Cello-oligosaccharides Research Articles

Biosynthesis and one-step enrichment process of potentially prebiotic cello-oligosaccharides produced by β-glucosidase from Fusarium solani.

Cello-oligosaccharides (COS) become a new type of functional oligosaccharides. COS transglycosylation reactions were studied to enhance COS yield production. Seeking the ability of the free form of Fusarium solani β-glucosidase (FBgl1) to synthesize COS under low substrate concentrations, we found out that this biocatalyst initiates this reaction with only 1g/L of cellobiose, giving rise to the formation of cellotriose. Cellotriose and cellopentaose were detected in biphasic conditions with an immobilized FBgl1 and when increased to 50g/L of cellobiose as a starter concentration. After the biocatalyst recycling process, the trans-glycosylation yield of COS was maintained after 5 cycles, and the COS concentration was 6.70 ± 0.35g/L. The crude COS contained 20.15 ± 0.25g/L glucose, 23.15 ± 0.22g/L non-reacting substrate cellobiose, 5.25 ± 0.53g/L, cellotriose and 1.49 ± 0.32g/L cellopentaose. A bioprocess was developed for cellotriose enrichment, using whole Bacillus velezensis cells as a microbial purification tool. This bacteria consumed glucose, unreacted cellobiose, and cellopentaose while preserving cellotriose in the fermented medium. This study provides an excellent enzyme candidate for industrial COS production and is also the first study on the single-step COS enrichment process.

Purification and fractionation of cellooligosaccharides synthesized from controlled cellulose hydrolysis by sulfuric acid using nanofiltration

Cellooligosaccharides (COS) as potential prebiotics have drawn increasing attention. They can be synthesized from cellulose via controlled hydrolysis by sulfuric acid (SA). However, it is challenging to efficiently separate COS from the hydrolysate. In this study, nanofiltration (NF) with commercial membranes was investigated to separate COS from cellulose hydrolysate by removing SA and non-prebiotic sugars (e.g., glucose) and further fractionating COS by size. Based on water permeability, pore size, and acid stability, two membranes (Trisep® XN45 and Synder® NFG) were selected from four candidates. The effects of operation conditions (membrane and applied pressure) and feed concentrations (sugars and SA) on the COS purification and fractionation were studied. It was found that the XN45 membrane with an estimated average pore size of 0.61 nm was efficient in removing SA and glucose from COS, while NFG with a larger pore size of 0.84 nm can be used to fractionate and further purify COS based on size (degree of polymerization, DP). Four sequential batch NF operations of cellulose hydrolysate through XN45 (×3) and NFG (×1) membranes recovered 80 % of the total COS in the hydrolysate and removed 99 % of SA and 95 % of glucose. NFG membrane can roughly separate COS into two fractions, one (retentate) with more larger COS (DP ≥ 9) and another (permeate) with more smaller COS (DP 2–8). This study successfully demonstrated the feasibility of NF to purify and fractionate COS from cellulose hydrolysate.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.

Production of cellooligosaccharides from oil palm bunch in bio-based choline chloride deep eutectic solvents and MALDI-TOF MS analysis of COS mixture

Lignocellulosic biomass pretreatment and cellulose dissolution are principal challenges for its conversion to high-value chemicals, materials and fuels. This work focused on production of soluble cellooligosaccharides (COS) from palm bunch for future use as nutraceutical supplement to enhance human's immune and digestive systems. Deep eutectic solvents (DESs) have been found as eco-friendly, renewable and promising solvents selective for lignin and hemicellulose removal from biomass. Additionally, the investigation of most suitable DES for both cellulose dissolution and subsequently cleavage of cellulose to COS is crucial. In the study, palm empty fruit bunch (EFB) was pretreated in different DES for screening of most favorable cellulose inter- and intra-molecular structural dissolution in a combination with beta,1–4 glycosidic bond cleavage by elevated reaction temperature for COS production. ChCl/urea was found to be the most promising DES for dissolving 76.83% of EFB constituents. The highest COS production yield at 90.1%w/w based on treated EFB fraction (74.7% COS yield based on cellulose in native EFB) was achieved in ChCl/urea DES from cellulose fraction extracted from EFB at 240 °C for 20 min. The study additionally provided insightful characterization and elucidation of COS molecular structure using 2D-HSQC and MALDI-TOF/MS techniques. MALDI-TOF/MS was a promising and rapid technique for simultaneously qualitative and quantitative analysis of COS at different degrees of polymerization in COS product.

