Synthesis and sensory characteristics of kokumi ?-[Glu]n-Phe in the presence of glutamine and phenylalanine: Glutaminase from bacillus amyloliquefaciens or aspergillus oryzae as the catalyst

Article abstract of DOI:10.1021/acs.jafc.7b03419

The transpeptidase activity of glutaminase from Bacillus amyloliquefaciens (GBA) and Aspergillus oryzae (GAO) to yield ?-[Glu]n-Phe peptides were verified for the first time. In the presence of Gln and Phe, ?-Glu-Phe and ?-Glu-?-Glu-Phe were synthesized by GAO, and ?-Glu-Phe, ?-Glu-?-Glu-Phe, ?-Glu-?-Glu-?-Glu-Phe, ?-Glu-?-Glu-?-Glu-?-Glu-Phe, and ?-Glu-?-Glu-?-Glu-?-Glu-?-Glu-Phe were synthesized by GBA. The Km values for the transpeptidation catalyzed by GBA and GAO were 47.88 and 153.92 mM (Phe as the acceptor), 84.89 and 236.47 mM (?-Glu-Phe as the acceptor), indicating that GBA had a greater affinity than GAO for Phe and ?-Glu-Phe in the transpeptidation reaction. The Km values for the transpeptidation catalyzed by GBA against acceptors, Phe and ?-[Glu](1≤n<5)-Phe (47.88-206.47 mM), increased with an elevated number of ?-glutamyl residue within the acceptor. The optimal conditions for ?-[Glu]n-Phe synthesis were pH 10 and 37 °C for 3 h, 300 mM Gln, 100 mM Phe, 0.05 U/mL GBA. All the ?-[Glu](1≤n≤5)-Phe exhibited astringency in water and imparted a kokumi taste to commercial soy sauce and model chicken broth. The astringent threshold values (2.5-3.92 mM) were approximately 3-fold of the kokumi threshold concentrations (0.78-1.53 mM). ?-[Glu]n-Phe or the post-enzymatic reaction mixture enhanced the umami intensity of commercial soy sauce and model chicken broth.

Full text of DOI:10.1021/acs.jafc.7b03419

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Article
Synthesis and sensory characteristics of kokumi #-[Glu]n-Phe in
the presence of glutamine and phenylalanine: Glutaminase from
Bacillus amyloliquefaciens or Aspergillus oryzae as the catalyst
Juan Yang, Dongxiao Sun-Waterhouse, Chun Cui, Keming Dong, and Wei Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03419 • Publication Date (Web): 13 Sep 2017
Downloaded from http://pubs.acs.org on September 13, 2017
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Synthesis and sensory characteristics of kokumi γꢀ[Glu]n-Phe in the presence of
glutamine and phenylalanine: Glutaminase from Bacillus amyloliquefaciens or Aspergillus
oryzae as the catalyst
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a
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Juan Yang , Dongxiao SunꢀWaterhouse , Chun Cui *, Keming Dong , Wei Wang
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School of food science and engineering, South China University of Technology, Guangzhou
510640, China
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Guangdong Weiwei Biotechnology Co., LTD., Guangzhou 510640, China
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Corresponding author: Chun Cui, PhD. Tel: +86 20 87114954; Fax: +86 20 87114954;
Eꢀmail:cuichun@scut.edu.cn
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First author: Juan Yang, PhD. Tel: +86 20 87114954; Fax: +86 20 87114954;
Eꢀmail:yangjuanꢀsky@163.com.cn
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ABSTRACT:
The transpeptidase activity of glutaminase from Bacillus amyloliquefaciens (GBA) and
Aspergillus oryzae (GAO) to yield γꢀ[Glu]nꢀPhe peptides were verified for the first time. In the
presence of Gln and Phe, γꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀPhe were synthesized by GAO, and
γꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe and
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe were synthesized by GBA. The Km values for the
transpeptidation catalysed by GBA and GAO were 47.88 and 153.92 mM (Phe as the acceptor),
84.89 and 236.47 mM (γꢀGluꢀPhe as the acceptor), indicating that GBA had a greater affinity than
GAO for Phe and γꢀGluꢀPhe in the transpeptidation reaction. The K
m
values for the
transpeptidation catalysed by GBA against acceptors, Phe and γꢀ[Glu](1 n<5)ꢀPhe (47.88 ~ 206.47
≦
mM), increased with an elevated number of γꢀglutamyl residue within the acceptor. The optimal
conditions for γꢀ[Glu]nꢀPhe synthesis were pH 10 and 37°C for 3 h, 300 mM Gln, 100 mM Phe,
≦n≦
0.05 U/mL GBA. All the γꢀ[Glu](1 5)ꢀPhe exhibited astringency in water and imparted a kokumi
taste to commercial soy sauce and model chicken broth. The astringent threshold values (2.5~3.92
mM) were approximately threeꢀfold of the kokumi threshold concentrations (0.78~1.53 mM).
γꢀ[Glu]nꢀPhe or the postꢀenzymatic reaction mixture enhanced the umami intensity of commercial
soy sauce and model chicken broth.
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KEYWORDS: Enzymatic synthesis, γꢀ[Glu]nꢀPhe, Kokumi, Astringency, Glutaminase
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INTRODUCTION
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Taste of food plays an important role in food choice. As a result, research has been directed
towards not only the flavoring compounds (which can impart the five basic tastes i.e. sweet, salty,
sour, bitter and umami), but also the kokumi substances (which that can modify the basic tastes,
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mouthfulness, thickness or continuity of foods at these effective concentrations). Recently,
γꢀglutamyl peptides with kokumiꢀimparting properties through activating the calciumꢀsensing
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receptor in human cells have attracted increasing attention.
