[Expression of a laccase gene from Pleurotus ostreatus in Pichia pastoris and characterization of the recombinant enzyme].
A Pleuotus ostreatus laccase gene was cloned by RT-PCR and designated as lccPol. Its sequence was submitted to GenBank with the accession number AY450404 obtained. The open reading frame was transformed into three Pichia pastoris strains GS115, KM71 and SMD1168, respectively, under control of the AOX1 promoter by using the vector pHBM906. LCCPo1 can be expressed by all three P. pastoris recombinant strains. Three different strategies for shake-flask cultures were compared: (1) (25 degrees C, 1.0% methanol), (2) (20 degrees C, 1.0% methanol), (3) (20 degrees C, 0.5% methanol). The laccase activity could be improved by increasing the methanol concentration befittingly. The results showed that the cultivation temperature had a marked effect on the production of active heterologous laccase. 2 - 6 folds higher laccase activities were obtained when the cultivation temperature was kept at 20 degrees C instead of 25 degrees C. The highest activities, 3.19U/mL [GS115 (pHBM565)], 2.56U/mL [KM71 (pHBM565)], and 2.49U/mL [SMD1168 (pHBM565)], were gotten when the induction were performed at 20 degrees C with 1.0% (V/V) methanol supplied. The temperature and pH optimum for the recombinant laccase produced by three strains were 60 degrees C and pH4.2, respectively.
Zhang
Yin-Bo
YB
Molecular Microbiology Lab, Hubei University, Wuhan 430062, China. ybzhang04@yahoo.com
Jiang
Mu-Lan
ML
Hu
Xiao-Jia
XJ
Zhang
Gui-Min
GM
Ma
Li-Xin
LX
D001483:Base Sequence; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000235:genetics*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature
D011994:Recombinant Proteins; D042845:Laccase