Selective utilization of gluco-oligosaccharides by lactobacilli: A mechanism study revealing the impact of glycosidic linkages and degree of polymerization on their utilization.

Gluco-oligosaccharides (GlcOS) are potential prebiotics that positively modulate beneficial gut commensals like lactobacilli. For the rational design of GlcOS as prebiotics or combined with lactobacilli as synbiotics, it is important to establish the structure requirements of GlcOS and specificity toward lactobacilli. Herein, the utilization of 10 GlcOS with varied degrees of polymerization (DP) and glycosidic linkages by 7 lactobacilli strains (Levilactobacillus brevis ATCC 8287, Limosilactobacillus reuteri ATCC PTA 6475, Lacticaseibacillus rhamnosus ATCC 53103, Lentilactobacillus buchneri ATCC 4005, Limosilactobacillus fermentum FUA 3589, Lactiplantibacillus plantarum WCFS1, and Lactobacillus gasseri ATCC 33323) was studied. L. brevis ATCC 8287 was the only strain that grew on α/β-(1→4/6) linked disaccharides, whereas other strains showed diverse patterns, dependent on the availability of genes encoding sugar transporters and catabolic enzymes. The effect of DP on GlcOS utilization was strain dependent. β-(1→4) Linked cello-oligosaccharides (COS) supported the growth of L. brevis ATCC 8287 and L. plantarum WCFS1, and shorter COS (DP 2-3) were preferentially utilized over longer COS (DP 4-7) (consumption ≥90% vs. 40%-60%). α-(1→4) Linked maltotriose and maltodextrin (DP 2-11) were effectively utilized by L. brevis ATCC 8287, L. reuteri ATCC 6475, and L. plantarum WCFS1, but not L. fermentum FUA 3589. Growth of L. brevis ATCC 8287 on branched isomalto-oligosaccharides (DP 2-6) suggested preferential consumption of DP 2-3, but no preference between α-(1→6) and α-(1→4) linkages. The knowledge of the structure-specific GlcOS utilization by different lactobacilli from this study helps the structural rationale of GlcOS for prebiotic development.

Enzymatic production of cello-oligosaccharides with potential human prebiotic activity and release of polyphenols from grape marc

Grape marc, the main winemaking byproduct, is an excellent source of bioactive polyphenols, such as anthocyanins, resveratrol and quercetin. An enzyme-catalysed treatment of marc was developed using endo-1,4-β-d-glucanase to release polyphenol O-glucosides and simultaneously generate the optimal concentration of water-soluble cello-oligosaccharides (COS), including cellopentaose, cellotriose, and cellobiose from the marc matrix. The prebiotic properties of marc hydrolysate rich in COS was assessed using human probiotic monocultures of Lactobacillus spp. and Bifidobacterium spp. strains, and invitro human faecal fermentation. The COS-rich hydrolysate showed excellent prebiotic effect in both studies, successfully supporting the growth of beneficial probiotic strains, and was highly fermentable by faecal microbiota producing gas and short chain fatty acids. Acetate and propionate production were the highest when faecal bacteria fermented COS-rich solution compared with standard substrates. For the first time, COS was shown to be fermented by faecal microbiota, demonstrating the potential benefits of valorised grape marc.

