Study On The Systhesis Of 2KGA And GA By Resting Cell Of Gluconobacter Oxydans

2-keto-D-gluconic acid(2KGA)and gluconic acid(GA)are widely used as chemical intermediates in food,pharmaceutical,construction and other fields.Currently,the methods used to produce 2KGA and GA are mainly microbial fermentation,but the fermentation method has many problems,such as by-products,substrates and product inhibition and so on.In this study,G.oxydans DSM 2003 and G.oxydans#1 obtained previously in our laboratory were overexpressed respectively by using homologous overexpression technique,and then high yield strains for production of 2KGA/GA were screened from recombinant strains.Biocatalysis conditions of 2KGA or GA production by resting cells of recombinant strains were optimized,a new process of production of 2KGA with high efficiency by using resting cell catalysis method was established.Main research results and conclusions in the synthesis of 2KGA and GA were summarized as follows:The first part,the main results and conclusions about the catalytic synthesis of 2KGA1.Construction and screen of a high-yield strain of 2KGA(1)G.oxydans DSM 2003 obtained previously in our laboratory was overexpressed by using homologous overexpression technique,and a high-yield strain,G.oxydans_tufB_ga2dh for production 2KGA was successfully screened.The catalysis efficiency of the recombinant strain was 97%higher than that of the control strain.(2)The transcript levels of gene coding gox1230,gox1231 and gox1232 in the three subunits of gluconate-2-dehydrogenase in the recombinant strain,were 162 times,44 times and 59 times of that of the corresponding gene in control strains,respectively.This shows that the ga2dh gene was overexpressed successfully.(3)In the 7L fermenter,the cell concentration is 30 g/L and GA concentration is 320 g/L,productivity of 2KGA in recombinant strain was increased by 111%compared with the wild-type strain.2.Optimization of 2KGA production from GA by recombinant strains resting cells(1)Under the condition of air supply,the optimized catalytic conditions is:cell concentration 30 g/L,the GA concentration of 320 g/L,pH5.8,temperature 30?.The corresponding substrate conversion rate is about 100%,and the productivity is 12.76 g/L/h,2KGA concentration is 318 g/L,and the yield is 99%.When the substrate concentration was higher than 320 g/L,the reaction rate decreased with the increase of GA concentration,and the high concentration of substrate inhibited the cell catalytic activity significantly.(2)Dissolved oxygen is an important factor that affects the conversion of GA to 2KGA.Insufficient oxygen will significantly restrict the catalytic reaction rate.However,too high dissolved oxygen conditions lead to the loss of catalytic activity of resting cells in the reaction system.(3)Under the condition of the oxygen supply,480 g/L of GA can be completely converted o 2KGA in 45h by 60 g/L resting cells.2KGA concentration of 453 g/L achieved,the yield of 95.3%obtained,productivity is 10.07 g/L/h.Compared with the results reported in the literature,the yield of our research reached leading level.(4)The preliminary purification process of the product was established.The colorless transparent crystal was obtained.The crystal was 2KGA(sodium salt)and the purity was 98.9%.3.Optimization of the conditions of 2KGA production from glucose by resting cells of recombinant strains(1)Under the condition of air supply,the cell concentration was 60 g/L and the glucose concentration was 200 g/L,all the substrates were converted to 2KGA in 21h by using the recombinant strain,the highest space-time yield was 11.2 g/L/h,2KGA concentration reached 234.6 g/L,the yield of 98%obtained,space-time yield is increased by 365%compared with the wild-type strain.(2)The effects of the fed-batch and continuous fermentation on the catalytic reaction were investigated.The results show that space-time yield is obviously improved by fed-batch mode.When the cell concentration was 60 g/L,the optimized substrates concentration is between 250 g/L and 300 g/L,the space-time yield can be increased by 15%and 24%respectively.(3)Under the condition of oxygen supply,270 g/L glucose can be converted to 2KGA by 30 g/L resting cells in 21h,2KGA concentration reaches 316g/L,the yield of 97%is obtained,and space-time yield reaches 15 g/L/h.Compared with the existing literature,we achieved the highest level in space-time yield of 2KGA.The second part,the main results and conclusions on the synthesis of GA1.A high-yield strain of GA was successfully constructed and screenedBased on the strain G.oxydans#1 screened in our laboratory previously,the high GA yield strain G.oxydans#1_PtufB_gdh was successfully constructed and screened by using gene homologous overexpression technology.The space-time yield of GA by the recombinant strain was 14%higher than that of the wild-type strain.2.The effect of glucose concentration on production of GA by resting cell of recombinant strains was investigated.Under the conditions of aeration and cell concentration of 40 g/L,the appropriate substrate glucose concentration determined ranges from 250 g/L to 300g/L.Under optimized conditions,all glucose was consumed completely in 20h by resting cells of recombinant strain,and the GA concentration of 313 g/L was obtained,and the 2KGA concentration of 38 g/L was achieved,space-time yield of GA was 15.65 g/L/h and yield was 89%.Space-time yield of GA is increased by 115%compared with the wild-type strain.3.Using the fed-batch method,glucose of 400 g/L was converted to 371 g/L GA and 40 g/L 2KGA by 40 g/L resting cells in 32h,space-time yield of GA was 11.6 g/L/h,and the selection rate was 93%.