Expression of a laccase cDNA from Trametes sp. AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation.
A laccase cDNA from Trametes sp. AH28-2 was expressed in Pichia pastoris, with the highest expression level of 4.0 mg L-1 (1360 U mg-1). The apparent Km (24.6 microM) for ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) and the carbohydrate content of the recombinant laccase A (rLacA) are approximately identical to those of the native LacA (nLacA). However, the two enzymes differed in the pH optimum when both ABTS and guaiacol served as substrates. The optimum pH for enzyme stability is 5.5 for rLacA. Thermal stability was also investigated. The mutagenesis of rLacA utilizing low-energy nitrogen ion implantation resulted in the isolation of a yeast clone that produced 7.7 mg L-1 (1085 U mg-1) of laccase, 92.5% more than the nonirradiated control (4.0 mg L-1). Compared with rLacA, the mutant LacA (mLacA) with five amino-acid residue changes in the coding sequence showed a slight change in its catalytic ability but superior thermal stability.
Hong
Yuzhi
Y
School of Life Sciences & Modern Experiment Technology Center, Anhui University, Hefei, China.
Xiao
Yazhong
Y
Zhou
Hongmin
H
Fang
Wei
W
Zhang
Min
M
Wang
Jun
J
Wu
Lijun
L
Yu
Zengliang
Z
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000235:genetics*; D008970:Molecular Weight; D016296:Mutagenesis; D009584:Nitrogen; D010843:Pichia / Q000235:genetics*; D020072:Polyporales / Q000201:enzymology*
D018076:DNA, Complementary; D042845:Laccase; D009584:Nitrogen