New Method To Produce Kokumi Seasoning from Protein

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New Method to Produce Kokumi Seasoning from Protein Hydrolysates Using Bacterial Enzymes Hideyuki Suzuki, Yuko Nakafuji, and Tomoki Tamura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03690 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Journal of Agricultural and Food Chemistry

Title: New Method to Produce Kokumi Seasoning from Protein Hydrolysates Using Bacterial Enzymes

Authors: Hideyuki Suzuki*, Yuko Nakafuji, and Tomoki Tamura

Affiliations: Division of Applied Biology, Kyoto Institute of Technology, Matsugasaki Sakyo-ku, Kyoto 606-8585, Japan

*Corresponding: Hideyuki Suzuki, email: [email protected], Fax: 81-75-724-7766

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ABSTRACT

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In the present study, we demonstrate a novel use for a commercially available

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glutaminase that can be used as a γ-glutamyltranspeptidase in kokumi seasoning

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production.

5

Bacillus licheniformis. The resulting protein hydrolysates were γ-glutamylated with a

6

γ-glutamyltranspeptidase, which is sold as a glutaminase from B. amyloliquefaciens, to

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produce kokumi seasonings.

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glutamine was added to the reaction mixture.

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for enzymatic proteolysis were optimized to liberate glutamine from gluten in large

Soy protein and gluten were hydrolyzed using a protease isolated from

For γ-glutamylation of soy protein hydrolysate, On the other hand, reaction conditions

10

amounts and the addition of glutamine was not required for γ-glutamylation of gluten

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hydrolysate.

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products, were subjected to taste evaluation.

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Although γ-glutamylation significantly reduced bitterness, the taste was still considered

14

unfavorable.

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significantly enhanced thickness, kokumi, and umami tastes, with moderate increase in

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saltiness.

The soy protein and gluten hydrolysates, as well as their γ-glutamylated Soy protein hydrolysates were bitter.

γ-Glutamylated gluten hydrolysate is the most preferable sample and had

17 18 19

Keywords: γ-glutamyltranspeptidase, γ-glutamylpeptide, glutaminase, protease

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INTRODUCTION In early days umami seasoning was manufactured from hydrolyzed gluten with hot

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hydrochloric acid.

Glutamine residue of the major gluten proteins, gliadins (α, β, γ, ω),

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and low and high molecular weight glutenin subunits, is nearly 40% of their total amino

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acid residues,1 while that of the major soy proteins, glycinin and β-conglycinin, is less

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than 10%.

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residues and hot hydrochloric acid cleaves not only the peptide bonds but also the amide

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bonds of the γ-carboxyl group of glutamine, the resulting hydrolysate contains very high

29

amounts of glutamic acid.

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eventually made the acid hydrolysis method to produce glutamate obsolete.2

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Nonetheless, various kinds of proteins from plants and animals have been hydrolyzed to

32

produce umami seasonings.

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various amino acids.

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for food manufacturers and widely added to processed food to increase the complexity

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of umami taste.

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hydrolyzed into amino acids, thereby producing very strong umami taste and are

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considered superior umami seasonings.

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raw material proteins is converted into chloropropanols in the presence of hydrochloric

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acid at high temperature.

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3-dichloro-2-propanol are listed as harmful chemicals by the Ministry of Agriculture,

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Forestry, and Fisheries of Japan (MAFF) and are marked for priority management.3

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the late 1970’s, 3-chloropropan-1, 2-diol was detected in several protein hydrolysates

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produced by acid hydrolysis and has since become a social problem.

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demonstrated that the amounts of chloropropanol could be reduced to below tolerable

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intake levels by alkaline treatment.

Since gluten consists of an exceptionally high percentage of glutamine

However, the invention of direct fermentation of glutamate

In this case, of course, the products are the mixtures of

In Japan, these protein hydrolysates are commercially available

Protein hydrolysates produced by acid hydrolysis are completely

However, contaminating glycerol found in

Among chloropropanols, 3-chloropropan-1, 2-diol and 1,

Studies have

As a result, in 2008, the MAFF gave 3

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manufacturers administrative direction for its thorough alkaline treatment.4 An alternative and milder method of producing protein hydrolysates is enzymatic

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hydrolysis of proteins.

