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.

N G. 1998. An acid proteinase from Neosartorya fischeri var. spinosa IBT 4872 was purified 38-fold with a yield of 11% by ultrafiltration, ammonium sulphate fractionation, Sephadex-G200 gel filtration, DEAE-Sephadex anion exchange chromatography, and hydroxyapatite chromatography. The enzyme was most active at pH 3·0 and 50°C and had a molecular weight of 45 kDa, as determined by SDS-PAGE. It was stable over a pH range of 3·0 to 6·0 and exhibited thermal stability up to 50°C. The Km value for haemoglobin was 0·44% (w/v). The activity was inhibited by pepstatin, suggesting that the enzyme is an aspartic proteinase.

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...

No abstract

No abstract