title: [Expression and characterization of Coprinus cinereus peroxidase].
abstract: OBJECTIVE
  The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently.
  
  
  METHODS
  We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115.
  
  
  RESULTS
  We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol.
  
  
  CONCLUSIONS
  The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.
authors.forename: Bingxue
authors.initials: B
authors.lastname: Dong
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Niu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Sanchun
authors.initials: S
authors.lastname: Geng
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Wanli
authors.initials: W
authors.lastname: Yuan
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Gong
authors.forename: Kewei
authors.initials: K
authors.lastname: Liang
mesh_terms: D003302:Coprinus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D009195:Peroxidase