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

Abstract

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.

Site-directed Mutagenesis of a β-Glycoside Hydrolase from Lentinula edodes

“…It utilizes the error-prone PCR or DNA shuffling technique in combination with the high-throughput screening method to continuously accumulate the dominant mutations with improved characteristics of the enzyme [5,11,12,13,14,15]. Rational design is conducted based on the understanding of the catalytic mechanism or the enzyme structure in which the stereo-structure can sometimes be predicted by protein homology modeling technique [16,17,18,19,20,21]. The key amino acids that may affect the enzyme properties can be chosen for site-directed mutagenesis, which includes the single site-directed mutation, multiple site-directed mutations, and saturation mutation.…”