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LWT - Food Science and Technology 43 (2010) 828835

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LWT - Food Science and Technology

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Characterization of NAD-dependent alcohol dehydrogenase enzymes of strawberrys achenes (Fragaria x ananassa cv. Elsanta) and comparison with respective enzymes from Methylobacterium extorquens
Panagiotis Koutsompogeras a, Adamantini Kyriacou b, Ioannis Zabetakis a, *
a b

Laboratory of Food Chemistry, Department of Chemistry, University of Athens, GR-157 71 Athens, Greece Department of Dietetics and Nutritional Science, Harokopio University of Athens, GR-176 76 Athens, Greece

a r t i c l e i n f o
Article history: Received 25 June 2009 Received in revised form 19 January 2010 Accepted 19 January 2010 Keywords: Achenes Alcohol dehydrogenase (ADH) Methylobacterium extorquens Flavour Symbiosis Strawberry

a b s t r a c t
Achenes were isolated from strawberries fruits, while the methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The Alcohol Dehydrogenase (ADH) enzymatic activities of the achenes were assessed and the optimum pH for ADH activity was found to be pH 10.0. Enzyme assays were carried out in order to dene the best substrate specicity at pH 10.0. The best substrates were found to be ethanol (Km 5.950 mM) and methanol (Km 12.610 mM). Only enzymes from the bacterium showed capability of using aldehydes especially formaldehyde as substrates. A wide variety of metals as well as EDTA and NaN3 were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE Electrophoresis experiments showed Molecular Weight of 47.0 kDa for the Alcohol Dehydrogenase from achenes of strawberries (Fragaria x ananassa). Additional experiments were conducted in order to dene certain thermodynamic properties of the enzymes, by using the dehydrogenation activities of these three enzyme sources which were calculated by measuring the absorption of NADH at 340 nm, in the Arrhenius equation. 2010 Elsevier Ltd. All rights reserved.

1. Introduction 2,5-Dimethyl-4-hydroxy-2H-furan-3-one (DMHF) is one of the most important strawberry avour components (Zabetakis, Gramshaw, & Robinson, 1999) and its biosynthesis has been studied by our group for over ten years (Zabetakis & Holden, 1996). The formation of DMHF by cell-free extracts of Methylobacterium extorquens and strawberry (Fragaria x ananassa cv. Elsanta) was recently reported (Koutsompogeras, Kyriacou, & Zabetakis, 2007). The authors showed that no doubt existed on the cooperation between the plant and the bacterial cells in the formation of DMHF. The food industrys interest in methylotrophy began to rise after new applications of methylotrophs were demonstrated (Anthony, 1982), such as the production of avouring or gelling agents. In a very important review on Pink Pigmented Facultative Methylotrophs (PPFMs) (Holland & Polacco, 1994), the intimate plant microbe interactions were described and particular emphasis was paid to the common benets of both plant and microbial cells.

* Corresponding author. Tel.: 30 210 7274 663; fax: 30 210 7274 476. E-mail address: izabet@chem.uoa.gr (I. Zabetakis). 0023-6438/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.01.017

Methylotrophic bacteria are involved in many important in vivo interactions between plants and bacteria (Holland & Polacco, 1994). The role of PPFMs and their enzymes have been studied in the use of reduced carbon compounds containing one or more carbon atoms but containing no carboncarbon bonds (Chistoserdova, Chen, Lapidus, & Lidstrom, 2003) and also in the oxidation of C1 substrates, such as methanol dehydrogenase and methylamine dehydrogenase (Chistoserdova, Laukel, Portais, Vorholt, & Lidstrom, 2004). The species of PPFM is very signicant biologically because of its close relationship with the plant (Holland & Polacco, 1994). Such a relationship was studied between strawberry and M. extorquens in callus cultures where the bacteria converted 1,2-propanediol to lactaldehyde (Zabetakis, 1997). A possible cooperation between strawberry and M. extorquens has been suggested (Zabetakis, Moutevelis-Minakakis & Gramshaw, 1999), regarding their enzyme system, which affects the dehydrogenation (oxidation) of certain alcohols. A further investigation on the alcohol dehydrogenation activity of the strawberry and the bacterium has been reported (Koutsompogeras, Kyriacou, & Zabetakis, 2006). Hence the purication of the alcohol dehydrogenases (ADHs) from bacterium and strawberries would prove to be extremely valuable in order to study this plantmicrobe interaction. Another similar symbiosis between