Their sequences mainly refer to
γꢀGluꢀVal, γꢀGluꢀMet, γꢀGluꢀGlu, γꢀGluꢀGln, γꢀGluꢀGly, γꢀGluꢀLeu, γꢀGluꢀHis, γꢀGluꢀCysꢀGly,
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γꢀGluꢀValꢀGly, which were usually identified from edible beans, cheese and soy sauces as the
flavor contributors in these food. To the best of our knowledge, the sequence like
γꢀGluꢀγꢀGluꢀAmino acid, γꢀGluꢀγꢀGluꢀγꢀGluꢀAmino acid, γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀAmino acid, et
al., have not yet been reported as one of the kokumi compounds.
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A largeꢀscale process for production of γꢀglutamyl peptides materials is critically needed to
ensure commercial use of these kokumi substances and avoid the dependence on agriculture and
influence of environmental and processing conditions. Biocatalytic synthesis of γꢀglutamyl
peptides using enzymes could be a costꢀeffective alternative. LꢀGlutaminase (Lꢀglutamine
amidohydrolase EC 3.5.1.2) is widely distributed in microorganisms including bacteria, yeast and
fungi. The enzyme catalyzes mainly the hydrolysis of glutamine to glutamic acid and ammonia and
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also the γꢀglutamyl transfer reaction. The glutaminase from Escherichia coli
and P.
can catalyze the hydrolysis and then the transfer of γꢀglutamyl moiety. The
glutaminase from P. nitroreducens can catalyze γꢀglutamyl transfer to ethylamine or methylamine
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Aeruginosa
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with repressed hydrolysis of glutamine. The glutaminase from Pseudomonas nitroreducens IFO
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12694 and Aspergillus oryzae were reported to possess transpeptidase activity and have been
used to catalyze the synthesis of Lꢀtheanine and a large number of γꢀglutamyl peptides,
respectively. To our knowledge, several microbial glutaminases might have transpeptidase activity
but no systematic research has been conducted to verify.
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The γꢀglutamyl transfer reaction is mainly catalyzed by bacterial γꢀglutamyltranspeptidase
(GGT, EC 2.3.2.2) and a few microbial Lꢀglutaminases involve in the transfer of γꢀglutamyl
moiety from glutathione, glutamine or other γꢀglutamyl compounds to other amino acids or
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peptides.
The feasible sequences for the synthetic γꢀglutamyl peptides might be γꢀGluꢀamino
acid, γꢀGluꢀγꢀGluꢀamino acid, γꢀGluꢀγꢀGluꢀγꢀGluꢀamino acid, etc. For instance, γꢀGluꢀγꢀGluꢀTau
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was found as
a
byproduct of the γꢀGluꢀTau synthesis catalysed by GGT;
and
γꢀglutamylꢀglutamine and other polyꢀglutamylated species were identified as the byproducts of the
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synthesis of γꢀglutamylꢀpeptides catalysed by GGT.
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Gamma–glutamylization of bitter amino acids has been proven a powerful approach to
improve the taste of bitter amino acids. For instance, Phe is known as a bitter amino acid, while
γꢀGluꢀPhe exhibited a slightly sour, salty, metallic taste, and an intense and complex brothy which
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was believed close to recently termed “Kokumi”. γꢀGluꢀPhe was perceived as sourness and
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astringency with the taste threshold concentration as 2500 ꢁmol/L for astringency and 200ꢀ500
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mg/L for sourness, although its kokumi taste threshold concentration has not been disclosed yet.
Moreover, γꢀglutamylization of Phe was accomplished by Suzuki et al. (2002) using the GGT as
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the catalyst with the conversion rate of Phe against γꢀGluꢀPhe reaching 70%. Accordingly, it is of
high interest to verify that γꢀglutamylation of Phe (a substrate for transpeptidation of glutaminases)
could attenuate bitterness perception and the products might impart kokumi properties. This study
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aimed to evaluate (1) the transpeptidase activity of two glutaminases (from Bacillus
amyloliquefaciens (GBA) and Aspergillus oryzae (GAO)); (2) the synthesis of the Pheꢀcontaining
γꢀglutamyl peptides; (3) the affinity of glutaminases for different acceptors (Phe and γꢀGluꢀPhe)
during the synthesis of γꢀglutamyl peptides; (4) the sensory characteristics of γꢀ[Glu]nꢀPhe and
postꢀenzymatic reaction mixture catalysed by glutaminases.
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MATERIALS AND METHODS
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Reagents and enzymes. LꢀGlutaminase from Bacillus amyloliquefaciens was acquired from
Amano Enzyme China Ltd. (Shanghai, China) (termed as “GBA” herein). LꢀGlutaminases from
Aspergillus oryzae and Aspergillus niger were from our lab (termed as “GAO” and “GAN”,
respectively, herein). Commercial peptides γꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe were purchased from
Peptide Biological Technology Co., LTD (Nanjing, China). Commercial amino acids Gln and Phe
were purchased from CapitalBio Corporation (Shanghai, China). GlyꢀGly and
γꢀLꢀglutamylꢀpꢀnitroanilide (γꢀGpNA) were obtained from SigmaꢀAldrich (Shanghai, China).
Solvents and chemicals including acetonitrile and formic acid, were of HPLC grade and purchased
from CapitalBio Corporation (shanghai, China).
