Preparation Of Gln-Bound Peptide With High Efficiency

Glutamine is a conditionally essential amino acid, it has very important role in metabolism. The instability and low solubility of free Gin hamper its use as a nutrition substrate hi routine clinical and stockbreeding. Then the Gin-bound peptide uch as Gly-Gln and Ala-Gin were generally applied. But these two synthesis dipeptides were expensive and used extremely limited in stockbreeding. Gln was rich in the wheat gluten. In this study, the wheat gluten was hydrolyzed by protease to produce Gin-bound peptide in order to find a substitute for free Gin.1 Selection of hydrolytic enzymes of glutenThe food grade proteases such as Pepsin, Trypsin, Protamex? Alcalase3.0T, Neutrase1.5Mg and Flavourzyme TM1000Mg were used to measure the hydrolytic capability of enzyme to amide group. The enzymes were used in proper condition to hydrolyze wheat gluten for 4 hours and the hydrolysates were tested. Gin bounded peptides was measured under the protection of Bis(trifluoroacetoxy)iodo]benzen(BTI) by HPLC. A pre-column deviation procedure of O-phthaldialdelhyde( OPA) was used for the determination of Gin by reversed-phase high performance liquid chromatography(HPLC). A phosphate buffer at pH 6.85 was used as the mobile phase A and methanol was B. Gradient elution was adopted and detected by ultraviolet detector( A. =340nm). The result of the study showed Pepsin, Trypsin and Protamex?had less hydrolytic capacity to amide group than the others. The hydrolytic capacity to amide group of Trypsin, Pepsin and Protamex?was 2.79%, 1.90%, 5.80% respectively. While the hydrolytic capacity to amide group of Neutrase1.5Mg, Alcalase3.0T and Flavourzyme?000Mg was 7732.25%, 26.55%, 16.38% respectively.The study showed that Trypsin, Pepsin and Protamex?could be used to prepare the Gin-bound peptides.2 Preparation of Gin-bound peptidesTrypsin, Pepsin and Protamex?were used to determine their proper initial pH value, temperature, enzyme concentration and substrate concentration for hydrolyzing wheatgluten. The hydrolytic condition of enzymes were as follows: Trypsin, pH8.0, 50C, enzyme concentration(short for E%) was 9%(w/w), substrate concentration (short for S%) was 12.8(w/v); Pepsin, pH2.0, 40C, E%=5%(w/w), S%=16%(w/v); Protamex? pH7.0, 40C,E%=8%(w/w), S%=12.8%(w/v). The wheat gluten hydrolyzed with Pepsin, Trypsin, Protamex?and their combination was studied. The samples were collected in degree of hydrolysis(DH) tests and the ammo groups were determined in hydrolysates. Then the average ammo acid number of peptides was calculated. The combination of Trypsin and Pepsin performed a better hydrolytic effect on gluten. Its conditions were as follows: Trypsin at pH8.0, 50C, S%=12.8%(w/v), E%=9%(w/w) for 6 hours, then Pepsin at pH2.0, 40C, E%=5%(w/w) for 5 hours and final average amino acid number of peptides in the hydrolysate was 2.20.3 Determination of Gln in peptidesThe samples were collected in degree of hydrolysis(DH) tests and the harvest of Gln was determined by HPLC. The wheat gluten was hydrolyzed by Trypsin, Pepsin and ProtamexT and the harvest of Gln was 77.86%, 87.70%, 69.58%, respectively. The harvest of Gin in hydrolysate of Trypsin mixed Protamex?was 39.52% which was the lowest. While the harvest of Gln in peptides of Trypsin combined with Pepsin was 60.43% which was the highest.