Amino Acid Converting Enzymes AACEs

Amino acids are the precursors of various volatile cheese flavor compounds that have been identified in cheese (93,94). They can be converted in many different ways by enzymes such as deaminases, decarboxylases, transaminases (aminotransferases), and lyases (Fig. 7). Transamination of amino acids results in the formation of a-keto acids that can be converted into aldehydes by decarboxylation and, subsequently, into alcohols or carboxylic acids by dehydrogenation. Many of these components are odor-active and contribute to the overall flavor of the dairy product. Moreover, other reactions may occur—for example, by hydrogenase activity toward a-keto acids resulting in the formation of hydroxyacids, which do hardly contribute to the flavor.

Using biochemical and genetic tools, various flavor-forming routes from amino acids and enzymes involved have recently been identified, mostly in L. lactis [see (95,97,106) for a review].

Aromatic amino acids, branched-chain amino acids, and methionine are the most relevant substrates for cheese flavor development. Conversion of aromatic amino acids can result in formation of undesirable flavors, so-called off-flavors, such as p-cresol, phenyl-ethanol, phenylacetaldehyde, indole, and skatole, which contribute to putrid, fecal, or unclean flavors in cheese (100). Many of these reactions can also occur under cheese conditions and are highly dependent on the strain used (107). This implies that by target selection of starter bacteria the formation of undesirable flavors can be avoided. A similar strain dependency is also found for enzyme activities that result in the formation of desired flavor compounds, indicating that a strong potential for starter improvement exists (see below).

Conversion of tryptophan or phenylalanine can also lead to benzaldehyde formation. This compound is found in various hard-type and soft-type cheeses and contributes positively to the overall flavor (94,96). In Lb. plantarum as well as in other lactic acid bacteria, the formation of benzaldehyde out of phenylalanine is initiated by an aminotransferase reaction followed by a chemical conversion of the intermediate phenylpyruvic acid into benzaldehyde (108,109). The latter reaction requires the presence of manganese, for which an efficient uptake system was found (110). This chemical conversion occurs at a high pH and in the presence of oxygen, so it is not very likely a main conversion pathway in cheese.

Branched-chain amino acids are precursors of various aroma compounds such as isobutyrate, isovalerate, 3-methylbutanal, 2-methylbutanal, and 2-methylpropanal. These compounds are found in various cheese types. Several enzymes that are able to convert these amino acids have been detected in L. lactis [see (95,97,106) for a review]. The aromatic aminotransferases can convert aromatic amino acids, but also leucine and methionine; the branched-chain aminotransferases can convert the branched-chain amino acids leucine, isoleucine, and valine, but also methionine, cysteine, and phenylalanine.

Volatile sulfur compounds derived from methionine, such as methanethiol, dimethyl-sulfide, and dimethyltrisulfide, are regarded as essential components in many cheese varieties (111). In fact, a Gouda cheese-like flavor can be generated by incubation of methionine with cell extracts of L. lactis (93). Conversion of methionine can occur via a aminotrans-ferase-initiated pathway by branched-chain or aromatic aminotransferases, or via an a,g-elimination of methionine by the lyase activities of cystathionine h-lyase (CBL), cystathionine g-lyase (CGL), or methionine g-lyase (MGL) (97,107,112-121).

It was found that the amount of a-keto acids determines the rate of the first step in the conversion of amino acids. Overproduction of the transaminases alone did not lead to a strong increase in amino acid conversion without a simultaneous addition of keto acids as co-substrate (122). The introduction of a glutamate dehydrogenase gene from Pepto-streptococcus in L. lactis resulted in a similar effect (123). However, whether this activity also results in a strong increase in the desired flavor components remains to be determined.

Ayad et al. (20) found that also the presence of enzymes required for subsequent conversions might be of crucial importance (Fig. 8).

Although cystathionine lyases are active under cheese-ripening conditions (112), their activity towards methionine could not be detected using 13C nuclear magnetic resonance (107). With this technique, only the aminotransferase-initiated pathway was observed suggesting that this pathway is most prominent in methionine catabolism to produce meth-anethiol. On the other hand, strains that overproduce cystathionine h-lyase, where found to be able to degrade methionine, indicating the potential of this enzyme in the production of sulfury flavors (120,121). The specificity of CBL (110) is a particular advantage in this respect, since one might expect that only sulfury flavor components would increase in strains with high activity. In case of overproduction of other less-specific enzymes such as transaminases, more pathways will be influenced at the same time.

Biosynthesis and degradation of some amino acids are intricately coupled pathways. For instance, the above mentioned cystathionine h-lyase can convert methionine to various volatile flavor compounds, but in bacteria its physiological function is the conversion of cystathionine to homocysteine, which is the penultimate step of methionine biosynthesis (97). This means that many of the AACEs are in fact involved in the biosynthesis of amino acids. It is well known that biosynthesis of amino acids is highly regulated, and therefore the growth conditions of the starter cultures might affect their flavor-forming capacities. For instance, in L. lactis the expression of the gene coding for cystathionine h-lyase is strongly influenced by the amounts of methionine and cysteine in the culture medium (120,121). High concentrations of these amino acids completely abolish transcription and result in L. lactis cells almost deficient of cystathionine h-lyase activity.

Similarly, it is found that the branched-chain aminotransferase is also regulated at the transcriptional level in L. lactis (124). The physiological role of branched-chain ami-notransferases in bacterial metabolism is to catalyze the last step in the biosynthesis of branched-chain or aromatic amino acids. Several enzymes can thus been considered as being involved in both biosynthesis and degradation of amino acids, and a-keto acids are intermediates in both directions. These examples illustrate that the choice of culture conditions can strongly influence the flavor-forming capacities of starter cultures like L. lactis.

Transaminase Decarboxylase activity activity

Figure 8 Variation in enzyme activities between industrial (SK110) and wild L. lactis (B1173 and B1157) strains in the two-step conversion of leucine towards 3-methyl aldehyde. The largest difference was found in step 2: the decarboxylation of the ketoacid towards the aldehyde.

Transaminase Decarboxylase activity activity

Figure 8 Variation in enzyme activities between industrial (SK110) and wild L. lactis (B1173 and B1157) strains in the two-step conversion of leucine towards 3-methyl aldehyde. The largest difference was found in step 2: the decarboxylation of the ketoacid towards the aldehyde.

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