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Applications of Nanotechnology to Bioprocessing
Published in Yubing Xie, The Nanobiotechnology Handbook, 2012
Susan T. Sharfstein, Sarah Nicoletti
A type of manipulation that is being explored is substrate channeling. Substrate channeling occurs when the intermediate product from an enzyme is transferred to another neighboring enzyme without allowing the intermediate to equilibrate in the bulk phase (Spivey and Ovádi 1999). This process can occur in vivo, ex vivo, or in vitro naturally, but there are many benefits to constructing synthetic complexes for substrate channeling for biotechnology purposes (Zhang et al. 2011). Substrate channeling allows for faster reaction rates when compared to free floating enzymes, as well as protecting unstable intermediates, obstructing substrate competition among different pathways, and enhancement of biocatalysis by avoiding unfavorable energetics of substrates. Some of these synthetic complexes include multifunctional fusion proteins, complexes linked with scaffolding, synthetic cellulosomes and recombinant cellulolytic microorganisms, and the co-immobilization of multiple enzymes, all of which can be seen in Figure 15.3. A co-immobilized enzyme complex was formed by coupling the enzymes to the surface of isocyanopeptides and styrene for use as nanoreactors (Van Dongen et al. 2009). These nanoreactors were multistep, first converting glucose acetate to glucose, and then oxidizing glucose into gluconolactone with hydrogen peroxide as a byproduct. At the end of the study it was concluded that the co-immobilized enzyme cascade allowed for a faster reaction rate of these conversions.
Biological Strategies in Nanobiocatalyst Assembly
Published in Grunwald Peter, Biocatalysis and Nanotechnology, 2017
Ian Dominic F. Tabañag, Shen-Long Tsai
The research group of Douglas et al. has engineered an in vivo one-pot heterologous expression-assembly system for the programmed encapsulation of fusion proteins in a high copy number utilizing the capsid and scaffold machinery of the VLPs derived from Salmonella typhimurium P22 bacteriophage (O’Neil et al., 2011). The P22 bacteriophage VLPs is composed of a coat protein that assembles into an icosahedral capsid with the aid of a scaffolding protein (SP) which is incorporated on its interior surface via non-covalent association. Moreover, the P22 VLPs undergo irreversible structural changes upon heating that lead to a significant increase of its internal volume and porosity and in turn increases the external accessibility to its hollow interior (Parker et al., 1998; Teschke et al., 2003; Parent et al., 2010). The engineered heterologous expression system contains the SP141 gene (truncated form of SP encoding amino acids 141–303 located at the C-terminal region (Parker et al., 1998; Weigele et al., 2005)) fused upstream to the P22 coat protein (CP). The protein of interest is then fused upstream of the SP141 to form the protein-SP141–CP fusion, and upon expression and self-assembly, a VLP containing the protein of interest is obtained. Using this expression system, enzymes such as alcohol dehydrogenase (AdhD), β-glucosidase (CelB), and even a multiple enzyme cascade of β-glucosidase (CelB), galactokinase (GALK), and glucokinase (GLUK) which catalyze sequential reactions in sugar metabolism, have been encapsulated inside P22 VLPs (Patterson et al., 2012a; Patterson et al., 2012b; Patterson et al., 2014). For the case of the encapsulated AdhD, the functional VLPs exhibited a very low retained enzymatic activity (around 15% of the free AdhD) due to the high enzymatic loading (~250 enzymes present in the capsid corresponding to a confinement molarity of 7.6 mM) inside the VLPs which caused a crowding effect (Patterson et al., 2012a). However, for the case of CelB encapsulation, the encapsulated CelB retained 94% of the free enzyme activity with an enzyme loading of ~84 enzymes per capsid (corresponding to a confinement molarity of 2.4 mM) which suggests that the quaternary structure of the enzyme was not hampered by the assembly and encapsulation (Patterson et al., 2012b). Lastly, the encapsulation of a multiple enzyme cascade consisting of CelB, GALK, and GLUK in P22 VLPs showed that the arrangement of the enzymes within the enzyme-SP141 fusion had a significant effect on the overall enzymatic activity. The kinetic studies have validated the presence of substrate channeling and it was reported that channeling between sequential enzymes is dependent on the inter-enzyme distance and the balance between their kinetic parameters (Patterson et al., 2014).
Fusion of cellobiose phosphorylase and potato alpha-glucan phosphorylase facilitates substrate channeling for enzymatic conversion of cellobiose to starch
Published in Preparative Biochemistry & Biotechnology, 2022
Xinyu Liu, Huawei Hou, Yapeng Li, Sen Yang, Hui Lin, Hongge Chen
Creating a bifunctional enzyme, which catalyzes two sequential reactions, from a single gene is an important strategy in accomplishing substrate channeling observed in nature.[13] The intermediate produced by the bifunctional enzyme has a higher chance of being channeled between the two catalytic sites than that by two separate enzymes owing to the spatial proximity of the catalytic sites. Glutamine phosphoribosylpyrophosphate amidotransferase (GPATase) from E. coli[14] and dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Leishmania major,[15] both having two different catalytic domains on a single polypeptide chain, are remarkable examples of naturally occurring bifunctional enzymes with substrate channeling, as evidenced by the presence of a 20 Å tunnel connecting the two active sites in GPATase and an electrostatic “highway” linking the two active sites in DHFR-TS. Many artificially constructed bifunctional fusion enzymes[16,17] have also exhibited enhanced catalytic efficiency, thus demonstrating the importance of enzyme fusion in engineering efficient cascade reactions; notably, few of these works have provided accurate evidence for substrate channeling due to their focus on practical applications.