Alcohol Acyltransferase Activity Research Articles

Effects of blanching and pickling process on the alcohol acyltransferase (AAT) activity, myristicin content and quality parameters of pickled nutmeg (Myristica fragrans).

Myristicin, a potential toxicant, is a major ester present in the pericarp of nutmeg (Myristica fragrans) and alcohol acyltransferase (AAT) is responsible for its generation. The objective of the study was firstly to estimate the effect of blanching and pickling process on the inactivation of AAT and production of myristicin in nutmeg pericarp. Secondly, the effect of pickling process on the physical, microbial, antioxidant and sensory properties of nutmeg was evaluated. The nutmeg fruit pericarp was water-blanched for 0, 3, 5, 7 and 9min. The increased blanching time significantly (p < 0.05) reduced the AAT activity (3.478 U/mg protein) compared to fresh nutmeg (39.034 U/mg protein). The reduction of myristicin was so efficient that it could not be detected after 9min of blanching. Similarly, the pickling process significantly (p < 0.05) inhibited the AAT activity and lowered the myristicin content. However, the blanching significantly (p < 0.05) lowered the total phenol content and reduced the free radical scavenging capacity of pickled nutmeg (BP) when compared with commercial pickled nutmeg. Blanched pickled nutmeg (BP) presented the same color characteristics as of commercial one, although significant reduction in hardness was observed. No growth of yeast and mold was noticed for all the blanched pickled nutmeg. The sensory analysis data demonstrated that blanching up to 7min maintained the overall acceptability of pickled nutmeg as of commercial pickled nutmeg. Thus, the study suggests that blanching and pickling treatment improved the quality of nutmeg effectively by inhibiting the AAT activity and reducing the myristicin content.

Eat1-Like Alcohol Acyl Transferases From Yeasts Have High Alcoholysis and Thiolysis Activity.

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2–C10) but only a small range of acyl donors (C2–C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.

The effects of blanching pretreatment and immersion of sodium metabisulfite/citric acid solution on the myristicin content and the quality parameter of nutmeg (Myristica fragrans) pericarp

Pickled nutmeg which is made from the osmotic dehydration process is one of the well-known and a trademark delicacy in Penang, Malaysia. Despite of the high demand from the consumers, the toxicant volatile compound of myristicin that is produced along the making process has become the limitation of the pickled nutmeg consumption. Hence, pre-treatments of different blanching period prior to osmotic dehydration process were suggested to mitigate the issue. Analysis of the texture, myristicin content, and alcohol acyltransferase (AAT) activity were undergone to the fresh (control), pretreated nutmeg pericarp, and post-pretreated pickled nutmeg. The nutmeg samples were prepared and subjected to various blanching period (0, 3, 5, 7, and 9 min) at 100 ℃ in distilled water, followed by immersing the samples into sodium metabisulfite/citric acid (SMB/CA) solution for 30 min. Then, the pretreated nutmeg pericarp underwent the osmotic dehydration process by using food grade sodium chloride. The increase in blanching period resulted in the reduction of texture hardness, myristicin content, and AAT activity of the pretreated nutmeg pericarp compared to the control. In overall, 7 min blanching period was preferable, as the myristicin level in nutmeg sample could not be detected and the AAT activity was at its lowest level.

Seasonal and Developmental Dynamics of the Essential Oil Content versus Catalytic Activity of Alcohol Acyltransferase in Foliage of Citronella

Citronella (Cymbopogon winterianus Jowitt) is an aroma grass cultivated as a cash crop for its foliage essential oil that is widely used in cosmetics/perfumery, insect-repellents, as source of synthagens etc. The oil comprises of mainly acyclic monoterpenols (geraniol and citronellol) and their esters (geranyl acetate and citronellyl acetate), besides citronellal. This study was undertaken to profile dynamics of the oil content and composition as a function of ontogeny and annual pattern of seasons and climate, including with reference to the volatile esters and catalytic activity of the enzyme involved in their synthesis, a BAHD-alcohol acetyltransferase, citronellol; acetyl coenzyme A, acetyltransferase. This may be the first report on arriving at average patterns from a three years reiterated study and on any enzymatic study of secondary metabolism of the grass. The oil ester content appeared to be governed by limitation of the acetyltransferase activity rather than availability of substrates (citronellol and geraniol).

Engineering Escherichia coli for the production of butyl octanoate from endogenous octanoyl-CoA.