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Enzymatic synthesis of γꢀglutamyl peptides. Water solutions containing Gln and Phe (both at
200 mM, pH 10.0) were prepared in duplicate to which a glutaminase (GBA or GAO, final
concentration, 0.05 U/mL) was added to initiate the reaction. The mixtures were subjected to a 2ꢀh
incubation at 37 ºC and then heating at 90 ºC for 10 min (to inactivate the enzyme). The resultant
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reaction mixtures in capped containers were cooled in a water bath or in the air and stored in
refrigerator at 4 ºC before use (termed as “postꢀenzymatic reaction products”). In separate
experiments, the molar ratio of substrates and reaction time were altered to evaluate, their effects
on the yield of γꢀglutamyl peptides. The experiment was carried out in triplicate. The yield of
γꢀglutamyl peptides was determined using the following equation: Yield = The amount of a
specific γꢀglutamyl peptide / The initial amount of Phe (mM) ×100%.
Analysis of post-reaction products using UPLC–Q-TOF–MS/MS. The identification of the
γꢀglutamyl peptides containing phenylalanine residue at Cꢀterminus was conducted using an ultra
performance liquid chromatography coupled with quadrupole timeꢀofꢀflight mass spectrometry
(UPLC–QꢀTOFꢀMS/MS) system. UPLC separation was performed using an Agilent 1290 series
UPLC system (Agilent Technologies) with an Agilent ZORBAX RRHD SBꢀC18 column (2.1 mm
× 50 mm, 1.8 ꢁm; maintained at 30 ºC). The mobile phase consisted of solvents (A) water with
0.1% formic acid and (B) acetonitrile with 0.1% formic acid and eluted at a constant flow rate of
0.3 mL/min using the following gradient: A:B = 100:0; 0–5 min, A:B from 100:0 to 90:10; 5–10
min, A:B from 90:10 to 85:15; 10–12 min, A:B from 85:15 to 100:0. Injection volume was 5 ꢁL.
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MS detection for the parent and fragment ions was performed using a maXis Impact QꢀTOF
MS/MS system (Bruker Daltonics, Beijing, China) fitted with an electrospray ionization (ESI)
probe. The sourceꢀheated electrospray ionization (HꢀESI) mass spectrometer was operated in
positive ion mode with capillary voltage at 4500 V, capillary temperature at 180
ꢀ
C and nitrogen
as curtain and collision gas (0.3 bar). Mass spectra were acquired at the rate of 1 spectra/s over the
mass/charge (m/z) range of 50ꢀ1000. Quantitative calculation of each of the identified products
was conducted by comparing the peak area of the extracted ions chromatograph (EIC) with
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corresponding external calibration curves. Software DataAnalysis, version 4.1 was used for
identification and quantification of products.
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Measurement of transpeptidase activity. The transpeptidase activity of GBA, GAO and
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GAN was measured following a previous method with some modifications. Substrates γꢀGpNA
and GlyꢀGly and the enzyme of interest were mixed in a 50 mM borateꢀNaOH buffer solution (pH
10.0) before an incubation at 37 ºC for 30 min. The reaction was then terminated by adding 0.1 M
HCl. One unit (U) of enzyme was defined as the amount of the enzyme that released 1 ꢁmol of
pꢀnitroaniline per min from γꢀGpNA through the transpeptidation reaction (which was monitored
at 410 nm by a UVꢀvisible spectrophotometer (UV765, Shanghai Youke Instrument Co., Ltd.,
Shanghai, China)). The pH and temperature for transpeptidation of glutaminases used in this assay
were altered in separate experiments to investigate pH and temperature effects.
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Enzymatic kinetic analysis of GBA or GAO catalysis for the synthesis of γꢀglutamyl
peptides and hydrolysis of γꢀ[Glu]n-Phe. GBA or GAO catalyzes the cleavage of the γꢀglutamyl
linkage of Gln and the transfer of the γꢀglutamyl moiety to water (to release glutamate via
hydrolysis) or to other amino acids and peptides (to form γꢀglutamyl compounds via
transpeptidation). In this study, the Michaelis–Menten constant (K ) for GBAꢀ or GAOꢀcatalyzed
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synthesis of Pheꢀcontaining γꢀglutamyl peptides through transpeptidation using Gln and Phe as
substrates was determined under the following conditions: GBA or GAO, 0.0025 U/mL; substrate
(Phe, or γꢀ[Glu](n<5)ꢀPhe), 5ꢀ50 mM; Donor substrate (Gln, 50 mM (fixed)); pH 10.0, 37 ºC,
m
reaction time 30 min. In the meantime, the Michaelis–Menten constant (K ) for the hydrolysis of
γꢀ[Glu]nꢀPhe was also determined through measuring the decrease in the amount of γꢀ[Glu]nꢀPhe
over the concentration range of 5ꢀ50 mM under the following conditions: pH 10.0, 37 ºC, reaction
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time 30 min; GBA or GAO, 0.0025 U/mL.
Analysis of sensory characteristics of γꢀglutamyl peptides
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Sensory characteristics profiling. Fifteen panelists (8 males, 7 females, aged 30–40 years),
who did not have taste disorders and have been trained in similar sensory experiments at regular
intervals for at least 2 years, were chosen. Each of the commercial γꢀ[Glu]nꢀPhe products and each
of the single samples were reconstituted in water (5 mM), commercial soy sauce (2 mM) and
model chicken broth (2 mM). These γꢀglutamyl peptides are an acidity in solution for the more
than one carboxylic acid groups in their molecules. Therefore, the pH values of these solutions
were adjusted to 6.5 using sodium hydroxide solution (1.0 mM), and using a very small amount of
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diluted formic acid for the slightly excessive NaOH according to published methods.
