Investigation of the substrate specificity of glutamyl endopeptidase using casein substrates

Abstract

Electrophoretically and chromatographically pure P-casein was prepared from acid caseinate exploiting the differential solubility of different caseins in the presence of Ca2+ at 4°C and 30°C. The yield of P-casein was approximately 5%. The p-casein purification process also yielded a co-fraction enriched in a-casein.GE was purified and characterized from Alcalase™ 2.4 L. Various protocols were investigated to purify GE before selecting a protocol employing hydrophobic interaction (HIC) and cation exchange (IEX) chromatography. The yield of GE obtained was approximately 42%. The purified P-casein and the enriched a-casein fractions were digested with GE at 37°C and 50°C over 4 h. The results show that GE was highly specific and hydrolysed the peptide bond predominantly on the carboxy terminal of Glu residues. GE also appeared to hydrolyze bonds on the carboxy terminal o f Asp. The results indicated that Met residues were poorly preferred at the P| ’ position. Whereas, Pro residues were not preferred at the P |’ position. It was also observed that the hydrolysis of Glu-X bond in Glu-Glu-X and in Glu-Glu-Glu-X sequences (X= Arg, Asn, lie and Lys) was preferred in comparison to Glu-Glu, suggesting that at the P i’ position Glu residues were poorly preferred. Non-specific cleavages were observed at the carboxy terminal of Phe (51), Thr (128) and Gin (188) on incubation of P-casein with GE. Non-specific cleavages at the carboxy terminal of Phe (24) and Gin (131) in a sicasein were observed on incubation of the enriched a-casein with GE only at 50°C. Synthetic peptides corresponding to specific sequences of P-casein were incubated with GE at 37°C for 120 min. The LC-MS analysis on the hydrolysed sample showed that multiple phosphorylated residues near the scissile bond did not influence the substrate specificity of GE. Furthermore, these studies showed that the Glu-Pro and the Glu-Met bonds were hydrolysed.