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1.
Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins such as tofu, boiled fish paste, and sausage. Other transglutaminases (TGases) are expected to be used in the same way, and also to extend the scope of industrial applications to materials, drugs, and so on. The TGases have great diversity, not only in amino acid sequence and size, but also in their substrate specificities and catalytic activities, and therefore, it is quite difficult to estimate their reactivity. We have developed an NMR-based method using the enzymatic labeling technique (ELT) for simultaneous analysis of the substrate specificities and reaction rates of TGases. It is quite useful for comparing the existing TGases and for screening new TGases or TGases variants. This method has shown that MTG is superior for industrial use because of its lower substrate specificity compared with those of guinea pig liver transglutaminase (GTG) and red sea bream liver transglutaminase (FTG). We have also found that an MTG variant lacking an N-terminal aspartic acid residue has higher activity than that of the native enzyme.  相似文献   

2.
Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.  相似文献   

3.
Conjugation of the milk protein sodium caseinate and a protein-containing polysaccharide, gum arabic, was achieved through the use of the cross-linking enzyme transglutaminase. The extent of conjugation was monitored by size exclusion separation coupled with a multiangle laser light scattering detector. The elution times of gum arabic solutions incubated with transglutaminase were unchanged over time, whereas incubation of sodium caseinate with transglutaminase resulted in shorter elution times as reaction time increased, indicating the formation of cross-linked caseinate polymers. However, when mixtures of caseinate and gum arabic were incubated with transglutaminase, the elution times were decreased markedly, indicating conjugation between the protein and polysaccharide. The molecular masses of the conjugates increased from approximately 950 to 1600 kDa. This method of protein-polysaccharide conjugation offers noticeable advantages over previously used methods, and the conjugates produced may exhibit unique functional properties.  相似文献   

4.
Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.  相似文献   

5.
The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.  相似文献   

6.
The ability of phaseolin to act as an acyl donor and acceptor substrate of transglutaminase was studied by using an enzyme isolated from Streptoverticillium mobarense. Phaseolin, a trimeric storage protein from Phaseolus vulgaris L., was shown to possess both glutamine and lysine residues reactive for the enzyme. The extent of transglutaminase-catalyzed cross-linking has been studied in function of both incubation time and enzyme concentration. Native- and SDS-PAGE demonstrated that phaseolin is intra- and intermolecularly cross-linked by transglutaminase and gives rise to different polymers as well as to modified forms of the protein having a similar molecular weight but lower Stokes radius if compared to unmodified phaseolin. Cross-linked phaseolin was found to be more resistant to proteolytic cleavage than the unmodified counterpart, as demonstrated by in vitro trypsin and pepsin digestion experiments. This behavior could suggest novel possible uses of the transglutaminase-modified phaseolin.  相似文献   

7.
A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using <10 mg of the same enzyme, illustrating the capacity of immobilized enzymes to be used more frequently in applications of this nature.  相似文献   

8.
Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.  相似文献   

9.
The feeding of ruminant proteins to ruminants is prohibited in most countries because the practice is thought to be responsible for the spread of bovine spongiform encephalopathy. However, currently available methods to detect ruminant blood products in rendered feedstuffs are inadequate because they lack species specificity, tissue specificity, and are not based on a thermostable analyte. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for this study that provides reliable and sensitive (0.05-0.5% v/v) detection of bovine blood materials in animal feed. The new sandwich ELISA employs two previously developed monoclonal antibodies (MAbs), Bb6G12 as the capture antibody and biotinylated MAb Bb3D6 as the detecting antibody, and is bovine-specific and blood-specific. The assay is based on the detection of a 60 kDa thermostable protein in bovine blood and provides a useful regulatory tool for monitoring fraudulent labeling or contamination of bovine blood in both heat-processed feedstuffs and unprocessed raw materials. Keywords: Bovine; blood; monoclonal antibody; sandwich ELISA.  相似文献   

10.
Improvement in the water stability and other related functional properties of thin (<50 μm) kafirin protein films was investigated. Thin conventional kafirin films and kafirin microparticle films were prepared by casting in acetic acid solution. Thin kafirin films cast from microparticles were more stable in water than conventional cast kafirin films. Treatment of kafirin microparticles with heat and transglutaminase resulted in slightly thicker films with reduced tensile strength. In contrast, glutaraldehyde treatment resulted in up to a 43% increase in film tensile strength. The films prepared from microparticles treated with glutaraldehyde were quite stable in ambient temperature water, despite the loss of plasticizer. This was probably due to the formation of covalent cross-linking between free amino groups of the kafirin polypeptides and carbonyl groups of the aldehyde. Thus, such thin glutaraldehyde-treated kafirin microparticle films appear to have good potential for use as biomaterials in aqueous applications.  相似文献   

