residues. This study examined the feasibility of the use of the transglutaminase reaction for fortifying food proteins bythe covalent attachment of limiting essential ...
Agric.
Biol.
Chem.,
45 (ll),
2587~2592,
1981
2587
Incorporation of AminoAcids into Food Proteins
by Transglutaminase*
Koji Ikura, Masaaki Yoshikawa, Ryuzo Sasaki and Hideo Chiba
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received May 25, 1981 Transglutaminase
catalyzes
the formation
of e-(y-glutamyl)lysine
crosslinks
and the sub-
stitution of a variety of primary amines for the y-carboxamide groups of protein-bound glutaminyl residues.
This
study examined the feasibility
of the use of the transglutaminase
reaction
for
fortifying food proteins by the covalent attachment of limiting essential amino acids. In the reactions using L-methionineethyl ester as a substrate, the methionine content of asland /J-caseins, and soybean 7S and 1 IS proteins increased to 200, 150, 240 and 350% of the starting materials, respectively. With wheat gluten, incorporation of L-lysine was tested and a 5.1-fold increase in lysine content was observed. These results suggest that transglutaminase could be a useful tool for improving the amino acid composition of food proteins.
Transglutaminase (glutaminyl-peptide yglutamyltransferase, i?-glutaminyl-peptide: amine y-glutamyl-transferase, EC 2.3.2.13)
catalyzes a calcium-dependent acyl-tranfer
re-
action in which the y-carboxamide groups of peptide-bound glutaminyl residues are the acyl donors. Primary amino groups in a variety of compoundsmayact as acyl acceptors with the
subsequent formation of monosubstituted yamides of peptide-bound glutamic acid, eAmino groups of the appropriate lysyl residues in proteins can also serve as substrates, generating intra- or intermolecular e-(y-
glutamyl) lysine crosslinks,
which are isopep-
epidermal keratinocytes,4)
stiffening of the ery-
throcyte membrane,5* wound healing,6) and receptor-mediated endocytosis of some proteins and polypeptide hormones.7) The biological importance of transglutaminases has been the subject of a recent review.8* The ability of transglutaminase to mediate the attachment of chemical labels or probes to specific proteins, and to introduce crosslinks
between them, has already been used for biochemical
studies
concerning the structure
and organization of various membraneproteins.9~13) Recently, a transglutaminasemediated
procedure
for the photochemical
tide bonds (for reviews, see refs. 1 and 2). Transglutaminases are widely distributed in
labeling of peptides and for the production of cleavable crosslinks between protein molecules nature, and several of them have been shown have also been reported.14* It has become very important, because of or suggested to perform biologically impor-
tant functions by catalyzing the formation of the exploding world population, to develop e-(y-glutamyl) lysine crosslinks with protein substrates. These functions include stabilization of the fibrin structure during hemo-
new methods for the effective utilization of food proteins to overcome the crisis due to food protein deficiency. Modifying proteins
stasis,3) formation of a cornifying envelope in
with enzymes is promising as a method of
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
improving the functional
properties
and nu-
tritive value of currently available food proteins,15 ~18) Wehave demonstrated, in previous
2588
K. Ikura,
M. Yoshdcawa, R. Sasaki and H. Chiba
papers, that transglutaminase can be a useful tool for polymerizing food proteins through the formation of intermolecular crosslinks.19'20) These polymerized proteins are expected to be good materials for textured products. The aim of this work was to investigate the feasibility of the use of a transglutaminase reaction for improving the amino acid composition of food proteins by the covalent attachment of limiting essential amino acids. We attempted the incorporation of methionine into bovine casein components and soybean proteins, and of lysine into wheat gluten using the guinea pig liver enzyme. MATERIALS AND METHODS
Transglutaminase reaction for the incorporation of an amino acid into proteins. The reaction mixture contained, in a total volume of 15ml, 0.1 m Tris-HCl buffer (pH 7.5), l ~5mMCaCl2, 10mM dithiothreitol, 1.5~ 10mg/ml substrate protein, 20 ~ 68 him amino acid (L-methionine ethyl ester
or L-lysine),
and 23~92/zg/ml
transglutaminase.