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scotch pine and M. extorquens has also been reported (Pirttila, Laukkanen, Popsiech, Myllyla, & Hohtola, 2000). Moreover, strawberry fruits are very popular because of their pleasant avour as well as their nutrient content. Its avour is an attractive one as part of the fruit or as an added avouring in many manufactured food products. Many metabolic changes take place throughout strawberry fruit growth and ripening, such as the degradation of chlorophyll, the accumulation of anthocyanin, softening, the metabolism of organic acid and sugars, and the production of avour compounds (Bood & Zabetakis, 2002). It is also known (Koutsompogeras et al., 2006) that a large group of metal ions, especially heavy metals such as lead, deactivate ADHs, mostly by reacting with SH residues of the enzymatic amino acid structure. These ions may also interact with free carboxyl groups of the enzyme, thus altering the enzymes conformation, which will also lead to enzymes partial deactivation. Another important category of enzyme effectors is chelates (e.g. EDTA), which also affect the activation of the enzymes by blocking a metal (usually zinc), which possesses a central place into the enzymes active site (Georgatsos, 1991). Additional experiments were conducted in order to dene certain thermodynamic properties of the enzymes with the use of Arrhenius equation based on the dehydrogenation activities of these 3 enzyme sources by measuring the absorption of NADH at 340 nm. The extent of the participation of each enzyme can be calculated by using the data from these comparisons. The aim of this study was to fully characterize and compare the plant and bacterial NAD-dependent alcohol dehydrogenases especially the enzyme derived from strawberrys achenes in order to enlighten to what extent the oxidation of the fragrance and avour related alcohols to the corresponding aldehydes is performed by the plant or bacterial enzymes or a combination of them. 2. Material and methods 2.1. Organisms, cultivation and cell extract preparations The strain which was used was M. extorquens (laboratory collection, strain IMI 369321 (CABI registration numbers), isolated from strawberry callus (Zabetakis, 1997)). M. extorquens, was grown in a nutrient medium containing 0.75% (v/v) 1,2-propanediol, 0.25% (v/ v) methanol and 1.0% (w/v) peptone, as described previously (Koutsompogeras et al., 2007). Cells were harvested at the end of their exponential phase then centrifuged (using a Gallenkamp centrifuge) at 7000 rpm for 10 min, washed twice with 0.1 M phosphate buffer (pH 8.0) containing 2% w/v soluble polyvinylpyrrolidone (PVP) as a phenolic scavenger, 1 mM ZnSO4, 10 mM b-mercaptoethanol (BME) as a reducing agent, and the following protease inhibitors: 0.8 mM phenylmethylsulfonyl uoride (PMSF), 1 mM benzamidine hydrochloride (BHC), and 5 mM 3-amino-n-caproic acid (ACA). They were then recentrifuged and stored in 50 ml of the same buffer. Small amounts of the bacterial pellet (2 g in 50 mL of the previous buffer) were prepared by ultrasonic disintegration (using a Branson Ultra sound device) of cells under ice cooling. The cell extract, which was used for enzyme assay, was stored at 2  C. The enzyme solution from strawberries was produced by placing 200 g of strawberries (Fragaria x ananassa) into an OMNI Mixer Speed 2 programme for 10 min. The pulp was then ltered under vacuum (with a 401 Whatman lter paper) and the ltrate was stored at 2  C. The following procedure was performed for the achenes. Seeds were removed from strawberries (ripe strawberries), washed with distilled water, then placed on a Whatman chromatography paper (No 3) and air-dried. Seeds were ground in 1.5 mL micro centrifuge tubes using a glass tissue grinder in a chilled (4  C) extraction buffer (in the ratio of 1 mL of buffer per 100 mg of seeds). The composition of the

