Dataset Information

Peptide-modified surfaces for enzyme immobilization.

ABSTRACT:

Background

Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.

Methodology/principal findings

A method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to ?-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.

Conclusions/significance

A simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.

SUBMITTER: Fu J

PROVIDER: S-EPMC3073003 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Peptide-modified surfaces for enzyme immobilization.

Fu Jinglin J Reinhold Jeremy J Woodbury Neal W NW

PloS one 20110408 4

<h4>Background</h4>Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise ...[more]

PMID: 21494577

Similar Datasets

Project description:Biocatalysis is a useful strategy for sustainable green synthesis of fine chemicals due to its high catalytic rate, reaction specificity, and operation under ambient conditions. Addressable immobilization of enzymes onto solid supports for one-pot multistep biocatalysis, however, remains a major challenge. In natural pathways, enzymes are spatially coupled to prevent side reactions, eradicate inhibitory products, and channel metabolites sequentially from one enzyme to another. Construction of a modular immobilization platform enabling spatially directed assembly of multiple biocatalysts would, therefore, not only allow the development of high-efficiency bioreactors but also provide novel synthetic routes for chemical synthesis. In this study, we developed a modular cascade flow reactor using a generalizable solid-binding peptide-directed immobilization strategy that allows selective immobilization of fusion enzymes on anodic aluminum oxide (AAO) monoliths with high positional precision. Here, the lactate dehydrogenase and formate dehydrogenase enzymes were fused with substrate-specific peptides to facilitate their self-immobilization through the membrane channels in cascade geometry. Using this cascade model, two-step biocatalytic production of l-lactate is demonstrated with concomitant regeneration of soluble nicotinamide adenine dinucleotide (NADH). Both fusion enzymes retained their catalytic activity upon immobilization, suggesting their optimal display on the support surface. The 85% cascading reaction efficiency was achieved at a flow rate that kinetically matches the residence time of the slowest enzyme. In addition, 84% of initial catalytic activity was preserved after 10 days of continuous operation at room temperature. The peptide-directed modular approach described herein is a highly effective strategy to control surface orientation, spatial localization, and loading of multiple enzymes on solid supports. The implications of this work provide insight for the single-step construction of high-power cascadic devices by enabling co-expression, purification, and immobilization of a variety of engineered fusion enzymes on patterned surfaces.

Dataset Information

Peptide-modified surfaces for enzyme immobilization.

Background

Methodology/principal findings

Conclusions/significance

Publications

Peptide-modified surfaces for enzyme immobilization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets