Investigation of mucor miehei rennet dextran sulphate conjugates

Chymosin (calf rennet)– extracted from the fourth stomach (abamosum) of the young calveshas been used worldwide in cheese production for years and it has been preferred for its high quality. However, nowadays there has been a shortage of the chymosin in the world market for decades, so proteolytic enzymes from different sources gained more importance. But its high thermostability and their other properties causes problems and prevents the useage of byproducts in food industry. In this study, covalent conjugates of mucor miehei rennet with dextran sulfate, mucor miehei rennet is commonly used in manufactured cheese, was synthesized and investigated for decrease its thermal resistance. In this study, fõrst purchased mucor miehei rennet was purified by column chromatography using Sephadex G–50 column. Then dextran sulphate was oxidized to aldehyde derivative with sodium periodate and the occurence of derivative confirmed with FTIR spectroscopy. Conjugates of different mole ratios of mucor miehei rennet and dextran sulphate were synthesized and purified again using G–100 column. Activities of purchased and purified mucor miehei rennet at different pHs and temperatures were determined. The activities of conjugates at different temperatures were also measured at pH 5,0. The characteristics of synthesized conjugates were investigated with HPLC, GPC (Viscotek) and FTIR. As a result, conjugates displayed decrease in the activities with the decrease of rennet: dextran molar ratios in all tested temperatures. Physicaly mixed (non covalent) mucor miehei rennetdextran sulphate mixtures showed the same activity with purified enzyme. These results demonstrated that, covalent conjugation of mucor miehei rennet with aldehyde dextran sulphate caused the decrease of thermal resistance of this enzyme and decrease in thermostability is caused by covalent bonding of mucor miehei rennet–dextran sulphate conjugates. Key words: Mucor miehei rennet, Dextran sulphate, Cojugate, Milk clotting activity, UV, GPC, FTIR, HPLC.