Development and validation of a screening system for a beta-galactosidase with increased specific activity produced by directed evolution

作者：Rentschler Eva; Schwarz Thilo; Stressler Timo^*; Fischer Lutz

来源：European Food Research and Technology, 2016, 242(12): 2129-2138.

DOI：10.1007/s00217-016-2709-x

• beta-Galactosidase
• Directed evolution
• Screening system
• Specific activity
• Lactose
• Metagenome

A powerful tool in enzyme engineering is directed evolution. The design of a suitable screening system to identify enzyme variants with the feature desired is one of the most important points in directed evolution experiments. In this article, we developed and validated a four-step activity-based screening system with a high throughput rate to identify a variant of the metagenome beta-galactosidase, called M1, with improved specific activity. An Escherichia coli host-vector system was chosen to realize a high production of the recombinant enzyme in microtiter plate format. Industrial-like conditions of milk processing in dairy industry (substrate lactose, solved in synthetic milk buffer, pH 6.75, and 8 A degrees C) were used for the activity screening. With a coefficient of variance of 15.6 % and a Z' factor value of 0.53, the assay was reproducible and suitable to identify enzyme variants with the requirement desired. A first-generation library of beta-galactosidase variants, constructed by error-prone PCR, was analyzed using the high-throughput screening developed, and one variant, called M1'K2, was identified as having a distinctly improved specific activity. Therefore, this variant was produced in shaking flasks, and after purification via metal affinity chromatography, the improved specific activity was confirmed. In detail, the variant M1'K2 showed a more than twofold enhanced specific activity toward lactose at 8 A degrees C compared to the parental beta-galactosidase M1.

• 出版日期2016-12