F.
Kazenwadel
*,
M.
Franzreb
and
B. E.
Rapp
Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. E-mail: franziska.kazenwadel@kit.edu
First published on 22nd April 2015
A sustainable alternative to traditional chemical synthesis is the use of enzymes as biocatalysts. Using enzymes, different advantages such as mild reaction conditions and high turnover rates are combined. However, the approach of using soluble enzymes suffers from the fact that enzymes have to be separated from the product post-synthesis and can be inactivated by this process. Therefore, enzymes are often immobilized to solid carriers to enable easy separation from the product as well as stabilization of the enzyme structure. In order to mimic the metabolic pathways of living cells and thus to create more complex bioproducts in a cell-free manner, a series of consecutive reactions can be realized by applying whole enzyme cascades. As enzymes from different host organisms can be combined, this offers enormous opportunities for creating advanced metabolic pathways that do not occur in nature. When immobilizing this enzyme cascades in a co-localized pattern a further advantage emerges: as the product of the previous enzyme is directly transferred to its co-immobilized subsequent catalyst, the overall performance of the cascade can be enhanced. Furthermore when enzymes are in close proximity to each other, the generation of by-products is reduced and obstructive effects like product inhibition and unfavorable kinetics can be disabled. This review gives an overview of the current state of the art in the application of enzyme cascades in immobilized forms. Furthermore it focuses on different immobilization techniques for structured immobilizates and the use of enzyme cascade in specially designed (microfluidic) reactor devices.
There are different approaches for the realization of enzyme cascades. Enzymes can be co-immobilized by different techniques in a more or less specific pattern, which leads to close proximity to each other and facilitates substrate channeling. Biocatalysts can also be separated into different reaction compartments that the product stream passes subsequently. As in separated reaction compartments the process parameters can be adapted, this approach is favored if the selected enzymes differ in their requirements concerning optimal process conditions.
A second important field, in which enzyme cascades are used, is the production of electricity using enzyme based biofuel cells. Enzymes used for this type of application normally belong to the family of oxidoreductased. The topic was very well reviewed in 2007 by Minteer et al. highlighting the advantages and disadvantages of enzyme fuel cells compared to microbial fuel cells. Advantages are the higher power densities that can be achieved by the immobilization of the biocatalysts and their higher specificity. However, they suffer from short life times (7–10 days) and only partly oxidized fuel substrates.20 The first enzyme cascade for electricity generation was already applied in 1998 by Palmore et al. The authors used alcohol dehydrogenase, aldehyde dehydrogenase and formate dehydrogenase for the oxidation of methanol to carbon dioxide.21 Another work was published by Akers et al. in 2004, where ethanol was oxidized in a two-step reaction to acetate by combining an alcohol dehydrogenase and an aldehyde dehydrogenase.
Tag name | Binding partner | Selected immobilization example |
---|---|---|
Poly-histidine-tag | Transition metals (Cu(II), Co(II), Zn(II), Ni(II) complexed to nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) | 39, 40 |
Poly-arginine-tag | Cation exchange material | 41 |
Biotin | Avidin/streptavidin | 42 |
Strep-tag | Strep-tactin | 43 |
FLAG-tag | Anti-flag monoclonal antibody | 44 |
Cellulose-binding domain | Cellulose | 45, 46 |
Ribosomal protein L2 (“Si-tag“) | Silica | 47 |
SNAP-tag | benzylguanine | 48 |
Halo-tag | Halo-tag-ligand | 49 |
![]() | ||
Fig. 1 Site-specific immobilization of polyhistidine-tagged proteins to a Ni-substrate by dip-pen-nanolithography. Reprinted (adapted) with permission from.39 Copyright (2015) American Chemical Society. |
![]() | ||
Fig. 2 Co-immobilization of three enzymes, triosephosphate isomerase (TIM), aldolase (ALD) and fructose 1,6-bisphosphatase (FBP) fused to three different dockerin-domaines (TIM-CTDoc, ALD-CCDoc, FBP-RFDoc) and coupled in a site-directed fashion to a protein scaffold (CBM3) carrying three cohesion domains (CTCoh, CCCoh, RFCof) for specific binding of the respective dockerin domain. Reprinted from ref. 13 with permission of John Wiley and Sons. |
![]() | ||
Fig. 3 Site-specific immobilization of HRP and GOx in defined distances ranging from 10 to 65 nm. Close proximity of the enzymes leads to an enhanced performance of the cascade due to substrate channeling. Reprinted with permission from.58 Copyright (2015) American Chemical Society. |
![]() | ||
Fig. 4 Site-specific immobilization of an enzyme cascade in polymersome nanocontainers: Candida antarctica Lipase B (CalB) is embedded in the polymersome membrane, glucose oxidase (GOx) is entrapped in the inner lumen of the container and horseradish peroxidase (HRP) is attached to the outer polymersome surface by a Click chemistry approach. Reprinted from ref. 17 with permission of John Wiley and Sons. |
![]() | ||
Fig. 5 Co-immobilization of β-galactosidase (βGal, green), glucose oxidase (GOx, here GOD, yellow) and horseradish peroxidase (HRP, red) in a microfluidic channel by two approaches: (A) immobilization to microbeads that are subsequently packed in the channel. (B) direct attachment to the inner surface of the microchannels. The packed bed reactor (A) proved to be the more efficient approach. Reprinted from ref. 18 with permission of The Royal Society of Chemistry. |
Technique | Immobilization chemistry | Support | Advantages | Disadvantages | Example |
---|---|---|---|---|---|
Random co-immobilization | |||||
Crosslinking to solid surfaces | Crosslinking agents, click chemistry approaches | Solid supports: surfaces, particles, fibres etc. | Fast and easy, co-localization of enzymes | Only statistical distribution, no site-specificity | 28 |
Encapsulation | Encapsulation in polymers | Polymers, surfaces coated with polymers | Less enzyme inactivation, fast and easy, co-localization | Diffusional limitations, as substrate has to enter support, no side-specificity | 29–32 |
Supportless crosslinking | Crosslinking agents, click chemistry approaches | — | High specific activities, no dilution by support | Only statistical distribution, no site specificity | 35 |
![]() |
|||||
Site-specific co-immobilization | |||||
Immobilization to protein scaffolds | Protein tags, click chemistry approaches | Scaffold protein | High control of localization, exploitation of substrate channeling | Protein tagging necessary (genetic engineering), time-consuming and elaborate | 13 |
Immobilization to DNA scaffolds | Protein tags, click chemistry approaches | Single stranded scaffold DNA, DNA-origami structures | High control of localization, exploitation of substrate channeling | Protein tagging necessary, time-consuming and elaborate | 54, 57 |
Immobilization in nanocontainers | Encapsulation, embedding, crosslinking, click chemistry approaches | Porous polymersomes | High control of localization by using different immobilization, compartments, product is soluble but separable by filtration | Time-consuming and elaborate | 17 |
Immobilization in microfluidic devices | Crosslinking agents, protein tags, click chemistry | Surfaces in microfluidic devices | Compartmentalization comparable to living cells, high level of control of fluidic etc. | Elaborate, special equipment needed | 18, 19 |
In this review an overview over different immobilization techniques has been given. The focus was on the random or site-specific immobilization of enzyme cascades leading to highly active multi-enzyme complexes with enhanced stability and activity. A great variety of techniques and different supports with sophisticated features exists nowadays in order to provide an optimal solution for the realization of enzyme cascades in many fields of application.
This journal is © The Royal Society of Chemistry 2015 |