Isolation of clt Genes Encoding milk clotting enzyme and characterization of milk clotting enzyme from new isolate Streptomyces pseudogriseolusNRC-15

Abstract

Research focused on isolation and characterization on new strain of Streptomyces pseudogrisiolus from Egyptian soil have proteinases which possesses activity (MCE) identified by 16S rRNA with universal primer and submit in genbank with accession number AB739622. In this work, study optimizes the extraction of milk-clotting enzyme (MCE) according to its adequacy to be employed as substitute of rennin. The organism grew well in medium 3 after 4days of incubation at temperatures of 35° ±2, pH 6.0. Cultural and environmental conditions for milk clotting enzyme production by Streptomyces were studied. Glucose (3%, w/v) and ammonium sulphate (0.2%w/v), were found to be the best C and N sources for milk clotting enzyme production respectively. Partially purified and characterized. MCE was obtained by fractional precipitation with ammonium sulphate, followed by the chromatography of the most active fraction on Sephadex G-100 with 20.39 purification fold and recovery about 80%. The maximum enzyme activity was at pH (6.5) and 45ºC. The clotting activity of the purified enzyme was stimulated with increasing CaCl 2 concentration up to 10mM. However, a gradual reduction of the activity was observed by increasing NaCl concentration between 4-8%. Whereas, Cu +2 and Zn +2 significant inhibition it. Interestingly MnCl 2 and MgSO 4 were found to have stimulatory effect, which has been rarely reported. Also study includes some kinetics calculation about MCE. Gene control MCE expression was detected by specific primer design using DNA star program, sequence illustrate gene found clt a and clt b, and submitted in genbank as new sequence with accession number AB739621. The milk clotting enzyme (MCE) obtained from S. pseudogrisiolus has potential as calf rennet substitutes in dairy products and it can be used in lypholyzed form as tablets in pharmaceutical products for patients with digestive system.