CORE Metadata, citation and similar papers at core.ac.uk Provided by Università degli Studi di Napoli Federico Il Open Archive EXTRACTION AND IMMOBILIZATION OF VEGETABLE ASPARTIC PROTEASES FOR CHEESE MAKING Marilena Esposito Dottorato in Scienze Biotecnologiche – XXVII ciclo Indirizzo Biotecnologie per le Produzioni Vegetali Università di Napoli Federico II Dottorato in Scienze Biotecnologiche – XXVII ciclo Indirizzo Biotecnologie per le Produzioni Vegetali Università di Napoli Federico II EXTRACTION AND IMMOBILIZATION OF VEGETABLE ASPARTIC PROTEASES FOR CHEESE MAKING Marilena Esposito Dottorando: Marilena Esposito Relatore: Prof. Prospero Di Pierro Coordinatore: Prof. Giovanni Sannia Ora dunque rimangono queste tre cose: la fede, la speranza e la carità. Ma la più grande di tutte è la carità. 1 Corinzi 13:13 INDEX SUMMARY ............................................................................................................................................. III RIASSUNTO ........................................................................................................................................... V 1. Introduzione..................................................................................................................................... V 2. Risultati ........................................................................................................................................... VI Estrazione di attività coagulante dalle foglie di carciofo (Cynara scolymus) ................................. VI Estrazione di attività coagulante dai fiori di cardo (Carduus defloratus) ...................................... VII Immobilizzazione di enzimi con attività coagulante ..................................................................... VIII 1. INTRODUCTION .................................................................................................... 1 1.1 Enzyme global market situation .................................................................................................... 3 1.2 Proteases ...................................................................................................................................... 3 1.2.1 Aspartic proteases ................................................................................................................. 4 1.2.2 Milk clotting aspartic proteases.............................................................................................. 5 1.2.3 Plant proteases as milk clotting enzyme for cheese making ................................................. 6 1.3 Overview of enzyme immobilization .............................................................................................. 7 1.3.1 Epoxy support ........................................................................................................................ 9 1.4 Aim of the thesis .......................................................................................................................... 10 1.5 References .................................................................................................................................. 10 2. RESULTS ............................................................................................................. 15 2.1 Extraction and characterization of milk clotting proteases from artichoke (Cynara scolymus, L.) leaves and alpine thistle (Carduus defloratus subsp. carlinifolius, Lam.) flowers ................. 17 2.1.1 Introduction .............................................................................................................................. 17 2.1.2 Materials and methods ............................................................................................................. 18 2.1.2.1 Materials ........................................................................................................................... 18 2.1.2.2 Methods ............................................................................................................................ 18 2.1.2.2.1 Protein extraction ........................................................................................................... 18 2.1.2.2.2 SDS-PAGE analysis ...................................................................................................... 19 2.1.2.2.3 Two-dimensional electrophoresis (2DE) ........................................................................ 19 2.1.2.2.4 Purification steps ........................................................................................................... 19 2.1.2.2.5 Milk-clotting activity ........................................................................................................ 19 2.1.2.2.6 Inhibition studies ............................................................................................................ 20 2.1.2.2.7 Statistical analysis ......................................................................................................... 20 2.1.3 Results and Discussion ............................................................................................................ 20 2.1.3.1 Cynara scolymus .............................................................................................................. 20 2.1.3.2 Carduus defloratus subsp. carlinifolius, Lam. ................................................................... 25 2.1.4 Conclusions .............................................................................................................................. 27 2.1.5 References ............................................................................................................................... 28 2.2 Immobilization of aspartic proteases with milk-clotting activity from Mucor miehei and Carduus defloratus L. subsp. carlinifolius ....................................................................................... 30 2.2.1 Introduction .............................................................................................................................. 30 2.2.2 Materials and Methods ............................................................................................................. 31 2.2.2.1 Materials ........................................................................................................................... 31 2.2.2.2 Methods ............................................................................................................................ 31 2.2.2.2.1 Enzyme purification ....................................................................................................... 31 2.2.2.2.2 Immobilization procedure ............................................................................................... 31 2.2.2.2.3 Determination of protein concentration .......................................................................... 32 2.2.2.2.4 Clotting activity assay .................................................................................................... 32 2.2.2.2.5 Determination of immobilized activity ............................................................................ 32 2.2.2.2.6 Reusability of the immobilized enzymes ........................................................................ 32 2.2.2.2.7 Statistical analysis ......................................................................................................... 33 I 2.2.3 Results and Discussion ............................................................................................................ 33 2.2.3.1 Mucor miehei protease immobilization ............................................................................. 33 2.2.3.2 Carduus defloratus protease immobilization .................................................................... 36 2.2.4 Conclusions .............................................................................................................................. 38 2.2.5 References ............................................................................................................................... 39 3. CONCLUSIONS ................................................................................................... 41 Appendix .............................................................................................................................................. 45 ACKNOWLEDGMENTS ....................................................................................................................... 46 II SUMMARY The worldwide increase in cheese production and consumption, together with the reduced supply and consequent price increase of calf rennet, has led to the search of new milk coagulant agents. Much research interest has been thus directed towards discovering milk clotting enzymes which would satisfactory replace calf rennet in cheese manufacture. Microbial rennet produced by genetically engineered bacteria has proven to be a suitable substitute even though the consumer constraints on the use of animal rennet either for religion reasons (Judaism and Islam) or diet habits (vegetarianism) or consumer concerns regarding genetically engineered foods (Germany, Netherlands and France forbid the use of recombinant calf rennet) have led to a growing interest toward vegetable coagulants. Therefore, several plant sources of milk-clotting enzymes have been investigated. Unfortunately, most of the “plant rennets” were found unsuitable because they produced extremely bitter dairy products due to a strong proteolysis.