Bart Brouwer

Nature Biotechnology, Published online: 08 May 2023; doi:10.1038/s41587-023-01773-0

Foldseek speeds up protein structural search by four to five orders of magnitude.

A new protein purification system, designated CSAP (chitin- and streptavidin-mediated affinity purification), has been developed for enzyme library screening. The system utilizes chitin powder as a chromatography matrix to purify Strep-tag II fusion proteins. Given the cost of chitin and the availability of Strep-tag II, the CSAP system offers a powerful platform for the screening of purified proteins.

Abstract

Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.

This Minireview summarizes the different strategies used in the design and engineering of novel enzymes that accommodate iminium catalysis. The advantages and challenges of developing enzymes for this catalysis mode are discussed. Recent developments in iminium biocatalysis showcase the tremendous power of combining chemomimetic biocatalyst design and directed evolution to create useful biocatalysts for new-to-nature transformations.

Abstract

The application of biocatalysis in conquering challenging synthesis requires the constant input of new enzymes. Developing novel biocatalysts by absorbing catalysis modes from synthetic chemistry has yielded fruitful new-to-nature enzymes. Organocatalysis was originally bio-inspired and has become the third pillar of asymmetric catalysis. Transferring organocatalytic reactions back to enzyme platforms is a promising approach for biocatalyst creation. Herein, we summarize recent developments in the design of novel biocatalysts that adopt iminium catalysis, a fundamental branch in organocatalysis. By repurposing existing enzymes or constructing artificial enzymes, various biocatalysts for iminium catalysis have been created and optimized via protein engineering to promote valuable abiological transformations. Recent advances in iminium biocatalysis illustrate the power of combining chemomimetic biocatalyst design and directed evolution to generate useful new-to-nature enzymes.