title: [Overexpression and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei].
abstract: OBJECTIVE
  Expression, purification and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei.
  
  
  METHODS
  The strong promoter and terminator of cellobiohydrolase I (cbh1) gene from T. reesei were amplified by PCR and inserted into pBluescriptIISK(+) to form vector pSKCST. The laccase gene from Pleurotus ostreatus was de novo synthesized according to T. reesei condon bias and cloned into the vector pLacdt resulting in the expression vector pSKLDT. The linearized pSKLDT was introduced into T. reesei strain Tu6 by protoplast-mediated transformation. The screened laccase expression transformants were grown in shake flasks on minimal medium and the recombinant laccase was purified and characterized.
  
  
  RESULTS
  Transformants were isolated in selective screening medium plate and identified by PCR. The enzyme activity of laccase in transformant LC-7 was 237.134 U/mL which was 28.6 -fold higher than that in P. ostreatus. The specific activity of the purified enzyme was 9852 IU/mg. Enzymatic assay revealed that the optimum temperature for its activity was 50 degrees C and pH was 3.0. The optimum substrate was ABTS and the K(m) and V(max) for ABTS were 7.58 x 10(-2) mmol/L and 9.752 x 10(-3) mmol/L/min. Metal ions like Cu2+, Zn2+, Fe3+, Mn2+, Ba2+, Mg2+ and Fe2+ had different inhibitory effect on purified laccase.
  
  
  CONCLUSIONS
  Under the regulation of cbh1 promoter and cbh1 signal peptide, heterologous laccase was successfully overexpressed in T. reesei.
authors.affiliation: College of Life Science, Engineering Research Center of Industrial Microbiology, Fujian Normal University, Fuzhou 350108, China. xinrui403@126.com
authors.forename: Xinrui
authors.initials: X
authors.lastname: Dong
authors.forename: Lina
authors.initials: L
authors.lastname: Qin
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Huang
authors.forename: Zhiyang
authors.initials: Z
authors.lastname: Dong
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D004795:Enzyme Stability; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature; D014242:Trichoderma / Q000235:genetics*
chemical_list: D008670:Metals; D042845:Laccase