Incubation was performed at 37°C, and 2.7ml aliquots were taken at intervals. The reaction was terminated by adding 0.27ml of0.4m EDTA(pH 8.0). The control zerotime product was prepared by mixing the reaction mixture with EDTAbefore addition of the enzyme. In the case of the reaction using wheat gluten as substrate, the reaction vessel was shaken continuously to disperse the insoluble gluten aggregates. With the terminated reaction mixtures, released ammonia was measured to determine the progress of the transglutaminase reaction and SDSpolyacrylamide gel electrophoresis was carried out to detect the product polymerized through the intermolec-
ular cross-linking catalyzed by the transglutaminase.
For
determination of the amino acid composition of the product, the terminated reaction mixtures were dialyzed Transglutaminase. Transglutaminase was purified from extensively against 10mM potassium phosphate buffer fresh guinea pig liver by the method of Connellan et al.21) (pH 7.6).
During the purification, enzyme activity was determined by a colorimetric hydroxamate procedure using an Determination of ammonia. Ammoniareleased during ethanol extract of a tryptic digest of casein as substrate.22) The prepared enzyme exhibited 95% of the reported the transglutaminase reaction was determined enzymically by the method described previously.19) specific activity upon assay by hydroxamate formation with 7V-carbobenzoxy-L-glutaminylglycine,23) and gave a SDS-polyacrylamide gel electrophoresis. Polyacrylsingle band on SDS*-polyacrylamide gel electrophoresis. amide gel electrophoresis in SDS was carried out on a This sample exhibited no proteolytic activity. The enzyme slab gel using the SDS-Tris-glycine discontinuous bufwas stored at -80°C in 0.4ml aliquots. An extinction fered system described by Laemmli.28) The gel was stained coefficient of E\%° Qnm=15.821) was used to determine with 0.1% Coomassie brilliant blue in 50% trichlorothe enzyme concentration.
acetic acid, and destained in 7% acetic acid.
Substrate food proteins. asl- and /^-Caseins were prepared from and individual Holstein cow's milk by theDetermination ofamino acid composition. A sample was hydrolyzed with 6n HC1 in a deaerated tube at 110°C for methods of Zittle et al.24'25) and Hipp et al.,26) respec24hr. The hydrolysate wasanalyzed for aminoacids with tively. Crude soybean IS and 1 1S proteins were prepara Hitachi KLA-5 apparatus. ed from defatted soybean flour, a gift from Fuji Oil Co., Ltd.* by the method of Thanh and Shibasaki.27) The prepared caseins and soybean proteins were dialyzed RESULTS AND DISCUSSION against distilled water, and lyophilized. Wheat gluten was purchased from Nakarai Chemicals, Ltd.
Stability of transglutaminase
Chemicals. L-Methionine ethyl ester hydrochloride and Stability of the transglutaminase activity, unbovine liver L-glutamate dehydrogenase (Type II) were der conditions simulating those used for the obtained from Sigma Chemical Co. L-Lysine of guaranteed reagent grade was from Nakarai Chemicals, Ltd.amino acid-incorporating reaction, was examined. Figure 1 shows that about 70% of the A tryptic digest of casein (N-Z-Amine Type E) was from initial activity remained after 4hr of incuHumko Sheffield. 7V-Carbobenzoxy-L-glutaminylglycine was from Vega Biochemicals. Other reagents of guaranbation. Both in the absence and in the presteed reagent grade were purchased from wako Pure ence of Ca2+, an essential co factor of the Chemical Industries, Ltd. and Nakarai Chemicals, Ltd. transglutaminase reaction, enzyme activity
SDS, sodium dodecyl sulfate.
was lost at the same rate, which indicates that the observed inactivation was not due to progress of the transglutaminase reaction using
2589
Incorporation of AminoAcid into Protein by Transglutaminase
'0
Time(hr) Fig. 1. Stability of Transglutaminase. The incubation mixture contained 0. 18 mg/ml transglutaminase, 10mM dithiothreitol, and 5mM CaCl2 (#) or no CaCl2 (O) in 0. 1 m Tris-HCl buffer (pH 7.5). The temperature was 37°C. At various times, an aliquot was removed to determine transglutaminase activity. A colorimetric hydroxamate procedure22) was used as the enzyme assay system. Percent activity was based on the activity observed before the start of incubation.
