Study Of Cloning And Expression Of A α-Hydroxynitrile Lyase CDNA From Cassava

Different stereo isomers of chiral compounds have different biological activity, a lot of medicines are required to be in form of unitary compounds. α-Hydroxynitrile Lyase(E.C.4.1.2.10, HNL) from the c-yanogenic crop cassava as a kind of biocatalyst could catalyze reversi-bly the condensation of hydrocyanic acid with aldehydes or ketone into (s) -cyanohydrins, which are valuable starting materials for various optically active compounds such as hydroxy compound, amine compound, carboxyl group compound and so on, and further they can convert into multi - chiral drugs. On the other hand, usage of chemical method for chiral resolution for compounds has many disadvantages such as complexity of the process, increasing the cost of production. So HNL is an increasing focus of studying in the field of asymmetric synthesis.Recently many studies about enzymatic reaction mechanism, gene cloning and expression of HNL have been reported. Gene of cDNA of from several plants were cloned, HNL cDNA from Heveabrasiliensis have been expressed in S.cerevisiae and Escherichia coli. And HNL cDNA from cassava have been expressed in E.Coli. Interiorly it is only reported that HNL cDNA from cassava were expressed in ￡ Coli, but no one report that HNL cDNA from cassava were expressed in Pichia pastoris.The cDNA of a -HNL were obtained by RT-PCR and cloned. The sequencing result for the cDNA showed that the sequence encoded for the HNL was not fully consistent with those published. The full sequences analysis demonstrated that the highest homology of cDNA sequence was about 99.1 % and the highest homology of amino acid sequence about 98.8 % to other four reported HNL genes from cassava. The cDNA was cloned into an expression vector pPIC3.5K by PCR and recombinant DNA technique. After the transformation of pPIC3.5K - HNL, screening for high copy transfor-mahts^ induction with methano^ SDS-PAGE analysis^ enzymatic activity assay, the HNL was, for the first time, efficiently expressed in Pichia pastoris and reached over 5040 units/L of culture.In order to more efficiently express HNL and reduce cost of producing, the cDNA of HNL was expressed in ￡. Coli. On the basis of cloning and sequencing Hydroxynitrile Lyase cDNA from Cassava, the cDNA fragment was cloned into expression vector pBV220, which was then transformed into K Coli DH5 by the method of CaCl2 transformation. Recombinants were obtained by LA plates and confirmed by restriction analysis. The recombinant protein was induced to express by changing temperature and existed in form of inclusion body. The expression product of HNL gene was analyzed by SDS-PAGE and enzyme activity, the results indicated t-hat HNL gene was expressed successfully in E coli and it reached over 4540 units/L of culture.what is said above laid the solid groundwork for getting a great of HNL, studying mechanism of HNL action and developing commercial production in the future.