Effects of dietary supplementation of lignocellulose-derived cello-oligosaccharides on growth performance, antioxidant capacity, immune response, and intestinal microbiota in rainbow trout (Oncorhynchus mykiss)

This study evaluated the prebiotic potential of cello-oligosaccharides (COS) produced from birch (Betula pendula), an under-utilised lignocellulosic source from the forestry industry, on growth performance, mucosal immunity, gut microbiota composition, and antioxidant capacity of juvenile rainbow trout (Oncorhynchus mykiss). In a 45-day trial, the fish were fed with diets containing 0%, 0.1%, 0.5% and 1.5% COS, while a diet containing fructo-oligosaccharides (0.5% FOS) was used as a positive control. Fish fed with the 0.5% and 1.5% COS diets showed significantly (P < 0.05) higher abundance of Ruminococcaceae, Bacillaceae and Lactobacillaceae, in the faecal microbiota. The COS diets also induced higher antioxidant capacity in the gut and serum, but there were no treatment effects (P > 0.05) on growth of rainbow trout. Gene expression analysis of the intestine showed significant elevation (P < 0.05) in expression of complement (c3 and c-type lectin) and receptor (tlr2) genes of the innate immune system in COS-fed fish. However, for cytokine and adaptive immune genes, no significant differences (P > 0.05) in gene transcripts were observed between the COS/FOS diets with the control diet. These results suggest that dietary cello-oligosaccharides can be a useful feed supplement for rainbow trout, which can modulate intestinal microbial communities, innate immune response and antioxidant capacity of the host.

Induction of plant disease resistance by mixed oligosaccharide elicitors prepared from plant cell wall and crustacean shells.

Basal plant immune responses are activated by the recognition of conserved microbe-associated molecular patterns (MAMPs), or breakdown molecules released from the plants after damage by pathogen penetration, so-called damage-associated molecular patterns (DAMPs). While chitin-oligosaccharide (CHOS), a primary component of fungal cell walls, is most known as MAMP, plant cell wall-derived oligosaccharides, cello-oligosaccharides (COS) from cellulose, and xylo-oligosaccharide (XOS) from hemicellulose are representative DAMPs. In this study, elicitor activities of COS prepared from cotton linters, XOS prepared from corn cobs, and chitin-oligosaccharide (CHOS) from crustacean shells were comparatively investigated. In Arabidopsis, COS, XOS, or CHOS treatment triggered typical defense responses such as reactive oxygen species (ROS) production, phosphorylation of MAP kinases, callose deposition, and activation of the defense-related transcription factor WRKY33 promoter. When COS, XOS, and CHOS were used at concentrations with similar activity in inducing ROS production and callose depositions, CHOS was particularly potent in activating the MAPK kinases and WRKY33 promoters. Among the COS and XOS with different degrees of polymerization, cellotriose and xylotetraose showed the highest activity for the activation of WRKY33 promoter. Gene ontology enrichment analysis of RNAseq data revealed that simultaneous treatment of COS, XOS, and CHOS (oligo-mix) effectively activates plant disease resistance. In practice, treatment with the oligo-mix enhanced the resistance of tomato to powdery mildew, but plant growth was not inhibited but rather tended to be promoted, providing evidence that treatment with the oligo-mix has beneficial effects on improving disease resistance in plants, making them a promising class of compounds for practical application.

Fractionation of functional oligosaccharides produced from sugarcane straw using serial nanofiltration membranes and their influence on prebiotic potential

Functional oligosaccharides are non-digestible by human gut enzymes and provide health benefits as fibers and prebiotics. The cello-oligosaccharides (COS) and xylooligosaccharides (XOS) are functional oligosaccharides obtained from xylan and cellulose, respectively, and are present in lignocellulosic material. The serial NF membranes process was performed to investigate the impact of the fractionation process on the prebiotic activity of oligosaccharides from xylan and cellulose. The NP030 (weight cut-off of 500–600 Da) and DK (weight cut-off of 150–300 Da) NF polymeric membranes were employed using defined operational conditions. The diafiltration (DF) was also investigated and it was determined that only a 1-time DF for NP030 was a more suitable strategy and improved the performance indices for short DP oligosaccharides. The short DP fractions obtained favored cell density for probiotic strains, which presented an increase on the optical density of up to 25 % after the fractionating process; enabling the use of short purified fractions in the food and pharmaceutical industry as a prebiotic ingredient.

