Characterizing NAD-dependent alcohol dehydrogenase enzymes of Methylobacterium extorquens and strawberry (Fragaria x ananassa cv. Elsanta)

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 microbial biotransformation of 1,2-propanediol to 2-hydroxypropanal (lactaldehyde) was studied. The bacterial alcohol dehydrogenase (ADH) enzymatic activities were assessed, and the optimum pH for ADH activity was found to be pH 6.0. Enzyme assays were carried out for both the bacterial and the strawberry extracts to define the best substrate specificity. For Methylobacterium extorquens, the best substrates were found to be methanol (Km = 0.78 mM) and 1,2-propanediol (Km = 15.84 mM), whereas for strawberries, 1-propanol (Km = 3.54 mM) and ethanol (Km = 6.66 mM) were the best substrates. A wide variety of metals as well as EDTA were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE experiments showed molecular weights of 45.0 and 24.6 kDa for the alcohol dehydrogenases of Methylobacterium extorquens and Fragaria x ananassa, respectively.     

Carbohydrate differences in strawberry crowns and fruit (Fragaria x ananassa) during plant development

Carbohydrates accumulation and mobilization are highly relevant in plants because they have been related to yield and quality. Therefore, the aims of this work were to determine soluble carbohydrates and starch in strawberry (Fragaria x ananassa cv. Camarosa) crown sections (basal, middle, and upper) at three different plant growth stages (vegetative, blooming, and fruiting), and in fruit varieties (cv. Camarosa, Seascape, and Oso Grande) grown on the same field and in a different geoclimate. The main soluble carbohydrates found were glucose, sucrose, and fructose. Concentration differences were found among crown sections and time. The lowest levels of glucose, fructose, and sucrose were present at the beginning of fruit formation (6.2, 1.8 mg/g, and trace, respectively). Starch increased in basal and middle sections at the same time (8.6 to 109.6 and 6.6 to 93.5 mg/g, respectively). There appears to be a relationship between crown and fruit soluble carbohydrates. The most abundant fruit monosaccharides in all varieties were glucose (160-190 mg/g), fructose (90-180 mg/g), and sucrose (30-120 mg/g), followed by myo-inositol (10-23 mg/g). Strawberry crowns are an important source of carbohydrates and they might play a role during plant development specifically related to fruit sweetness. Fruit quality is highly influenced by a combination of several factors such as genotype, geoclimate, and probably carbon partitioning.     

Aroma biosynthesis in strawberry: s-adenosylmethionine:furaneol o-methyltransferase activity in ripening fruits

Among the most important volatile compounds in the aroma of strawberries are 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methoxy derivative (methoxyfuraneol, mesifuran). Three strawberry varieties, Malach, Tamar, and Yael, were assessed for total volatiles, Furaneol, and methoxyfuraneol. The content of these compounds sharply increased during fruit ripening, with maximum values at the ripe stage. An enzymatic activity that transfers a methyl group from S-adenosylmethionine (SAM) to Furaneol sharply increases during ripening of strawberry fruits. The in vitro generated methoxyfuraneol was identified by radio-TLC and GC-MS. The partially purified enzyme had a native molecular mass of approximately 80 kDa, with optimum activity at pH 8.5 and 37 degrees C. A high apparent K(m) of 5 mM was calculated for Furaneol, whereas this enzyme preparation apparently accepted as substrates other o-dihydroxyphenol derivatives (such as catechol, caffeic acid, and protocatechuic aldehyde) with much higher affinities (K(m) approximately 105, 130, and 20 microM, respectively). A K(m) for SAM was found to be approximately 5 microM, regardless of the acceptor used. Substrates that contained a phenolic group with only one OH group, such as p-coumaric and trans-ferulic acid, as well as trans-anol and coniferyl alcohol, were apparently not accepted by this activity. It is suggested that Furaneol methylation is mediated by an O-methyltransferase activity and that this activity increases during fruit ripening.     

