Study On The Effect Of Inducible Expression Of Hyaluronidase On Yield Of Hyaluronic Acid

Hyaluronate,also known as hyaluronic acid,is a high molecular weight,linear mucopolysaccharide.It is widely used in cosmetics,pharmaceuticals and other industries.Streptococcus zooepidemicus is the predominant strain of HA production industrially.HA is the main component of the capsular.Currently,the production process of hyaluronic acid are animal tissue extraction and microbial fermentation.Microbial fermentation method is more popular.However,the high viscosity and low fermentation dissolved oxygen transfer rate is major limiting factors for microbial fermentation.Streptococcus zooepidemicus lack of complete electron transport chain,and therefore can not aerobic phosphorylation,resulting in a very low capacity levels.If we can improve the level of dissolved oxygen,it will increase the HA production.In this study,Streptococcus zooepidemicus ATCC39920 as the research object,by cleavage of hyaluronic acid-induced gene expression and cellular research to improve the fermentation of exocrine status quo;and the use of the laboratory has established a controllable two-step fermentation mode production of HA.The main findings are as follows:1.Construction and analysis of different sucrose inducible expression systemPlasmid pLH243 that has been completed stably replicated as a starting carrier and insert.scrR-P_scrA regulatory elements to build carrier pLH256.Then insert S_amyA-hylA,S_amyA-hylB,ShylB-hylB behind scrR-P_scrA regulatory elements in order to build sucrose-inducible expression vector pLH268?pLH278?pLH279.By analyzing the inducible expression system shows:In the expression system with plasmid pLH278 that the HA has the highest yield,higher than the wild-type strain of HA nearly 17%.2.Establish a controlled two-step fermentation mode to produce HAThe pLH268?pLH278?pLH279 were transformed into wild type strain of Streptococcus zooepidemicus.According to the two-step fermentation pattern paper proposes to:In 0-10 hours,added to an initial concentration of 30 g/L glucose in order to obtain a high cell density.In 10-24 hours,added to an initial concentration of 20 g/L of sucrose,in order to induce the expression of hyaluronan lyase.With the propdse two-stage culture strategy,HA production of strain S239 was 4.13 g/L,and HA yield of strain S12 was 3.52 g/L.From the results we can see that HA production increased by 17.3%.