Hydrothermal pretreatment for the production of prebiotic oligosaccharides from tobacco stem

Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response surface methodology to evaluate the effects of temperature (161.72 to 218.3 °C) and solid load (SL) (2.93 to 17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The result indicated yield of 96% w[XOS]/w[xylan] for 190 °C-2.93% SL. The highest value for COS and total oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 °C-17.07% SL. The mass balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

Cello-oligosaccharides production from multi-stage enzymatic hydrolysis by lignocellulosic biomass and evaluation of prebiotic potential

Cello-oligosaccharides (COS) are very recent promising water-soluble dietary fibers, which favors several application in food, chemical and pharmaceutical industries. A three-stage enzymatic hydrolysis of cellulose extracted from agroindustrial wastes (sugarcane straw and coffee husk) with the 1,4-β-endoglucanase cellulase of glycoside hydrolase family (GH12) was developed with a processing time of 48 h. Finds evidenced that removal of products (glucose and cello-oligosaccharides) at each stage provided an increase in cello-oligosaccharides enzymatic hydrolysis in yield of approximately 65%. COS promoted growth of Lactobacillus acidophilus and Lactobacillus brevis after 24 h of fermentation, although they have presented low efficiency with Bifidobacterium sp. This multi-stage enzymatic hydrolysis has potential as a successful strategy to reduction enzyme requirements for COS production with prebiotic properties. Industrial relevanceThis work proposes an innovative technology of multi-stage enzymatic hydrolysis using the enzyme adsorbed on the cellulose surface obtained from renewable sources (sugarcane straw and coffee husk). This strategy is potential to increase the COS yield production and possibly make its cost-viable. The study is also relevant due to accessing the growth promotion of a representative selection of probiotic Lactobacillus and Bifidobacterium strains in order to evidence their prebiotic potential to be used as a possible functional ingredient for the pharmaceutical and food industries.

Comparing 13C methyl and deuterated methyl isotopic labeling for the quantification of methyl cellulose patterns using mass spectrometry

The methyl substitution along and among the polymer chains of methyl cellulose (MC) is commonly analyzed by ESI-MS after perdeuteromethylation of the free-OH groups and partial hydrolysis to cello-oligosaccharides (COS). This method requires a correct quantification of the molar ratios of the constituents belonging to a particular degree of polymerization (DP). However, isotopic effects are most pronounced for H/D since their mass difference is 100%. Therefore, we investigated whether more precise and accurate results could be obtained for the methyl distribution of MC by MS of 13CH3 instead of CD3-etherified O-Me-COS. Internal isotope labeling with 13CH3 makes the COS of each DP chemically and physically much more similar, reducing mass fractionation effects, but at the same time requires more complex isotopic correction for evaluation. Results from syringe pump infusion ESI-TOF-MS with 13CH3 and CD3 as isotope label were equal. However, in the case of LC-MS with a gradient system, 13CH3 was superior to CD3. In the case of CD3, the occurrence of a partial separation of the isotopologs of a particular DP resulted in slight distortion of the methyl distribution since the signal response is significantly dependent on the solvent composition. Isocratic LC levels this problem, but one particular eluent-composition is not sufficient for a series of oligosaccharides with increasing DP due to peak broadening. In summary, 13CH3 is more robust to determine the methyl distribution of MCs. Both syringe pump and gradient-LC-MS measurements are possible, and the more complex isotope correction is not a disadvantage.Graphical abstract