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proteins into amino acids, resulting in weaker umami taste.

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products often taste bitter because of residual peptides.

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However, enzymatic hydrolysis does not completely hydrolyze Moreover, the resulting

In a previous study, we showed that γ-glutamylation can dramatically reduce the

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bitterness of amino acids.5

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γ-glutamyl peptides were reported to have strong kokumi taste.6-9

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originally defined that a kokumi substance as itself has a weak aroma and sweetness,

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but a small amount of its addition to the dishes enhances their flavor characters, such as

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continuity, mouthfulness, and thickness.10

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food scientists today.6-9

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“Koujien” from Iwanami Shoten, explains kokumi as a deep and dense taste.

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think mouthfulness and continuity are not essential factors of kokumi for ordinary

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Japanese.

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Furthermore, several γ-glutamyl amino acids and Ueda et al.

Their definition is widely accepted among

However, the most authoritative Japanese dictionary, And we

Commercially available kokumi seasonings usually contain various combinations

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of broths of various foods, yeast extracts, Maillard-reacted peptides, and protein

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hydrolysates, depending on manufacturers.

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used because they contain a lot of glutathione (γ-Glu-Cys-Gly) which has been known

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as a kokumi substance.6

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compounds by the Ministry of Health, Labor and Welfare (MHLW) of Japan, pure

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glutathione is not allowed to use as a food additive in Japan.

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was listed in food additives by the MHLW and was commercialized as a kokumi

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seasoning.

Among them, yeast extracts are usually

However, since glutathione is listed in pharmaceutical


Recently, γ-Glu-Val-Gly

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γ-Glutamyltranspeptidase (GGT, EC 2.3.2.2) catalyzes the transfer of γ-glutamyl

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group from γ-glutamyl compounds to amino acids and peptides, and the hydrolysis of

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γ-glutamyl compounds to generate glutamic acid.11

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reactions have different optimal pH conditions, selective catalysis can be achieved by

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adjusting the reaction pH.12 We have developed the enzymatic method to produce

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various γ-glutamyl compounds using bacterial GGT as a catalyst and glutamine as a

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γ-glutamyl donor.2, 12, 16

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residues are hydrolyzed to glutamic acid as described above.

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glutamine residues remain as glutamine when it is hydrolyzed by a protease and the

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released glutamine can be used as a γ-glutamyl donor in the transpeptidation reaction.

80 81

Considering that these two

If gluten is hydrolyzed by hot hydrochloric acid, its glutamine On the other hand,

In this study, we developed a new method to produce kokumi seasoning by γ-glutamylation of protein hydrolysates made by enzymatic proteolysis.

82 83 84

MATERIALS AND METHODS

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Proteins and enzymes

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Gluten (Fumerit A) was a gift from Fresh Food Service (Tokyo, Japan) and soy

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protein (Fujipro F) from Fuji Oil (Izumisano, Osaka, Japan).

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licheniformis (Protin SD-AY10), protease from B. amyloliquefaciens (Protin SD-NY10),

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papain (W-40), and GGT from B. amyloliquefaciens (Glutaminase Diwa SD-C100S)

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were gifts from Amano Enzyme (Nagoya, Japan).

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sold as a glutaminase from Amano Enzyme, but it has GGT activity as shown in

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RESULTS AND DISCUSSION section.

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Protease from Bacillus

Glutaminase Diwa SD-C100S is

Escherichia coli GGT for hydrolysis of γ-glutamyl peptides was purified from



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strain CY6, which harbors plasmid pCY213 in strain SH641 (F- ∆ggt-2 rpsL recA

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srl300::Tn10).14

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N-terminal region under the control of T5 promoter and Lac operator-repressor system.

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His-tagged E. coli GGT was purified from the cell-free extracts of strain CY6, using a

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column packed with Ni Sepharose 6 Fast Flow (GE Healthcare Bio-Sciences; Pittsburgh,

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PA).13

Plasmid pCY2 expresses E. coli GGT containing a His-tag on the

E. coli GGT for Western blot analysis was purified from strain SH642, which

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harbors plasmid pSH10114 in strain SH641.

