Focused directed evolution of β-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening

Mazura, Pavel; Filipi, Tomáš; Souček, Přemysl; Brzobohatý, Břetislav

doi:10.1016/j.carres.2010.08.016

Cited by 3 publications

(2 citation statements)

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“…Positive clones were screened for activity directly on Petri dishes by a procedure developed to identify clones with β-glucosidase activity using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-O-β-D-glucopyranoside (XGlc, supplied by Biosynth AG, Staad, Switzerland), which yields a blue reaction product [2]. A 40 mg L −1 dose of XGlc was found to be sufficient and cultivation at 37°C for 12 h, followed by 12 h at 16°C, gave the best results.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, ideally parameters describing the performance of mutagenesis methods should be empirically estimated and incorporated into the formulas to avoid underestimation of required screening efforts [2]. The screening is usually the most expensive part of the overall effort, so it is highly desirable to identify the most appropriate screening scale.…”

Section: Introductionmentioning

confidence: 99%

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Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

et al. 2014

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Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins’ structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations’ effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

et al. 2014

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Engineering the cytokinin-glucoside specificity of the maize β-d-glucosidase Zm-p60.1 using site-directed random mutagenesis

et al. 2012

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Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

Feng

Liu

Yang

et al. 2013

Chemistry Central Journal

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BackgroundFor screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity.ResultsDuring uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity.ConclusionSpecific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

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Focused directed evolution of β-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening

Cited by 3 publications

References 18 publications

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

Engineering the cytokinin-glucoside specificity of the maize β-d-glucosidase Zm-p60.1 using site-directed random mutagenesis

Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

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