Medium chain esters produced from fruits and flowering plants have a number of commercial applications including use as flavour and fragrance ingredients, biofuels, and in pharmaceutical formulations. These esters are typically made via the activity of an alcohol acyl transferase (AAT) enzyme which catalyses the condensation of an alcohol and an acyl-CoA. Developing a microbial platform for medium chain ester production using AAT activity presents several obstacles, including the low product specificity of these enzymes for the desired ester and/or low endogenous substrate availability. In this study, we engineered Escherichia coli for the production of butyl octanoate from endogenously produced octanoyl-CoA. This was achieved through rational protein engineering of an AAT enzyme from Actinidia chinensis for improved octanoyl-CoA substrate specificity and metabolic engineering of E. coli fatty acid metabolism for increased endogenous octanoyl-CoA availability. This resulted in accumulation of 3.3 + 0.1 mg/L butyl octanoate as the sole product from E. coli after 48 h. This study represents a preliminary examination of the feasibility of developing E. coli platforms for the synthesis single medium chain esters from endogenous fatty acids.

Screening of adjunct cultures and their application in ester formation in Camembert-type cheese

The ethanol content and esterase and alcohol acyltransferase activities are the limiting factors in the synthesis of ethyl esters in Camembert-type cheeses. This study aimed to investigate the effects of alcohol, esterase and alcohol acyltransferase activities on ethyl ester formation in Camembert-type cheeses. Five experimental cheeses were prepared with three adjunct cultures with different enzyme activities and two levels of ethanol content (400 or 800 μg/g). The cheeses were aged for 4 weeks and analysed weekly for basic physicochemical, textural, volatile and sensory properties. The results showed that both the enzyme activity and ethanol content were limiting factors in the synthesis of ethyl esters in the Camembert-type cheeses. Variation in the esterase synthesis activity was observed among lactic acid bacteria, and the starter culture Lactococcus lactis MA 14 LYO distinguished itself through its high acidifying and esterase hydrolysis abilities. The addition of CCFM 12, a lactic acid bacteria strain with high esterase and alcohol acyltransferase activity, along with 400 or 800 μg/g of ethanol, notably enhanced the generation of ethyl esters and the corresponding fruity flavour, without causing dramatic changes in the basic physicochemical indices and microbial profile. In addition, cohesiveness was influenced by the addition of 400 and 800 μg/g of ethanol, and more resilience with 800 μg/g of ethanol had been found. The results showed that the addition of CCFM12 with 400 and 800 μg/g of ethanol may be applied in the production of Camembert cheese to enhance its fruity flavour.

The Relationship between CmADHs and the Diversity of Volatile Organic Compounds of Three Aroma Types of Melon (Cucumis melo).

Alcohol dehydrogenase (ADH) plays an important role in aroma volatile compounds synthesis of plants. In this paper, we tried to explore the relationship between CmADHs and the volatile organic compounds (VOCs) in oriental melon. Three different aroma types of melon were used as materials. The principle component analysis of three types of melon fruit was conducted. We also measured the CmADHs expression level and enzymatic activities of ADH and alcohol acyl-transferase (AAT) on different stages of fruit ripening. An incubation experiment was carried out to investigate the effect of substrates and inhibitor (4-MP, 4-methylpyrazole) on CmADHs expression, ADH activity, and the main compounds of oriental melon. The results illustrated that ethyl acetate, hexyl acetate (E,Z)-3,6-nonadien-1-ol and 2-ethyl-2hexen-1-ol were the four principal volatile compounds of these three types of melon. AAT activity was increasing with fruit ripening, and the AAT activity in CH were the highest, whereas ADH activity peaked on 32 DAP, 2 days before maturation, and the ADH activity in CB and CG were higher than that in CH. The expression pattern of 11 CmADH genes from 24 to 36 day after pollination (DAP) was found to vary in three melon varieties. CmADH4 was only expressed in CG and the expression levels of CmADH3 and CmADH12 in CH and CB were much higher than that in CG, and they both peaked 2 days before fruit ripening. Ethanol and 4-MP decreased the reductase activity of ADH, the expression of most CmADHs and ethyl acetate or hexyl acetate contents of CB, except for 0.1 mM 4-MP, while aldehyde improved the two acetate ester contents. In addition, we found a positive correlation between the expression of CmADH3 and CmADH12 and the key volatile compound of CB. The relationship between CmADHs and VOCs synthesis of oriental melon was discussed.