11.
Peanut protein isolate (PPI) was treated by high-pressure microfluidization (40, 80, 120, and 160 MPa) and/or transglutaminase (TGase) cross-linking. It was found that individual microfluidization at 120 MPa was more effective in improving the solubility, emulsifying properties, and surface hydrophobicity of PPI than at other pressures (e.g., 40, 80, or 160 MPa). Individual TGase cross-linking also effectively changed the physicochemical and functional properties of PPI. Microfluidization (120 MPa) or TGase cross-linking caused the unfolding of PPI structure, resulting in the decrease of α-helix and β-turns levels and the increase of β-sheet and random coil levels, as proved by Fourier transform infrared (FTIR) and circular dichroism (CD) spectra. Compared with individual treatments, microfluidization followed by TGase cross-linking significantly (p < 0.05) improved the emulsion stability during long-term storage (20 days). Moreover, the combined treatments led to looser structure of PPI and resulted in more obvious changes in physicochemical properties.  相似文献   

12.
Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.  相似文献   

13.
Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.  相似文献   

14.
To investigate the site specificity of two transglutaminases (TGases), that is, the enzymes from guinea pig liver (GTGase) and Streptoverticillium (MTGase), the acyl acceptor and donor sites in alpha-lactalbumin were determined. Alpha-lactalbumin was cross-linked in the presence of dithiothreitol by GTGase and MTGase for 15 and 30 min, respectively. Cross-linked alpha-lactalbumins by GTGase and MTGase were digested with lysylendopeptidase followed by the separation of the resulting peptides using reverse-phase HPLC. By the sequence analysis of the peptide fragments containing two N termini, which indicates the presence of cross-linked peptide, the lysine residues targeted by TGases were identified as follows: for GTGase, Lys16, Lys93, and Lys122; for MTGase, Lys5. These peptide fragments were further digested by V8 protease. Separation and sequence analyses of the resultant peptides were performed to identify glutamine residue involved in cross-linking. It was found that Gln54 was cross-linked to lysine residues by GTGase and MTGase in common. It is suggested that the difference in the numbers of lysine residues targeted by GTGase and MTGase may be responsible for the difference in the polymerization process of alpha-lactalbumin between GTGase- and MTGase-catalyzed systems.  相似文献   

15.
A rapid and convenient method for the precise quantification of epsilon-(gamma-glutamyl)lysine isopeptide in lyophilized proteolytic digests of cross-linked plant protein samples was developed. The isopeptide was baseline-separated from three other isomers containing lysyl and glutamyl residues by reverse-phase high-performance liquid chromatography after exhaustive proteolytic digestion of the samples cross-linked by a microbial transglutaminase (MTG). Highly selective detection was performed by electrospray mass spectrometry in MS/MS mode. Demonstrating the applicability of the suggested analytical procedure, enzymatic cross-linking of protein isolates from soy [Glycine max (L.) Merr.], pea [Pisum sativum L.], and the sweet lupin species Lupinus albus L. and Lupinus angustifolius L. was investigated after incubation with 0.01 g of MTG/100 g of protein for 0-240 min at 40 degrees C. The liquid chromatography-mass spectrometry (LC-MS) method was successfully applied to monitor the kinetics of epsilon-(gamma-glutamyl)lysine isopeptide formation. Since the calculated initial levels of epsilon-(gamma-glutamyl)lysine in the genuine leguminous protein isolates were between 40 and 77 micromol/100 g, an isopeptide detection limit of 0.5 microg/mL, corresponding to approximately 50 micromol/100 g of protein, was shown to suffice for quantifying the cross-linking rate enzymatically induced by MTG. Concentrations of epsilon-(gamma-glutamyl)lysine in the texturized proteins ranged from 100 to 500 micromol/100 g of protein.  相似文献   

16.
Transglutaminase-catalyzed cross-linking of interfacial proteins in oil-in-water has been shown to influence physical stability, but little is known about how this reaction impacts lipid oxidation. Therefore, this study evaluated the influence of transglutaminase-induced interfacial protein cross-linking on the oxidative stability of casein-stabilized menhaden oil-in-water emulsions. Interfacial casein in menhaden oil-in-water emulsions cross-linked by transglutaminase (pH 7.0) produced a cohesive interfacial protein layer that could not be removed from the emulsion droplet by Tween 20. Although transglutaminase cross-linked the interfacial casein, these emulsions did not show increased oxidative stability when compared to untreated emulsions as determined by measurement of lipid hydroperoxides and thiobarbituric acid reactive substances. These results indicate that increasing the cohesiveness of proteins at the interface of oil-in-water emulsions does not inhibit lipid oxidation. This could be due to the ability of prooxidative species such as iron to diffuse through the cross-linked protein layer where it could promote the decomposition of lipid hydroperoxides into free radicals that could oxidize unsaturated fatty acids in the emulsion droplet core.  相似文献   

