Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymatic biosystem provided an alternative method for the utilization of cellulosic biomass rather than cellulolytic enzyme hydrolysis to fermentative monomeric sugars followed by microorganism fermentation, showing potentials in the production of biocommodities such as hydrogen, rare sugars, and electricity from cellulosic biomass.
Cell-free synthetic enzymatic biosystem is emerging to expand the traditional biotechnological mode by utilizing a number of purified/partially purified enzymes and coenzymes in a single reaction vessel for the production of desired products from low-cost substrates. Here, a cell-free synthetic biosystem containing minimized number of reactions was designed for the conversion of d-glucose to l-lactate via pyruvate. This NADH-balanced biosystem was comprised of only 5 thermophilic enzymes without ATP supplementation. After optimization of enzyme loading amounts, buffer concentration and cofactor concentration, d-glucose was converted to l-lactate with a product yield of ∼90%. Our study has provided an emerging platform with potentials in producing pyruvate-derived chemicals, and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.
cXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol ؊1 versus 94.7 U nmol ؊1 ) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (T m ), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions. X ylanases (endo-1,4--xylanase; EC 3.2.1.8) hydrolyze the -1,4 bond in the xylan backbone of hemicellulose, which yields short xylooligosaccharides or xylose. They are distributed in several glycoside hydrolase families (GH), such as GH5, GH7, GH8, GH10, GH11, and GH43, and most of them belong to GH10 and GH11 according to the CAZy (Carbohydrate Active Enzymes) database (1). Xylanase families differ in their physicochemical properties (molecular mass and isoelectric point [pI]), three-dimensional structures, and catalytic mechanisms (2). Enzymes from GH family 11 have a relatively low molecular weight and a high pI, and they possess a -jelly roll secondary structure, a double displacement catalytic mechanism, and two glutamates acting as catalytic residues (3). In contrast, members of GH10 typically have a high molecular mass and a low pI and display an (␣/) 8 barrel fold. Xylanases have been found in many organisms, such as bacteria, fungi, algae, and protozoa (4). Xylanases from fungi or mesophilic bacteria generally share a low optimum temperature and poor thermal stability (5). Thermostable xylanases have been characterized from some thermophilic microorganisms, such as Clostridium thermocellum (6...
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