extraction buffer was 100 mM TrisHCl (pH 7.5) including 2% w/v soluble polyvinylpyrrolidone (PVP) as a phenolic scavenger, 1 mM ZnSO4, 10 mM b-mercaptoethanol (BME) as a reducing agent, and the following protease inhibitors: 0.8 mM phenylmethylsulfonyl uoride (PMSF),1 mM benzamidine hydrochloride (BHC), and 5 mM 3-aminon-caproic acid (ACA). The extract was centrifuged at 13,600 g for 15 min and the supernatant was collected as a crude enzyme source.

2.2. Enzyme assays Arrhenius equation protein measurement The enzyme assays are based on measuring the absorption at 340 nm for NADH (Georgatsos, 1991). All enzyme experiments were conducted in three independent replicates. Values presented here are the mean values standard deviation for 95% condence levels. Three different and independent bacterial cultures or fruit batches were assessed for each experiment. Batches were formed by pooling six different fruits. The standard dehydrogenation assays contained: 35 mM phosphate buffer or 35 mM (trishydroxyamino-methane) (Tris) depending on the pH prole, 1 mM NAD, 0.1 mL enzyme extract, in a total volume of 3 mL. Injecting the respective substrate each time started the reaction. The exact substrate concentration was: methanol (30 mM), ethanol (25 mM), 1-propanol (25 mM), 2-propanol (25 mM), glycerol (20 mM), 1,2propanediol (25 mM), 1-butanol (5 mM), benzyl alcohol (0.5 mM), methanal (20 mM), ethanal (20 mM), propanal (20 mM), butanal (20 mM). In order to dene certain thermodynamic properties of the enzymes based on the Arrhenius equation 1,2-propanediol (5 mM), was used as a substrate for the enzyme source. The inuence of metal chelators (EDTA) and metal ions were tested by preincubation of the enzymatic extract with the compound for 5 min. In the case of metal ions especially, instead of a phosphate buffer an acetate buffer 35 mM was used. Moreover the bacterial cell free extract was diluted with the acetate buffer 0.5 M, pH 6 instead of a phosphate buffer. Protein was measured using bovine serum albumin as a standard (Bradford, 1976). More specically, proteins were stained with Coomassie Brilliant Blue R-250. Moreover, the enzymes activity was measured at 30  C, 40  C, 50  C and 60  C respectively. From the additional experiments, which were conducted in order to dene certain thermodynamic properties of the enzymes based on the Arrhenius equation, the following parameters were determined: free energy change, proexponential factor of the Arrhenius equation, activation energy, enthalpy change and entropy change. The following equations were used:

logA=Ao k=2303$t ;

DG R$T $lnkh=K $T ;
lnk Ea=R$T lnA;

DH Ea R$T ; DG DH T $DS:
where, Ao / The enzymes initial activity, A / The enzymes nal activity after heating for t(s) time, t / Heating Time (s), k / Velocity constant for the enzymes thermal deactivation, R / Universal gas constant (8,3145 K mol1 K1), T / Temperature (K), h / Planck constant (6,6261 1034 J s), K / Boltzman constant (1,3806 1023 J K1), DG/ Free energy change, A/ Proexponential factor of the Arrhenius equation, Ea / Activation energy, DH/ Enthalpy change, DS/ Entropy change. Dehydrogenase activity (Ao) was measured at 25  C.

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Achenes - pH Effect

Table 1B Apparent Km values of alcohol dehydrogenase from M. extorquens at pH 10, having aldehydes as substrates. Substrate Formaldehyde Ethanal Propanal Butanal Km (mM) 4460 18,985 279,224 327,902

100% 80%

% Activity

60% 40% 20% 0% 0 2 4 6 8 10 12 14

pH
Fig. 1. Effect of pH on alcohol dehydrogenase activity for the achenes of strawberry. The activity at pH 10.0 was considered as 100%. The error bars correspond to replicate measurements.