80
120
40
Time(min) Fig. 3. Incorporation of Methionine into Caseins and Soybean Proteins. Samples were obtained from the reactions shown in Fig. 2. The determination of amino acid composition is described in Materials and Methods.
(#) asl-Casein; protein.
(O) ^-casein;
(A) 7S protein;
(A) US
Relative content of methionine was based on the amount of methionine obtained with each starting material (asl=2.7,
j3=2.9,
Incorporation
7S=1.1,
ratios
and
11S=1.O
[(incorporated
methionine
mol%).
ethyl
ester/added methionine ethyl ester) x 100] obtained after the incubation for 120 min were 2.1% for asl, 1.2% for /?, 1.1% for IS, and 1.0% for US.
the enzymeprotein as substrate.
Incorporation of methionine into bovine casein componentsand soybeanproteins Methionine
80 120
40
Time(min) Fig. 2. Transglutaminase Methionine Incorporation Proteins.
Reaction in the System for into Caseins and Soybean
(à") asl- and ( O) jS-Caseins; the reaction mixture contained 5 mg/ml casein, 10mMdithiothreitol, 5mMCaCl2, 50mM L-methionine ethyl ester, and 23 jug/ml transglutaminase in 0.1 m Tris-HCl buffer (pH 7.5). (A) Soybean IS protein; the reaction mixture contained 5 mg/ml
IS
protein,
10mM dithiothreitol,
5mM CaCl2,
68 mML-methionine ethyl ester, and 92 /ig/ml transglutaminase in 0.1 m Tris-HCl buffer (pH 7.5). (A) Soybean US protein; the reaction mixture contained 1.5 mg/ml 115 protein, 10mM dithiothreitol, 1 mMCaCl2, 68 mML-methionine ethyl ester, and 92 /xg/ml transglutaminase in 0.1 M Tris-HCl buffer (pH 7.5). Other conditions of the reaction and the determination of ammonia were described in Materials and Methods.
is the limiting
essential
amino
å acid in milk and soybean proteins. Since the presence of a-carboxyl groups in free amino acids prevent their a-amino groups from being acyl-acceptor
substrates
in the transgluta-
minase reaction,1'29* L-methionine ethyl ester was used as substrate in the reaction for
incorporation of methionine into bovine ca-
sein components and soybean proteins. The progress of each transglutaminase reaction was followed by determining the ammonia released during the reaction and it was confirmed that all of the tested proteins could serve as a substrate (Fig. 2). The ammonia released during the incubation comes from two reactions catalyzed by transglutaminase;
the incorporation of methionine into proteins, and the formation of inter- or intramolecular
M. Yoshikawa, R. Sasaki and H. Chiba
B
K. Ikura,
å å »w
D
2590
»å¼å
Fig. 4. SDS-Polyacrylamide Gel Electrophoresis
Patterns of Caseins and Soybean Proteins during
Methionine Incorporation Catalyzed by Transglutaminase. Samples were obtained from the reactions shownin Fig. 2. 15^g of protein was applied to each lane. Electrophoresis was carried out in a 3% stacking gel and a 12% separating gel at constant voltages (40V for 1.5hr, and 120V for the next 5.0hr). Migration is from top to bottom.
(A) asl-casein;
(B) jS-casein; (C) 75 protein; (D) 115 protein.
Samples on each gel are, from left, 0, 5, 10, 60, and 120 min products. e-(y-glutamyl)
lysine
crosslinks
between pro-
teins. Therefore, in order to determine the
amount of methionine incorporated into the
protein, amino acid analysis was carried out on the protein products obtained from the reaction system at intervals. Figure 3 shows that, with increasing incubation time, the methionine content of each protein product increased. After 120 min, the methionine content of asl-casein was 2.0 times that of its starting material and for /?-casein, IS protein, and 1 1S protein, the values were 1.5, 2.4, and 3.5 times those of their starting materials, respectively. To detect the formation of intermolecular crosslinks, changes in the molecular size of
protein products during the transglutaminase reaction
were analyzed
by SDS-polyacryl-
amide gel electrophoresis. For all of the proteins, it can be seen that with increasing incubation time there is a progressive disappearance of the bands detected in the starting
material (Fig. 4). Concurrently new protein
bands, somewith slower electrophoretic mobilities and someunable to enter the separating gel, appeared, indicating that in spite of the presence of a high concentration of a primary amine (methionine ethyl ester), polymerization
of peptide chains occurred through intermolecular crosslinking. Compared with soybean 75 and 1 15 proteins, asl- and /?-caseins exhibited higher substrate effectiveness for the polymerization
through
intermolecular
cross-
linking.
