Establishment of a high-throughput screening method for 3-sterone-Δ1-dehydrogenase mutant library
[Objective]To establish a fast and high-throughput method for screening the mutant library of 3-sterone-Δ1-dehydrogenase(KstD).[Method]Based on the crystal structure of published KstD protein,saturated mutation primers were designed,mutant library was constructed,protein expression conditions were optimized,and crude enzyme solution was obtained by freeze-thaw method combined with lysozyme method.Based on the principle of spectrophotometry,the detection reaction system was optimized,and the change of absorbance was detected by enzyme marker to reflect the enzyme activity.[Result]The mutant library was successfully constructed using saturated mutant primers.The optimal conditions for expression of KstD were 0.3 mmol/L final concentration of IPTG and induction at 20 ℃ for 20 h.When the concentration of lysozyme in buffer was 0.5 mg/mL,the target protein content of supernatant was the highest.The optimized enzyme activity detection conditions are as follows:Tris-HCl buffer solution consisted of 50 mmol/L,pH 8.0,0.4 mmol/L DCPIP,1.5 mmol/L phenazine methyl sulfate(PMS),0.7 mmol/L stenodione and appropriate enzyme solution at 30 ℃ and reaction time of 5 min,and was determined at 600 nm wavelength.[Conclusion]A fast and high-throughput method for screening the mutant library of 3-sterone-Δ1-dehydrogenase(KstD)was successfully established.
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