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The chiral drug industry has soared in recent years,now represent close to one-third of alldrug sales worldwide.Many of these drugs are peptides.The precursors for synthesizingthose peptide drugs are often chiral non-natural amino acids,which are expensive tomanufacture.Enzymes offer an alternative to chiral chemical catalysis,but since thesubstrates are artificial compounds,enzyme specificity is often a limitation.Among theenzymes able to produce chiral amino acids are the aminotransferases,which catalyse thereversible transfer of an amino group to a ketoacid,usually from L-glutamate as donor.Escherichia coli TGI houses four such aminotransferases.The overall objective will be aset of four mutant transaminases from E.coli,which efficiently produce a wide range ofnon-natural amino acids for pharmaceutical industry.
Four transaminase genes have been cloned and then four enzymes (TyrAT、AspAT、BCAT and AvtAT) were successfully over-expressed and purified with four-steppurification(ammonium sulphate precipitation;iron exchange chromatography;hydrophobic interaction chromatography and gel filtration).Purified enzymes wereobtained in all 3.525mg TyrAT,4.2mg AspAT,2.43mg BCAT and 4.6mg AvtAT,respectively.Subsequently,substrate range and kinetic parameters for each transaminase scheduled to bedefined carefully using non-natural amino acids.However,there is no common method fordeterminating activity and substrate specificity for all aminotransferases.Thus,threecoupling systems and new modified HPLC method was developed.Due to the significantproduct inhibition and low activity of the coupled enzyme,three coupling system are notsuitable for accurate kinetic measurement,but still could be used in screening.Themodified HPLC method has been developed utilizing mobile phase of 97％ M.Q.water/3％ methanol (both of the solvent added with 0.1％ TFA);and running time of 40 min.Thevelocity of aromatic transaminase was 1.2 μM/min determined by HPLC.
Since directed evolution depends on an efficient screening procedure,an effective highthroughput screening method for aminotransferases by coupling with glutamatedehydrogenase (GDH) has been developed.NADH production was linked to the reductionof a tetrazolium dye,forming a formazan to be detected spectrophotometrically.Next,coupling GDH screening was used to determine the substrate specificity of eachtransaminase towards thirty-six non-natural amino acids.Very excitingly,the specificityappears to be extensive,for instance TyrAT shows high activity towards a series ofstructure derivative of L-phenylalanine.Likewise,BCAT displays activity to both L-tert-leucine and D-alanine.In order to confirm the results obtained,further experiments need tobe done by HPLC to acquire more precise parameters.