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coli GGT from its own promoter at 20°C.

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the periplasmic fraction of strain SH642 by ammonium sulfate precipitation and

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Chromatofocusing as described previously.14

Plasmid pSH101 expresses wild-type E. Wild-type E. coli GGT was purified from

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Protein concentration and GGT activity measurements Protein concentration was measured by the method of Bradford (Bio-Rad; Hercules,

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CA) using BSA as a standard.

GGT activity was measured by the colorimetric method

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using γ-glutamyl-p-nitroanilide (γ-GpNA) (Wako Pure Chemicals; Osaka, Japan) and

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Gly-Gly (Nakarai Tesque; Kyoto, Japan) as described previously15 with slight

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modifications.

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increase in absorbance at 410 nm was measured using an automated recorder, and the

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enzymatic activity was calculated based on the initial reaction rate.

Instead of terminating the reaction by the addition of acetic acid, the

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Measurement of amino acids and peptides Amino acids and peptides were measured with an HPLC system (model LC-20A;

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Shimadzu; Kyoto, Japan) equipped with a Shim-pack Amino Na column (Shimadzu) as

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described previously.16 Samples were deproteinized by mixing with 1/10 volume of

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100% TCA solution.

Samples were then passed through a membrane filter


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(Millex-LH Syringe Driven Filter Unit, pore size 0.45 µm, Merck Millipore; Billerica,

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MA) and then subjected to HPLC analysis.

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SDS-polyacrylamide gel electrophoresis and Western blot analysis SDS-PAGE was performed as described previously.17

Western-blotting was

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performed as described,18 but using anti-rabbit immunoglobulin, horseradish

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peroxidase-conjugated anti-rabbit IgG(H+L) whole IgG from goat (Jackson Immuno

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Research; West Grove, PA) as a second antibody and the peroxidase immuno-stain kit

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from Wako Pure Chemicals (Osaka, Japan).

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Taste evaluation of seasoning samples The produced seasoning samples were evaluated for taste characteristics by ten

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panel members.

The panels consist of 6 females and 4 males from our laboratory

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members and students of nearby laboratories with the average of 26 years old.

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were not trained because we aimed to produce kokumi seasonings acceptable by

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ordinary Japanese.

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samples.

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samples they tasted.

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dissolved in 50 ml of thin taste bouillon soup, and panel members tasted a teaspoonful

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of the soup.

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(Nestle Japan; Kobe, Japan) was dissolved in 600 ml of hot water and cooled down to

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the room temperature where it is recommended to dissolve in 300 ml of hot water.

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Bitterness, saltiness, umami, kokumi, and thickness were rated based on a five-point

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category scale, and the intensities of the taste are shown as none, no taste perceived; +,

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slightly perceived; ++, weakly perceived; +++, perceived; and ++++, strongly perceived.

They

Members were informed about the purpose of the test and the

Written informed consents were obtained, but the testers were blind to which For taste evaluation, lyophilized seasoning samples (0.5 g) were

To make thin taste bouillon soup, one cube of Western taste bouillon



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Preference was also determined by ranking the samples from the best to the worst.

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RESULTS AND DISCUSSION

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Glutaminase Daiwa is, or at least contains, GGT

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GGT can hydrolyze the amide bond of glutamine.

This is the same reaction

150

catalyzed by glutaminase.

151

molecular structures. Moreover, glutaminase has never been reported to transfer

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γ-glutamyl residues from a γ-glutamyl donor to amino acids and/or peptides.

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However, GGT and glutaminase (EC 3.5.1.2) have different

Glutaminase Daiwa SD-C100S is sold as a salt-tolerant glutaminase from Amano

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Enzyme as a food additive.

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salt-tolerant glutaminase shown in the patent (JP 2001046075) from Daiwa-kasei, which

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is now part of Amano Enzyme, is almost the same as that of WP_013352483.1, a

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prospective GGT of B. amyloliquefaciens DSM 7.