Effects of intermittent warming on aroma-related esters of 1-methylcyclopropene-treated ‘Nanguo’ pears during ripening at room temperature

Refrigeration combined with 1-methylcyclopropene (1-MCP) treatment is an effective method for the long-term storage of climacteric fruit, but this benefit is offset by a loss of aroma when the fruit is then ripened at room temperature. The pear cv. ‘Nanguo’ (Pyrus ussuriensis Maxim.) was treated with 1-MCP and continuously stored at 0±0.5°C or exposed to the same storage temperature alternated with 20±1°C for 1 d every 20 d during storage (namely, intermittent warming [IW]). Changes in aroma-related esters, levels of fatty acids, and the activity and gene expression of alcohol acyltransferase (AAT) and lipoxygenase (LOX) were investigated. Changes in the gene expression of the enzymes (1-aminocyclopropane-carboxylase (ACC) synthase (ACS) and ACC oxidase (ACO)) involved in ethylene biosynthesis and the expression of genes involved in ethylene signal transduction were also studied. Significantly higher ester content was found in IW-treated fruit. Increased activity and gene expression of AAT, LOX, and accumulated polyunsaturated fatty acids were associated with enhanced aroma quality in IW-treated fruit. The increased translation levels of PuACS1 and PuACO1 contributed to the elevated ethylene levels in IW-treated fruit. IW treatment seemed to have no significant effect on the induction of the expression of PuETR1 and merely stimulated the expression of PuCTR1 during the prior period of the shelf life at 20°C. Treatment with IW effectively prevented the loss of aroma-related esters from 1-MCP-treated ‘Nanguo’ pears, and possible mechanisms for this were discussed.

Low temperature conditioning prevents loss of aroma-related esters from ‘Nanguo’ pears during ripening at room temperature

Refrigeration delays ripening and reduces decay of pear fruit, but this benefit is offset by a loss of aroma when the fruit is then ripened at room temperature. Low temperature conditioning (LTC) has been used to alleviate chilling injury (CI) of fruit, however, its effect on aroma remains unknown. Pear fruit cv. ‘Nanguo’ (Pyrus ussuriensis Maxim.) were subjected to LTC (namely, exposed to 10°C, decreased by 1°C every 3 days to 5°C, then decreased by 1°C per day to 0±0.5°C) and changes in aroma-related esters, levels of fatty acids, the activity and gene expression of alcohol acyltransferase (AAT), lipoxygenase (LOX), and alcohol dehydrogenase (ADH) were investigated. Changes in the activity and gene expression of enzymes involved in ethylene biosynthesis and the expression of genes involved in ethylene signal transduction were also studied. Significantly higher ester contents and levels of linoleic and linolenic acids were found in LTC-treated fruit. Levels of PuAAT1, PuLOX1, and PuADH3 transcripts were promoted by LTC treatment and the activity of AAT, LOX and ADH was higher in LTC-treated fruit during the shelf-life at room temperature. The increased levels of the expression and activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) contributed to the elevated ethylene levels in LTC-treated fruit. The expression of genes involved in ethylene signal transduction (PuETR1 and PuCTR1) was also promoted by LTC treatment. Treatment with LTC effectively prevented the loss of aroma-related esters from refrigerated ‘Nanguo’ pears and possible mechanisms were discussed.

Development of flavor-related metabolites in cherimoya (Annona cherimola Mill.) fruit and their relationship with ripening physiology

Flavor is one of the most important attributes of fresh fruit for the consumer, and is affected by several factors, including genotype, maturity stage, and environmental conditions. Flavor-related metabolites were characterized in two important cherimoya varieties, cv. Concha Lisa and cv. Bronceada, during fruit ripening. The most important sugars present were glucose, fructose and sucrose, and only fructose and glucose increased during ripening. The most important acids were tartaric, malic and citric acids, and all increased as ripening progressed. Overall aroma profile was mainly determined by esters and terpenes in both varieties. Ester compounds such as ethyl hexanoate, butyl butyrate and hexyl propanoate increased during ripening. The activity of alcohol acyl transferase also increased during fruit ripening concomitant with ester accumulation. Terpenes, such as α and β-pinene, showed a reduction during ripening, whereas others, such as myrcene and limonene, increased.

