Cloning And Expression Of Thermomyces Lanuginosus ZJB09222 Lipase And Its Application In Production Of (3S)-Cyano-2-Earboxyethyl-5-Methylhexanoieacid

As a new generation of anti-epileptic drug,pregabalin,with more toleration and higher safety,was more extensively used in industry than others.The production of pregabalin through chemoenzymatic approaches has been a hot issue.Meanwhile,it also enhanced the development of intermediates,such as(3S)-cyano-2-carboxyethyl-5-methylhexanoic acid which was the most representative intermediate of pregabalin.The existing production process,which was easy to be copied,always used Lipolase(from Novozymes)as the biocatalysis.Thus,we focused on the construction of recombinant plasmid carrying Thermomyces lanuginosus ZJB09222 lipase to realize the transformation from racemic of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to(3S)-cyano-2-carboxyethyl-5-methylhexanoic acid.It's consistent with green-chemistry and the request for sustainable development as a new process.Based on published DNA sequence,the full length cDNA of the lipase was cloned using RT-PCR method.It revealed that the cDNA of the T.lanuginosus lipase gene encoded a protein lipase of 292 amino acid residues.The open reading frame of the cDNA was cloned into the plasmid pET28b,which was then transformed into E.coil BL21(DE3)strain.The Mrof the expression product was 32 ku.The recombinant E.coli BL21/pET28b-TLL exhibited highest activity of 41 U/mL at 28? for 7 h induction with 0.lmmol/L IPTG after screening of the best medium.In addition,the biotransformation of racemic of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to(3S)-cyano-2-carboxyethyl-5-methylhexanoie acid was investigated using recombinant lipase.Influences of reaction conditions on lipase activity and enantioselectivity were also investigated.Results indicated that optimal working pH of the lipase was 7.5 in 50 mM Tris-HCI buffer.The lipase showed highest activity at 45?,but its enantiomeric ratio decreased with increasing of temperature.MnCl2 was the optimal addition agent.It has reached the end of reaction in 4 hours,with 150 mM MnCl2,which had saved reaction time.Addition of cosolvent,n-hexane and THF,had significant effect not only on enzyme activity but also on enantioselectivity.The E-value increased to 162 by addition of 7.5%(v/v)n-hexane.It conformed that the higher concentration of n-hexane had improved enantiomeric ratio and membrane permeability by pretreating the reaction with 5%and 8%of n-hexane within different time.But it would cause the lost of enzyme activity for a long pretreat time.Further more,the enzyme activity arrived at 30.9 U/g,which was 3 times more than before,under the optimized conditions.