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reaction of Glutaminase Diwa with antibody generated against E. coli GGT.

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Glutaminase Diwa and E. coli GGT purified from strain SH642 were subjected to

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SDS-PAGE (Fig. 1A), followed by Western blot analysis using rabbit anti-E. coli GGT

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antibody as the first antibody, as described previously (Fig. 1B).19

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We realized that the amino acid sequence of the

To verify this, we tested the

Based on the DNA sequence of E. coli ggt gene and the amino acid sequences,20 the

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calculated molecular weights of the large and small subunits are 39,198 and 20,010,

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respectively.

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under the accession number NC_01455.1.

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WP_013353848.1, which were coded in genes 9781192 and 9778114, respectively, were

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both annotated as GGT.

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subunit, but GGT is coded in a single gene.

The sequence of the B. amyloliquefaciens DSM 7 genome is available The proteins WP_013352483.1 and

Mature GGT consists of one large subunit and one small While it is translated and exported from


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the cell membrane, the signal peptide of bacterial GGT is cleaved off.

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inactive precursor is subjected to an autocatalytic proteolytic processing to become an

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active hetero-dimeric mature enzyme.21, 22

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newly made N-terminal Thr residue is the catalytic nucleophile of GGT23 and the

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N-terminal of the small subunit is well conserved.20 We predicted that

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WP_013352483.1 is GGT because it contains the typical bacterial GGT sequence, with

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TTH at the prospective N-terminal of the small subunit.

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subtilis 168 GGT showed that Thr405 of B. amyloliquefaciens GGT is the N-terminal of

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the small subunit.

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by the Sosui program.24

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subunits were calculated to be 41,398 and 19,976, respectively.

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Then, an

The oxygen atom of the side chain of the

Homology searching with B.

The first 25 amino acids were predicted to encode a signal peptide Therefore, the molecular weights of the large and small

As shown in Fig. 1A, E. coli GGT consists of the large and small subunits (lane 2),

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whereas Glutaminase Diwa showed at least seven major bands (lane 3).

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band between the 48K marker and the large subunit of E. coli GGT from pSH101 as

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well as a 20K band (lane 3).

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positive reactions with anti-E. coli GGT antibody (lane 6).

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We detected a

The proteins corresponding to these two bands showed

We also tested whether Glutaminase Diwa can catalyze the transpeptidation

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reaction of γ-GpNA and Gly-Gly to form γ-Glu-Gly-Gly.

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containing 0.5 mM γ-GpNA, 60 mM Gly-Gly, 50 mM Imidazole-HCl pH 10, and 1.8

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µg/ml (20 µg/ml including additives) of Glutaminase Diwa was incubated at 37°C for 1

189

h and subjected to HPLC analysis.

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retention time similar to that of γ-Glu-Gly-Gly (Bachem; Bunbendorf, Switzerland) was

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observed in the reaction mixture (data not shown).

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The reaction mixture

After 1 h of incubation, a new peak with a

The above findings indicate that Glutaminase Diwa is, or at least contains, GGT. Hereafter, Glutaminase Diwa is called B. amyloliquefaciens GGT since we used it as a 9 ACS Paragon Plus Environment


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GGT.

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γ-Glutamylation of soy protein hydrolysates As the first attempt, we tried soy protein as a starting protein.

Since a large

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proportion of vegetable cooking oil is extracted from soybeans, a large amount of

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soymeal arises and soy protein extracted from soymeal is commercially available.

200

protein was dissolved in distilled water at the final concentration of 5%, and the pH was

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adjusted to 8.5 with NaOH.

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concentration of 3.8 mg/ml (20 mg/ml including the additives) and incubated at 45°C

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for 5 h.

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centrifuged at 12,000 rpm for 5 min, and the supernatant was used as protein

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hydrolysate.

206

after which glutamine and B. amyloliquefaciens GGT were added at the final

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concentrations of 5 mM and 1.8 µg/ml (20 µg/ml including additives), respectively.

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The reaction mixture was incubated at 37°C for 5 h and subsequently terminated by

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incubation at 80°C for 10 min.

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evaluation.

