Affiliation:
1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
Abstract
ABSTRACT
Laccase from
Myceliophthora thermophila
(MtL) was expressed in functional form in
Saccharomyces cerevisiae
. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in
k
cat
, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in
S. cerevisiae
is 30% higher than that of the same enzyme expressed in
M. thermophila
(110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This
S. cerevisiae
expression system makes MtL available for functional tailoring by directed evolution.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
244 articles.
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