Sensory
characteristics including mouthfulness, thickness, continuity, umaminess, saltiness, sourness,
astringency, and bitterness were evaluated in dual test using a 5ꢀpoint intensity scale (from 0, (not
detectable) to 5, (strongly detectable). Kokumi is a Japanese expression for complicated sensory
characteristics of food. Kokumi does not refer to an independent taste but encompasses thickness,
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continuity and mouthfulness with tasteꢀenhancing characteristics.
Each of these descriptors
was tested separately in the sensory room at 25 2 °C through placing an aliquot (10 mL) of each
ꢀ
sample in the mouth, swirling in the mouth for 10 s, swallowing at once and recording
corresponding scores based on the whole 25 s taste experience. The samples for evaluation were
placed in glass cups (20 mL) coded with threeꢀdigit numbers, covered with lid and served in a
rationalized order. Bottled water was used to rinse the mouth between tasting trials. Thickness
refers to rich complexity and was expressed as the increased taste intensity evaluated 10 s after
food tasting; Continuity refers to the unbroken and consistent existence to give longꢀlasting
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sensory effects or increased aftertaste, and was measured as persistent taste intensity 25 s after
food tasting; Mouthfulness describes neither taste nor aroma but an overall perception associated
with food structure, texture and morphological complexity, that is, food sensory reinforcement or
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increment of taste sensation throughout the whole mouth (not just on the tongue).
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Determination of taste recognition threshold concentrations for astringency and kokumi
activity. The threshold values of the γꢀ[Glu]nꢀPhe in water, commercial soy sauce and model
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chicken broth were measured using a threeꢀalternative forcedꢀchoice test. The effects of
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γꢀ[Glu]nꢀPhe concentrations were evaluated through doseꢀresponse experiments, in which
samples with elevated γꢀ[Glu]nꢀPhe concentrations were prepared by adding increasing amounts
of peptides (from 1 to 50 mmol/L) to solutions of model chicken broth, and presented in order of
increasing concentrations to the trained panel.
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Evaluation of taste-enhancing effect of post-enzymatic reaction products. The
postꢀenzymatic reaction products prepared in this study were added at a concentration of 2 mg/mL
to solutions of commercial soy sauce and model chicken broth, and subjected to panel evaluation
on the tasteꢀenhancing effects. Sensory characteristics including mouthfulness, thickness,
continuity and umami quality were evaluated using a 5ꢀpoint intensity scale (0, not detectable; 5,
strongly detectable).
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Statistical analysis. All experiments and analyses were conducted in triplicate, and the data
were collected and reported as “means ± standard deviation”. Analysis of variance and
significance of difference were performed using SPSS 16.0 statistical software (SPSS Inc.,
Chicago, USA). Duncan’s new multiple range test was performed to determine if the difference
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between data within 99% confidence level.
RESULTS AND DISCUSSION
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Transpeptidase activity of glutaminase. The transpeptidase activities of GBA and GAO were
detected with a specific activity of 100 and 10 U/g, respectively, at the optimal pH of 10.0 and
optimal temperature of 37 ºC. However, GAN exhibited no transpeptidase activity. These results
indicate that GBA and GAO possessed transpeptidase activity and could catalyze a γꢀglutamyl
transfer reaction to yield Pheꢀcontaining γꢀglutamyl peptides in the presence of Gln and Phe.
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Analysis of peptide products by UPLC-QTOF-MS/MS after catalysis by glutaminase.
γꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀPhe were both detected in the two postꢀreaction mixtures catalyzed by
GBA and GAO, whilst γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe and
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe were detected only in the postꢀreaction mixture catalyzed by
GBA. The parent and fragment ion m/z values, and likely peptide sequences in GBAꢀcatalyzed
postꢀreaction mixture were listed in Table 1. The molecular ion signals of m/z 295.1295, 424.1718,
553.2141, 682.2568 and 811.2996 in positive ESI mode were likely ionized γꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe
and
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, which are a series of γꢀ[Glu]
n
ꢀPhe short peptide chains with a
Cꢀterminal Phe residue. As shown in Table 1, fragmentation of γꢀGluꢀPhe led to the generation of
+
a series of fragment ions i.e. mainly [MꢀPheꢀCOOH] (m/z = 84.0446, bꢀCO
2
type ion),
+
+
2
[PheꢀCOOH] (m/z =120.0810, yꢀCO type ion), [Phe+H] (m/z = 166.0860, y type ion). The ion
fragments of γꢀ[Glu](1< n 5)ꢀPhe were y type and b1 type ions (bꢀCO /NH type ion). The number
2 3
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of y type ions was the same as that for the γꢀglutamyl residue in the molecule, with the m/z of y1,
y2, y3, y4 and y5 being 166.0863, 295.1283, 424.1709, 553.2134 and 682.2550, respectively. The
m/z values of the parent and fragment ions for the γꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀPhe in the
postꢀreaction mixture catalyzed by GAO were almost identical to those for GBA (data not shown).
These findings revealed that Pheꢀcontaining γꢀglutamyl peptides can be synthesized through a
γꢀglutamyl transfer reaction catalyzed by glutaminases with Gln and Phe as substrates, and
synthetic γꢀ[Glu](nꢀ1)ꢀPhe can be used as the acceptor to form the γꢀ[Glu]
n
ꢀPhe via the γꢀglutamyl
The mass spectrometer operated in positive mode employed extracted ion
chromatogram (EIC) for detection and quantitation of γꢀ[Glu]nꢀPhe products (Fig. 1). The
concentrations of the γꢀGluꢀPhe, γꢀ[Glu] ꢀPhe, γꢀ[Glu] ꢀPhe, γꢀ[Glu] ꢀPhe and γꢀ[Glu] ꢀPhe
23, 26
transfer reaction.
