Directed Evolution Of Soybean Peroxidase

Soybean peroxidase （SBP） belongs to class III of the plant peroxidasesuperfamily（EC1.11.1.7）. Compared with the similar peroxidase，it has obviousadvantage, such as wider substrate scope, higher heat resistance, better acid-basestability, no pollution, higher safety coefficient and scource easy to get. At present,SBP has broad prospect of application in food processing, biomedical testing,industrial wastewater treatment and high polymer materials synthesis area. However,as the natural protein enzymes, its catalytic efficiency, substrate affinity and stabilityhave to be further improved. In this thesis, we use directed evolution technology,combining gene mutation and high-throughput screening to enhance catalytic activityof SBP. By doing so, SBP can be better adapted to industrial application requirements.Prone PCR become the most widely used means of the evolution because of itsadvantages of simple and effective. The research content and results are as follows:1. Using Pichia pastoris expression systems provided by Invitrogen Corporation,recombinant plasmids pPICZα-A-sbp were transformed into host cells Pichia pastorisGS115by means of electric transformation, and successfully expressed bioactive SBPwith methanol induced.In order to make Pichia pastorios express SBP as high as possible, the testconducted a preliminary comparative study in different inoculum size, methanolconcentration, pH and induction time, and get the optimal conditions: inoculum size100ml/100ml, methanol adding amount0.75%, inductive medium pH6.0, theinduction time48h. The SBP enzyme activity is19.7U·ml-1. It lays the foundation forscreening SBP mutants of high catalytic activity effectively, and provides a referencefor future mass production of SBP in fermenters.2. In order to improve enzymatic activity of soybean peroxidase（SBP） and studythe correlation between its primary structure and enzymatic activity, we use twosequential error-prone PCR method on soybean peroxidase genes（sbp）. The secondround error-prone PCR products and plasmid vectors pPICZα-A are double enzymedigested respectively. After the PCR products ligated into the vectors by T₄DNAligase, recombinant plasmids pPICZα-A-sbp are transformed into Escherichia coli DH5α through calcium chlorid method. A random mutation library is constructed, andthe capacity is about1.77×10⁶cfu.3. After the random mutation library is constructed, how to screen beneficialmutant efficiently from the library has become the key to the enzyme in vitroevolution experiment. In this experiment, we use microplate screening method greatlyimproving sample processing speed, increasing the flux of the screening and savingthe time of screening. So far, we have screened576mutants，and have not yet foundthe SBP mutant that the catalytic activity significantly increased.