Immobilization Of Transaminase And Its Application On Sitagliptin Production

Sitagliptin is the active ingredient in Januvia,a leading drug for the treatment of type 2 diabetes.Merck,in collaboration with those from Codexis,have recently reported on their process research towards the industrial manufacture of sitagliptin phosphate,which is the fourth generation of sitagliptin manufacture involving an asymmetric amination from an important intermediate,sitagliptin ketone,to sitagliptin using an immobilized transaminase.The production process shows many drawbacks including adding a lot of organic solvent,bad operational stability.In this work,We will develop a strategy for immobilization of the engineered transaminase from E.coli,which can catalyse the production of Sitagliptin.This paper was focused on the process of immobilization,the characteristics of immobilized transaminse and the possibilities of using immobilized transaminse to produced Sitagliptin in a packed bed reactor.Firstly,we researched on the enzyme immobilization process.After screening of immobilization support in our Lab,a domestic epoxy resin made of methyl methacrylate polymer,ES-103b,was chosed as carrier to immobilize the Sitagliptin transaminse by multipoint covalent binding.The optimal conditions for immobilization were determined as follows:25?,the resin and enzyme solution was 1:10,enzyme lyophilized powder was added to the 1 M of potassium phosphate solution?adjusted to pH7.0?to form the immobilization system?5.2 U/mL,2m M PLP?.The immobilization system was incubated for 24 h and got the immobilized enzyme with 78 U/g wet support.Then,this immobilized enzyme was placed on an incubator with pH9.0 100 m M potassium phosphate buffer?containing 20%glycerol?at 30?for 30 h.At last,the resin was incubated in 3 M Glycine-NaoH Buffer at 25?for 12 h and finally got the immobilized enzyme with 45.6 U/g wet support.In the above condition,we scaled up the production of immobilized transaminase to500 mL system and the activity of immobilized enzyme was about 52.7U/g wet support with activity recovery of 49.3%.Then we investigated the characteristic of the immobilized transaminase.The optimal reaction temperature was 50?,the half-time of the immobilized transaminase at 40?,50?and 60?were 26.04day,16.18 day and 7.99 day,respectively.Compared with free enzyme,the optimal reaction pH didn't change.The activity changed little when the pH increased from 7.0 to 9.0,while it had a drastic drop above pH9.0.The enantioselectivity of product was above 99.9%.Before the immobilized transaminase was employed to synthesis Sitagliptin,some reaction conditions such as amine donor and its concentration,cosolvent,effect of subsrate/product concentration on enzyme acitivity were well investigated.When the concentration of substrate was 30g/L,space-time yield reached the highest(0.81 g.L^-1h^-1g^-1)with a conversion of 52%in 20 hours,while the reaction stopped and began reverse when the product concentration was above 15 g/L.We selected DMSO as the cosolvent for the highst conversion of 40%in 4hours.We choose isopropylamine as the amine donor with the concentration of 50 mM.Finally,a packed bed reactor?PBR?was used to produce Sitaglitin.The optimized operation parameters were examined as follows:200 mL,30g/L of substrate was recycled through the PBR with a feeding rate of2.6 mL/min,the H/D of the PBR was 11 and the immobilized transaminase load was 30.0 g.The 50%conversion of substrate only needed about 15 h in the optimized condition.The trasamination catalysed by immobilized transaminase in PBR was operated for 10batches and the conversion of substrate was almost unchanged.