Acta Scientific Biotechnology Volume 2 Issue 7 November 2021 Efficient Immobilization of Milk Clotting Enzyme Produced by Rhizomucor miehei on Tricalcium Phosphate Maysa Elsayed Moharam*, Magda A El- Bendary and Amira M Roshdy Microbial Chemistry Department, Biotechnology Institute, National Research Centre, Elbohouth street, Dokki, Giza, Egypt *Corresponding Author: Maysa Elsayed Moharam, Microbial Chemistry Department, Biotechnology Institute, National Research Centre, Elbohouth street, Dokki, Giza, Egypt. Research Article Received: October 10, 2021 Published: October 23, 2021 © All rights are reserved by Maysa Elsayed Moharam., et al. Abstract Milk clotting enzyme produced by Rhizomucor miehei NRRL 2034 under solid state fermentation was efficiently immobilized on Tricalcium phosphate (TCP) with immobilization degree of 90 % and activity retained of 93%. Optimum pH value for enzyme im- mobilization was pH 4. Stirring time of 30 min. was most suitable for the process at 30 o C. 3 mg protein concentration was the best for ideal immobilization. Immobilized enzyme reacted optimally at pH 5 which represents the optimum value for the free enzyme preparation. Optimum temperature raised to 70°C for immobilized enzyme preparation in comparison to 60°C for free enzyme. Im- mobilized enzyme showed improved thermal stability. Keywords: Milk Clotting Enzyme; Rhizomucor miehei; Immobilization; Tricalcium Phosphate Introduction Enzymes are biocatalysts that increasing the reaction rates of different chemical and biological processes [1]. Milk coagulation is the main step in cheese manufacturing. Calf rennet, is an aspar- tic protease enzyme (E.C. 3.4.23.4) and renin enzyme excreted by the calves fourth stomach. All cheese types are prepared by milk coagulated by renin like enzyme [2]. Specificity for K- casein cleav- age is required for good coagulation process [3]. Obtaining natural milk coagulants is a challenge because of the huge amount of milk coagulants required for dairy industries requirements for milk coagulants worldwide [4]. Milk clotting enzyme (MCE) of animal origin are expensive, and their consumption has been restricted due to religious or dietary reasons. Therefore, searching for rennet substitutes and decreasing their cost is encouraged [5]. Among different rennet sources, Microbial rennet is considered a good source of calf rennin substitute. Many research articles had investigated fungal microbial rennet like enzymes. Rhizomucor miehei MCE production conditions and its properties [6] and re- cently, Aspergillus oryzae [7]. The protease produced by Rhizomu- cor miehei is an acid-asparatate protease with molecular weight of about 38.000. It consists of a poly peptide chain which is similar to calf rennet in 3-D-structure and its properties [4,8]. It is similar to the true calf rennet in its high specificity in splitting similar peptide bonds in kappa-casein, high milk coagulating activity, calcium re- quirement and high cheese quality [8]. Immobilized enzymes leads to easy and feed back of technol- ogy processes. Also they are more specific, are easier to separate the product than the soluble enzyme systems and can be reused or applied in continuous process [9,10]. These advantages of immo- bilized enzymes are important in the food industry. Furthermore, the immobilization may improve the enzyme characteristics such as the activity, thermal stability, selectivity and specificity to sub- strates and reduction of the inhibition and dissociation by reaction products and metal ions [1]. Citation: Maysa Elsayed Moharam., et al. “Efficient Immobilization of Milk Clotting Enzyme Produced by Rhizomucor miehei on Tricalcium Phosphate". Acta Scientific Biotechnology 2.7 (2021): 13-18.