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Counteracting expression deficiencies by anticipating posttranslational modification of PaHNL5-L1Q-A111G by genetic engineering

Paper ID Volume ID Publish Year Pages File Format Full-Text
25250 43563 2007 9 PDF Available
Title
Counteracting expression deficiencies by anticipating posttranslational modification of PaHNL5-L1Q-A111G by genetic engineering
Abstract

(R)-2-chloromandelic acid represents a key pharmaceutical intermediate. Its production on large scale was hampered by low turnover rates and moderate enantiomeric excess (ee) using enzyme as well as metal catalysts. The cloning and heterologous overexpression of an (R)-hydroxynitrile lyase from Prunus amygdalus opened a way to large-scale production of this compound. Especially the rationally designed mutation of alanine to glycine at amino acid position 111 of the mature protein tremendously raised the yield for enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandelonitrile, which can be hydrolysed to the corresponding alpha hydroxy acid. However, expression of this mutein was less efficient than for the unmodified enzyme. Subsequent LC/MS/MS-analysis of the protein sequence revealed that mutation A111G triggered the posttranslational deamidation of the neighbouring residue asparagine (N110) to aspartic acid. This finding on the one hand could explain the decreased secretion efficiency of the mutant as compared to the wildtype enzyme, but on the other hand raised the question which of the two residues was truly accountable for the enhanced conversion. The muteins N110D, A111G and N110DA111G were constructed and compared in terms of protein productivity and performance in chemical syntheses. The expression level of the double mutein was augmented significantly and the enantioselectivity remained high. Reduced protein expression of mutein PaHNL5-L1Q-A111G was remedied by mutational anticipation of posttranslational deamidation.

Keywords
Hydroxynitrile lyase; Posttranslational modification; Engineering; Secretion enhancement; Deamidation; PaHNL; Prunus
First Page Preview
Counteracting expression deficiencies by anticipating posttranslational modification of PaHNL5-L1Q-A111G by genetic engineering
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 1, 30 March 2007, Pages 30–38
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering