title: [High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes].
abstract: A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).
authors.affiliation: School of Life Sciences & Modern Experiment Technology Center, Anhui University, Hefei 230039, China.
authors.forename: Teng-Jiao
authors.initials: TJ
authors.lastname: Cui
authors.forename: Xiao-Tang
authors.initials: XT
authors.lastname: Wang
authors.forename: Hong-Min
authors.initials: HM
authors.lastname: Zhou
authors.forename: Yu-Zhi
authors.initials: YZ
authors.lastname: Hong
authors.forename: Ya-Zhong
authors.initials: YZ
authors.lastname: Xiao
authors.forename: Teng-Jiao
authors.initials: TJ
authors.lastname: Cui
authors.forename: Xiao-Tang
authors.initials: XT
authors.lastname: Wang
authors.forename: Chun-Lei
authors.initials: CL
authors.lastname: Pu
mesh_terms: D003001:Cloning, Molecular; D005285:Fermentation; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D042845:Laccase / Q000096:biosynthesis* / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D055454:Trametes / Q000201:enzymology* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D042845:Laccase