Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to 15 enzymes under mild reaction conditions (i.e. 37°C and atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmol of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.
The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the worlds future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agricultures environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.
Let enzymes work: H2 was produced from xylose and water in one reactor containing 13 enzymes (red). By using a novel polyphosphate xylulokinase (XK), xylose was converted into H2 and CO2 with approaching 100?% of the theoretical yield. The findings suggest that cell-free biosystems could produce H2 from biomass xylose at low cost. Xu5P = xylulose 5-phosphate, G6P = glucose 6-phosphate.
Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.
The discovery of stable and active polyphosphate glucokinase (PPGK, EC 220.127.116.11) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²? was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²? at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k (cat) and K(m) values of CBM-PPGK on glucose were 96.9 and 39.7 s?¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t(1/2)?=?120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.
One-step enzyme purification and immobilization were developed based on simple adsorption of a family 3 cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose (RAC). An open reading frame (ORF) Cthe0217 encoding a putative phosphoglucose isomerase (PGI, EC 18.104.22.168) from a thermophilic bacterium Clostridium thermocellum was cloned and the recombinant proteins with or without CBM were over-expressed in Escherichia coli. The rate constant (kcat ) and Michaelis-Menten constant (Km ) of CBM-free PGI at 60°C were 2,765 s(-1) and 2.89 mM, respectively. PGI was stable at a high protein concentration of 0.1 g/L but deactivated rapidly at low concentrations. Immobilized CBM (iCBM)-PGI on RAC was extremely stable at ?60°C, nearly independent of its mass concentration in bulk solution, because its local concentration on the solid support was constant. iCBM-PGI at a low concentration of 0.001 g/L had a half-life time of 190 h, approximately 80-fold of that of free PGI. Total turn-over number of iCBM-PGI was as high as 1.1×10(9) mole of product per mole of enzyme at 60°C. These results suggest that a combination of low-cost enzyme immobilization and thermoenzyme led to an ultra-stable enzyme building block suitable for cell-free synthetic pathway biotransformation that can implement complicated biochemical reactions in vitro.
The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. 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 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.
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