Process Study Of Sitagliptin Using Whole Cells And Its Process Design On A 200 Tons Per Year Scale

Sitagliptin is an efficient inhibitor of dipeptidyl peptidase IV(DPP-4),which can be used to protect and improve the phsiological function of the endogenous incretin.It has become the best selling oral hypoglycemic agents(OHA)for type II diabetes.The key step in sitagliptin production is the preparation of chiral amines,which can be prepared from sitagliptin precursor ketone via chemical and enzymatic method.As to the chemical method,expensive rhodium,chiral ligand and asymmetric hydrogenated amine are required,which results in the high cost and environment pollution.The aminotransferase based enzymatic method is more preferable due to the mild reaction condition and the excellent enantioselectivity.Therefore,it is more sustainable and has attracted a wide spread attention.To eastablish an efficient and ecofriend method for sitagliptin,the cultivation of ZJB ATA-1(a recombinant transaminase procucing Escherichia Coli strain)and biocatalysis process of the resulting transaminase were investigated and optimized.The optimized cultivation and biocatalysis process were further successfully scaled-up to 5000 L fermenter and 2000 L reactor,respectively.Based on the above results,an enzymatic 200 t/a sitagliptin process was designedFirstly,the cultivation processes of ZJB ATA-1were successfully scaled-up to 50 L,500 L and 5000 L fermenter from a 5 L fermenter with equal aeration ratio principle.Then the parameters for the 5000 L fermenter cultivation were optimized and the optimum conditions were as following: the first-class seed(shake flask)with 7?9 h inoculum age,the second-class seed(50 L)with 3?5 h inoculum age,the third-class seed(500L)2?3 h inoculum age,tap water,industrial glycerine,10%(v/v)inoculum size,0.6(v/v)medium loading and 12.5 g/L lactose.Under above conditions,after 12 h cultivation,the maximum biomass,specific enzyme activity and volumetric enzyme activity reached 12.7 DCW g/L,367.04 U/g and 4661.35 U/L,respectively.The effect of resting cells and cell lysate of ZJB ATA-1 on the biocatalysis was investigated and the resting cells were chosen for sitagliptin production.Moreover,the reaction process was optimized and the optimum parameters were as following: 0.2 M triethanolamine buffer system,pH 8.5,45?,1 mM PLP and wet cells 100 g/L.Under above conditions,100g/L substrate was completely converted to sitagliptin within 24 h.The e.e.value of the resulting sitagliptin was over 99.9%.Finally,based on the above results,a 200 t/a sitagliptin process was designed.