Effect of ultrasound pretreatment on structural,physicochemical, rheological and gelation properties of transglutaminase cross-linked whey protein soluble aggregates

A solution (10%, w/v) of whey protein soluble aggregates (WPISA) was pretreated with high-intensity ultrasound (HUS, 20 kHz) for different durations (10–40 min) before incubation with transglutaminase (TGase) to investigate the effect of HUS on the structural, physicochemical, rheological, and gelation properties of TGase cross-linked WPISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that HUS increased the amounts of high-molecular-weight polymers/aggregates in WPISA after incubation with TGase. HUS significantly increased (P < 0.05) the degree of TGase-mediated cross-linking in WPISA, as demonstrated by a reduction in free amino group contents. HUS significantly increased (P < 0.05) the particle size, intrinsic fluorescence intensity, and surface hydrophobicity of TGase cross-linked WPISA, but had no significant impact (P > 0.05) on the zeta-potential or total free sulfhydryl group content of TGase cross-linked WPISA. The apparent viscosity and the consistency index of TGase cross-linked WPISA were significantly increased by HUS (P < 0.05), which indicated that HUS facilitated the formation of more high-molecular-weight polymers. HUS significantly increased (P < 0.05) the water holding capacity and gel strength of glucono-δ-lactone (GDL)-induced TGase cross-linked WPISA gels. The results indicated that HUS could be an efficient tool for modifying WPISA to improve its degree of TGase-mediated cross-linking, which would lead to improved rheological and gelation properties.     

凝胶层析和离子交换层析结合法纯化高盐发酵液中谷氨酰胺转胺酶

采用凝胶层析和离子交换层析相结合的方法分离纯化高盐培养基中的谷氨酰胺转胺酶,优化的凝胶层析的条件,上样量6mL,流速为0.25 mL/min;离子交换层析的上样量50 mL,流速为3 mL/min.酶被纯化了4.22倍,比活力达17.33 U/mg蛋白,回收率为77.5％.液相色谱-串联质谱鉴定、蛋白质数据库比对结果表明,纯酶与AAN01353是同种蛋白质.     

Enzymatic determination of biogenic amines with transglutaminase

Tyramine, histamine, putrescine and cadaverine, the most common biogenic amines indicating the food quality, were studied in the transglutaminase-catalyzed reaction. Transglutaminase (protein-glutamine gamma-glutamyltransferase EC 2.3.2.13) catalyzes an acyl transfer reaction between a donor substrate and an acceptor substrate (e.g. biogenic amine) and forms a cross-linkage between substrates with a release of ammonia. The reaction can be monitored by measuring the ammonia produced in the reaction. The concentration of produced ammonia was found to be proportional to the concentration of biogenic amine and could hence be used to determination of biogenic amines in food matrixes.     

Effect of high intensity ultrasound on transglutaminase-catalyzed soy protein isolate cold set gel

The effects of high intensity ultrasound (HIU, 105–110 W/cm² for 5 or 40 min) pre-treatment of soy protein isolate (SPI) on the physicochemical properties of ensuing transglutaminase-catalyzed soy protein isolate cold set gel (TSCG) were investigated in this study. The gel strength of TSCG increased remarkably from 34.5 to 207.1 g for TSCG produced from SPI with 40 min HIU pre-treatment. Moreover, gel yield and water holding capacity also increased after HIU pre-treatments. Scanning electron microscopy showed that HIU of SPI resulted in a more uniform and denser microstructure of TSCG. The content of free sulfhydryl (SH) groups was higher in HIU TSCG than non-HIU TSG, even though greater decrease of the SH groups present in HIU treated SPI was observed when the TSCG was formed, suggesting the involvement of disulfide bonds in gel formation. Protein solubility of TSCG in both denaturing and non-denaturing solvents was higher after HIU pretreatment, and changes in hydrophobic amino acid residues as well as in polypeptide backbone conformation and secondary structure of TSCG were demonstrated by Raman spectroscopy. These results suggest that increased inter-molecular ε-(γ-glutamyl) lysine isopeptide bonds, disulfide bonds and hydrophobic interactions might have contributed to the HIU TSCG gel network. In conclusion, HIU changed physicochemical and structural properties of SPI, producing better substrates for TGase. The resulting TSCG network structure was formed with greater involvement of covalent and non-covalent interactions between SPI molecules and aggregates than in the TSCG from non-HIU SPI.     