2.3. pH prole Investigation for the pH prole of the enzyme was carried out by producing a series of buffer solutions, ranging from 2.0 to 12.0 with an increasing step of 2.0 pH units. For pH values ranging from 2.0 to 8.0 a 0.2 M phosphate buffer was used, whereas for pH values ranging from 10.0 to 12.0 a 0.2 M Trishydrochloride was applied. The pH dehydrogenation assay was performed with methanol (100 mM) as substrate. The pH was measured with pH-meter ORION 410 A. 2.4. Chromatographic separation/purication of ADHs The chromatographic separation/purication of ADHs was performed on a SEPHADEX C-50 [Pharmacia Sweden] column (Georgatsos, 1991). The procedure for the bacterium and the strawberry body has been previously described (Koutsompogeras et al., 2006). The procedure performed for the achenes has been described in paragraphs 2.1. The protein content of the crude extracts was 310 mg of protein for the achenes seeds of strawberry. Subsequently 20 fractions were collected during the rst purication step; the fraction with the highest dehydrogenation activity was put into the column (2 ml) one more time. 19 more fractions were produced by this method. The fraction with the highest dehydrogenation activity, which came out the second time, was used for further SDS-PAGE Electrophoresis. 2.4.1. SDS-PAGE Active fractions, which were collected from the chromatographic separation, were subsequently subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)(Georgatsos, 1991) in order to obtain their molecular weights (Sigma). In order to perform the electrophoresis, the following preparations were made: 12.5% Acrylamide gels were prepared according to Laemmli (Laemmli,

1970). Proteins were stained with Coomassie Brilliant Blue R-250. Protein Markers (High and Low Range), were used as standards to establish the unknown proteins Molecular Weight. The device used for the electrophoresis was a mini Protean by BioRad. Numbers in parentheses indicate Molecular Weight of Standard Proteins used in this experiment in (kDa): Myosin (205), b-Galactosidase from E. coli (116), Phosphorylase b from rabbit muscle (97), Fructose-6phosphate Kinase from rabbit muscle (84), Albumin bovine serum (66), Glutamic Dehydrogenase from bovine liver (55), Ovalbumin from chicken egg (45), Glyceraldehyde-3-phosphate Dehydrogenase (36), Carbonic Anhydrase from bovine erythrocytes (29), Trypsinogen, bovine pancreas (24), Trypsin Inhibitor from soybean (20), a-Lactalbumin from bovine milk (14.2), Aprotinin bovine lung (6.5). 2.4.2. Native-PAGE The same procedure which was carried out for SDS-PAGE electrophoresis was applied for native-PAGE electrophoresis with the exception that no SDS was added to the solutions. The protein markers which were used in this experiment were produced by the following procedure. 300 mg of bovine serum albumin and 50 mg of ADH from Saccharomyces cerevisiae were added to 100 mL of deionised water. Molecular Weight numbers in parentheses in

A 120%
100%
79% 83% 94% 98% 99% 100%

% Relative activity

80%
67%

60%
43%

40%
14%

20%

7%

9%

0%
Zr K r u a Pb Ba Fe Zn a N Bl an k C C C

B 120%
100% 100%

100% 80% 88% 66% 49%

% Relative activity

80% 60% 40% 20% 0% 0,00 10,50 Concentration [mM]

Table 1A Apparent Km values of alcohol dehydrogenase from achenes of strawberry (Fragaria x ananassa) at pH 10, having alcohols as substrates. Substrate Ethanol Methanol Glycerol 1-butanol 1,2propanediol 2propanol Benzyl-alcohol 1propanol Km (mM) 5950 12,610 18,943 15,987 31,010 35,839 103,140 60,947

21,00

Fig. 2. (A): Relative activities for the effect of metals on alcohol dehydrogenase activity for the achenes of strawberry. Control without addition of metal was considered as 100%. The error bars correspond to replicate measurements. (B): Relative % effect of EDTA (black bars) and NaN3 (white bars) on alcohol dehydrogenase activity for the achenes of strawberry. Control without addition of EDTA was considered as 100%. The error bars correspond to replicate measurements.