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Soy

Protease from B. licheniformis was added to a final

The reaction was terminated at 80°C for 10 min.

The reaction mixture was

The pH of the protein hydrolysate was adjusted to pH 10 with NaOH,

The resulting mixture was lyophilized for taste

As shown in Fig. 2A and B, new peaks appeared at the retention times ranging from

212

2 to 13 min after incubation of the protein hydrolysates with glutamine and B.

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amyloliquefaciens GGT.

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at pH 5.5, and glutamate was detected in large amounts (Fig. 2C).

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exclusively catalyzes the hydrolysis reaction of γ-glutamyl linkage at pH 5.5,12 these

216

findings indicate that the peaks observed at the retention times from 2 to 13 min after

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treatment with B. amyloliquefaciens GGT (Fig. 2B) were γ-glutamyl peptides, although

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the amino acids and/or peptides to which γ-glutamyl moieties were transferred were not

These peaks disappeared after hydrolysis using E. coli GGT


Since E. coli GGT

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determined.

220 221 222

γ-Glutamylation of gluten hydrolysate For γ-glutamylation of soy protein hydrolysate, 5 mM glutamine was added as a

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γ-glutamyl donor, since soy protein does not contain sufficient glutamine residues.

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the other hand, gluten consists of an exceptionally high percentage of glutamine

225

residues as mentioned above.

226

by protease can be optimized, enough amounts of glutamine is expected to be released

227

from gluten and the released glutamine can be used in the subsequent transpeptidation

228

reaction as a γ-glutamyl donor.

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(a) Optimization of proteolytic conditions of gluten

230

On

Therefore, if the reaction condition of gluten hydrolysis

The enzymatic activities of protease from B. licheniformis, protease from B.

231

amyloliquefaciens, and papain on gluten were compared.

232

producing glutamine from gluten using these proteases was pH 9.

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concentrations higher than 3% is difficult to dissolve uniformly.

234

solution was hydrolyzed with these three proteases at the final concentration of 3 mg/ml

235

(20, 50, and 6 mg/ml including additives, respectively).

236

production was compared under the reaction conditions of pH 9, temperature of 45°C,

237

and 8 h of incubation.

238

the highest amounts of glutamine compare to the other two proteases.

239

The optimum pH for Gluten at Therefore, 3% gluten

The efficiency of glutamine

As shown in Fig. 3, protease from B. licheniformis produced

To optimize the reaction conditions for glutamine production, the concentration of

240

protease from B. licheniformis, reaction temperature, reaction pH, and reaction time

241

were investigated (Fig. 4).

242

gluten solution with 3 mg/ml (20 mg/ml including additives) of protease from B.

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licheniformis at 45°C, pH 9 for 8 h.

The optimum reaction condition was: hydrolysis of 3%

The reaction produced 3.4 mM glutamine, which 11

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corresponds to the amount of glutamine added for γ-glutamylation of soy protein

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hydrolysates that contains little glutamine.

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8,000 rpm for 10 min, and the supernatant was subjected to the γ-glutamylation.

247

(b) Optimization of γ-glutamylation of gluten hydrolysate

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The reaction mixture was centrifuged at

The gluten hydrolysate was prepared as described above using protease from B.

249

licheniformis.

250

amyloliquefaciens GGT was added to the hydrolysate at the final concentration of 1.8

251

µg/ml (20 µg/ml including additives).

252

reaction time were determined.

253

peptides corresponds to each peak at the retention time from 2 to 13 min by HPLC

254

analysis is/are kokumi peptide(s), the conditions by which all or almost all of these

255

peaks become big were investigated (Fig. 5 is shown as an example).

256

reaction conditions were achieved at pH 9, reaction temperature at 45°C, and reaction

257

time of 6 h.

258

the taste test.

259

The pH of the reaction mixture was adjusted with NaOH, after which B.

The optimum reaction temperature, pH, and

Since we do not know which of the γ-glutamyl

The optimum

The reaction mixture (200 ml) was lyophilized and subsequently used for

The yield of γ-glutamylated soy protein hydrolysate was 54% starting from soy

260

protein, while that of γ-glutamylated gluten hydrolysate was 90% starting from gluten.

