[Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties].
The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.
Zhang
Wei
W
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. zhangwei7312@hotmail.com
Fan
Yun-liu
YL
Yao
Bin
B
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D043322:Lactase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D011994:Recombinant Proteins; D043322:Lactase