Intermittent warming alleviated the loss of peach fruit aroma-related esters by regulation of AAT during cold storage

Low temperature storage slows ripening and reduces decay of peach fruit, although storage life is limited due to chilling injury. Intermittent warming (IW) can be used to alleviate fruit chilling injury, however, its effect on aroma remains unknown. Yellow-fleshed peach (Prunus persica L. Batsch cv. Jinxiu) fruit (melting type) was stored at 5°C or exposed to 20°C for 1d every week during storage. Changes in fruit esters, alcohol acyltransferase (AAT) activity and gene expression were investigated. Flesh browning (FB) was observed on the third day of shelf-life at 20°C after 21d of storage at 5°C, while no FB was found in IW-treated fruit for up to 28d. Significant lower ester contents were found in peach fruit with flesh browning. Expression profiles of PpAAT1 were similar to AAT activity profiles, both of which increased during shelf-life of fruit treated with IW. As precursors of esters, levels of linoleic and linolenic acids were high in IW-treated peach fruit. Treatment with IW effectively alleviated the loss of aroma-related esters associated with FB, and high levels of AAT activity and PpAAT1 expression in IW-treated peach fruit contributed to the formation of the esters.

Characterisation of two alcohol acyltransferases from kiwifruit ( Actinidia spp.) reveals distinct substrate preferences

Volatile esters are key compounds of kiwifruit flavour and are formed by alcohol acyltransferases that belong to the BAHD acyltransferase superfamily. Quantitative RT-PCR was used to screen kiwifruit-derived expressed sequence tags with proposed acyltransferase function in order to select ripening-specific sequences and test their involvement in alcohol acylation. The screening criterion was for at least 10-fold increased transcript accumulation in ripe compared with unripe kiwifruit and in response to ethylene. Recombinant expression in yeast revealed alcohol acyltransferase activity for Actinidia-derived AT1, AT16 and the phylogenetically distinct AT9, using various alcohol and acyl-CoA substrates. Functional characterisation of AT16 and AT9 demonstrated striking differences in their substrate preferences and apparent catalytic efficiencies ( V max ′ K m - 1 ) . Thus revealing benzoyl-CoA:alcohol O-acyltransferase activity for AT16 and acetyl-CoA:alcohol O-acyltransferase activity for AT9. Both kiwifruit-derived enzymes displayed higher reaction rates with butanol compared with ethanol, even though ethanol is the main alcohol in ripe fruit. Since ethyl acetate and ethyl benzoate are major esters in ripe kiwifruit, we suggest that fruit characteristic volatile profiles result from a combination of substrate availability and specificity of individual alcohol acyltransferases.

Preharvest Aminoethoxyvinylglycine Plus Postharvest Heat Treatments Influence Apple Fruit Ripening after Cold Storage

The impact of heat plus aminoethoxyvinylglycine (AVG) treatments alone or in combination on ripening of four apple cultivars has been studied. A solution of AVG was applied to ‘Lodi’, ‘Senshu’, ‘Redchief Delicious’, and ‘Red Fuji’ apple trees ≈4 weeks before normal harvest at 124 g·ha−1 a.i. After harvest, half of each group of control and AVG-treated fruit was heated at 38 °C for 4 days and then stored at 4 °C for 30 days. After cold storage, AVG and heat individually suppressed ethylene production of ‘Senshu’ and ‘Redchief Delicious’ but not of ‘Lodi’ or ‘Red Fuji’. The combination of AVG with heat treatment reduced ethylene production the most consistently in each cultivar except ‘Lodi’, suggesting some additive effect of the treatments. The respiration rate after cold storage was not consistently affected by any treatment. AVG alone and with heat maintained firmness of ‘Lodi’, AVG plus heat maintained it in ‘Senshu’, but neither ‘Redchief Delicious’ nor ‘Red Fuji’ firmness responded to the treatments. AVG-treated ‘Lodi’ and ’Redchief Delicious’ fruit, heated fruit of all cultivars, and AVG plus heat in all had lower titratable acidity than controls after cold storage. Although there were no effects of any treatment on fruit soluble solids concentration, the combined treatment increased the soluble solids:titratable acidity ratio of all cultivars, although heat or AVG alone had no consistent effects. Total ester production by ‘Redchief Delicious’ peel tissue after cold storage was reduced 44% by AVG and 70% or more by heat and AVG plus heat. There were no differences in peel alcohol acyltransferase activity among the treatments, supporting the hypothesis that substrate availability was the limiting factor for ester synthesis in treated fruit. Overall, heat plus AVG treatment did not provide any advantage over each alone for maintaining apple fruit quality during short-term cold storage.