Comparative study of the products of the peroxidase-catalyzed and the polyphenoloxidase-catalyzed (+)-catechin oxidation. Their possible implications in strawberry (Fragaria x ananassa) browning reactions

The peroxidase- and polyphenoloxidase-catalyzed oxidations of (+)-catechin yield several products showing different degrees of polymerization, which are apparently responsible for the pigment decay and the associated browning reaction that occurs in processed strawberry fruits and their derived foods. In this work, we have purified both peroxidase and polyphenoloxidase from Oso Grande cv. strawberry fruits, and comparatively analyzed the products of their enzyme-mediated (+)-catechin oxidation. The joint analysis by reversed-phase and size-exclusion HPLC of the (+)-catechin oxidation products obtained with both enzymes indicate that they were qualitatively the same: dehydrodicatechin B4, a (+)-catechin quinone methide, dehydrodicatechin A, a (+)-catechin trimer, and a (+)-catechin oligomer with polymerization degree equal to or greater than 5. The main quantitative differences between the oxidative reactions were the great amount of oligomer formed in the case of the polyphenoloxidase-mediated reaction and the low amount of (+)-catechin reacted in the case of the peroxidase-mediated reaction. One of the possible reasons for such low levels of (+)-catechin consumption in the case of the peroxidase-mediated reaction was the possible inhibition by products of the enzyme-catalyzed oxidation. In fact, the peroxidase-mediated (+)-catechin oxidation was differentially inhibited by dehydrodicatechin A, showing a competitive type inhibition and a k(I) of 6.4 microM. In light of these observations, these results suggest that brown polymer formation, estimated as oligomeric compounds resulting from (+)-catechin oxidation, in strawberries is mainly due to polyphenoloxidase, and although peroxidase also plays an important role, it is apparently auto-regulated by product (dehydrodicatechin A) inhibition.     

Influence of cultivar on quality parameters and chemical composition of strawberry fruits grown in Brazil

Six strawberry cultivars grown on the same commercial plantation in Brazil were evaluated for their chemical composition and quality attributes at the ripe stage. The profiles of the main soluble sugars, ascorbic acid, and anthocyanins were also obtained during the developmental stages. Results showed significant differences among cultivars in all of the investigated parameters. Cv. Campineiro showed an average value for texture of 0.63 N, half the value found for cv. Oso Grande. Anthocyanin content ranged from 13 (cv. Campineiro) to 55 (cv. Mazi) mg/100 g. Total ascorbic acid found for cv. Campineiro (85 mg/100 g) was twice the amount found in cv. Dover (40 mg/100 g). Fructose was the predominant soluble sugar in almost all cultivars. The proportion among the main soluble sugars (fructose, sucrose, and glucose) was similar for Oso Grande and Toyonoka cultivars. The flavonol content (quercetin plus kaempferol derivatives) ranged from 2.7 to 7.1 mg/100 g, with a mean value of 6.1 mg/100 g, whereas free ellagic acid ranged from 0.9 to 1.9 and total phenolics varied from 159 to 289 (mean 221) mg/100 g.     

Acetyl-coa: alcohol acetyltransferase activity and aroma formation in ripening melon fruits

Melon varieties (Cucumis melo L.) differ in a range of physical and chemical attributes. Sweetness and aroma are two of the most important factors in fruit quality and consumer preference. Volatile acetates are major components of the headspace of ripening cv. Arava fruits, a commercially important climacteric melon. In contrast, volatile aldehydes and alcohols are most abundant in cv. Rochet fruits, a nonclimacteric melon. The formation of volatile acetates is catalyzed by alcohol acetyltransferases (AAT), which utilize acetyl-CoA to acetylate several alcohols. Cell-free extract derived from Arava ripe melons exhibited substantial levels of AAT activity with a variety of alcohol substrates, whereas similar extracts derived from Rochet ripe melons had negligible activity. The levels of AAT activity in unripe Arava melons were also low but steadily increased during ripening. In contrast, similar extracts from Rochet fruits displayed low AAT activity during all stages of maturation. In addition, the benzyl- and 2-phenylethyl-dependent AAT activity levels seem well correlated with the total soluble solid content in Arava fruits.     

Characterization of polyphenol oxidase and peroxidase and influence on browning of cold stored strawberry fruit

