The Conversion Of Citrulline By Streptococcus Faecalis

As one of the non-protein amino acids,citrulline plays important role in the urea cycle.Recent years many foreign researches show citrulline has a lot of important physiological functions such as radical-scavenging hydroxyl,indicator of the foreign exclusive effect,vasodilatation,stabilizing blood pressure, diagnosing rheumatoidal arthritis,antioxidation and so on. Citrulline is increasingly used in food,cosmetic and pharmaceutical formulations.At present,there are three methods for produceing citrulline:enzymatic conversion,fermention and chemical method. Enzymatic method,with arginine deiminase catalyzing and converting L-arginine to L-citrulline,is the most important process for the purpose.The thesis investigated the entire process for citrulline by enzymatic conversion.The process include isolate strains,fermention,conversion and separation.We trying to find a high yield,low cost,simple,high-quality product line for industrial production. The thesis first have found a high activity strain by original selection,the second selection and mutagenesis from watermelon soil and ascertain it industrial strain.The number is BT-13.Several factors affecting on the production of L-citrulline were studied in the fermention.The optimum conditions are as follows:the time is 15h,inoculation 2%,medium volumes 100ml/250ml,pH6.5,170rpm,37℃.In this conditions,100g/l L-arginine can be coverted 82.16g/1L-citrulline after 10h conversion.The conversion is the key to the whole process.Enzymatic production of L-citrulline by Streptococcus faecalis,with a step enzymatic reaction,avoid the complex feedback regulation of citrulline synthesis pathway and can accumulated higher concentration.Many factors affecting on the production of L-citrulline were studied.The optimum conditions are as follows:0.4mol/L acetate buffer,pH6.0,35℃, 120r/min,ρ(CTAB)=0.3g/L,0.4g organisms,30h,100g/LL-Arginine, 60ml/250ml.The rate is 98%.For the fermenter,L-citrulline concentrations up to 92.72g/l after 25h reaction and the rate is 92.19%.When the second conversion,L-citrulline concentrations up to 87.53 g/l and the rate is 87.03%.The thesis also researched on the process for the separation and purification of L-citrulline.We identified the conditions for citrulline purification by ion exchange method.The solution passed through a column packed with D-201 strong basic anion-exchange resin at 25℃. and adjusted to pH 10.0 with NaOH.After eluting with 1.0mol/l NaCl,the eluant was evaporated to crude crystals of citrulline by dissolved in ethanol.The elution rate was 90.69%.Finally,the purity of the product was 95.34%and the yield of citrulline in the whole process was 79.71%.