2
3
4
5
synthesized in the presence of GBA and GAO were 61.59 ± 2.54 mM and 20.31 ± 2.34 mM, 27.64
± 1.24 mM and 3.10 ± 0.82 mM, 14.09 ± 1.02 mM and “not detected”, 1.50 ± 0.34 mM and “not
detected”, and 0.13 ±0.04 mM and “not detected”, respectively. The yield of γꢀGluꢀPhe was higher
18
than that reported previously in the literature when the same type of GAO was used, whereas the
2
1
yield of γꢀGluꢀPhe catalysed by GBA was only 43% of that catalyzed by GGT. In both cases of
GBA and GAO, a lower yield was associated with the γꢀ[Glu]nꢀPhe product with a higher
molecular weight. γꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe in the postꢀreaction
mixture catalyzed by GBA accounted for 97.52% of the total γꢀ[Glu]nꢀPhe, whilst accounting for
11.70 % Phe and 13.56 % Gln in the case of GAO. The differences in the yield and type of
γꢀ[Glu]nꢀPhe products between GBA and GAO catalysis suggest that GAO seemed less efficient,
compared to GBA, in catalyzing the transfer of γꢀglutamyl moiety to a tripeptide or a higher
molecular weight peptide.
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Determination of the Michaelis–Menten constant (K ) of GBA and or GAO catalysis for
m
the synthesis of γꢀ[Glu]n-Phe and hydrolysis of γꢀ[Glu]n-Phe. A “PingꢀPong” mechanism might
be involved and led to the generation of a covalently bonded γꢀglutamyl acylꢀenzyme intermediate
involving substantial hydrogen bonding and charge interactions which could subsequently link to
34
Phe or existing peptides as acylꢀacceptor substrates to form a γꢀglutamyl peptides. The two
glutaminases, GBA and GAO, exhibited difference in affinity for acceptors (Phe and γꢀGluꢀPhe),
with GBA having a greater affinity than GAO for Phe and γꢀGluꢀPhe in the synthesis reaction
m
(Table 2). The K values for GBA against acceptor, Phe or γꢀ[Glu](n<5)ꢀPhe of GBA increased with
m
n value i.e. the lowest K value for Phe (n = 0, 47.88 mM) and the highest (206.47 mM) for
4
γꢀ[Glu] ꢀPhe. Thus, it became more difficult to synthesize γꢀ[Glu]nꢀPhe when the number of
γꢀglutamyl residues in the acceptor increased. Moreover, the K values (4.81 ~ 40.75 mM) for the
m
hydrolysis catalyzed by GBA were lower than that for the synthesis catalyzed by GBA. The
changing trend of K
m
values of GAO for the synthesis of γꢀ[Glu](n 2)ꢀPhe as compared to the K
m
≦
values for corresponding hydrolysis was similar to that of GBA. GAO could not catalyze the
transfer of γꢀglutamyl moiety to the tripeptide (γꢀ[Glu] ꢀPhe) or peptides with the number of
2
γꢀglutamyl residues greater than higher than 2 in the synthesis reaction (Table 2). These findings
suggest that GBA was superior to GAO in the synthesis of Pheꢀcontaining γꢀglutamyl peptides,
and the transfer of γꢀglutamyl moiety to a γꢀ[Glu]nꢀPhe would become more difficult with an
increase of number of γꢀglutamyl residues in the molecule.
2
2
2
49
50
51
Sensory characteristics of individual γꢀ[Glu]n-Phe. γꢀ[Glu]nꢀPhe in water (pH 6.5)
exhibited astringent sensation at 5 mM which was in agreement with the previous findings that
1
, 4, 5, 7
these kokumi compounds are either tasteless or astringent in water (Fig. 2).
When each of
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the γꢀ[Glu]nꢀPhe products was added at 2 mM to commercial soy sauce, enhanced continuity and
umami taste (p<0.05) were perceived. The increased taste intensity and longꢀlasting continuity, as
compared to the Control, caused by the addition of γꢀ[Glu]nꢀPhe to the commercial soy sauce were
further demonstrated in the inserted figure of Fig. 2. The enhancement of commercial soy sauce
with a continuity taste by γꢀ[Glu]nꢀPhe was similar to that caused by peptideꢀassociated Maillard
29,30
reaction in a umami solution.
Further, the addition of γꢀ[Glu]nꢀPhe at 2 mM to a model
chicken broth led to significant (p < 0.05) enhanced of mouthfulness, thickness and umami taste.
These findings were consistent with recent reports that γꢀglutamyl peptides such as γꢀGluꢀValꢀGly
3
,5,7
and γꢀGluꢀCysꢀGly, γꢀGluꢀCysꢀβꢀAla, γꢀGluꢀLeu, and γꢀGluꢀVal are kokumi flavor compounds.
In summary, γꢀ[Glu]nꢀPhe would exhibit varied flavor characteristics in different food. It is the
first time to describe the sensory characteristics of γꢀ[Glu](n>1)ꢀPhe in this article. γꢀ[Glu](n>1)ꢀPhe
compounds have similar sensory characteristics to that of γꢀGluꢀPhe, although their
tasteꢀenhancing scores were slightly lower than that of γꢀGluꢀPhe (Fig. 2) i.e. the taste score in
water and the tasteꢀenhancing scores in commercial soy sauce and model chicken broth decreased
progressively with an increase of the number of γꢀglutamyl residues. The taste effect of the
γꢀ[Glu]nꢀPhe would become weaker with an increased molecular weight. Accordingly, the type of
continuity, mouthfulness, thickness, and the enhancing umami taste of food taste perception were
defined as “kokumi” taste, thus all the γꢀ[Glu]nꢀPhe were confirmed as kokumiꢀactive peptides.