Polyphenol oxidase and peroxidase were extracted from two different varieties of strawberry fruit (Fragaria x ananassa D, cv. 'Elsanta' and Fragaria vesca L, cv. 'Madame Moutot') and characterized using reliable spectrophotometric methods. In all cases, the enzymes followed Michaelis-Menten kinetics, showing different values of peroxidase kinetics parameters between the two cultivars: Km = 50.68 +/- 2.42 mM ('Elsanta') and 18.18 +/- 8.79 mM ('Madame Moutot') mM and Vmax = 0.14 +/- 0.03 U/g ('Elsanta') and 0.05 +/- 0.01 U/g ('Madame Moutot'). The physiological pH of fruit at the red ripe stage negatively affected the expression of both oxidases, except polyphenol oxidase from 'Madame Moutot' that showed the highest residual activity (68% of the maximum). Peroxidase from both cultivars was much more thermolable as compared with PPO, losing over 60% of relative activity already after 60 min of incubation at 40 degrees C. The POD activation energy was much lower than the PPO activation energy (DeltaE = 97.5 and 57.8 kJ mol-1 for 'Elsanta' and 'Madame Moutot', respectively). Results obtained from d-glucose and d-fructose inhibition tests evidenced a decreasing course of PPO and POD activities from both cultivars as the sugar concentration in the assay medium increased. Changes in CIE L*, a*, b*, chroma, and hue angle values were taken as a browning index of the samples during storage at 4 degrees C. A decrease in L* was evident in both cultivars but more marked in 'Elsanta'. PPO and POD activities from cv. 'Elsanta' were very well-correlated with the parameter L* (r2=0.86 and 0.89, respectively) and hue angle (r2=0.85 and 0.93, respectively). According to these results, the browning of the fruit seemed to be in relation to both oxidase activities.     

Developmental and varietal differences in volatile ester formation and acetyl-CoA: alcohol acetyl transferase activities in apple (Malus domestica Borkh.) fruit

Apple (Malus domestica Borkh.) cultivars differ in their aroma and composition of volatile acetates in their fruit flesh and peel. Cv. Fuji flesh contains substantial levels of 2-methyl butyl acetate (fruity banana-like odor), while the flesh of cv. Granny Smith apples lacks this compound. Granny Smith apples accumulate mainly hexyl acetate (apple-pear odor) in their peel. Feeding experiments indicated that Fuji apples were able to convert hexanol and 2-methyl butanol to their respective acetate derivatives in vivo, while Granny Smith apples could only convert exogenous hexanol to hexyl acetate. Differential substrate specificities of the in vitro acetyl-CoA:alcohol acetyl transferase (AAT) activities were also detected among cultivars. In Granny Smith apples, the AAT activity was detected only in the peel, and its specificity was almost exclusively restricted to hexanol and cis-3-hexenol. In Fuji apples, the AAT activity was detected in both peel and flesh and apparently accepted a broader range of alcohols as substrates than the Granny Smith enzyme activity. Our data strongly suggest that different AAT activities are operational in apple tissues and cultivars and that these differences contribute to the variation observed in the accumulation of volatile acetates.     

Volatile compounds of selected strawberry varieties analyzed by purge-and-trap headspace GC-MS

Six strawberry (Fragaria x ananassa Duch.) varieties, Senga Sengana, Jonsok, Polka, Korona, Bounty, and Honeoye, were studied in relation to composition of their volatile headspace compounds collected by dynamic purge-and-trap concentration. Also, the influence of cultivation technique on the volatile compounds was tested by comparing conventionally and organically cultivated strawberry varieties. In addition, the effect of geographical origin on strawberry volatiles was investigated. The emphasis of this study was lay in the industrial utilization of strawberry. A total of 52 different volatile compounds were determined from frozen strawberries grown in 1997 and 1998. In addition to individual compounds, total areas of chromatogram profiles were compared. The major esters were found to be methyl butanoate, ethyl butanoate, methyl hexanoate, and ethyl hexanoate. With the application of principal component analysis and analysis of variance to the data matrix, the varieties were classified into three groups. Var. Honeoye was most different from the others. Organic cultivation had no effect on strawberry volatiles.     

Characterization of a benzyl alcohol dehydrogenase from Lactobacillus plantarum WCFS1

Aroma is an important sensory parameter of food products. Lactic acid bacteria have enzymatic activities that could be important in the modification of food aroma. The complete genome sequence from Lactobacillus plantarum WCFS1 shows a gene (lp_3054) putatively encoding a protein with benzyl alcohol dehydrogenase activity. To confirm its enzymatic activity lp_3054 from this strain has been overexpressed and purified. Protein alignment indicated that lp_3054 is a member of the family of NAD(P)-dependent long-chain zinc-dependent alcohol dehydrogenases. In lp_3054 all of the residues involved in zinc and cofactor binding are conserved. It is also conserved the residue that determines the specificity of the dehydrogenase toward NAD (+) rather than NADP (+) and, therefore, L. plantarum benzyl alcohol dehydrogenase is less active in the presence of NADP (+) than in the presence of NAD (+). The purified enzyme exhibits optimal activity at pH 5.0 and 30 degrees C. The kinetic parameters K m and V max on benzyl alcohol as a substrate were, respectively, 0.23 mM and 204 mumol h (-1) mg (-1). Besides its activity toward benzyl alcohol, it showed activity against nerol, geraniol, phenethyl alcohol, cinnamyl alcohol, and coniferyl alcohol, all of which are volatile compounds involved in determining food aroma. The biochemical demonstration of a functional benzyl alcohol dehydrogenase activity in this lactic acid bacteria species should be considered when the influence of bacterial metabolism in the aroma of food products is determined.     