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A
1,000

Enzyme Activity
14 12

Absorbance

0,800 0,600 0,400

10 8 6 4

Fig. 4A and B indicate Molecular Weight of Standard Proteins used in this experiment in (kDa): Albumin bovine serum (66), ADH from S. cerevisiae (43.2). An aliquot of 40 mg of protein was loaded in each sample well. 2.4.3. Extraction of proteins from native electrophoresis gel The gel was initially cut with a razor into horizontal lanes. Each lane was subsequently placed into a buffer solution (50 mM Tris HCl, pH 9) for 24 h at 04  C. The extracted proteins (from both the bacterium and the strawberry) were then tested for ADH activity in the way we previously described. 3. Results & discussion

0,200 0,000 0 5 10 15 20

2 0

# of Fraction

Enzyme Activity
0,100 0,090 0,080 14 12 10 8 6 4 2 0 21 26 31 36

pH

3.1. pH prole, physical and catalytic properties, enzyme assays The pH prole of the ADH enzyme from the achenes of strawberries is shown in Fig. 1. From this data, it is concluded that the pH optimum is pH 10.0 [with methanol (100 mM) as substrate]. All the subsequent measurements were performed at this pH. NAD (as sodium salt) was used as electron acceptor, while the produced NADH was measured spectrophotometrically at 340 nm (Georgatsos, 1991). One unit is the amount of enzyme that forms 1 mmol NADH min1. Apparent Km values for achenes of strawberries (Fragaria x ananassa), are presented in Table 1A. The respective Km values for aldehydes as substrates are shown in Table 1B. No dehydrogenase activity was observed when the enzyme extracts from the plants were used for both the body and achenes. The presence of: Na, Cr3, Zn2, K1, Zr2, Fe2, Ca2, Li, Pb2, Ba2, (all at 5 mM) resulted in relative activities which are: Na (99%), Cr3 (14%), Zn2 (83%), K1 (98%), Zr2

Absorbance

0,070 0,060 0,050 0,040 0,030 0,020 0,010 0,000

# of Fraction
Fig. 3. (A) Chromatographic separation of alcohol dehydrogenase from achenes of Fragaria x ananassa Step 1 (circles: absorbance at 340 nm, squares: absorbance at 280 nm and triangles: pH gradient). (B) Chromatographic separation of alcohol dehydrogenase from achenes of Fragaria x ananassa Step 2 (circles: absorbance at 340 nm, squares: absorbance at 280 nm and triangles: pH gradient).

Fig. 4. A) Alcohol dehydrogenase characterisation from achenes of strawberries by SDS-PAGE. Lane 1: Molecular weight markers (High Range). Lane 2: Alcohol dehydrogenase from achenes of Fragaria x ananassa. Numbers on the left of Molecular Weight Markers indicate Molecular weight of proteins in kDa. (B) Alcohol dehydrogenase characterisation from achenes of strawberries by native PAGE. Lane 1: Molecular weight markers. Lane 2: Alcohol dehydrogenase from achenes of Fragaria x ananassa. Numbers on the left of Molecular weight markers indicate Molecular weight of proteins in kDa.

pH

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A 60
58 56 54 52 50 48 46 44 kJ

Activation Energy

B 58000
59

Enthalpy Change

57 50

56000 54000 52000 50000 48000 46000 44000 42000

kJ

54453 47199

55898

St ra wb er ry

ba ct er iu m

et hy lo

Enzyme Source

C 100000
96000 92000 kJ 88000 84000 80000 76000 84415

Free Energy Change

D
96689

Entropy Change -110 -112 -114 -116 -118 -120 -122 -124
Achenes Methylobacterium Strawberry

