Studies On The Preparation Of 5'-deoxynucleotides By Enzymatic Hydrolysis Of DNA

Deoxynucleotide was mainly used clinically for the treatment of leukaemia, also effectively for the therapy of aplastic anemia , decrease of thrombocyte and hepatitis . Deoxynucleoside triphosphate, a widely used reagent , was got from the deoxynucleosidemonophosphate(deoxynucleotide) by enzyme. Deoxynucleoside can be got from deoxynucleotide by deoxynucleotidase, and deoxynucleoside was a significant produrg in the anti-cancer and anti-virus reach. It was main method to get deoxynucleoside monophosphate by using nuclease P1 to digest the heat-denatured DNA, nuclease P1 was easily got through fermentation by Penicillium citrinum .The earliest domestic repart about digesting the heat-denatured DNA by enzyme was published in 1970s, but till now, the number of reports about it has been less than ten . In order to change this status, we had developed this item.The central content of this paper included two parts, one was the screening and fermenting of Penicillium citrinum strains by nitrogen ion-beam injection mutagensis. and the other was the studies on enzymatic hydrolysis of the heat-denatured DNA by nuclease P1 . the content of first part is as follows:1. screened out a high-yield nuclease P1 strain from the Penicillium citrinum IM02 as initial strain.2. treated the initial strain with nitrogen ion-beam injection , then screening out the high-yield mutagenic strains .3. studied on the hereditary stability of the high-yield mutagenic strain.4. studied on the fermentation conditions of high-yield mutagenic strain by shaking flask.The conclusion of above studies was : after treated by nitrogen ion-beam injection , the activity of nuclease P1 fermented by the high-yield mutagenic strain elevated 61% than the initial strain, and the heredity of the high-yield mutagenic strain was stable.The studies on the conditions of the shaking flask fermentation, such as seeds age, seeds inoculated rate, initial pH, temperature and so on, revealed theeffects on the activity of nuclease PI.In the aspect of enzymatic hydrolysis of the heat-denatured DNA by nuclease PI of this paper, time course of 5'-deoxynucleotides' production have been firstly studied, the course showed that the velocity of the production of dGMP was the fastest one, dCMP was the slowest one. the concentration of the totle product was almost not varied after the hydrolysis passed 100 minutes. The effects of several factors that influence the products of hydrolysis , such as temperature , pH , the initial substrate concentration and the concentration of enzyme, was studied , the optimum hydrolytic condition was determined through orthogonal test . The optimum hydrolytic condition was : temperature 58°C , pH6.5 , the concentration of DNA 4.0gL"', the concentration of enzyme 4% , the yield rate is 92%. Based on the experimental date, the kinetics Michaelis constant was got.