Lipoxygenase and hydroperoxide lyase activities in ripening strawberry fruits

The enzymes lipoxygenase and hydroperoxide lyase have been identified in strawberry (Fragariax ananassa Duch.) var. Camarosa. Their subcellular localization, substrate preference, and product specificity were determined in mature strawberry fruits. The activity of both enzymes was located mainly in the microsomal fraction. Linolenic acid was the preferred substrate for strawberry lipoxygenase, forming 13- and 9-hydroperoxides of this acid in the proportion 70:30. The strawberry hydroperoxide lyase cleaves 13-hydroperoxide of linoleic (13% relative activity) and linolenic (100% relative activity) acids to form hexanal and (3Z)-hexenal, respectively. Both enzyme activities and endogenous content of volatile aldehydes formed by sequential action of lipoxygenase-hydroperoxide lyase were evaluated during strawberry development and ripening. A sequential enzymatic pathway for the formation of green odor compounds in strawberry is proposed.     

Functionality of bioactive compounds in Brazilian strawberry (Fragaria x ananassa Duch.) cultivars: evaluation of hyperglycemia and hypertension potential using in vitro models

Fruits of seven fully ripened strawberry cultivars grown in Brazil (Dover, Camp Dover, Camarosa, Sweet Charlie, Toyonoka, Oso Grande, and Piedade) were evaluated for total phenolics, antioxidant activity based on DPPH radical scavenging assay, and functionality such as inhibition of alpha-amylase, alpha-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potentially managing hyperglycemia and hypertension. The total phenolics content ranged from 966 to 1571 microg of gallic acid/g of fruit fresh weight for Toyonoka and Dover, respectively. No correlation was found between total phenolics and antioxidant activity. The major phenolic compounds in aqueous extracts of strawberries were ellagic acid, quercetin, and chlorogenic acid. Strawberries had high alpha-glucosidase inhibitory activity. However, alpha-amylase inhibitory activity was very low in all cultivars. This suggested that strawberries could be considered as a potential dietary source with anti-hyperglycemic potential. The evaluated cultivars had no significant ACE inhibitory activity, reflecting low anti-hypertensive potential.     

Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.     

Characterization of tyrosinase from the cap flesh of portabella mushrooms

Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.     

Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria x ananassa)

An octaploid (Fragaria x ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria x ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens. Out of 25 transgenic lines, nine lines showed a reduction in CHS mRNA accumulation of more than 50% as compared to the untransformed cv. Calypso control. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from one to four. The pigmentation of the fruit was only affected when less than 5% of the control CHS expression level was detected. A stable antisense phenotype over a period of 4 years was obtained in the primary transgenic lines at a rate of 1:20. As a consequence of the reduced activity of CHS, the levels of anthocyanins, flavonols, and proanthocyanidins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to highly increased levels of cinnamoyl glucose (520% of control), caffeoyl glucose (816% of control), and feruloyl glucose (1092% of control) as well as p-coumaryl alcohol (363% of control) and p-coumaryl-1-acetate (1079% of control), which occur only as trace components in untransformed control fruits. These results demonstrate that the introduction of an antisense CHS construct in strawberry results in an unpredictable biochemical phenotype, thereby confirming that CHS function is an important regulatory point of substrate flow between the flavonoid and the phenylpropanoid pathways.     

Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.     

Characterization of alcohol acyltransferase from olive fruit

Alcohol acyltransferase catalyzes the esterification of volatile alcohols with acyl-CoA derivatives to produce volatile esters typically present in the aroma of some fruits. This enzyme was detected in extracts from the pericarp tissues of ripe olive fruits using hexanol and acetyl-CoA as the substrates. Alcohol acyltransferase showed a very low activity level in these fruits, with an optimum pH value at 7.5 and high K(m) values for hexanol and acetyl-CoA. The substrate specificity of this enzyme for various alcohols was also studied. The involvement of the studied enzyme in the biogenesis of the volatile esters present in the aroma of virgin olive oil was discussed.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.