Production of recombinant camel chymosin enzyme with GAP promotor in Pichia pastoris

Chymosin is the major enzyme to used to make cheese in the dairy industry. This enzyme is secreted by abomasus of mammal and ıt has a milk clotting property. Nowadays commercial chymosin is acquired to four different sources. These are derived from animal, plant, microbial and recombinant production. Animal chymosin is acquired traditionally to abomasus of unweaned calf. Because of increasing demand in the world and slaughter to unweaned calf, alternative source of microbial, plant and recombinant were increased the tendecy for thıs industry is fall shorted. P. pastoris is commonly prefered that is an expression host for production of recombinant protein. This yield has high yield to production of heterologous protein. Despite Pichia pastoris has several promotors, the most extensively used promotors are AOX1 and GAP promotor. GAP promotor is responsible for region of expression of 3- phosphate dehydrogenase gene and is a constructional promoter. In this research; camel chymosin gene was expressed to Pichia pastoris which is a methylotrophic yeast, under the control of GAP promotor, using expression vector which is PGAPZαA. P. pastoris X33 strains which are transfered gene by electroporation. Clone copy number was detecded by Southern analysis. Chosen Single of copy strain with conditions of shaking flask, suitable pH (3-7) and temperature ( 20- 28 °C) was determined by factorial experimental design with 3 parallel, 2 repeat. According to statictical analysis, optımum of temperature is 24 and pH is 3 was determined. High production of fermentation ( 5 –L) was carried out ph 3 and 24°C. The stage of fermentation was maintained at GAP promotor which is efficient at hypoxic conditions through 103 hours. The highest enzyme activity was found approximately 20 IMCU/ mL in other word 2000 SoxhletUnıt/mL in 56.hours. Produced recombinant enzyme is 214.7 mg/ L in stage of fermentation. According to SDS-PAGE analysis, the molecular weight of the chymosin was found approximately 42 kDa. The optimum conditions of the enzyme were determined pH 3.0- 4.0 and temperature of 50°C. The enzyme is protected stability to 2 hours, ph 3- iii 6.5 and between 20°C - 65°C but increasing above of pH 4 and temperature of 50°C give rise decreasing of enzyme activity. The camel chymosin was activated by Fe+2 and Mg2+ ; respectively %129.58 and % 72.5 but inhibited by Cu+2, Zn+2. While this enzyme highest activity has between 20 Mm-40 Mm CaCl2 concentration, activity is lost with increasing concentration of calcium. The pickled cheese was produced to used of recombiant camel chymosin. Rendement of commercial recombinant chymosin with rendement of recombinant rennet were compared. While commercial bovine chymosin has %14.8 rendement, recombinant camel chymosin has % 15.45. According the results; recombinant camel chymosin determined effective rennet as a commercial chymosin. When optimization conditions of production of recombinant camel chymosin with increasing rendement and scale-up works were carried out, camel chymosin which has potential for production of commercial.