[High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes].
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).
Cui
Teng-Jiao
TJ
School of Life Sciences & Modern Experiment Technology Center, Anhui University, Hefei 230039, China.
Wang
Xiao-Tang
XT
Zhou
Hong-Min
HM
Hong
Yu-Zhi
YZ
Xiao
Ya-Zhong
YZ
Cui
Teng-Jiao
TJ
Wang
Xiao-Tang
XT
Pu
Chun-Lei
CL
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
D007527:Isoenzymes; D011994:Recombinant Proteins; D042845:Laccase