Optimization Of Fermentation Process And Cloning And Expression Of ?-D Glucuronidase Produced By Endophytic Fungus Chaetomium Globosum S108

Monoglyoxylic acid glycyrrhetic acid(GAMG)as a derivative of Glycyrrhizin(GL)has stronger bioavailability and physiological activity than GL,and its sweetness is 5 times that of GL,which has important application value.An endophytic fungus strain Chaetomium globosum S108 which can produce?-D-Glucuronidase(GUS)was screened from Dongxiang wild rice by our laboratory in previous work.The GUS can efficiently catalyze GL to produce GAMG.However,the low production of GUS enzyme by strain S108 has limited its industrial application.Therefore,in order to improve the activity of GUS produced by C.globosum S108 and reduce the cost of production of GAMG,this study provide the strategy for production of GAMG using microorganic fermentation.The effects of fermentation medium and fermentation conditions for production of GUS using C.globosum S108 were systematically investigated,including optimize the fermentation process of 250 m L shake flasks and 5 L fermenters.In addition,the GUS gene was also cloned and expressed using molecular cloning technology,and the enzymatic activity of the recombinase was investigated.The results were obtained as follows:1.The optimization of carbon source for producing GUS using fermentation of C.globosum S108 at shake flask culture was performed.The results showed that the optimal carbon source was sucrose.The enzymatic activity reached the maximum(198 U/m L)under the conditions of 5 g/L sucrose concentration.2.The optimization of nitrogen source was investigated for the production of GUS by C.globosum S108 in shake flask fermentation,and it was found that peptone was the most suitable nitrogen source,and the optimum concentration was 7 g/L.3.Under the optimal conditions of 5 g/L sucrose and 7 g/L peptone,C.globosum S108 was used to produce GUS.The result was about 3.3 times higher than the control,reaching 645 U/m L.Under the optimal conditions,the morphology of C.globosum S108 was further observed.It was found that under the conditions of peptone as nitrogen source,the morphology of C.globosum S108 was sparsely spheroidal,but there was no significant difference in carbon source bacterial cells.4.Based on the optimization of the shake flask fermentation process,a scale-up experiment of the fermentation tank was carried out,and the relevant control conditions of the fermentation tank were optimized.In the 5 L fermenter scale-upculture,the type of agitator and the stirring speed were optimized.Under the condition of enough dissolved oxygen,the results show that the type of the stirrer was arrow blade type,the rotation speed was 200 r/min,the fermentation result was the best,and the GUS enzymatic activity was reached at 792 U/m L.5.The gene cg-GUS encoding of GUS was successfully cloned from the genomic DNA of C.globosum S108 by PCR method.The gene contain 1893 bp,encode 631 amino acids,and the molecular weight is about 70 KDa.GUS prokaryotic expression vector p EASY-Blunt-gus8902 was constructed and transformed into E.coli BL21(DE3).The recombinant enzyme was induced for expressed,and the results showed that the activity of the recombinant enzyme was lower than the native GUS.