Tryptophanase Of Genetically Engineered Bacteria Construction And Expression

Using PCR technology, a 2.4kb DNA fragment, part of tryptopanase operon, containing a promoter, a regulator gene TnaC located downstream of the promoter and a desired tryptopanase gene TnaA which can be expressed by the promoter from E.coli k-12, was cloned to pMD18-T vector. The DNA sequence is the same as which was published on SCIENCE.To prove that the cloned DNA fragment can express tryptopanase,a new plasmid pET28C-TnaA , in which the cloned DNA fragment was located downstream of T7 promoter on pET28c was constructed and transformed into host BL21(DE3),a BL21 lysogen of bacteriophage DE3 in which the only promoter known to direct transcription of the T7 RNA polymerase gene is the lacuvS promoter ,which is inducible by IPTG. Addition of IPTG to growing culture of the lysogen induces T7 RNA polymerase ,which in turn transcribe the target DNA in the plasmid. In the presence of glucose and appropriate conditions such as temperature and concerntration of IPTG, a 52KD protein with tryptopanase activity was expressed. Temperature and the concerntration of IPTG are crucial factors for expressing tryptopanase with high activity. A conclusion can be drawn that tryptopanase expressed only by constructed plasmid during "catabolite repression".Induced either by IPTG or L-tryptophan in the absence of easily metabolized carbon such as glucose the strain BL21(DE3)/ PET28C-TnaA can express tryptopanase.The fermentation conditions were optimized respectively. In the presence of glucose and IPTG as induce agent, the concentration is the crucial factor for expression of active tryptophanase. If the IPTG concentration is less than 0.2mM, the optimum temperature is 37 Lower temperature is necessary to obtain active tryptophanase in the case of higher concentration of IPTG. The presence of Calcium is beneficial to obtain higher tryptophanase activity .The activity of tryptophanse expressed by addition of appropriate concerntration of IPTG is 91-fold higher than that of no induction. The highest specific activity was...