261

This is because a large amount of precipitate was removed after the hydrolysis of soy

262

protein.

263 264 265

Taste evaluation of seasoning samples Produced seasoning samples were evaluated by ten panel-members.

Seasoning

266

samples were added to thin bouillon, and tastes were compared with and without the

267

addition of each sample.

The pH of the reaction mixtures were adjusted to alkaline


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before proteolysis and γ-glutamylation, but during each reaction the pH shifted to

269

neutral.

270

The thickness, kokumi, bitterness, saltiness, and umami were evaluated based on a

271

five-point category scale.

272

was also determined by ranking the samples from the best to the worst (Fig. 7).

273

soy protein hydrolysate is quite bitter and had the least favorable taste.

274

bitterness was considerably reduced by γ-glutamylation, the preference of

275

γ-glutamylated soy protein hydrolysate ranked forth.

276

half of the panel members selected the γ-glutamylated gluten hydrolysate as the most

277

preferred sample.

278

γ-glutamylated gluten hydrolysate, but adding it to the thin bouillon obviously enhanced

279

thickness, kokumi, and umami taste.

280

hydrolysate also increased saltiness, although the effect was smaller compared to those

281

of the other tastes.

Therefore, the pH of tsere

The results are summarized in Fig. 6.

Sample preference The

Although

On the other hand, more than

Half of the panel members perceived slight bitterness in the

Addition of the γ-glutamylated gluten

282

In this study, we established a new method of producing kokumi seasoning from

283

gluten using protease from B. licheniformis and B. amyloliquefaciens GGT, which is

284

sold as a glutaminase.

285

Commercially sold kokumi seasonings are the mixture of various ingredients as

286

described in INTRODUCTION and various manufactures have been made great efforts

287

how to blend ingredients to make kokumi seasonings.

288

allowed to use as a food additives and was commercialized as a kokumi seasoning.

289

γ-Glu-Val-Gly can be synthesized from glutamine and Val-Gly using GGT as we

290

showed previously25, but in this case Val-Gly is required as a substrate and it is not

291

inexpensive.

Protein hydrolysates are fundamentally umami seasonings.

Recently, γ-Glu-Val-Gly was

On the other hand, our method does not require any pure amino acids or



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peptides, but gluten, protease, and GGT.

GGT for the use of food processing was not

293

commercially available.

294

SD-C100S sold as a glutaminase for food processing has GGT activity, all materials

295

necessary for kokumi seasoning production are available commercially.

296

of our method is that the price of GGT is not so inexpensive.

297

obtain bacterial GGT with higher transpeptidation activity and develop efficient

298

purification method to supply GGT more economically in order to generalize our

299

method to produce kokumi seasoning.

However, since we proved that Glutaminase Daiwa

The weakness

Our future goal is to

300 301

ACKNOWLEDGEMENTS

302

This research was supported by the grants from the Fuji Foundation for Protein

303

Research and from the Japan Food Chemical Research Foundation.

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Harvey, L., Eds.; Woodhead Publishing, Cambridge, UK; 2013; pp. 385-412.

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chloropropanols in food. (in Japanese)

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(http://www.maff.go.jp/j/syouan/seisaku/c_propanol/) (November 5, 2017).

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57, 1440-1448.

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9. Ohsu, T.; Amino, Y.; Nagasaki, H.; Yamanaka, T.; Takeshita, S.; Hatanaka, T.;

333

Maruyama, Y.; Miyamura, N.; Eto, Y.; Involvement of the calcium-sensing receptor

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in human taste perception. J. Biol. Chem., 2010, 285, 1016-1022.

335

10. Ueda, Y.; Sakaguchi, M.; Hirayama, K.; Miyajima, R.; Kimizuka, A.; Characteristic

336

flavor constituents in water extract of garlic.

337

163-169.

