Microbial Proline Racemase-proline Dehydrogenase Cascade For Efficient Preparation Of D-proline

D-proline is an important chiral intermediate of serotonin analogues such as antimigraine drug eletriptan.At present,the chemical asymmetric transformation technology is used for the industrial production of D-proline.This technology has the disadvantages of high production cost and large environmental pollution.The preparation of optically active compounds by bio-catalysis has become a research hot-pot because of its environmental friendliness.In this thesis,a cascade catalytic process of proline racemase-proline dehydrogenase is developed for the efficient preparation of D-proline,including the racemization of L-proline and the enantioselective degradation of L-proline in DL-proline.Bio-racemization of L-proline.A proline racemase engineering strain was constructed.The culture conditions of recombinant bacteria and the racemic reaction conditions were optimized respectively.The optimum culture conditions recombinant bacteria were as follows: LB medium added with 3 g/L glucose,culture temperature25℃,initial p H 7,IPTG 0.2m M,inducer time 18 h.The optimum racemic reaction conditions were as follows: cell concentration 1g wet weight cell/L,1%(v/v)toluene as permeabilizer,temperature 37℃,p H6.5.Within 48 h,L-proline almost to 1000 g/L could be racemized to DL-proline with 1 g/L of wet Escherichia coli cells expressing Pro R.Strains were screened for the preparation of D-proline by enantioselective degradation.The results showed that Pseudomonas pseudoalcaligenes XW-40 was the dominant strain.The fermentation conditions of Pseudomonas pseudolcaligenes XW-40 for the production of D-proline were optimized with DL-proline as the precursor.The optimal medium was as follows: peptone 3 g/L,beef extract 1g/L,sodium chloride 5g/L,yeast extract 3 g/L,DL-proline 12 g/L.The optimal culture conditions were as follows:p H7.0,temperature 30℃,culture time 48 hours.D-proline of 6 g/L was produced in the fermentation stage.In the bio-transformation stage,the feeding concentration of DL-proline in a single batch was 10 g/L,and the feeding times reached 5 batches.Through the cascade of fermentation-biotransformation,the D-proline was accumulated with 31 g/L and more than 99% enantiomeric excess.The product of L-proline was protected by Boc-group and obtained N-(tert-butoxycarbonyl)-5-hydroxy-L-proline(an alternative chiral intermediates of saxagliptin for treatment of type 2 diabetes mellitus),which confirmed that the enzyme catalyzed by the reaction was proline dehydrogenase.Preparation of D-proline by resting cell of Pseudomonas pseudolcaligenes XW-40.The optimal conditions were as follows: cell concentration 160 g wet weight cell/L,DL-proline 20 g/L,temperature 30℃,p H7.5.Using the Aeration-cell cycle strategy,the cells could be recycled in 9 batches,and the reaction time of each batch was shortened to one quarter of that without aeration.After separation,18 g DL-proline can produce8.2g D-proline,and the D-proline was obtained in 45.7% with an enantiomeric excess of99.6%.Moreover,N-boc-5-hydroxy-L-proline was also synthesized from L-glutamate semialdehyde,a dehydrogenated product of L-proline,in a 16.7% yield.Construction of proline dehydrogenase engineering strain and evaluation of its catalytic activity.A proline dehydrogenase engineering strain was constructed with E.coli BL21 as the host.The preliminary results showed that the recombinant cells could catalyze the enantioselective degradation of L-proline in DL-proline.Further research will lay a technical foundation for the industrial production of D-proline by genetically engineered proline racemase-proline dehydrogenase cascade.