The transpeptidase activity of glutaminase from Bacillus amyloliquefaciens (GBA) and Aspergillus oryzae (GAO) to yield γ-[Glu]n-Phe peptides were verified for the first time. In the presence of Gln and Phe, γ-Glu-Phe and γ-Glu-γ-Glu-Phe were synthesized by GAO, and γ-Glu-Phe, γ-Glu-γ-Glu-Phe, γ-Glu-γ-Glu-γ-Glu-Phe, γ-Glu-γ-Glu-γ-Glu-γ-Glu-Phe, and γ-Glu-γ-Glu-γ-Glu-γ-Glu-γ-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 (γ-Glu-Phe as the acceptor), indicating that GBA had a greater affinity than GAO for Phe and γ-Glu-Phe in the transpeptidation reaction. The Km values for the transpeptidation catalyzed by GBA against acceptors, Phe and γ-[Glu](1≤n<5)-Phe (47.88–206.47 mM), increased with an elevated number of γ-glutamyl residue within the acceptor. The optimal conditions for γ-[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 γ-[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). γ-[Glu]n-Phe or the post-enzymatic reaction mixture enhanced the umami intensity of commercial soy sauce and model chicken broth.Keywords: astringency; enzymatic synthesis; glutaminase; kokumi; γ-[Glu]n-Phe;

•High solid concentrations (28%, w/w) facilitate the enzymatic yeast cells hydrolysis.•Higher protein recovery, DH, reducing sugar contents and 5′-nucleotides contents can be achieved at 28% high solid.•The micromorphology of hydrolysed yeast cells can confirm the chemical analysis results.•The hydrolysis efficiency of alcalase for yeast cells is higher than that of papain.To investigate the effect of high solid concentrations (w/w) on the enzymatic hydrolysis of yeast cells, yeast cells were hydrolyzed by papain and Alcalase at 18% and 28% solid concentrations (w/w), respectively. High solid concentrations resulted in high protein recovery, degree of hydrolysis (DH) and contents of reducing sugar and 5′-nucleotides (5′-GMP, 5′-IMP), which indicated a positive effect on the release of yeast intracellular substances. High-performance liquid chromatography (HPLC) analysis showed negligible difference in the molecular mass distribution of peptides. The micromorphology of the hydrolysed yeast cells was studied by transmission electron microscopy (TEM). Result demonstrated that high solid concentrations accelerated the plasmolysis, deformation and lysis of yeast cells. Similarly, addition of NaCl or commercial yeast extract to the yeast cell suspension (18% w/w) improved the DH. Therefore, high concentrations of NaCl or yeast cell hydrolysates in solutions with high solid concentrations could damage the yeast cell membrane, leading to effective contact between the enzyme and the substrate and consequent increase in the hydrolysis efficiency. Nevertheless, the hydrolysis mechanism remains unknown and must be further investigated.Download high-res image (161KB)Download full-size image

Phytohemagglutin (PHA), purified from red kidney beans (Phaseolus vulgaris) by Affi-Gel blue affinity chromatography, was subjected to ultrahigh-pressure (UHP) treatment (150, 250, 350, and 450 MPa). The purified PHA lost its hemagglutination activity after 450 MPa treatment and showed less pressure tolerance than crude PHA. However, the saccharide specificity and α-glucosidase inhibition activity of the purified PHA did not change much after UHP treatment. Electrophoresis staining by periodic acid–Schiff (PAS) manifested that the glycone structure of purified PHA remained stable even after 450 MPa pressure treatment. However, electrophoresis staining by Coomassie Blue as well as circular dichroism (CD) and differential scanning calorimetry (DSC) assay proved that the protein unit structure of purified PHA unfolded when treated at 0–250 MPa but reaggregates at 250–450 MPa. Therefore, the hemagglutination activity tends to be affected by the protein unit structure, while the stability of the glycone structure contributed to the remaining α-glucosidase inhibition activity.

The effect of the structural features of hydrochloric acid-deamidated wheat gluten with different degrees of deamidation (DDs) on the susceptibility to enzymatic hydrolysis by pancreatin was investigated. The wheat gluten deamidated by hydrochloric acid with a DD of 55% revealed the highest susceptibility to enzymatic hydrolysis as evaluated by the hydrolysis degree and nitrogen solubility index of the hydrolysates. An increase of peptides with MW below 3000 Da was observed as the DD increased. Raman spectra in the 1740–1800 cm–1 and 521–530 cm–1 range suggested that wheat gluten had taken off the deamidation with different DDs and that the disulfide bond had disrupted the sulfhydryl groups with different intensities, respectively. Results from the deconvolution of the amide I region of FTIR spectra in the 1600–1700 cm–1 range showed that the content of the α-helix decreased and that the content of the β-turn and β-sheet increased with increasing DDs, which improved the molecular structure and flexibility of wheat gluten. A scanning electron microscope (SEM) revealed that the image of HDG-55% presented the smoothest surface and the least uniform pore, enabling the sample to be more susceptible to enzymatic hydrolysis. The above information will enable us to better understand the effect of structure on the susceptibility of deamidated wheat gluten.