Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10 000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.

Saturated mutagenesis

DNA shuffling

Thermostability

Saturation (graph theory)

Protein Engineering

10.1021/ja1030479

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Citations (226)

Ochratoxin A degrading enzymes ofStenotrophomonassp. 043-1a

FEMS Microbiology Letters (2023)

Christoph Gonaus Laura Wieland Gerhard Thallinger Shreenath Prasad

Abstract Ochratoxin A is a secondary metabolite that acts as a mycotoxin and is produced by Aspergillus, Penicillium, and other fungal species. It is a threat to animal and human health due to nephrotoxic, carcinogenic, and genotoxic properties and its widespread incidence in agricultural products. To reduce this threat, biological remediation processes are of growing interest. The aerobic gram-negative bacterium Stenotrophomonassp. 043-1a, isolated from soil, was previously shown to degrade ochratoxin A into the non-toxic ochratoxin α and l-phenylalanine (Schatzmayr et al. 2002). However, the enzyme or enzymes catalyzing this reaction in this strain remained elusive. Here, we report the targeted purification of Stenotrophomonassp. 043-1a lysate via ammonium sulfate precipitation, size-exclusion chromatography, and hydrophobic interaction chromatography to identify the ochratoxin A degrading enzymes by subsequent peptide fragment fingerprinting. The metallo-dependent hydrolase Chr1_3858681_3267 and a member of the peptidase S9 family, Chr1_3858681_771, were shown to degrade ochratoxin A. This was, to our knowledge, the first report of an ochratoxin A degrading enzyme from the peptidase S9 family.

Ochratoxins

Ammonium sulfate precipitation

Stenotrophomonas

10.1093/femsle/fnad028

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Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of Rhodococcus rhodochrous PA-34

Indian Journal of Microbiology (2007)

Shreenath Prasad Juliet Roshini Mohan Raj Tek Chand Bhalla

Rhodococcus rhodochrous

Nitrile hydratase

Bioconversion

Dry weight

10.1007/s12088-007-0007-9

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Citations (15)