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Browsing by Author "Sun, Fangfang"

Now showing 1 - 4 of 4
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    Development of Building Blocks - Thermostable Enzymes for Synthetic Pathway Biotransformation (SyPaB)
    Sun, Fangfang (Virginia Tech, 2012-04-25)
    Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes that microbes cannot do. One of the largest challenges is the high cost and instability of enzymes and cofactors. To overcome this obstacle, strong motivations have driven intensive efforts in discovering, engineering, and producing thermostable enzymes. In this project, ribose-5-phosphate isomerase (RpiB), one of the most important enzymes in the pentose phosphate pathway, was cloned from a thermophile Thermotoga maritima, and heterologously expressed in Escherichia coli, purified and characterized. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 80°C and pH 6.5-8.0. It had a half lifetime of 71 h at 60°C, resulting in its turn-over number of more than 2 x108 mol of product per mol of enzyme. Another two thermostable enzymes glucose-6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) and their fusion proteins G6PDH-DI and DI-G6PDH were cloned from Geobacillus stearothermophilus, heterologouely expressed in E. coli and purified through its His-tag. The individual proteins G6PDH and DI have good thermostability and reactivity. However, the presence of DI in fusion proteins drastically decreased G6DPH activity. However, a mixture of G6PDH and a fusion protein G6PDH-DI not only restored G6PDH activity through the formation of heteromultimeric network but also facilitated substrate channeling between DI and G6PDH, especially at low enzyme concentrations. My researches would provide important building blocks for the on-going projects: high-yield hydrogen production through cell-free enzymatic pathways and electrical energy production through enzymatic fuel cells.
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    Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii
    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Zhang, Y. H. Percival (Nature, 2015-08-20)
    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin – the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 °C for 30 min. This enzyme was characterized and required Mg²⁺ as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 °C in the presence of 0.5 mM MgCl₂, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.
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    A Hidden Transhydrogen Activity of a FMN-Bound Diaphorase under Anaerobic Conditions
    Collins, John; Zhang, Ting; Huston, Scott; Sun, Fangfang; Zhang, Y. H. Percival; Fu, Jinglin (PLOS, 2016-05-04)
    Background Redox cofactors of NADH/NADPH participate in many cellular metabolic pathways for facilitating the electron transfer from one molecule to another in redox reactions. Transhydrogenase plays an important role in linking catabolism and anabolism, regulating the ratio of NADH/NADPH in cells. The cytoplasmic transhydrogenases could be useful to engineer synthetic biochemical pathways for the production of high-value chemicals and biofuels. Methodology/Principal Findings A transhydrogenase activity was discovered for a FMN-bound diaphorase (DI) from Geobacillus stearothermophilus under anaerobic conditions. The DI-catalyzed hydride exchange were monitored and characterized between a NAD(P)H and a thio-modified NAD+ analogue. This new function of DI was demonstrated to transfer a hydride from NADPH to NAD+ that was consumed by NAD-specific lactate dehydrogenase and malic dehydrogenase. Conclusions/Significance We discover a novel transhydrogenase activity of a FMN-DI by stabilizing the reduced state of FMNH2 under anaerobic conditions. FMN-DI was demonstrated to catalyze the hydride transfer between NADPH and NAD+. In the future, it may be possible to incorporate this FMN-DI into synthetic enzymatic pathways for balancing NADH generation and NADPH consumption for anaerobic production of biofuels and biochemicals.
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    A high-energy-density sugar biobattery based on a synthetic enzymatic pathway
    Zhu, Zhiguang; Tam, Tsz Kin; Sun, Fangfang; You, Chun; Zhang, Y. H. Percival (Springer Nature, 2014-01)
    High-energy-density, green, safe batteries are highly desirable for meeting the rapidly growing needs of portable electronics. The incomplete oxidation of sugars mediated by one or a few enzymes in enzymatic fuel cells suffers from low energy densities and slow reaction rates. Here we show that nearly 24 electrons per glucose unit of maltodextrin can be produced through a synthetic catabolic pathway that comprises 13 enzymes in an air-breathing enzymatic fuel cell. This enzymatic fuel cell is based on non-immobilized enzymes that exhibit a maximum power output of 0.8 mW cm(-2) and a maximum current density of 6 mA cm(-2), which are far higher than the values for systems based on immobilized enzymes. Enzymatic fuel cells containing a 15% (wt/v) maltodextrin solution have an energy-storage density of 596 Ah kg(-1), which is one order of magnitude higher than that of lithium-ion batteries. Sugar-powered biobatteries could serve as next-generation green power sources, particularly for portable electronics.