Biomimicry Promotes the Efficiency of a 10‐Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate

Abstract

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. KeywordsBiomimicry; Enzymes; Glycolysis; Nanoparticles; Tethered Enzymes Multiple strategies have been used to interface biological components with inorganic surfaces. In engineering studies and industrial applications, carboxyl-amine binding chemistry is the most common attachment strategy [1] . However, this chemistry is not sitespecific and does not inherently impart proper molecular orientation-two factors that can * ajt32@cornell.edu.Supporting information for this article is given via a link at the end of the document. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript lead to significant reduction in function of the tethered enzyme [2] . Use of self-assembly templates can immobilize and orient enzymes; for instance sequential reactions of glucose oxidase and horseradish peroxidase have been demonstrated using a DNA scaffold to control enzyme spacing [3] . Recently, the sequential reaction of three enzymes conjugated to quantum dots was demonstrated [4] . Despite the different approaches available, the current state of the art remains limited to two-or three-step, coupled tethered reactions [5] . This is in stark contrast to multi-step reactions in solution, in which complex reactions have been demonstrated ranging up to a 13-step production of ethanol from glucose [6] .Inspired by the organization of glycolytic enzymes on the fibrous sheath of mammalian sperm [7] , we previously showed that replacement of germ cell-specific targeting domains with a bioaffinity tag provided oriented immobilization of glycolytic enzymes. This translated into significant advantages in specific activity of both individual enzymes and for sequential reactions when compared against the same enzymes tethered via random adsorption without a histidine tag or carboxyl-amine chemistry [8] These prior studies suggested that biomimetic oriented immobilization increased the activity of individual tethered enzymes for sev...

Cascades in Compartments: En Route to Machine‐Assisted Biotechnology

“…end, hexahistidine-tagged recombinant glycolytic enzymes were immobilized in an oriented fashion with respect to the surface of Ni 2+ -NTA-modified agarose beads.I nitial studies with two [55] or three [56] different enzymes demonstrated that surface-tethered pathway components indeed show sequential enzymatic activities.V ery recently,t his approach was extended to a1 0-step sequential enzymatic reaction of glycolytic enzymes,w hich converted glucose into lactate. [57] It was observed that, although the efficiency of the individual enzymes was higher in solution, the efficacyo ft he 10-step pathway was significantly higher when the enzymes were tethered on the particle surface (Figure 4). Thea uthors suggest that ac hanneling of intermediates within the hydration layer of proteins on the bead interface and aconcomitant reduced diffusional loss of an intermediate product might be ar eason for the increase in production efficiency.A lternatively,w eak attractive interactions between the substrate molecules and the bead surface even might create a" virtual compartment" which can accelerate the throughput.…”

Multienzymatic Cascade Reactions via Enzyme Complex by Immobilization

“…Mukai et al coimmobilized enzymes on Ni-NTA-functionalized silica nanoparticles (500 nm). 154 The efficacy of the 10-step pathway, as measured by conversion of glucose to lactate, was significantly higher with tethering, enabling a 3-fold increase in the complexity of the sequential tethered reactions, inducing an elegant, high-throughput glycolytic system. A macroporous silica foam (MSF) also was reported by Cao and coworkers.…”

Pursuing the Promise of Enzymatic Enhancement with Nanoparticle Assemblies

“…Related to this, the Travis laboratory reported on a 10-step enzyme pathway designed to convert glucose to lactate tethered to 500-nm-diameter microparticles. 134 Individual enzyme activity was found to be higher in solution; however, the overall conversion efficiency (defined as [lactate product]/ [glucose consumed]) of the entire pathway was higher when tethered. Although not accessing the NP-enhancement phenomena described here (as evidenced by a lack of enhancement of the individual enzymes), their results nevertheless suggested a benefit from colocalization that presumably would arise from some type of channeling effect.…”