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HERO ID
1000089
Reference Type
Journal Article
Title
Engineering pH-tolerant mutants of a cyanide dihydratase
Author(s)
Wang, L; Watermeyer, JM; Mulelu, AE; Sewell, BT; Benedik, MJ
Year
2012
Is Peer Reviewed?
Yes
Journal
Applied Microbiology and Biotechnology
ISSN:
0175-7598
EISSN:
1432-0614
Volume
94
Issue
1
Page Numbers
131-140
Language
English
PMID
21993481
DOI
10.1007/s00253-011-3620-9
Web of Science Id
WOS:000301747500013
Abstract
Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynD(pum)) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynD(pum) mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.
Keywords
Nitrilase; Cyanide; Bioremediation; Cyanide dihydratase; pH tolerance; Protein stability
Tags
IRIS
•
Ammonia
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