Effects of Aminoethoxyvinylglycine Plus 1-Methylcyclopropene on ‘Royal Gala’ Apple Volatile Production After Cold Storage

Both aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP) are useful tools for managing apple fruit ripening, and their impacts on apple volatile production have been independently assessed. In this work, their combined effect, as might occur in commercial production and postharvest storage, on ‘Royal Gala’ apple volatile production at harvest and after air storage was compared with the effect of each alone. An aqueous solution of AVG was applied to ‘Royal Gala’ apple trees 4 weeks before the normal harvest date (H1) at 124 g·ha−1/a.i. in 2004 and 2005. Control and AVG-treated fruit were treated at H1 or after harvest of AVG fruit 2 weeks later (H2) for 20 h at 30 °C with 1-MCP at a final headspace concentration of 1 μL·L−1. Fruit were ripened for 7 d at room temperature immediately after harvest and treatment or after treatment and then cold storage at 4 °C for 6 or 12 weeks. Peel and cortex tissue of control and AVG plus 1-MCP-treated fruit was provided with butanol or hexanol and ester production was quantified. The combination of AVG plus 1-MCP was more effective in reducing internal ethylene concentration than either alone. Both total volatile production and that of the major individual esters, including hexyl acetate, butyl acetate, and 2-methylbutylacetate, which are considered key constituents of ‘Gala’ aroma, were consistently repressed by the combination of AVG plus 1-MCP after harvest and up to 12 weeks of cold storage. The effects of AVG plus 1-MCP were evident even with H2 fruit when the effects of AVG alone on fruit ripening were at least partially lost. Because alcohol–acyl transferase activity was unaffected by AVG plus 1-MCP, AVG plus 1-MCP-treated peel and cortex samples had similar total ester production when they were provided butanol or hexanol. Total alcohols showed recovery in most treatments except AVG plus 1-MCP, so precursor availability was likely the major factor limiting ester production. The results indicated a sustained adverse effect of the AVG plus 1-MCP treatment on aroma volatile production that could impact consumer acceptability.

Prokaryotic expression, purification, and sub-cellular localization of a novel alcohol acyltransferase from apple

The coding region of the alcohol acyltransferase gene (MdAAT2) from apple was sub-cloned into expression vectors, pET30a and pET32a, and introduced into E. coli for expression. The purified pET30a/MdAAT2 fusion proteins were used to immunize rabbits following standard protocols. The partially soluble fusion proteins had alcohol acyltransferase activity and were detected only in the pET32a/Origami B(DE3) expression system. Immunolocalization analysis indicated that MdAAT2 is mainly in the cytoplasm, in agreement with the prediction of sub-cellular localization obtained by the LOCSVMpsi program. Western blot analysis indicated that ester biosynthesis in different apple cultivars was related positively to the accumulation of MdAAT2.

Development of molecular and biochemical tools to investigate fruit quality traits in strawberry elite genotypes

Molecular and biochemical studies were undertaken to elucidate gene/product relationships which influence key fruit quality traits in cultivated strawberry (Fragaria × ananassa Duch.). Comparative transcription profiling experiments in selected genotypes pointed out a number of differentially-expressed genes, possibly related to important fruit quality traits as aroma and fruit firmness. Some of the altered cDNAs encode putative cinnamyl alcohol dehydrogenase, cinnamoyl CoA reductase, cellulase and expansin genes, involved in the early steps of lignin biosynthesis and in cell wall structure, respectively. Parallel biochemical analyses studied the spectra of volatile compounds by Proton Transfer Reaction-Mass Spectrometry (PTR-MS) and alcohol acyl transferase (AAT) specific activity in red fruits. A correlation between the expression of an aat gene, total AAT activity and the presence of related esters in fruit headspace was found.