-117

et hy lo

Enzyme Source

-119 -128

Ac he ne

rry

(J/K)

92199

te riu

St ra w

-126 -128 -130

Enzyme Source

M et

hy

Enzyme Source

lo b

ac

E 2500000
2000000 1500000 1000000 500000 0 1919125

be

1570847 496540
m

Ac he ne

te riu

Fig. 5. A) Activation energy (kJ) of ADH for the 3 enzyme sources (achenes, bacterium and main body of strawberry). (B) Enthalpy change (kJ) of ADH for the 3 enzyme sources (achenes, bacterium and main body of strawberry). (C) Free Energy change (kJ) of ADH for the 3 enzyme sources (achenes, bacterium and main body of strawberry). (D) Entropy change (J/K) of ADH for the 3 enzyme sources (achenes, bacterium and main body of strawberry). (E) Pro exponential factor of the Arrhenius equation of ADH for the 3 enzyme sources (achenes, bacterium and main body of strawberry).

(67%), Fe2 (43%), Ca2 (94%), Pb2 (7%), Ba2 (9%) for achenes of strawberries (Fragaria x ananassa), (control without addition of metal was considered as 100%). The results are shown in Fig. 2A. Addition of metal chelators resulted in the following activities (control without addition of EDTA or NaN3 was considered as 100%): For EDTA (10.5 mM): 80%, EDTA (21 mM): 49%, while for NaN3 (10.5 mM): 88%, NaN3 (21 mM): 66%, for achenes of strawberries (Fragaria x ananassa). These results are shown in Fig. 2B. 3.2. Chromatographic separation/purication of ADHs The results are shown in Fig. 3A and B. The fraction with the highest dehydrogenation activity from the second elution was used for further SDS-PAGE Electrophoresis. Purication results for achenes of strawberries ADHs are shown in Table 2. Yield is the % fraction of the total activity (U) in purication step 1 towards the total activity (U) in purication step 2. Purication (fold) is the fraction of the specic activity [U (mg protein)1] in purication step 1 towards the specic activity [U (mg protein)1] in purication step 2.

M et

hy

Enzyme Source

lo b

3.2.1. SDS-PAGE The results for SDS-PAGE Electrophoresis are shown in Fig. 4A. Based on this gure (LANE 2) it is suggested that the molecular weight of the ADH from the achenes of strawberries is 45 kDa. 3.2.2. Native-PAGE The results for native-PAGE electrophoresis are shown in Fig. 4B. From these data, it can be suggested that the Molecular weight of

Table 2 Purication (fold) for achenes of strawberries (Fragaria x ananassa). Yield (%) is the % fraction of the total activity (U) in purication step 1 towards the total activity (U) in purication step 2. Purication (fold) is the fraction of the specic activity [U (mg protein)1] in purication step 1 towards the specic activity [U (mg protein)1] in purication step 2. Purication step 1 2 Total protein (mg) 156.4 53.1 Total activity (U) 110.2 93.3 Specic activity [U (mg protein)1] 0.7 1.8 Yield (%) 100 85 Purication (fold) 1 2.5

