Production and characterization of Acidothermus cellulolyticus endoglucanase in Pichia pastoris.
The endoglucanase (E1) from Acidothermus cellulolyticus has been used extensively in cellulase research. The goal of this work was to produce high levels of this enzyme in a system that facilitates purification. A codon-optimized synthetic gene for A. cellulolyticus E1 with a C-terminal histidine tag was cloned into the genome of Pichia pastoris. Strain KM71H expressed the most enzyme, with a yield of 550mg/L culture supernatant. The temperature optimum (80°C) and pH optimum (5.1) of the purified enzyme agree with previously determined values for the enzyme produced in other systems. Michaelis-Menten kinetic parameters were determined, using a fluorescent substrate (methylumbelliferyl-β-d-cellobioside) at various temperatures. This thermostable enzyme can be used in future cellulosic biofuels-related research.
Department of Chemical Engineering & Materials Science, University of California, Davis, CA 95616, USA. blindy@ucdavis.edu
Benjamin E
BE
Lindenmuth
Karen A
KA
McDonald
D000192:Actinomycetales / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D056804:Biofuels; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005813:Genes, Synthetic; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D001426:Bacterial Proteins; D056804:Biofuels; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; C056374:endoglucanase 1; D002480:Cellulase