17.
Surface hydrophobicity (SH) of milk proteins treated physicochemically (by heating and Maillard reaction) or modified enzymatically (by transglutaminase, lactoperoxidase, laccase, and glucose oxidase) was assessed in relation to their techno-functional properties. Heat-treatment increased SH of whey protein isolate and decreased SH of sodium caseinate and bovine serum albumin. Maillard reaction of milk proteins caused time-depended decreases of SH. Only for total milk protein reacting with glucose and lactose elevated SH-values were detected. Protein modification with transglutaminase, laccase, and lactoperoxidase strongly increased the SH of whey protein isolate and total milk protein. Incubation with glucose oxidase elevated SH values of sodium caseinate, whey protein isolate, and total milk protein. When correlating SH with techno-functional properties, a positive correlation was observed between SH and foam formation, and a negative correlation was observed between SH and foam stability as well as emulsion stability. No clear correlation was detected between SH and emulsifying activity, surface tension, viscosity, and heat stability of enzymatically and physicochemically treated milk proteins.  相似文献   

18.
Cysteine sulfoxides and alliinase activity of some Allium species   总被引：17，自引：0，他引：17  
The flavor precursors of 17 species belonging to the Alliaceae family were analyzed by HPLC, and results were evaluated with respect to the classification of species into their genus, subgenus, and section. Identification and quantification of these precursors were carried out by synthetic and natural reference materials. In addition, nine of these species were investigated in terms of their alliinase activity. Alliinase (EC 4.4.1.4) catalyzes the conversion of odorless (+)-S-alk(en)yl-L-cysteine sulfoxides into volatile thiosulfinates. Cysteine sulfoxides as well as alliinase activity were found in all investigated samples, and (+)-S-methyl-L-cysteine sulfoxide was most abundant. (+)-S-Propyl-L-cysteine sulfoxide was detected in only a few, not closely related, species. Analysis of the crude protein extract of nine species gave evidence that alliinase activities of samples were similar in terms of pH and temperature optimum, K(M) value, and substrate specificity. For all investigated protein extracts, the highest specific alliinase activity was found for (+)-S-(2-propenyl)-L-cysteine sulfoxide (alliin). The substrate specificity of these enzymes was not related to relative abundance of the cysteine sulfoxides. However, SDS-PAGE yielded some significant differences among species in terms of their total protein compositions. Species belonging to different subgenera exhibited a specific protein pattern with molecular masses between 13 and 35 kDa.  相似文献   

19.
Transglutaminase (epsilon-glutaminyl-peptide:amine gamma-glutaminyl-transferase, EC 2.3.2.13) (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins and peptides. The substrate specificity of TGase for primary amines was investigated to incorporate various functional groups into proteins and peptides. In this study, microbial transglutaminase and guinea pig liver transglutaminase were used. For the primary amines to be incorporated into benzyloxycarbonyl-L-Gln-Gly (Z-Gln-Gly), they were required to have more than four carbon chains without side chains between the functional groups. These results suggest that with appropriate primary amines as spacers, various functional groups, carboxyl groups, phosphate groups, saccharides, and so on, can be incorporated into proteins by using TGase.  相似文献   

20.
为了探究酶制剂对非发酵面团在冻融循环后的品质的影响,该文比较了2种酶制剂转谷氨酰胺酶(TGase)与木聚糖酶(xylanase)对非发酵面团冻融品质的改良效果。结果表明,与对照组相比,添加转谷氨酰胺酶和木聚糖酶均能改变冻融后面团蛋白各组分的含量。添加转谷氨酰胺酶使醇溶蛋白以及谷蛋白含量显著下降(P0.05)、谷蛋白大聚合体含量有所上升。添加木聚糖酶则使醇溶蛋白含量上升,使谷蛋白含量显著降低(P0.05)。转谷氨酰胺酶对面团中戊聚糖含量影响不显著(P0.05),而添加木聚糖酶使面团中可溶性戊聚糖含量显著上升(P0.05)。转谷氨酰胺酶的添加加速了冻融循环后非发酵面团的失水,木聚糖酶则显著降低了其失水率(P0.05)。除在10 g/kg转谷氨酰胺酶显著降至1.32 ms外,弛豫时间T2(1)基本维持在1.75 ms,而弛豫时间T2(2)均有所减小;添加转谷氨酰胺酶后,深层结合水相对含量呈逐渐上升趋势;而木聚糖酶则反之。木聚糖酶使冻融面团强韧性与对照组相比显著增强(P0.05),剪切力小幅下降,硬度显著下降(P0.05);与转谷氨酰胺酶相比,木聚糖酶更能缓解冻融面团的质构劣变;转谷氨酰胺酶改善冻融熟面坯的黏性、弹性与内聚性,木聚糖酶则改善"硬度上升,弹性下降"的劣变现象。在改善冻融面团流变特性方面,转谷氨酰胺酶效果更为显著(P0.05)。因此,添加转谷氨酰胺酶与木聚糖酶均能在一定程度上改善冷冻非发酵面团在冻融条件下的品质劣变,但它们的作用方面有所差异。研究结果为两者能够在冷冻非发酵面制品中被广泛地应用提供理论基础。  相似文献   

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