Transglutaminase surface recognition by peptidocalix[4]arene diversomers

A series of N-linked tetrakis(tetrapeptido)calix[4]arene diversomers, 3A-P, has been synthesized by coupling of a cone calix[4]arene tetracarboxylic acid chloride with tetrapeptides 1A-P obtained in a parallel fashion. The inhibition activity of 3A-P towards tissue and microbial transglutaminase was evaluated by in vitro assays with a labeled substrate. Kinetic analysis using one of the most active derivatives (3A) showed a noncompetitive inhibition with respect to the amino acceptor substrate and an uncompetitive inhibition with respect to amino donor substrate. Experimental results are in accordance with an inhibition due to a protein specific surface recognition on a region noncomprising the enzyme active site.     

两种微生物源谷氨酰胺转氨酶与底物特异性识别的全原子动力学模拟研究

微生物源谷氨酰胺转氨酶(Transglutaminase, TGase)能够催化蛋白间氨酰基转移促进蛋白交联,已经广泛应用于食品、轻工等多种领域.然而目前微生物源TGase与底物间基于长程作用力的分子特异性识别机制仍不清楚,理性改造困难.本研究以源自高茂源链霉菌(MTG)和源自枯草芽孢杆菌(BTG)的两种TGase为研究对象,使用分子对接技术构建酶与底物的结合复合体并使用分子动力学模拟解析两种TGase与底物识别的分子机制.结果显示两种TGase在底物识别过程高度类似,均由催化腔外周loop区域氨基酸残基通过范德华及静电作用固定底物整体,而后由位于催化腔底部催化三联体内Asp(MTG)或Glu(BTG)通过羧基侧链稳定反应发生局部空间构象并拉近底物Gln内γ-酰胺基与核心催化残基Cys间距,以便后续反应过程中TGase-底物中间体的生成.上述结果阐明了TGase与底物活性基团的分子识别模式,提出了两种TGase内作用于底物识别的氨基酸残基,为以后TGase的理性改造研究提供了理论基础.     

Screen-printed electrodes as impedimetric immunosensors for the detection of anti-transglutaminase antibodies in human sera

The concentration of anti-transglutaminase antibodies in human sera is an important analytical marker for the diagnosis of the autoimmune disorder celiac disease. In this work, an immunosensor for the electrochemical detection of anti-transglutaminase antibodies in human sera was developed. The immunosensor is based on the immobilization of transglutaminase onto screen-printed gold electrodes which were covered with a polyelectrolyte layer of poly (sodium-4-styrensulfonic acid). The antigen-antibody interaction was evaluated using an amplification step: incubation with peroxidase (POD)-labeled immunoglobulins and subsequent biocatalytic oxidation of 3-amino-9-ethylcarbazole (AEC). Changes in the interfacial properties of the sensor electrode were determined by electrochemical impedance spectroscopy (EIS). Impedance spectra could be fitted to a Randles equivalent circuit containing a constant phase element (CPE). Furthermore, it was shown that impedance measurements could be simplified by performing EIS at only two selected frequencies, without loss of reliability. Incubation of these disposable immunosensor chips with various anti-transglutaminase antibody concentrations resulted in changes in their charge transfer resistance (R_ct). Thereby, a calibration graph could be established. Finally, immunosensors were used for characterizing different human sera with respect to their anti-transglutaminase autoantibody concentration of the IgG and IgA type.     

Substrate specificity of <Emphasis Type="Italic">Streptomyces</Emphasis> transglutaminases

Transglutaminase (TGase) is a multifunctional enzyme vital for many physiologic processes, such as cell differentiation, tissue regeneration, and plant pathogenicity. The acyl transfer function of the enzyme can activate primary amines and, consequently, attach them onto a peptidyl glutamine, a reaction important for various in vivo and in vitro protein crosslinking and modification processes. To understand better the structure-function relationship of the enzyme and to develop it further as an industrial biocatalyst, we studied TGase secreted by several Streptomyces species and Phytophthora cactorum. We purified the enzyme from S. lydicus, S. platensis, S. nigrescens, S. cinnamoneus, and S. hachijoensis. The pH and temperature profiles of S. lydicus, S. platensis, and S. nigrescens TGases were determined. The specificity of S. lydicus TGase toward its acyl-accepting amine substrates was characterized. Correlation of the electronic and steric features of the substrates with their reactivity supported the mechanism previously proposed for Streptomyces mobaraensis TGase.