Studies On Enzymatic Glycosylation Of Diosgenin And Biosynthesis Of Related Compounds

Trillin is a glucoside of diosgenin which is an important kind of steroidal saponins. It is the active ingredient in medicinal plants of dioscoreaceae. Although it can be isolated from plants, this is extremely difficult and uneconomic, especially in large amounts due to the the amount of trillin in plant is rather limited. Thus, glycosylations in vitro are important for the synthesis of steroidal saponins for the pharmaceutical industry. Chemical synthesis would provide a feasible route to obtain homogeneous saponins, however, the protection and the deprotection of hydroxy groups are unavoidable and it requires organic solvents. So the more economic and cleaner route should be enzymatic synthesis. In this work, a glucosyltransferase cloned from potato bud was used to glucosylate steroidal sapogenins and steroidal alkaloids, in which UDP-glucose was provided as an indispensable sugar donor. But UDP-glucose is extremely expensive and unstable as a kind of energy-rich compound. So avoiding the use of UDP-glucose can greatly increase predominance for enzymatic synthesis of trillin. A multiple-enzymes system for enzymatic synthesis of trillin is artificially designed, the glycosylation reactions in vitro, in which the low-cost maltodextrin, UTP, phosphate were used as substrates. UDP-glucose and glucose-1-phosphate are not only vital intermediates in the one-pot bioreaction system as mentioned above, but also high value-added energy-rich sugar donors in the reaction catalyzed by enzymes. This dissertation describes the biosynthesis of UDP-glucose and glucose-1-phosphate using the enzymes covalently immobilized on amino-functionalized magnetic nanoparticles. In addition, galactose-1-phosphate was also synthesized using a recombinant galactokinase cloned from E. coli in this work for the purpose of diosgenin glycorandomazation in the future.The genes of malpase, ugpase,ppase, udpgdh from E. coli K12 and the gene of sgtl.1 from the bud of potato have been cloned. They were individually expressed with activity in E. coli BL21 (DE3) or E. coli DH5a. The expression condition for production of these recombinant proteins were optimized. And the recombinant enzymes were prepared and stored in the state of lyophilized powder.The one-pot system for biocatalytic synthesis of diosgenyl-β-d-glucopyranoside contained maltodextrin phosphorylase (E1, MalPase), uridine diphosphate glucose pyrophosphorylase (E2, GIPTTase), inorganic pyrophosphatase (E3, PPase), solanidine glucosyltransferase (E4, SGT1.1) and maltodextrin, UTP, phosphate, diosgenin. To increase the yield of trillin, some factors such as cosolvents, reaction conditions and substrate concentration were optimized. Under the optimum conditions:the reaction mixture containing 6.58 U El/1,125 U E2/1,7 U E3/1,0.75 U E4/1,5 mM MgCl2,100 mM phosphate buffer (pH 9), maltodextrin (5%, m/v), 10 mM UTP and 0.1 mM diosgenin was incubated in a thermomixer at 200 rpm and 40℃for 48 h, giving trillin in 28% yield (ca.15.8 mg/l).The amino-functionalized Fe3O4 nanoparticles prepared by chemical coprecipitation are an ideal material for immobilizing various enzymes which are needed for synthesis of UDP-glucose. The Fe3O4 nanoparticles activated by glutaraldehyde was found to be able to tightly bind with maltodextrin phosphorylase (E1), uridine diphosphate glucose pyrophosphorylase (E2) and inorganic pyrophosphatase (E3) respectively. The conditions for one-pot biocatalysis were optimized:18 U E1/l,100 U E2/l,25 U E3/l; maltodextrin,5% (m/v); UTP,2 mM; and MgCl2,10 mM; in 100 mM sodium phosphate buffer, pH 7.5; at 30℃for 10 h. Under the optimum conditions, the immobilized multiple-enzyme biocatalyst could be easily recovered and reused for five times in repeated syntheses of UDP-Glc. After purified by SAX column and ODS column orderly, approximately 630 mg of crystallized UDP-Glc was obtained from 1 litre of reaction mixture, with a moderate yield of 53.3% (based on UTP conversion).The optimal reaction conditions for glucose-1-phosphate synthesis by immobilized MalPase was:MalPase,24 U/l; maltodextrin,5%(m/v); in 100 mM sodium phosphate buffer, pH 7.5; and reacting at 30℃for 8 h. The immobilized MalPase could be easily recovered and reused for eight times in repeated synthesis of glucose-1-phosphate. After simple purification, approximately 440 mg of a crude product was afforded, with a moderate isolation yield of 70.5%.The genes of galk from E. coli K12 had been cloned and overexpressed in E. coli BL21(DE3). The reaction conditions for galactose-1-phosphate synthesis by galactokinase (GalK) was optimized as follows:the reaction mixture was composed of 36 U GalK/ml,22 mM ATP,20 mM galactose and 3.5 mM MgCl2 in 100 mM sodium phosphate buffer (pH 7.5), and incubated at 30℃for 8 h. Under the optimal conditions,0.54 g crude product was isolated from a 100-ml system, with a yield of 88.2%.