The Preparation Of Mimic Enzyme Copper (Ⅱ) Complex And Its Hydrolytic Effect On Peptide Bonds

The hydrolysis of peptide bonds can be applied to analysis of protein sequence, preparation of functional peptides and peptide drugs, and so on. In order to obtain desired flavor and functional properties in the industrialized production of food, we need to hydrolyze protein moderately. Under this process, solubility, emulsifying and foaming properties of hydrolyzed proteins are changed. The technology of peptide bond hydrolysis mainly divided into four types:enzymatic hydrolysis, acid and alkali hydrolysis, small inorganic molecules and organic molecules hydrolysis, metal complexes hydrolysis. However, they have several shortcomings, such as strong volatility, toxic, harsh reaction conditions, and so on. Therefore, it is significant to synthesize protein mimic enzyme, which has natural enzyme properties, without above drawbacks.In this paper, we used methanol, formaldehyde, ethylene diamine, ethanolamine and other reagents to synthesize the mimic enzyme aza crown ether copper (Ⅱ) complex based on template synthesis method. The complex possessed effective hydrolysis activity of peptide bonds and was characterized using FTIR and UV-vis spectrophotometer. Meanwhile the stability of complex in different conditions was carried out. SDS-PAGE, HPLC, MALDI-TOF-MS, LC-MS and other methods were used to investigate hydrolysis activity, hydrolysis sites and hydrolysis process of peptide bonds. The results are as follows:(1) Four kinds of different proteins (myoglobin, lysozyme, ovalbumin, bovine serum albumin), varying in spatial structure were used as substrates for hydrolysis. The hydrolysis of peptide bonds in proteins using the aza crown ether copper (Ⅱ) complex was studied. SDS-PAGE results showed that the peptide bonds of myoglobin, ovalbumin, bovine serum albumin were hydrolyzed to some extent (degree of hydrolysis:bovine serum albumin> ovalbumin> myoglobin) at the condition of 60℃, the peptide bonds of lysozyme was not hydrolyzed. Where:the hydrolysis of bovine serum albumin and ovalbumin have exopeptidase characteristic, the hydrolysis of myoglobin has endopeptidase characteristic.(2) Selecting myoglobin and bovine serum albumin as substrates which have different hydrolysis characteristic. The hydrolysis acitivity and sites in proteins using the aza crown ether copper (II) complex was studied. SDS-PAGE results showed that aza crown ether copper (Ⅱ) complex respectively hydrolyzed bovine serum albumin and myoglobin were over 70% and 40% at the condition of 60℃ for 3 d. MALDI-TOF-MS datas showed that aza crown ether copper (Ⅱ) complex selectively disrupted the peptide bonds of Lys17-Val18, Lys79-Lys80 and Lys97-His98 in myoglobin. The hydrolysis process of aza crown ether copper (Ⅱ) complex demanded for the various parts coordination of the substrates, the hydrolysis reaction was consistent with Michaelis-Menten equation between aza crown ether copper (Ⅱ) complex and myoglobin and bovine serum albumin. Calculated by Linewaver-Burk method:Vmax for hydrolysis myoglobin was 5.56×10-5 M·h-1 and Km was 7.18×10-3 M; Vmax for hydrolysis bovine serum albumin was 1.00×10-4 M·h-1 and Km was 4.36×10-3M.(3) According to the hydrolysis sites, Q peptide (G-K-H-Q-S-H-A-T) containing these sites was synthesized. Bacitracin and Q peptide were used as the substrates.The hydrolysis activity and process in peptides using the aza crown ether copper (Ⅱ) complex was studied. HPLC results showed that aza crown ether copper (Ⅱ) complex respectively hydrolyzed bacitracin and Q peptide were over 80% and 85% at the condition of 60℃ for 3 d. The optimum pH of hydrolysis bacitracin was 6.5. LC-MS datas showed that the complex promoted the hydrolysis of peptide bonds, while also promoted dehydration condensation reaction of amino and carboxyl in bacitracin molecular and formed new peptide bonds. Aza crown ether copper (Ⅱ) complex could notablely hydrolyzed Q peptide, which from another point of view proved the capability of the complex hydrolyzed myoglobin. The hydrolysis reaction was consistent with Michaelis-Menten equation between aza crown ether copper (Ⅱ) complex and bacitracin and Q peptide. Calculated by Linewaver-Burk method:Vmax for hydrolysis bacitracin was 1.72×10-4 M·h-1 and Km was 3.50×10-3 M; Vmax for hydrolysis Q peptide was 1.44×10-4 M·h-1 and Km was 3.29×10-5 M.In conclusion, the aza crown ether copper (Ⅱ) complex, as a peptide mimic enzyme, can effectively promote the hydrolysis of peptide bonds. It will provide theoretical guidance and technical support for further study in food and biotechnology industry.