338 339 340

Agric. Biol. Chem., 1990, 54,

11. Tate, S.; Meister, A.; γ-Glutamyl transpeptidase: catalytic, structural and functional aspects. Mol. Cell. Biochem. 1981, 39, 357-368. 12. Suzuki, H.; Yamada, C.; Kato, K.; γ-Glutamyl compounds and their enzymatic

341

production using bacterial γ-glutamyltranspeptidase.

342

333-340.

343

Amino Acids, 2007, 32,

13. Yamada, C.; Kijima, K.; Ishihara, S.; Miwa, C.; Wada, K.; Okada, T.; Fukuyama,

344

K.; Kumagai, H.; Suzuki, H.

345

acid acylase activity of a bacterial γ-glutamyltranspeptidase.

346

Microbiol., 2008, 74, 3400-3409.

347

Improvement of the glutaryl-7-aminocephalosporanic Appl. Environ.

14. Suzuki, H.; Kumagai, H.; Echigo, T.; Tochikura, T.; Molecular cloning of

348

Escherichia coli K-12 ggt and rapid isolation of γ-glutamyltranspeptidase.

349

Biochem. Biophys. Res. Commun., 1988, 150, 33-38.

350 351 352 353

15. Suzuki, H.; Kumagai, H.; Tochikura, T.; γ-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization.

J. Bacteriol., 1986, 168, 1332-1335.

16. Suzuki, H.; Izuka, S.; Minami, H.; Miyakawa, N.; Ishihara, S.; Kumagai, H.; Use of bacterial γ-glutamyltranspeptidase for enzymatic synthesis of γ-D-glutamyl


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354 355


compounds.

Appl. Environ. Microbiol., 2003, 69, 6399-6404.

17. Suzuki, H.; Kumagai, H.; Tochikura, T.; γ-Glutamyltranspeptidase from Escherichia

356

coli K-12: purification and properties. J. Bacteriol., 1986, 168, 1325-1331.

357

18. Hashimoto, W.; Suzuki, H.; Yamamoto, K.; Kumagai, H.; Effect of site-directed

358

mutations on processing and activity of γ-glutamyltranspeptidase of Escherichia coli

359

K-12.

360

J. Biochem., 1995, 118, 75-80.

19. Hashimoto, W.; Suzuki, H.; Nohara, S.; Tachi, H.; Yamamoto, K.; Kumagai, H.;

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Subunit association of γ-glutamyltranspeptidase of Escherichia coli K-12. J.

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Biochem., 1995, 118, 1216-1223.

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20. Suzuki, H.; Kumagai, H.; Echigo, T.; Tochikura, T.; DNA sequence of the

364

Escherichia coli K-12 γ-glutamyltranspeptidase gene, ggt.

365

5169-5172.

366 367

J. Bacteriol., 1989, 171,

21. Suzuki, H.; Kumagai, H.; Autocatalytic processing of γ-glutamyltranspeptidase. J. Biol. Chem., 2005, 277, 43536-43543.

368

22. Okada, T.; Suzuki, H.; Wada, K.; Kumagai, H.; Fukuyama, K.; Crystal structure of

369

the γ-glutamyltranspeptidase precursor protein from Escherichia coli: Structural

370

changes upon autocatalytic processing and implications for the maturation

371

mechanism.

372

J. Biol. Chem., 2007, 282, 2433-2439.

23. Inoue, M.; Hiratake, J.; Suzuki, H.; Kumagai, H.; Sakata, K.; Identification of

373

catalytic nucleophile of Escherichia coli γ-glutamyltranspeptidase by

374

γ-monofluorophosphono derivative of glutamic acid: N-terminal Thr-391 in small

375

subunit is the nucleophile.

376 377

Biochemistry, 2000, 39, 7764-7771.

24. Gomi, M.; Sonoyama, M.; Mitaku, S.; High performance system for signal peptide prediction: SOSUIsignal. Chem-Bio Info. J., 2004, 4, 142-147.



378

25. Suzuki, H.; Yamada, C.; Improvement of the flavor of amino acids and peptides using

379

bacterial γ-glutamyltranspeptidase. In Recent highlights in flavor chemistry &

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biology; Hofmann, T.; Meyerhof, W.; Schieberle, P., Eds.; Deutsche

381

Forschungsanstalt für Lebensmittelchemie, Garching, Germany; 2008, pp.227-232.

