Purification and characterization of heterologously expressed nitrilases from filamentous fungi
Language English Country Germany Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Aminohydrolases chemistry genetics isolation & purification metabolism MeSH
- Escherichia coli genetics MeSH
- Gene Expression MeSH
- Fungal Proteins chemistry genetics isolation & purification metabolism MeSH
- Fungi enzymology genetics MeSH
- Kinetics MeSH
- Cloning, Molecular MeSH
- Hydrogen-Ion Concentration MeSH
- Molecular Chaperones genetics metabolism MeSH
- Recombinant Proteins chemistry genetics isolation & purification metabolism MeSH
- Enzyme Stability MeSH
- Substrate Specificity MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Aminohydrolases MeSH
- Fungal Proteins MeSH
- Molecular Chaperones MeSH
- nitrilase MeSH Browser
- Recombinant Proteins MeSH
Nitrilases from Aspergillus niger CBS 513.88, A. niger K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. crassa nitrilase exhibited the highest yield of 69,000 U L(-1) culture. Co-expression of chaperones (GroEL/ES in G. moniliformis and P. marneffei; GroEL/ES and trigger factor in N. crassa and A. niger CBS 513.88) enhanced the enzyme solubility. Specific activities of strains expressing the former two enzymes increased approximately fourfold upon co-expression of GroEL/ES. The enzyme from G. moniliformis (co-purified with GroEL) preferred benzonitrile as substrate (K(m) of 0.41 mM, V(max) of 9.7 μmol min(-1) mg(-1) protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest V(max) of 7.3 μmol min(-1) mg(-1) protein in cell-free extract, but also a high K(m) of 15.4 mM, for 4-cyanopyridine. The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (K(m) of 3.4 and 2.0 mM, respectively; V(max) of 10.6 and 17.5 μmol min(-1) mg(-1) protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher K(m) values. Significant amounts of amides were only formed by the G. moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.
Appl Microbiol Biotechnol. 2013 Oct;97(20):9263-4 PubMed
References provided by Crossref.org
Exploring the potential of fungal arylacetonitrilases in mandelic acid synthesis
A comparative study of nitrilases identified by genome mining
