Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

• Klíčová slova
• enzyme kinetics, fluorescence, glucose, glycoside hydrolases, lab automation, β-glucosidase,
• MeSH
• automatizace MeSH
• beta-glukosidasa chemie   MeSH
• katalýza MeSH
• kinetika MeSH
• kukuřice setá enzymologie   MeSH
• rostlinné proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-glukosidasa MeSH
• rostlinné proteiny MeSH

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.

• MeSH
• aktivace enzymů MeSH
• beta-glukosidasa genetika   metabolismus   MeSH
• databáze proteinů MeSH
• genová knihovna MeSH
• hydrolýza MeSH
• kodon MeSH
• kukuřice setá genetika   metabolismus   MeSH
• mutageneze MeSH
• proteinové inženýrství * metody   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• substrátová specifita MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-glukosidasa MeSH
• kodon MeSH
• rostlinné proteiny MeSH