Functional expression and stabilization of horseradish peroxidase by directed evolution inSaccharomyces cerevisiae

2001 ◽  
Vol 76 (2) ◽  
pp. 99-107 ◽  
Author(s):  
Birgit Morawski ◽  
Sara Quan ◽  
Frances H. Arnold
Keyword(s):  
Horseradish Peroxidase ◽  
Directed Evolution ◽  
Functional Expression

scholarly journals Directed Evolution Reveals Requisite Sequence Elements in the Functional Expression of P450 2F1 inEscherichia coli

2012 ◽  
Vol 25 (9) ◽  
pp. 1964-1974 ◽  
Author(s):  
James B. Y. H. Behrendorff ◽  
Chad D. Moore ◽  
Keon-Hee Kim ◽  
Dae-Hwan Kim ◽  
Christopher A. Smith ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Functional Expression ◽  
Inescherichia Coli ◽  
Sequence Elements

New Approaches for Functional Expression of Recombinant Horseradish Peroxidase C InEscherichia Coli

1999 ◽  
Vol 17 (5) ◽  
pp. 359-379 ◽  
Author(s):  
Vitaly Grigorenko ◽  
Tatiana Chubar ◽  
Yury Kapeliuch ◽  
Torsten Börchers ◽  
Friedrich Spener ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Functional Expression ◽  
Inescherichia Coli ◽  
Recombinant Horseradish Peroxidase ◽  
New Approaches

scholarly journals Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

2003 ◽  
Vol 69 (8) ◽  
pp. 5037-5037 ◽  
Author(s):  
Thomas Bulter ◽  
Miguel Alcalde ◽  
Volker Sieber ◽  
Peter Meinhold ◽  
Christian Schlachtbauer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Functional Expression ◽  
Fungal Laccase

scholarly journals Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

2003 ◽  
Vol 69 (2) ◽  
pp. 987-995 ◽  
Author(s):  
Thomas Bulter ◽  
Miguel Alcalde ◽  
Volker Sieber ◽  
Peter Meinhold ◽  
Christian Schlachtbauer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Enzyme Stability ◽  
Expression System ◽  
Fold Increase ◽  
Total Activity ◽  
Functional Expression ◽  
Glycosylation Site ◽  
C Terminus ◽  
Improved Stability

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.

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