Purpose: Recently discovered Anabaena variabilis PHENYLALANINE AMMONIA LYASE (Av PAL) proved to be a good candidate for enzyme replacement therapy of phenylketonuria. Outstanding stability properties of a mutant version of this enzyme, produced already in our laboratory, have led us to the idea of culture conditions optimization for soluble expression of this therapeutically valuable enzyme in E. coli.Methods: In the present study, the gene encoding mutant version of Av PAL was cloned into the pET28a expression vector. Different concentrations of IPTG, induction period, growth temperature, shaking speed, as well as different types of culture media were examined with respect to the amount of recombinant protein produced and specific activity of the enzyme.Results: Based upon our findings, maximum amount of active mutant enzyme was attained by addition of 0.5 mM IPTG at 150 rpm to the TB culture media. The yield of active enzyme at cluture tempreature of 25 oC and induction period of 18 hour was the highest.Conclusion: The results of this study indicated that the yield of mutant Av PAL production in E. coli can be affected mainly by culture temperature and inducer concentration.