20702-56-1

Upstream product

• 106927-48-4 4-nitrophenyl β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside 
• 2106-10-7 1-deoxy-1-fluoro-α-D-glucose 
• 528-50-7 cellobiose 
• 59-56-3 α-D-glucopyranosyl-1-phosphate 
• 9004-34-6 cellulose 
• 1184-46-9 D-maltohexaose 
• 171609-69-1 maltotetraosyltrehalose 
• 1980-14-9 maltoheptaose 

Downstream Products

• 15548-43-3 O-β-D-mannopyranosyl-(1->4)-D-mannopyranose 
• 3458-28-4 D-Mannose 
• 6401-81-6 mannotriose 
• 132314-20-6 3,5-dimethylphenylcarbamate of cellotetraose 
• 50-99-7 D-glucose 
• 67-47-0 5-hydroxymethyl-2-furfuraldehyde 
• 492-61-5 β-D-glucose 
• 78797-67-8 O-β-D-glucopyranosyl-(1->4)-O-β-D-glucopyranosyl-(1->4)-1,6-anhydro-β-D-glucopyranose 
• 73356-26-0 β-D-glucopyranosyl-2-glycolaldehyde 
• 498-07-7 levoglucosan 
• 35405-71-1 1,6-anhydro-β-D-cellobiose 
• 13360-52-6 Cellobiose 
• 145072-72-6 o-chloro-p-nitrophenyl β-maltotetraoside 
• 66767-99-5 α-D-glucopyranosyl-(1->4)-α-D-glucopyranosyl-(1->4)-α-D-glucopyranosyl-(1->4)-D-glucitol 

Relevant academic research and scientific papers

Supercritical water treatment for cello-oligosaccharide production from microcrystalline cellulose

Microcrystalline cellulose was treated in supercritical water at 380 °C and at a pressure of 250 bar for 0.2, 0.4, and 0.6 s. The yield of the ambient-water-insoluble precipitate and its average molar mass decreased with an extended treatment time. The highest yield of 42 wt % for DP2-9 cello-oligosaccharides was achieved after the 0.4 s treatment. The reaction products included also 11 wt % ambient-water-insoluble precipitate with a DPw of 16, and 6.1 wt % monomeric sugars, and 37 wt % unidentified degradation products. Oligo- and monosaccharide-derived dehydration and retro-aldol fragmentation products were analyzed via a combination of HPAEC-PAD-MS, ESI-MS/MS, and GC-MS techniques. The total amount of degradation products increased with treatment time, and fragmented (glucosyln-erythrose, glucosyln-glycolaldehyde), and dehydrated (glucosyln-levoglucosan) were identified as the main oligomeric degradation products from the cello-oligosaccharides.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH?7.0 and 60?°C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5–8.5) and temperatures (up to 50?°C) for extended periods of time (over 48?h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8?kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Hydrolysis behaviors of sugarcane bagasse pith in subcritical carbon dioxide-water

The aim of this study was to describe the hydrolysis behavior of sugarcane bagasse pith (SCBP) in subcritical CO2-water. The hydrolysis was carried out in a batch reactor using different temperatures (160 to 260 °C), liquid to solid ratios (20:1 to 100:1), CO2 pressures (0 to 7.3 MPa), stirring speeds (0 to 500 rpm) and reaction times (0 to 40 min). The highest total reducing sugar yield (43.6%) was obtained at 200 °C, liquid to solid ratio 30:1, 2 MPa CO2, 500 rpm and 50 min. Two-dimensional heteronuclear single quantum coherence (2D HSQC) nuclear magnetic resonance (NMR), scanning electron microscopy (SEM) and Fourier transform infrared spectrometry (FT-IR) were used to help elucidate the physical and chemical characteristics of the raw material and residual solid particles, with results consistent with the removal of hemicellulose during hydrolysis. The changes in the concentration of products with time were analyzed to understand product distribution through high-performance liquid chromatography (HPLC) and to infer the reaction mechanism.

Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides

In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86-95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.

Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis

The microbial deconstruction of the plant cell wall is a key biological process that is of increasing importance with the development of a sustainable biofuel industry. The glycoside hydrolase families GH5 (PaMan5A) and GH26 (PaMan26A) endo-β-1,4-mannanases from the coprophilic ascomycete Podospora anserina contribute to the enzymatic degradation of lignocellulosic biomass. In this study, P. anserina mannanases were further subjected to detailed comparative analysis of their substrate specificities, active site organization, and transglycosylation capacity. Although PaMan5A displays a classical mode of action, PaMan26A revealed an atypical hydrolysis pattern with the release of mannotetraose and mannose from mannopentaose resulting from a predominant binding mode involving the -4 subsite. The crystal structures of PaMan5A and PaMan26A were solved at 1.4 and 2.85 A resolution, respectively. Analysis of the PaMan26A structure supported strong interaction with substrate at the -4 subsite mediated by two aromatic residues Trp-244 and Trp-245. The PaMan26A structure appended to its family 35 carbohydrate binding module revealed a short and proline-rich rigid linker that anchored together the catalytic and the binding modules.

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-d-glucopyranosyl-[(1→4)- β-d-glucopyranosyl]n-(1→2)-d-glucopyranose, and β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]n- (1→3)-d-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85°C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85°C. The kcat values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s-1, respectively, whereas the kcat values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s-1, respectively. The KM values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.

Increased transglycosylation activity of Rhodotorula glutinis endo-β-glucanase in media containing organic solvent

The transglycosylation of p-nitrophenyl-β-D-cellotrioside to cellotetraose catalyzed by endo-1,4-β-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50°C in the presence of 40% acetone.

Study of the action of human salivary alpha-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocyanate

The degradation mechanism of a synthetic substrate, 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G3), by human salivary alpha-amylase (HSA) was investigated by kinetic and product analyses. It was observed that the enzyme attacked the various CNP-maltooligosaccharides (CNP-G3, to CNP-G6) releasing free CNP. Addition of 500 mM potassium thiocyanate (KSCN) was also found to greatly increase the rates of CNP-release. It was the fastest with CNP-G3, and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G5). On the other hand, addition of KSCN decreased the rate of cleavage between glucan-glucan bonds in maltopentaose. Product analysis showed that KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G3, and that product distribution of free CNP was largely dependent on substrate concentration. Formation of CNP-G6, a larger product than the original substrate CNP-G3, was found to be present in the digest at high concentrations of substrate and in the presence of KSCN. Based on these results, a degradation pathway for CNP-G3 involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN would result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as well as the enhancement of the catalytic efficiency.