2
2
2
2
70
71
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Threshold concentrations and the taste intensity of γꢀ[Glu]n-Phe. The astringent and
kokumi taste threshold concentrations of the γꢀ[Glu]nꢀPhe were determined (Table 3).
γꢀ[Glu]nꢀPhe in water (pH 6.5) exhibited astringent sensation with corresponding threshold
concentrations being 2.5, 3.34, 3.58, 3.69 and 3.92 mM, respectively. In comparison, γꢀ[Glu]nꢀPhe
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90
in commercial soy sauce and model chicken broth (pH 6.5) only exhibited a kokumi flavour, with
no astringent sensation detected and corresponding threshold concentrations in the range of
0.78ꢀ1.42 mM. No significant difference (p<0.05) was detected in the threshold concentrations
between the γꢀ[Glu]nꢀPheꢀfortified commercial soy sauce and γꢀ[Glu]nꢀPheꢀfortified model
chicken broth, and the threshold concentrations increased progressively with an increased
molecular weight. The kokumi threshold concentrations of γꢀGluꢀPhe were approximately
5
twoꢀfold lower than that of γꢀ[Glu] ꢀPhe, which agreed with the results of their taste profile
analysis (i.e. the taste effect of these γꢀ[Glu]nꢀPhe weakened progressively with an increase of
molecular weight). Further, the threshold concentration of each γꢀ[Glu]nꢀPhe compound in the
commercial soy sauce and model chicken broth was significantly lower (p<0.05) than that in water.
γꢀGluꢀPhe had a high astringent taste in water (2.5 mM) but a significantly lower threshold value
in a commercial soy sauce or model chicken broth (0.93 and 0.78 mM, respectively). Such
differences between in water and in model chicken broth were in accordance with those of
3
γꢀGluꢀLeu, γꢀGluꢀVal γꢀGluꢀCysꢀβꢀAla and γꢀGluꢀCysꢀGly in water and chicken broth.
γꢀ[Glu]nꢀPhe only exhibited a kokumi taste at a low concentration, but an astringent sensation
would be perceived when its concentration was increased to almost threeꢀfold of the kokumi
threshold value.
2
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2
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92
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The doseꢀdependent effect of γꢀ[Glu]nꢀPhe on the model chicken broth was further evaluated
(Fig. 3). The highest tasteꢀenhancing score of the umaminess, thichness and mouthfulness (the so
called “kokumi” taste) intensity occurred at about 5 mM, above which these tastes sensation began
to decrease with an increased peptide dose. γꢀ[Glu]nꢀPhe at a concentration over 3 mM would also
induce an astringent sensation, besides different degrees of these “kokumi” sensory attributes.
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Thus, it was possible that the presence of astringency might suppress the kokumi sensation. A
29
similar change of flavor was also reported for the kokumiꢀactive leucyl dipeptides.
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99
00
01
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13
Optimization of the yield of γꢀ[Glu]n-Phe. This set of experiments only employed GBA as
catalyst. As shown by the reaction timeꢀcourse (Fig. 4A), the yields of γꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe
rapidly increased to 27.02, 9.01, 4.25, 1.12 and 0.043 %, respectively, after 1 h reaction, then
st
rd
increased at a lower rate to 32.09, 14.34, 7.76, 1.31, and 0.065 % respectively, from the 1 to 3 h
before leveling off. The yield of γꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe generally increased with an
elevated proportion of Glu, except for a valley value for all the γꢀ[Glu]nꢀPhe at Glu/Phe of 1.5 and
a dropꢀoff for γꢀ[Glu] ꢀPhe and γꢀ[Glu] ꢀPhe at Glu/Phe of 4.0 (Fig. 4B). The optimal yield of
4
5
γꢀ[Glu]nꢀPhe occurred at a Glu/Phe proportion of 3.0 (Glu:Phe = 300 mM:100 mM) i.e. 49.64%
for γꢀGluꢀPhe, 25.66% for γꢀGluꢀγꢀGluꢀPhe, 19.54% for γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, 3.75% for
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe and 0.13% for γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, with the
consumption of 58.40% Gln and 98.70% Phe. Therefore, the most suitable conditions for the
synthesis of γꢀ[Glu]nꢀPhe were likely 300 mM Gln, 100 mM Phe, 0.05 U/mL GBA at pH 10 and
37°C for 3 h. An excess of LꢀGln in the reaction mixture would benefit the formation of
γꢀglutamylꢀenzyme intermediate and facilitate the subsequent transpeptidation reaction.
3
3
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Effect of the post-reaction mixture under optimal conditions on the sensory
characteristics of commercial soy sauce and model chicken broth. Under the aboveꢀmentioned
optimal conditions, 98.70% Phe was consumed as an acceptor to synthesize γꢀ[Glu]nꢀPhe, the
bitterness was found to be reduced and even disappeared whilst kokumi taste was present.
