Application of Bacterial Î³-Glutamyl-Transpeptidase to Improve the

Chapter 15

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Application of Bacterial γ-Glutamyl-Transpeptidase to Improve the Taste of Food Hideyuki Suzuki and Hidehiko Kumagai Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan

Bacterial γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) was applied to improve the taste of food. We found that the bitterness of amino acids was reduced, sourness produced, and preference increased with γ-glutamylization. An enzymatic method for the synthesis of γ-glutamyl amino acids involving GGT was developed. An enzymatic method involving GGT for the synthesis of theanine, which is the major umami component of tea, was developed. The major umami component of soy sauce is glutamate (Glu), and the effective conversion of Gln to Glu by glutaminase during its fermentation in the presence of 18% NaCl is a matter of concern. Purified Bacillus subtilis GGT was salt-tolerant and 76% of its original glutaminase activity remained even in the presence of 18% NaCl.

© 2004 American Chemical Society In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

223


224 GGT is the key enzyme in glutathione metabolism and is widely distributed in living things (1-4). GGT consists of one large subunit and one small subunit. The molecular weight of the large subunit is about 40.000 and that of the small subunit is about 20.000. It was known that the large and small subunits of mature GGT are generated from a common enzymatically inactive precursor through post-translational proteolytic processing (5-13). We recently showed that this processing is an autocatalytic event and that the catalytic nucleophile for the processing is the oxygen atom of the side-chain of the Thr residue that is going to be the N-terminal of the small subunit after processing (14). This oxygen atom is also the nucleophile for the enzymatic reaction (15). GGT catalyzes two reactions (Figure 1). The reactions catalyzed by GGT proceed via a γ-glutamyl-enzyme intermediate. If this intermediate undergoes nucleophilic substitution by amino acids or peptides, it is the transpeptidation reaction yielding new γ-glutamyl compounds. However, if the intermediate undergoes nucleophilic substitution by water, it is the hydrolysis reaction, which releases glutamic acid. When the original γ-glutamyl compound is glutamine, the hydrolysis reaction is a "glutaminase" reaction. The pH optima of the two reactions are different. Therefore, by adjusting the reaction pH, we can make the enzyme catalyze the transpeptidation reaction selectively. Employing various γ-glutamyl acceptors, we can synthesize various γ-glutamyl compounds using GGT.

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Figure 1. The mechanisms of the enzymatic reactions catalyzed by GGT Why are we interested in γ-glutamyl compounds? One reason is that their solubility in water is increased by γ-glutamylization. Another reason is that the γ-glutamyl linkage is resistant to attack by peptidases in serum. Therefore, γglutamyl compounds can possibly be used as pro-drugs for specific organs that express GGT, as in the cases of γ-Glu-DOPA (16-19) and γ-Glu-dermorphin (20). Another reason is that some γ-glutamyl compounds taste good. In this

In Challenges in Taste Chemistry and Biology; Hofmann, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

225 chapter, we describe the application of bacterial GGT to improve the taste of food using both its transpeptidation and hydrolysis activities.


Utilization of the Transpeptidation Reaction of GGT to Improve the Taste of Food Glutathione, which is one of the major γ-glutamyl compounds in living things, tastes sour, but has a preferable and refreshing lemon-like sourness. We found that the taste of γ-Glu-Phe was strikingly different from that of Phe. γGlu-Phe tasted just like glutathione.

Effect of γ-Glutamylization of Bitter Amino Acids Some L-amino acids taste bitter. Aromatic amino acids, basic amino acids, and branched-chain amino acids taste bitter. Unfortunately, many bitter amino acids are essential ones. Therefore, when we take an amino acid mixture orally, the bitterness of these amino acids is a crucial problem. These days amino acid mixtures consisting of branched-chain amino acids, Phe, Arg, and/or Lys, are very popular in Japan as supplements. However, they taste extremely bitter, and thus are hard to take orally without sweetening and flavoring. Phe is an essential amino acid and one of the most bitter ones. First, the taste of Phe and γ-Glu-Phe was evaluated by eight panel members. As shown in Figure 2, various members evaluated Phe to be bitter at 15 mM. However, γGlu-Phe did not show any bitterness at all. Phe was practically not sour, but the sourness of γ-Glu-Phe was obvious. Also the preference for γ-Glu-Phe was better than that for Phe. Therefore, it was evident that γ-glutamylization of Phe improved its taste. Several panel members said γ-Glu-Phe had a refreshing lemon-like sourness, and we think this was the reason why they preferred γ-GluPhe. When an equimolar amount of glutamic acid was added to Phe, the mixture was sourer than Phe alone, however, there was no change in the bitterness or preference. This indicates that the γ-glutamyl linkage is necessary for the effect and that the existence of Glu is not enough. The sourness of a mixture of Phe and Glu was not refreshing. Moreover, when Glu was added, the taste became harsh. This might be the reason why the addition of Glu was not effective. There was no significant difference in taste between Phe and γ-GluPhe neutralized with NaOH. This suggests that the sourness of γ-Glu-Phe is critical. The effect of γ-glutamylization on the taste of other bitter amino acids was also examined, and similar results were obtained for Val, Leu, and His (Figure 3).