382


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383

FIGURE CAPTIONS

384

Fig. 1

385

blot analysis.

386

indicate the molecular weights; Wide View Marker III, Wako Pure Chemicals, Osaka,

387

Japan); lanes 2, 5: E. coli GGT (4.3 µg-protein); lanes 3, 6: Glutaminase Daiwa (72

388

µg-protein).

389

respectively.

(A) SDS-PAGE stained with Coomassie brilliant blue R-250, (B) Western Lane 1, 4: molecular weight marker (numbers in the left column

L and S indicate the positions of the large and small subunits,

390 (A) The hydrolysates

391

Fig. 2

392

added with 5 mM Gln. (B) After γ-glutamylation of the hydrolysates by Glutaminase

393

Daiwa at pH 10.

394

γ-glutamylγ-glutamylated hydrolysate by E. coli GGT at pH 5.5.

HPLC analysis of the soy protein hydrolysates.

(C) After cleavage of

395 396

Fig. 3

397

from (1) Bacillus licheniformis, (2) B. amyloliquefaciens, and (3) papain.

398

of gluten solution (3%) was hydrolyzed with these three proteases.

399

glutamine formation was compared.

400

mean and the standard deviation of three independent experiments.

Comparison of glutamine formation activity from gluten among proteases Thirty ml

The efficiency of

Glutamine concentrations were expressed as the

401 Concentrations of

402

Fig. 4

403

released glutamine in 30 ml of the reaction mixtures were expressed as the mean and

404

standard deviation of three independent experiments.

405

protease from B. licheniformis were used, namely 1.5, 3, and 4.5 mg/ml (10, 20, and 30

406

mg/ml including additives).

407

(C) The pH of the reaction mixture was varied from 8 to 10.

Optimization of proteolytic conditions of gluten.

(A) Various concentrations of

(B) Reaction temperature was varied from 32 to 55°C.


(D) The amounts of


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408

glutamine produced with prolonged reaction times were measured.

The reaction was

409

initiated with 30 ml of the reaction mixture, and 1 ml of the reaction mixture was

410

sampled at each sampling time.

411 Optimization of reaction temperature of γ-glutamylation of gluten

412

Fig. 5

413

hydrolysate.

414

B. amyloliquefaciens GGT was added to the solution at the final concentration of 1.8

415

µg/ml (20 µg/ml including additives).

416

incubated with reciprocal shaking at 120 rpm for 6 h at the respective temperatures

417

indicated in the figure.

The pH of the gluten hydrolysate (30 ml) was adjusted to 9 with NaOH.

The mixture was placed in a 100-ml flask and

418 S: soy protein hydrolysate or its γ-glutamylated product; G:

419

Fig. 6

420

gluten hydrolysate or its γ-glutamylated product.

421

of the taste: none, no taste perceived; +, slightly perceived; ++, weakly perceived; +++,

422

perceived; and ++++, strongly perceived.

423

panel members who evaluated the samples.

424

bouillon only, bouillon with the addition of protein hydrolysate, and bouillon with the

425

addition of γ-glutamylated protein hydrolysate, respectively.

Taste evaluation.

Vertical axes indicate the intensities

Horizontal axes indicate the number of the White, gray, and black bars indicate

426 Preferences for protein hydrolysates and their γ-glutamylated products.

427

Fig. 7

428

Vertical axes indicate the preferences, and horizontal axes indicate the number of the

429

panel members who evaluated the samples.

430

γ-glutamylated product; G: gluten hydrolysate or its γ-glutamylated product. White,

431

gray, and black bars indicate bouillon only, bouillon with the addition of protein

S: soy protein hydrolysate or its


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432

hydrolysate, and bouillon with the addition of γ-glutamylated protein hydrolysate,

433

respectively.



Fig. 1


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Fig. 2



Fig. 3


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Fig. 4



Fig. 5


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Fig. 6 27 ACS Paragon Plus Environment


Fig. 7


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TOC Graphic


New Method To Produce Kokumi Seasoning from Protein

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