High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes.
- Author:
Teng-Jiao CUI
1
;
Xiao-Tang WANG
;
Hong-Min ZHOU
;
Yu-Zhi HONG
;
Ya-Zhong XIAO
;
Teng-Jiao CUI
;
Xiao-Tang WANG
;
Chun-Lei PU
Author Information
1. School of Life Sciences & Modern Experiment Technology Center, Anhui University, Hefei 230039, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Fermentation;
Isoenzymes;
biosynthesis;
genetics;
Laccase;
biosynthesis;
genetics;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
Trametes;
enzymology;
genetics
- From:
Chinese Journal of Biotechnology
2007;23(6):1055-1059
- CountryChina
- Language:Chinese
-
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).
