3 kDa, cytochrome c = 12.4 kDa
concentration compared with nil activation by the same concentration of NaCl. NaCl, however, at concentration as high as 200 mM activated PME about 113%. Nighojkar et al. (1994) have also reported the divalent cations Ca?? and Mg?? to be the activators of PME. According to them, activation of PME by metallic ions appears to be due mainly to interaction of ions with the substrate rather than with the
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86
Acta Physiol Plant (2009) 31:81–87 450
Table 2 Effect of organic solutes and inhibitors on PME activity
400
S. No
Substance
Conc
Relative activity (%)
1
Control
–
100
0.007
300
0.006 -1
1/v (ug galA/ml )
ug galA released ml-1
350
250 200 150
-5 -3 -1/Km
100
Km = 3.1g/l
2
Glycine
1M
93
0.004
3
Glycerol
1M
71
0.003
4
Glucose
1M
75
5
Maltose
1M
63
6
Sucrose
1M
43
7 8
0
5, 5 -Dithiobis-2-nitrobenzoic acid Iodine
1 mM 1 mM
96 5
9
N-acetylimidazole
5 mM
55
10
Di-ethylpyrocarbonate
2 mM
44
0.005
0.002 0.001 0 -1
1
3
5
1/[S] (% pectin)
7
9
11
-1
50 0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Fig. 6 Effect of varying pectin concentration on activity of partially purified PME
Fig. 7 Dixon’s plot for determination of Ki for D-galacturonic acid inhibition of PME. The standard reaction mixture in addition contained: 1 (open triangle). 0.5 (filled circle), 0.3 (open cirlce) and 0.25 (filled triangle) per cent pectin
enzyme. They suggest that metallic ions release the enzyme molecule trapped in the free carboxylic groups of the substrate enabling them to reactivate. However, contrary to this, there are evidences which clearly demonstrate that Na? and Ca??, like other metallic ions can bind with enzyme causing conformational changes to more active forms (Marcus and Schejter 1983; Alonso et al. 1995). Polygalacturonic acid (PGA) is found to inhibit the action of PME on pectin competitively. Dixon plot (Fig. 7) reveals the inhibition by PGA to decrease on increasing the pectin concentration and gives the Ki value for inhibitor constant to be 1.97 mM. These observations are in accordance with the earlier reports (Nighojkar et al. 1994; Nari et al. 1991; Alonso et al. 1997). The responses of a number of solutes at high concentration are recorded in Table 2. The results clearly demonstrate that at 1 M concentration, all the solutes tested
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were inhibitory but to varying degree of effectiveness. The relative order of effectiveness as indicated by relative inhibition (per cent of control) was glycine \ glucose \ glycerol \ maltose \ sucrose. Except for glycerol, the order corresponds directly to the molecular weight, suggesting that the inhibition was the response to per cent concentration of the solute rather than the molar concentration. Higher the per cent concentration of the solute, higher was the inhibition. The banana enzyme is similarly inhibited by a variety of low molecular weight polyols (Brady 1976). It is suggested that the inhibition may probably be the result of several factors including an environment of lowered water activity which is less suitable for enzymic action (Chang et al. 1965), polyol–pectin interaction (Jansen et al. 1960) and perhaps some degree of interference of the enzyme active site (Rouse et al. 1960). Inhibitors for certain specific groups are generally employed to identify groups present at the active centre of the enzymes (Markovic and Jornvall 1986). Iodine at 1 mM concentration completely inhibited the enzyme while diethyl pyrocarbonate and N-acetyl imidazole inhibited the enzyme by 56 and 45%, respectively, suggesting the involvement of histidine alongwith tyrosine at the active site of the enzyme. 5, 50 -Dithiobis-2-nitrobenzene (DTNB), however, had no effect on enzyme activity thus ruling out the possibility of –SH group involvement in the activation. Active site of guava PME seems to be similar to that of tomato PME as Markovic and Patoka (1977) also observed iodine to strongly inhibit the tomato PME and DTNB not to show any inhibition.
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