Continuous process technology for bottom-up synthesis of soluble cello-oligosaccharides by immobilized cells co-expressing three saccharide phosphorylases

BackgroundContinuous processing with enzyme reuse is a well-known engineering strategy to enhance the efficiency of biocatalytic transformations for chemical synthesis. In one-pot multistep reactions, continuous processing offers the additional benefit of ensuring constant product quality via control of the product composition. Bottom-up production of cello-oligosaccharides (COS) involves multistep iterative β-1,4-glycosylation of glucose from sucrose catalyzed by sucrose phosphorylase from Bifidobacterium adeloscentis (BaScP), cellobiose phosphorylase from Cellulomonas uda (CuCbP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCdP). Degree of polymerization (DP) control in the COS product is essential for soluble production and is implemented through balance of the oligosaccharide priming and elongation rates. A whole-cell E. coli catalyst co-expressing the phosphorylases in high yield and in the desired activity ratio, with CdP as the rate-limiting enzyme, was reported previously.ResultsFreeze-thaw permeabilized E. coli cells were immobilized in polyacrylamide (PAM) at 37–111 mg dry cells/g material. PAM particles (0.25–2.00 mm size) were characterized for COS production (~ 70 g/L) in mixed vessel with catalyst recycle and packed-bed reactor set-ups. The catalyst exhibited a dry mass-based overall activity (270 U/g; 37 mg cells/g material) lowered by ~ 40% compared to the corresponding free cells due to individual enzyme activity loss, CbP in particular, caused by the immobilization. Temperature studies revealed an operational optimum at 30 °C for stable continuous reaction (~ 1 month) in the packed bed (volume: 40 mL; height: 7.5 cm). The optimum reflects the limits of PAM catalyst structural and biological stability in combination with the requirement to control COS product solubility in order to prevent clogging of the packed bed. Using an axial flow rate of 0.75 cm− 1, the COS were produced at ~ 5.7 g/day and ≥ 95% substrate conversion (sucrose 300 mM). The product stream showed a stable composition of individual oligosaccharides up to cellohexaose, with cellobiose (48 mol%) and cellotriose (31 mol%) as the major components.ConclusionsContinuous process technology for bottom-up biocatalytic production of soluble COS is demonstrated based on PAM immobilized E. coli cells that co-express BaScP, CuCbP and CcCdP in suitable absolute and relative activities.

Modified dietary fiber from cassava pulp affects the cecal microbial population, short-chain fatty acid, and ammonia production in broiler chickens

The objective of this study was to investigate the effects of modified dietary fiber from cassava pulp (M-DFCP) supplementation in broiler diets on cecal microbial populations, short-chain fatty acids (SCFAs), ammonia production, and immune responses. A total of 336, one-day-old male broiler chicks (Ross 308) were distributed over 4 dietary treatments in 7 replicate pens (n=12 chicks) using a completely randomized design. Chicks were fed the control diet and 3 levels of M-DFCP (0.5, 1.0, and 1.5%) for an experimental duration of 42 d. The M-DFCP contained total dietary fiber (TDF), soluble dietary fiber (SDF), insoluble dietary fiber (IDF), cello-oligosaccharides (COS), and xylo-oligosaccharides (XOS) of approximately 280.70, 22.20, 258.50, 23.93, and 157.55 g/kg, respectively. The 1.0 and 1.5% M-DFCP supplementation diets showed positive effects on stimulating the growth of Lactobacillus spp. and Bifidobacterium spp., enhancing SCFAs (acetic, propionic, butyric acid, and branched SCFAs) and lactic acid concentrations during growing periods. Broilers fed 1.0 and 1.5% M-DFCP also exhibited a significant increase in caecal Lactobacillus spp. and lactic acid concentrations during the finisher period as well. In addition, M-DFCP also reduced cecal digesta and excreta ammonia production in broilers over both periods (0-21 and 22-42 d of age). However, M-DFCP did not exhibit any effect on total serum immunoglobulin (Ig) or lysozyme activity. In conclusion, this study shows that M-DFCP can be used as a dietary fiber source in broiler diets, with a recommended level of approximately 1.0%.