The higher effectiveness of soybean proteins as substrates in the methionine-incoporation reaction, and the higher reactivity of casein in
the intermolecular crosslinking reaction, may be attributed to their conformational features. The unfolded structure of caseins30)
would make them more available for intermolecular cross-linking, while soybean proteins, because of their relatively rigid structure,31* would resist intermolecular crosslinking, which would favor the incorporation of methionine.
Incorporation of lysine into wheat gluten Lysine is the limiting essential amino acid in wheat gluten, and therefore lysine fortification
was attempted using transglutaminase. The transglutaminase reaction was carried out with continuous shaking, because gluten is almost completely insoluble in this reaction system. Gluten exhibited high substrate effec-
2591
Incorporation of AminoAcid into Protein by Transglutaminase
Fig. 5.
Transglutaminase
Timean(min) Reaction
in the System
for
Lysine Incorporation into Gluten. The reaction mixture contained 10 mg/ml gluten, lOmM Fig. 7. SDS-Polyacrylamide Gel Electrophoresis dithiothreitol, 5 mMCaCl2, 20mML-lysine, and 61 fig/ml transglutaminase in 0.1 m Tris-HCl buffer (pH 7.5). Other Patterns of Gluten during Lysine Incorporation Catalyzed conditions of the reaction and the determination of am- by Transglutaminase. monia were described in Materials and Methods. Samples were obtained from the reaction shown in Fig. 5.
About 10/ig of protein was applied to each lane. Conditions for electrophoresis were the same as described in the legend to Fig. 4. Samples on the gel are, from left, 0, 10, 60, and 150 min products.
substrate for lysine incorporation catalyzed by transglutaminase.
This might
be due to the
high glutamine content of gluten, which is the 120
80
Time(min)
site of lysine attachment. These results suggest that the transgluta-
minase reaction can be used for improving the
Fig. 6. Incorporation of Lysine into Gluten. Samples were obtained from the reaction shownin Fig. 5. The determination of amino acid composition is described in Materials and Methods. Relative content of lysine
amino acid composition of food proteins through the covalent attachment of limiting essential amino acids. Determination of the optimal conditions for amino acid incorpo-
was based on the amount of lysine obtained with the starting material (1.5 mol%). Incorporation ratio [(in-
ration into evaluation proteins by oftransglutaminase, and nutritional the new proteins,
corporated lysine/added lysine) x 100] obtained after the incubation
for 150 min was 32%.
tiveness, comparable to that of asl-casein, for
the transglutaminase reaction measured by ammonia release (Figs. 2 and 5). Figure 6 shows that the lysine content of gluten in-
remain to be done. Methionine
incorporated into food proteins by the action of transglutaminase appear to be biologically available from the following observations: (i) £-(L-y-Glutamyl)-L-lysine
can replace
lysine
in
supporting the growth of rats and chicks.32'33) (i )
Two enzymes, y-glutamylamino
creased with increasing incubation time and clotransferase34 clotransferase,36) by 150min the content was 5.1 times that of the starting material. The polymerization of gluten through intermolecular crosslinking was also observed (Fig. 7). Although gluten is almost completely insoluble in the reaction system, it is a good
and lysine
acid
cy-
~36) and y-glutamylamine cyhave been found in kidney
and other mammaliantissues. The former can catalyze the conversion of L-y-glutamyl-Lmethionine into L-methionine and 5-oxo-lproline, and the latter, £-(L-y-glutamyl)-L-
lysine into L-lysine and 5-oxo-L-proline.
2592
K. Ikura,
M. Yoshikawa, R. Sasaki and H. Chiba
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