St ra w

ac

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the ADH from the achenes of strawberries is 45 kDa. Based on the electrophoresis results (Fig. 4A and B), both the native and the reduced forms of the enzymes exhibit similar molecular weights. This suggests that the plants ADH exists as a single polypeptide chain similar to the ADH found in strawberry pulp (fruit without achenes) (Koutsompogeras et al., 2006). The molecular weight of ADH from strawberrys body was reported to be 24.6 kDa while the respective enzyme from M. extorquens had a molecular weight of 45.1 kDa (Koutsompogeras et al., 2006). This implies the fact that ADH from the strawberry enzyme from the main body is a simpler one and may evolve from partial hydrolysis of the achenes ADH enzyme. Furthermore there is a possible cooperation between the achenes and the bacterial enzyme. 3.2.3. Activity of extracted proteins from native electrophoresis gel The activity for the extracted proteins (ADH) had the value of 22.03% for the achenes of the strawberry. The percentage (%) value refers to the ratio of the ADH activity prior and after the electrophoresis respectively. 3.3. Enzyme assays Arrhenius equation The results which were obtained are presented in Fig. 5AE. With regard to the thermodynamic results that are derived from Arrhenius equation having 1,2-propanediol 100 mM as substrate for the enzyme the following results were obtained. The activation energy (Ea) of enzyme from the achenes of strawberry corresponds to the 85% of the activation energy of the corresponding enzyme from the strawberry while the corresponding Ea of the enzyme from M. extorquens is similar to that of the enzyme from the strawberry. Moreover the change of enthalpy (DH) for the enzyme from the seeds of strawberry corresponds to the 84% of DH of the corresponding enzyme from the strawberry while the corresponding DH of the enzyme from M. extorquens is roughly the same with the DH of the enzyme from strawberry. Respectively, the change of free energy (DG) for the enzyme from the seeds of strawberry corresponds to 87% of the corresponding enzyme from the strawberry while the corresponding DG of the enzyme from M. extorquens is similar to that of the enzyme from strawberry. The value of the factor of frequency (A) for the enzyme from the strawberry corresponds to the 26% of the corresponding value for the seeds of strawberry, while the value of A for the enzyme from M. extorquens corresponds to the 82% of corresponding value for the seeds of strawberry. Based on the above data, it may be assumed that since the activation energy (Ea) of the enzyme from the seeds of strawberry is smaller than the respective one from the enzyme of the body of the strawberry or the bacterium one, the dehydrogenation process takes place more easily in the achenes section. Respectively since the change of free energy (DG) for the enzyme from the seeds is smaller than the two aforementioned enzymes, the oxidation process takes place more easily in the achenes section. It appears as though a preparation for the enzymatic activity of dehydrogenation takes place rst in the achenes and then the whole process proceeds in the main body of the fruit of strawberry. The enzyme from achenes seems to become quite easily thermically deactivated compared to the other two enzymes since it almost loses all of its activity at 60  C as it is shown in Fig. 6. By comparing the Km values of the three enzymes, it could be suggested that ethanol and methanol are the best substrates for the enzyme derived from achenes of strawberry. ADH from achenes does not seem to have a tendency to oxidize benzyl alcohol or 1-propanol. The comparison of Km values presented here with Km values previously reported (Koutsompogeras et al., 2006) suggests

Temperature Deactivation 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Temperature [ C]

Fig. 6. Temperature Deactivation of ADHs from the three sources of the enzyme (diamonds: achenes, squares: strawberry and triangles: methylobacterium).

that while 1-propanol is the second best substrate for the achenes enzyme, this alcohol (1-propanol) is the best substrate for the strawberry enzyme. This is a remark, which shows the degree of the diversication of each enzyme depending on which part of the plant exists in. A possible cooperation between the ADH from the body of the fruit and the ADH of achenes is also suggested here. On the other hand, methanol, which is the best substrate for the bacterium, is the second best substrate for the achenes enzyme. A hint for a potential plantmicrobe interaction can be assigned here (Fig. 7A). In addition, a comparison between the Km values obtained at pH 6.0 with the ones obtained at pH 10.0 reveals that the values of the second group correspond to 49% of the rst group (Fig. 7B), which means that generally ADH perform better at pH 10.0.