The oxidized cellooligosaccharides confer thermotolerance in Arabidopsis by priming ethylene via heat shock factor A2.

Global climate change, especially heatwaves and aridity, is a major threat to agricultural production and food security. This requires common efforts from the scientific community to find effective solutions to better understand and protect the plant's vulnerabilities to high temperatures. The current study demonstrates the potential of cellooligosaccharides (COS), which are native and oxidized signaling molecules released by lytic polysaccharide monooxygenases (LPMO) enzymes during cell wall degradation by microbial pathogens. The extracellular perception of COS leads to the activation of damage-triggered immunity, often protecting the plant against biotic stress. However, how these signaling molecules affect abiotic stress tolerance is poorly understood. Here, we show that native COS and oxidized COS (oxiCOS) perception increase the transcript levels of several HEAT SHOCK FACTORS (HSFs) and HEAT SHOCK PROTEINS (HSPs) genes in Arabidopsis plants. However, only oxiCOS treatment triggers ethylene priming and increases thermotolerance. Furthermore, the function of the transcription factor HSFA2 is required for these processes. Altogether, our results indicate that the perception of Damage-Associated Molecular Patterns (DAMPs) may improve tolerance to adverse abiotic conditions, like exposure to high temperatures.

Enzymatic generation of short chain cello-oligosaccharides from Miscanthus using different pretreatments

Enzyme combinations producing short-chain cello-oligosaccharides (COS) as major bio-products from cellulose of Miscanthus Mx2779 accessed through different pretreatment methods were compared. Over short hydrolysis times, processive endoglucanase TfCel9a produced a high percentage of cellotetraose and cellopentaose and is synergistic with endoglucanase CcCel9m for producing short oligomers from amorphous cellulose but had low activity on untreated Miscanthus. Hydrolysis of the latter improved when these were combined with a mutant cellobio/triohydrolase OsCelC7(−105) and a lytic polysaccharide monooxygenase TrCel61a, a combination which also produced the highest COS yields from phosphoric acid swollen cellulose. Steam explosion pretreatment of Miscanthus increased COS yields, with/without phosphoric acid swelling, while increased swelling time (from 20 to 45 min) also increased yields but decreased the need for TrCel61a. The highest COS yields (933 mg/g glucan) and most stable product profile were obtained using ionic liquid [C2mim][OAc] pretreatment and the three enzyme mixture TfCel9a, Cel9m and OsCel7a(−105).

Steering the formation of cellobiose and oligosaccharides during enzymatic hydrolysis of asparagus fibre

The enzymatic conversion of cellulosic-rich waste streams of white asparagus into cellobiose and cello-oligosaccharides (COS) is proposed for producing a natural carrier agent. The enzyme cocktail ‘Celluclast’ was used to steer towards maximum conversion and minimum formation of monosaccharides to obtain an enzymatic hydrolysate with a high glass transition temperature (Tg). Different enzyme loadings and hydrolysis times were tested in combination with a sodium hydroxide pre-treatment of the asparagus fibre. A loading of 700 nkat/g substrate and 7 h of hydrolysis time resulted in the best yield/purity combination, namely a conversion of 36 g/100 g cellulose with 81% celllobiose/COS. The same hydrolysis conditions were tested in a larger bench-scale experiment (conversion of 45 g/100 g cellulose) and the soluble hydrolysates were concentrated and spray-dried. The high Tg (108 °C) of the spray-dried hydrolysates of asparagus fibre proves its potential usage as a carrier agent for spray drying.