226

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Figure 2. Comparison of the taste of Phe and γ-Glu-Phe at 15 mM. (A) Bitterness, (B) sourness, and (C) preference. The vertical axes indicate the number of people. The horizontal axes in (A) and (B) indicate the intensity of the taste: 0, did not feel; l,felt slightly; 2, felt weakly; 3, felt; 4, felt strongly. The horizontal axes in (C) indicate the preference on a 5-point scale. (Adapted with permission from reference 21. Copyright 2002 American Chemical Society.)


227

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21 Ο

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Figure 3. Effect of γ-glutamylization of Val Leu, and His. (A) Bitterness, (B) sourness, and (C) preference. The axes indicate the same as in Figure 2. The concentrations used were 50 mMfor Val and Leu, and their γ-glutamyl derivatives, and 70 mM for His and γ-Glu-His. (Reproduced with permission from reference 21. Copyright 2002 American Chemical Society.)



228 Kirimura et al. (22) compared the taste of various dipeptides, and reported that α-Glu-Phe was bitter and sour, while γ-Glu-Phe was not bitter but sour. However, they also reported that all γ-glutamyl amino acids they tested were astringent. As far as we examined, γ-Glu-Phe, y-Glu-Val, γ-Glu-Leu, and γ-GluHis were not astringent at all, which is inconsistent with their results. In conclusion, γ-glutamylization reduced the bitterness, and increased the sourness of and preference for bitter amino acids. y-Glutamylization should be a powerful method for improving the taste of bitter amino acids. The question may arise as to how γ-glutamyl amino acids are taken up in the intestines of humans and then metabolized. One possibility is that the γglutamyl linkage is cleaved by highly abundant GGT on the brush borders of the columnar epithelial cells on the tips of the villi of the small intestine (23, 24), and the released Glu and amino acids are taken up through regular transport systems for amino acids. This is similar to the mechanism of glutathione transport that Inoue suggested (25). Another possibility is that γ-glutamyl amino acids are taken up intact and the γ-glutamyl linkage is cleaved by GGT existing in the kidneys.

Enzymatic Production of γ-Glutamyl Amino Acids Using Bacterial GGT Among the bitter amino acids we tested, the effect of γ-glutamylization was most remarkable for Phe. Therefore, the reaction conditions for the synthesis of γ-Glu-Phe involving bacterial GGT were optimized recently (21). Besides, we have already developed an enzymatic method for synthesizing various γglutamyl amino acids involving bacterial GGT as a catalyst (26-32). Bacterial GGTs are superior to eukaryotic GGTs, because bacterial GGTs are either periplasmic (33) or extracellular enzymes (34), and can be purified as soluble enzyme preparations from tank cultures, whereas eukaryotic GGTs are membrane-bound enzymes (1, 2). The characteristics of our method can be summarized as follows: • • • • •

A less expensive γ-glutamyl donor, L-glutamine, can be used effectively as well as glutathione, unlike in the case of mammalian GGT. Since GGT is a transferase and not a synthetase, it does not require any energy source such as ATP. The substrate specificity of GGT for γ-glutamyl acceptors is quite broad and thus various γ-glutamyl compounds can be synthesized. The transpeptidation reaction can occur at various pHs. A lot of GGT is readily available because both E. coli and B. subtilis GGTs


229

•

can be purified by simple two-step purification procedures from overexpressing strains (34-36). Unlike a chemical synthetic method, the protection and deblocking of reactive groups of the substrates are not required.


The optimum reaction conditions and yields of various γ-glutamyl compounds synthesized with our method are summarized in Table I.

Table I. The Reaction Conditions and Yields of Various γ-Glutamyl Amino Acids Synthesized with Our Method

γ-Glutamyl Amino Acid Concentration Synthesized L-Gln Acceptor GGT (mM) (mM) (mU/ml)

y-Glu-L-DOPA

Application of Bacterial Î³-Glutamyl-Transpeptidase to Improve the

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