Remaining Enzyme Activity %

A 120,0
100,0

103,1

Km [mM]

80,0

60,9
60,0 40,0 20,0 0,0

31,0 6,0 12,6 16,0 18,9

35,8

no

ha no

ta n

G ly ce

ed

an

an

Et ha

2Pr op

1Pr op

1Bu

M et

pa n

Pr o

B 160
Km [mM]

140 120 100 80 60 40 20 0

ol ro pa no l m et ha no l ro pa no l ce ro l oh ol l ro pa ne di 1 p 2 p nz yl 1bu ta no l an o -a lc gl y et h

1, 2 p

1, 2

Substrate

Fig. 7. (A) Km values [mM] from ADHs of achenes for certain alcohols, which where used as substrates. (B) Km values [mM] from ADHs of achenes at pH 6.0 (dark bars) and pH 10.0 (white bars).

be

Be

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Al

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ho l

ol

ro l

ol

io l

ol

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120%

100% % Relative activity 80%

100%

100%

100%
36%
Et ha no l

47%

37%

60% 3 1%

25%

17% 23%

40% 20%

22%

31%

30%

19%

18%

18%

19%

47%

6% 10%

10%

0%
ho l ol G ly ce ro l l l io l 1Pr op 2Pr op 1Bu ha no an o an o ed co ta n l

pa n

Al

3%

nz yl

Be

1, 2

Pr o

Substrate
Fig. 8. Relative % Km Values (dark bars: achenes, white bars: bacterium and striped bars: strawberries). For achenes the Km value of ethanol is divided with all the Km values of the other alcohols and the nal result is expressed in (%) percentage activity. For the bacterium, the Km value of methanol is divided with all the Km values of the other alcohols and the nal result is expressed in (%) percentage activity, whereas for the strawberry the Km value of 1-propanol is divided with all the Km values of the other alcohols and the nal result is expressed in (%) percentage activity respectively. The error bars correspond to replicate measurements.

4. Conclusions The presented data in this work reveal that the cooperation between plant and microbial cells is a matter of intense interest and should be viewed more closely. Alcohol Dehydrogenases (ADHs) contain a metal in their active center usually Zn but they are also strongly inhibited by Zn2, as well as by other metals. From the presented data it was found that certain metals especially heavy metals cause a certain amount of deactivation to the enzyme for both the bacterium and the strawberry itself. The fact that EDTA reduces the enzymatic activity, for both the bacterium and the strawberry, is an additional proof that the enzyme either possesses a metal in its active center or it is activated by metal ions, which are blocked by the chelate action. The ADH enzymatic activity was also observed after native electrophoresis and extraction of the protein bands. It can thus be concluded that the obtained puried bands after SDS electrophoresis correspond to ADH enzymes. From our experiments, it may also be concluded that ADHs from the strawberrys body and achenes do not use aldehydes as substrates. Only the bacterial enzymes use these substances as substrates especially formaldehyde (Table 1B). Thus, it could be deduced that there is no signicant need for the plants enzymes to use these molecules as substrates, or maybe that they need an external enzyme source to facilitate their use. The results reported here suggest an enzymatic collaboration between the bacterium and the strawberry (Fig. 8). The initial alcohols can be converted into the respective aldehydes with the aid of enzymes from M. extorquens, which in turn are transformed into DMHF with the use of enzymes from Fragaria x ananassa. The aforementioned enzyme (ADH) could possibly play a crucial role in the formation or improvement of the strawberry fruit. The role of PPFMs in the biosynthesis of strawberry avour can not be easily understood since those kind of plant-microbe interactions may be disguised as part of the plants activities, thus being observed as plant biochemical reactions and not bacterial ones. It has though been demonstrated that PPFMs can enhance the avour

biosynthesis in strawberry callus cultures while the bacteria can nd a niche in strawberry by consuming the alcohols of the plant (e.g. 1,2-propanediol) (Zabetakis and Gramshaw, 1998). Our current work focuses on the interactions of bacterial (M. extorquens) and strawberry (Fragaria x ananassa cv. Elsanta) cells in order to further highlight the role of the bacteria in the biosynthesis of DMHF and investigate the use of biotechnological methods to enhance the formation of DMHF. Acknowledgements The nancial support from the Special Account for Research Grants (University Of Athens Special Account for Research Grants) is kindly acknowledged. References
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