The EstA esterase is responsible for the main capacity of Lactococcus lactis to synthesize short chain fatty acid esters in vitro

190

159

4Lactic acid bacteria (LAB) have been widely used as starter or nonstarter cultures in the dairy industry for over a thousand years. They play an essential role in flavor formation during the fermentation of dairy products. Several metabolic routes can lead to the formation of flavor compounds when LAB are growing in milk. One of the main precursors for flavor compounds in milk is casein, although they can also be derived from fatty acids and sugars. The proteolytic system of LAB degrades casein into its constituent amino acids, which can be converted to flavor compounds. Although amino acid catabolism by LAB has been well researched (5, 31, 65, 78), many flavor-forming routes are yet to be discovered.Over 20 LAB genomes have been fully sequenced (1,11,12,16,19,41,44,45,53, 68,71). The available genomic information provides us with new opportunities to study the flavorforming potential of LAB. However, one of the main problems that one encounters while reconstructing flavor-forming routes based on the genomic information stored in the public databases is the inconsistency in the functional annotation for many of the relevant genes. These genes are mostly members of larger protein families. Moreover, even when the functional annotation in databases is appropriate, it sometimes reflects only part of the protein's full functional potential, since broad substrate specificities are often not taken into consideration in the annotation.We have now improved the functional annotation of the key enzymes in the formation of flavor compounds from amino acids by applying comparative genomics approaches that have been developed within our group to specify the annotation of homologous proteins, by combining phylogeny, gene context, and experimental evidence (32). We focused especially on the enzymes involved in the metabolism of sulfur-containing amino acids since these are known to be precursors of many flavor compounds in dairy fermentations. Comparative analysis of the various sequenced LAB species and strains resulted in an overall view of differences in their flavor-forming capacities.

Section: Enzymes Involved In the Transamination Routementioning

confidence: 93%

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria

Liu

Nauta

Francke

et al. 2008

190

159

“…Part of the work performed in this study was verifying that the thermostable esterase EST2 has the typical synthetic activities found in the esterases/lipases from lactic acid bacteria, as studies have shown that ester and thioester synthesis is catalyzed by esterases/lipases and is important for the development of the characteristic cheese flavors during ripening (5,11,26,28). Nardi et al (38) demonstrated that EstA is the main enzyme responsible for ester synthesis in L. lactis. Therefore, in this study a new system was developed to measure ester synthesis spectrophotometrically in an aqueous environment under conditions that simulated milk-and cheese-like conditions.…”

Section: Resultsmentioning

confidence: 59%

“…By using esterase purification (4) and genome sequencing (3), it has been shown that this bacterium contains a single esterase (EstA) and has no lipase or alcohol acetyltransferase homologous to the enzymes found in yeast (14). EstA is probably a key enzyme in the development of food flavor through the degradation of esters and lipids and the synthesis of esters and thioesters (38). Studies using an EstAnegative mutant partially confirmed this (12).…”

mentioning

confidence: 88%

“…Recently, enzymes with key activities, alone or in combination with starters, have been used to enhance cheese flavor (10,(21)(22)(23)45). The aims of this study were to determine if thermophilic esterase EST2 (30) from Alicyclobacillus acidocaldarius, a bacterium first isolated from an acidic creek in Yellowstone National Park (6), exhibits high esterase activity in milk and in a standardized cheese model and to determine if this enzyme has thioesterase and ester-synthesizing activities like those of Lactococcus lactis EstA (38).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Alicyclobacillus acidocaldariusThermophilic Esterase EST2's Activity in Milk and Cheese Models

Mandrich

Manco

Rossi

et al. 2006

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30°C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.

“…This background octyl acetate production activity may be caused by the indigenous L. lactis esterase EstA, which is capable of catalyzing ester production through the condensation of acid and alcohols or through transacylation reactions (39,47) We also included the L. lactis NZ9000(pNZ7601pNZ7610) strain producing tRNA 1 Arg and L. lactis NZ9000(pNZ7630) carrying the codon-optimized SAAT gene in these experiments. The octyl acetate production rates with induced and noninduced cultures of these clones were similar to production rates with their L. lactis NZ9000(pNZ7601) counterparts (Table 3).…”

Section: Vol 73 2007 Expression Of Plant Flavor Genes In Lactococcumentioning

confidence: 99%

Expression of Plant Flavor Genes in Lactococcus lactis

Hernández

Molenaar

Beekwilder³

et al. 2007

Lactic acid bacteria, such as Lactococcus lactis, are attractive hosts for the production of plant-bioactive compounds because of their food grade status, efficient expression, and metabolic engineering tools. Two genes from strawberry (Fragaria x ananassa), encoding an alcohol acyltransferase (SAAT) and a linalool/nerolidol synthase (FaNES), were cloned in L. lactis and actively expressed using the nisin-induced expression system. The specific activity of SAAT could be improved threefold (up to 564 pmol octyl acetate h ؊1 mg protein ؊1 ) by increasing the concentration of tRNA 1 Arg , which is a rare tRNA molecule in L. lactis. Fermentation tests with GM17 medium and milk with recombinant L. lactis strains expressing SAAT or FaNES resulted in the production of octyl acetate (1.9 M) and linalool (85 nM) to levels above their odor thresholds in water. The results illustrate the potential of the application of L. lactis as a food grade expression platform for the recombinant production of proteins and bioactive compounds from plants.