Production of cello‐oligosaccharides through the biorefinery concept: A technical‐economic and life‐cycle assessment

AbstractProducers look for co‐products to increase the value chain and production flexibility. With a high market aggregate value, the production of cello‐oligosaccharides (COS) as functional oligosaccharides from cellulosic substrates has been investigated. As COS/cellopentaose production process are rarely described in the literature, this study estimated the cellopentaose production cost and performed a life‐cycle assessment of the cellopentaose production process previously developed using sugarcane straw as a substrate. The results demonstrated that, through the six scenarios studied, it was possible to obtain a cellopentaose unit production cost varying between USD 0.40 and 1.15/mg, and that optimization in the upstream sector can reduce the total plant direct cost and the overall life‐cycle impact. The results also suggested that a better understanding of the hydrolysis solid/liquid proportion is necessary to reduce the bulk material cost. An environmental impact reduction of between 16.2% and 19.9% was also observed. In terms of the integrated biorefinery concept, sugarcane straw fermentation represents a prospective technology for the production of COS. © 2021 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Kinetic modeling of phosphorylase-catalyzed iterative \u03b2-1,4-glycosylation for degree of polymerization-controlled synthesis of soluble cello-oligosaccharides

BackgroundCellodextrin phosphorylase (CdP; EC 2.4.1.49) catalyzes the iterative β-1,4-glycosylation of cellobiose using α-d-glucose 1-phosphate as the donor substrate. Cello-oligosaccharides (COS) with a degree of polymerization (DP) of up to 6 are soluble while those of larger DP self-assemble into solid cellulose material. The soluble COS have attracted considerable attention for their use as dietary fibers that offer a selective prebiotic function. An efficient synthesis of soluble COS requires good control over the DP of the products formed. A mathematical model of the iterative enzymatic glycosylation would be important to facilitate target-oriented process development.ResultsA detailed time-course analysis of the formation of COS products from cellobiose (25 mM, 50 mM) and α-d-glucose 1-phosphate (10–100 mM) was performed using the CdP from Clostridium cellulosi. A mechanism-based, Michaelis–Menten type mathematical model was developed to describe the kinetics of the iterative enzymatic glycosylation of cellobiose. The mechanistic model was combined with an empirical description of the DP-dependent self-assembly of the COS into insoluble cellulose. The hybrid model thus obtained was used for kinetic parameter determination from time-course fits performed with constraints derived from initial rate data. The fitted hybrid model provided excellent description of the experimental dynamics of the COS in the DP range 3–6 and also accounted for the insoluble product formation. The hybrid model was suitable to disentangle the complex relationship between the process conditions used (i.e., substrate concentration, donor/acceptor ratio, reaction time) and the reaction output obtained (i.e., yield and composition of soluble COS). Model application to a window-of-operation analysis for the synthesis of soluble COS was demonstrated on the example of a COS mixture enriched in DP 4.ConclusionsThe hybrid model of CdP-catalyzed iterative glycosylation is an important engineering tool to study and optimize the biocatalytic synthesis of soluble COS. The kinetic modeling approach used here can be of a general interest to be applied to other iteratively catalyzed enzymatic reactions of synthetic importance.

Synthesis of cello-oligosaccharides by depolymerization of cellulose: A review

Cello-oligosaccharides (COS) are small carbohydrate molecules derived from cellulose through partial hydrolysis. COS exert physiological influence on plants, animals and humans when externally dosed or consumed. Their potential as a prebiotic for humans and animals and as an elicitor for plants has generated interest in their synthesis and purification. Currently, their lack of availability, even at scales necessary for research, has restricted the progress in this field. In this review, we explore the methods for depolymerization of cellulose with a focus on catalytic partial hydrolysis to COS. Aspects of cellulose depolymerization using enzymes, homogeneous catalysts and heterogeneous catalysts are described. Methods for qualitative and quantitative characterization of COS are discussed along with future challenges in COS synthesis. Role of mechanocatalysis and other technologies in branched oligosaccharide synthesis is also explored for solvent free cellulose depolymerization.