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Subsequently, the lyophilized postꢀenzymatic reaction mixture (300 mM Gln/100 mM Phe as the
substrates) was added at a concentration of 2 mg/mL (mainly including 1.6 mM γꢀGluꢀPhe, 0.8
mM γꢀGluꢀγꢀGluꢀPhe, 0.6 mM γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe) to commercial soy sauce and model
chicken broth for sensory evaluation. The greatest change in score brought by adding
postꢀenzymatic reaction mixture was found for the intensity of continuity (2.23→3.03), followed
by umami taste (3.72→4.21) in the case of commercial soy sauce. Enhancement of mouthfulness
(1.99→2.74), thickness (2.55→3.24) and umami taste (3.69→4.02) was perceived in model
chicken broth. These results were similar to the findings associated with commercial γꢀ[Glu]nꢀPhe
products, suggesting that both γꢀ[Glu]nꢀPhe and the postꢀenzymatic reaction mixture can be used
as food additives in commercial soy sauce and model chicken broth. Glutaminases are known to
be used in the fermentation process of soy sauce to improve the content of glutamate. It is possible
that kokumiꢀactive γꢀglutamyl peptides would be synthesized by the glutaminase during the
production of soy sauce. The findings that γꢀ[Glu]nꢀPhe exhibited only sourness and astringent
sensation in water but could impart a kokumi taste at a low concentration to commercial soy sauce
and model chicken broth, along with the obtained sensory information on the series kokumi
γꢀ[Glu]nꢀPhe synthesized by GBA or GAO, are undoubtedly useful to the food industry.
3
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39
In this study, the transpeptidase activity of GBA and GAO to yield γꢀ[Glu]nꢀPhe peptides were
verified and the Pheꢀcontaining γꢀglutamyl peptides were synthesized for the first time using GBA
and GAO as the catalysts. The identification of γꢀGluꢀPhe and γꢀGluꢀγꢀGluꢀPhe in the
postꢀreaction mixture catalyzed by GAO, and γꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe,
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe in the postꢀreaction mixture
m m
catalyzed by GBA, along with the lower K values for the hydrolysis by GBA than the K values
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for synthesis, indicates the feasibility for industrial production of these Pheꢀcontaining γꢀglutamyl
peptides. GBA was more suitable for the synthesis of Pheꢀcontaining γꢀglutamyl peptides than
m m
GAO based on their respective K values for the transpeptidation reaction. The fact that the K
values of GBAꢀcatalyzed transpeptidation reaction against acceptors (Phe and γꢀ[Glu](n>1)ꢀPhe)
increased with an elevated number of γꢀglutamyl residues, further suggest the greater difficulty to
synthesize γꢀ[Glu]nꢀPhe with high molecular weights.
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ACKNOWLEDGEMENTS
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The authors are grateful for the financial support by Open Funding Project of the State Key
Laboratory of Biochemical Engineering (No. 2015KFꢀ04), the Science and technology Plan
Project of Guangzhou (No. 201604020067), the Fundamental Research Funds for the Central
Universities (2017ZD093) and the Science and technology plan project of Guangdong Province
(No. 2017A010105002).
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occurring flavor enhancers. Journal of Molecular Catalysis B: Enzymatic, 2012, 84, 65ꢀ71.
(21) Suzuki, H.; Kajimoto, Y.; Kumagai, H. Improvement of the Bitter Taste of Amino Acids
through the Transpeptidation Reaction of Bacterial γꢀGlutamyltranspeptidase. Journal of
Agricultural and Food Chemistry, 2002, 50, 313ꢀ318.
(22) Suzuki, H.; Izuka, S.; Miyakawa, N.; Kumagai, H. Enzymatic production of theanine, an
“umami” component of tea, from glutamine and ethylamine with bacterial
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(23) Suzuki, H.; Miyakawa, N.; Kumagai, H. Enzymatic production of γꢀLꢀglutamyltaurine
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(24) Suzuki, H.; Kato, K.; Kumagai, H. Enzymatic Synthesis of γꢀGlutamylvaline to Improve the
Bitter Taste of Valine. Journal of Agricultural and Food Chemistry, 2004, 52, 577ꢀ580.
(25) Suzuki, H.; Yamada, C.; Kato, K. γꢀGlutamyl compounds and their enzymatic production
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transpeptidase and autotranspeptidase activities of Bacillus subtilis γꢀglutamyltransferase.
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quantification of thymol metabolites in plasma, liver and duodenal wall of broiler chickens
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γꢀGlutamyltranspeptidase from Bacillus subtilis SK11.004. Journal Agricultural of Food
Chemistry, 2011, 59, 6233ꢀ6238.
(29) Liu, J. B.; Song, H. L.; Liu, Y.; Li, P.; Yao, J.; Xiong, J. Discovery of kokumi peptide from
yeast extract by LCꢀQꢀTOFꢀMS/MS and sensomics approach. Journal of the Science of Food
and Agriculture, 2015, 95, 3183ꢀ3194.
(30) Ogasawara, M.; Katsumata, T.; Egi, M. Taste properties of maillardꢀreaction products
prepared from 1000 to 5000 Da peptide. Food Chemistry, 2006, 99(3), 600ꢀ604.
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(32) Glabasnia, A.; Hofmann, T. Sensoryꢀdirected identification of tasteꢀactive ellagitannins in
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properties, and biotechnological applications. Cellular & Molecular Life Sciences, 2012, 69,
3381–3394.
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Table 1. The peptides identified and corresponding m/z values of parent and fragment ions in postꢀenzymatic reaction mixture
catalyzed by glutaminase of B. Amyloliquefaciens (GBA)
Parent
ion (m/z;
Proposed peptides
Fragment ions (m/z)
+
[
M+H] )
+
+
+
2
4
95.1295
24.1718
γꢀGluꢀPhe
84.0446 [MꢀPheꢀCOOH] , 120.0810 [PheꢀCOOH] , 166.0860 [Phe+H]
+
8
4.0446, 120.0808, 166.0861, 232.0961 [M−γꢀglutamyl residue−NH
3 2
−CO +H] ,
γꢀGluꢀγꢀGluꢀPhe
+
+
2
78.1024 [M−γꢀglutamyl residue−NH
3
+H] , 295.1285 [M−γꢀglutamyl residue+H]
+
5
6
53.2141
82.2568
γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe
γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀG
luꢀPhe
84.0443,166.0859, 232.0964, 295.1277, 424.1692 [M−γꢀglutamyl residue+H] ,
+
130.0497 [GlnꢀNH
3
+H] , 166.0862, 232.0964, 295.1288, 424.1714, 553.2133
8
11.2996
130.0493 , 166.0863, 232.0964, 295.1283, 424.1709, 553.2134, 682.2550
Reaction conditions: 200 mM Gln, 200 mM Phe, pH 10, 37 °C and 2 h.Data obtained by ESIꢀQꢀTOFꢀMS/MS in positive mode.
