Integrated Bioprocess Of Cellulase-catalysed Conversion And Three-liquid-phase Extraction Of Diosgenin

Diosgenin is an important starting material in the steroidal hormone industry. In Dioscorea Zingibernsis C.H. Wright (DZW), diosgenin is connected with glycosyl as steroidal saponin. In industry, diogenin is prepared mainly using acid to hydrolyze DZW tubers. However, this process brings serious pollution. Enzymatic hydrolysis has many strong points, such as high selectivity, not violent reaction conditions, environmentally friendly and lower energy consumption. Conversion efficiency suffers from the substrate inhibition and the product inhibition. In order to solve this problem, we used the three-liquid-phase extraction (TLPE). TLPE is a kind of method which is a promising technology to separate different compounds in a complex system. It was effective for the separation of diosgenin and steroidal saponins as well as other active components with different polarity. Enzyme hydrolysis occurrs in the three-liquid-phase system (TLPS) to make full use of the characteristic of enzyme hydrolysis and TLPE. Coupling of extraction and cellulase-catalysed conversion of steroidal saponins to diosgenin would be desired to remove inhibition and obtain high yield of diosgenin.Firstly, the substrates and enzyme should be distributed in the different phases in the TLPS appropriately in order to achieve the coupling effect. In addition, the system should keep vitality of cellulase to achieve the conversion of steroidal saponins efficiently. We also investigated the influences of enzyme concentration and temperature on the reaction. After screening, the concentrations of 1,4-dioxane, ammonium sulfate and n-hexane in TLPS were determined as 16.1%(w/w),10.5%(w/w) and 21.0%(w/w), respectively.The other coupling conditions were determined:transformation temperature for 35℃; pH 5.0; enzyme concentration 0.051 U/g.Secondly, we used an TLPS to integrate bioprocess of cellulase-catalysed conversion and extraction of steroidal saponins and diosgenin. The steroidal saponins from DZW and cellulase are distributed in the middle phase (1,4-dioxane phase).74.2%of hydrolyzed glucose is distributed in the bottom phase (ammonium sulfate phase), which could effectively relieve the product inhibition to enzyme. Enzyme activity retains well during reaction in the TLPS:the residual activity of enzyme is still 54%after 96h, and the yield of diosgenin is 69.4%. The yield of diosgenin in the TLPS is double to the yield of diosgenin in the same concentration of 1,4-dioxane, and 27.6 times as in the aqueous phase.Finally, PEG was used to modify enzyme and improve the stability of cellulase in TLPS. The steroidal saponins from DZW and cellulase are distributed in the middle phase (1,4-dioxane phase).75.8%of hydrolyzed glucose is distributed in the bottom phase (ammonium sulfate phase), which could effectively relieve the product inhibition to enzyme. Enzyme activity retains well during reaction in the TLPS:the residual activity of enzyme is still 70%after 96h. And the yield of diosgenin is 85.2%, whichincreases by 16%. It turned out that the modification of cellulase is good for the conversion of steroidal saponins into diosgenin. This way provides a new approach for improving the integrated effect of cellulase-catalysed conversion and extraction of steroidal saponins and diosgenin using an TLPS, and could provide a possible method of conversion and extraction of natural products.