Genetically-modified R-ω-transaminase: purification and self-assembly facilitating interaction with substrate droplets.

Abstract:

OBJECTIVES:An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase. RESULTS:R-ω-Transaminase (R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP-R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-R-ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %. CONCLUSIONS:The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports.

journal_name

Biotechnol Lett

journal_title

Biotechnology letters

authors

Gao Q,Fu Y,Peng Y,Liu W,Feng W

doi

10.1007/s10529-015-1998-7

subject

Has Abstract

pub_date

2016-03-01 00:00:00

pages

489-94

issue

3

eissn

0141-5492

issn

1573-6776

pii

10.1007/s10529-015-1998-7

journal_volume

38

pub_type

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