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Table 2. The Km value (mM) of GBA and GAO for the synthesis and
hydrolysis of γꢀ[Glu]nꢀPhe (n=0ꢀ5)
Synthesis
GAO
47.88±0.47 153.92±5.47
Hydrolysis
GBA
GBA
ꢀ
GAO
LꢀPhe
ꢀ
γꢀGluꢀPhe
84.89±1.02 236.47±21.95 24.81±1.02
79.11±5.05
γꢀ[Glu]
γꢀ[Glu]
γꢀ[Glu]
γꢀ[Glu]
2
3
4
5
ꢀPhe 95.23±7.05
ꢀPhe 126.47±10.05
ꢀPhe 206.47±13.05
ꢀ
ꢀ
ꢀ
ꢀ
30.73±2.05
56.42±2.05
70.79±3.86
80.75±3.11
99.76±8.01
ꢀ
ꢀ
ꢀ
ꢀPhe
ꢀ
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Table 3. Taste threshold concentrations of the Pheꢀcontaining
γꢀglutamyl peptides
Taste threshold concentration (m mol/L) in
γꢀ[Glu]
n
ꢀPhe
Commercial
Model chicken
a
Water
b
b
soy sauce
broth
#
γꢀGluꢀPhe
2.5
0.89±0.064
1.01±0.047
1.22±0.053
1.34±0.037
1.42±0.093
0.78±0.052
0.96±0.054
1.10±0.020
1.25±0.032
1.33±0.026
γꢀ[Glu]
γꢀ[Glu]
γꢀ[Glu]
γꢀ[Glu]
2
3
4
5
ꢀPhe
ꢀPhe
ꢀPhe
ꢀPhe
3.34±0.097
3.58±0.27
3.69±0.21
3.92±0.12
a: astringency; b: kokumi described as continuity and enhanced
umami in commercial soy sauce; mouthfulness and thickness in
1
5
model chicken broth. #: a value obtained from literature . The
pH of all the solutions was adjusted to 6.5.
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Figure Captions
4
4
4
55
56
57
Fig. 1. Extracted ion chromatogram (EIC) of the Pheꢀcontaining γꢀglutamy peptides in the
postꢀenzymatic reaction mixture by GBA. A: γꢀGluꢀPhe, B: γꢀGluꢀγꢀGluꢀPhe, C:
γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, D: γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe, E: γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe.
4
4
4
58
59
60
Fig. 2. Taste profile analysis of γꢀ[Glu]nꢀPhe. All the γꢀ[Glu]nꢀPhe were dissolved in water,
commercial soy sauce (CSS), or model chicken broth (MCB), the pH is adjusted to 6.5. Inserted
Figure about the intensity of aftertaste as a function of continuity time in commercial soy sauce.
4
4
4
61
62
63
Fig. 3. The doseꢀdependent effect of the γꢀ[Glu]nꢀPhe (“ ”, “ ”, “ ”, “ ”, “ ” represented
2 3 4 5
γꢀGluꢀPhe, γꢀ[Glu] ꢀPhe, γꢀ[Glu] ꢀPhe, γꢀ[Glu] ꢀPhe, γꢀ[Glu] ꢀPhe respectively) on the umaminess,
thickness, mouthfulness and astringency sensations of model chicken broth.
4
4
4
4
64
65
66
67
Fig. 4. Optimization of the yield of γꢀ[Glu]nꢀPhe: (A), Effect of the reaction time on the synthesis
of γꢀglutamylation of Phe (200 mM Phe, 200 mM Gln, 0.05 U/mL glutaminase; pH 10, and 37 °C);
(B), Effect of the substrate molar concentration ratio (Gln/Phe) on the γꢀglutamylation of
phenylalanine (0.05 U/mL glutaminase; pH 10; 37 °C; 3 h).
4
4
68
69
Fig. 5. Tasteꢀmodulatory activity of the postꢀenzymatic reaction mixture obtained under optimal
conditions in commercial soy sauce (CSS) and model chicken broth (MCB).
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A: γꢀGluꢀPhe m/z=295.0000
B: γꢀGluꢀγꢀGluꢀPhe m/z=424.0000
Intens.
x10
Intens.
x10
1.0
7
7
1
1
0
0
0
0
.25
.00
.75
.50
.25
.00
0.8
0.6
0.4
0.2
0.0
2.5
5.0
7.5
10
2.5
5.0
7.5
10
C:γꢀGluꢀγꢀGluꢀγꢀGluꢀPhe m/z=553.0000
D:γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe m/z=682.0000
Intens.
x10
Intens.
x10
6
6
1
.25
.00
3
1
2
1
0
0.75
0
.50
.25
0
0.00
2.5
5.0
7.5
10
2.5
5.0
7.5
10
E:γꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀγꢀGluꢀPhe m/z=822.000
Intens.
5
x10
2
1
0
2.5
5.0
7.5
10
Retention time (min)
4
4
71
72
Fig. 1.
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Fig. 2.
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Fig. 3.
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