Effect of AVG and MCP on Volatile Production of `Gala' Apple

Our goal was to determine how aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene (MCP) interact to influence postharvest storability and volatile production of `Gala' apple. In 2004 and 2005, AVG was applied to `Gala' apple trees 4 weeks before harvest. After harvest, control and AVG-treated fruit were treated for 20 h at 30 °C with MCP, and fruit were ripened at ambient temperature immediately after harvest, after MCP treatment, or after storage at 4 °C for 6 and 12 weeks. For both seasons, control fruit reached the highest internal ethylene concentration (IEC) during ripening at ambient temperature immediately after harvest. After storage, control fruit had very low IEC in 2004, but the highest in 2005. In general, the combined treatment repressed ethylene production the most for all harvest dates and lengths of storage. AVG plus MCP-treated fruit consistently had the highest flesh firmness (FF) but also had the lowest total volatile production (TVP) by flesh or peel after 6 and 12 weeks in cold storage following both harvest dates. The activity of alcohol acyltransferase was affected by the treatments, but could not explain all the variation found in TVP. TVP was lower for flesh than peel of control and treated fruit, but feeding alcohol substrates to the fruit resulted in a significant increase in TVP, regardless of tissue type or treatment. The results indicate that the combination of AVG plus MCP maintained apple fruit FF in cold storage better than the treatments used alone, but also substantially reduced TVP. Substrate availability seemed to be the most limiting factor affecting TVP by flesh and peel of `Gala' apples treated with AVG plus MCP.

CHARACTERIZATION OF GENES INVOLVED IN THE FORMATION OF AROMA VOLATILES IN ´CHARENTAIS´ MELON FRUIT

Volatiles esters impart distinct characteristics to the fruit quality. 'Charentais' cantaloupe melon (Cucumis melo 'cantalupensis') is characterized by abundant sweetness and aromatic flavour. Plant alcohol acyl transferase (AAT) genes have been identified and shown to be involved in aromas production. Recently, two cDNAs (Cm-AA T 1 and Cm-AAT 2 ) putatively involved in the formation of aroma volatile esters have been isolated from melon fruit. Cm-AAT 1 protein exhibit alcohol acyl transferase activity while no such activity could be detected for Cm-AAT 2 . Two new cDNAs (Cm-AATj and Cm-AAT 4 ) have been isolated from melon fruit that showed 69% and 36% similarity, respectively, with Cm-AAT 1 . The percentage similarity over the whole amino acid sequence between them is 34%. Cm-AAT 3 and Cm-AAT 4 show the highest similarity to the tobacco Nt-HSR201 protein and a rose alcohol acyltransferase Rh-AAT 1 , respectively. All Cm-AATs genes, share three conserved regions common to the BAHD acyltransferase gene superfamily. Heterologous expression in yeast revealed that some of the encoded proteins have a wide range of specificity while others are specific to a narrow range of substrates.

Role of olive seed in the biogenesis of virgin olive oil aroma.

Results obtained in a set of experiments point to an effective participation of olive seeds in the biosynthesis of olive oil aroma through the lipoxygenase pathway during the extraction process to produce virgin olive oil. Data showed that olive seeds should contain enzymatic activities metabolizing 13-hydroperoxides other than hydroperoxide lyase, giving rise to a net decrease in the content of C6 unsaturated aldehydes during the olive oil extraction process. Olive seeds seem also to supply this process with alcohol dehydrogenase activity, being more specific for saturated C6 aldehydes and not acting on C5 alcohols. Moreover, olive seeds would be responsible for the biosynthesis of 30-50% esters during the olive oil extraction process of intact fruits. Thus, olive seeds would afford a load of alcohol acyltransferase activity that might be quite unspecific in terms of substrate, producing any kind of esters.

Catalytic properties of alcohol acyltransferase in different strawberry species and cultivars.

The substrate specificity of alcohol acyltransferase (AAT) enzymes from different strawberry varieties was studied. Proteins with AAT activity from fruits of Fragaria x ananassa Duch. cv. Oso Grande were purified to apparent homogeneity and used for kinetic studies with different straight-chain alcohols and acyl-CoAs. K(m) values obtained for Oso Grande enzyme with six different alcohols, using acetyl-CoA as cosubstrate, decreased with increasing length of the alcohol chain. In similar experiments the increase in the acyl-CoA carbon chain was also found to be correlated with a higher substrate specificity. Heptanol (K(m) = 0.73 mM) and hexanoyl-CoA (K(m) = 0.41 mM) were the best substrates for Oso Grande AAT. Comparative catalytic studies were carried out with AAT partially purified extracts from the wild type Fragaria vesca and five commercial strawberry varieties: Tudnew, Carisma, Camarosa, Sweet Charlie, and Eris. The specificities of these enzymes toward five selected alcohols and acyl-CoAs reflected interesting cultivar differences.