Yield Of Oligosaccharides Research Articles

Enhanced enzymatic hydrolysis of pretreated almond-tree prunings for sugar production

Almond-tree prunings (ATP), an agricultural residue largely available in Mediterranean countries, were pretreated with either hot water or dilute sulphuric acid at 180–230°C. Solids derived from hot water pretreatments were further submitted to alkaline peroxide delignification. In addition, all solids obtained from the three mentioned processes were hydrolysed by cellulases and β-glucosidases to investigate their enzymatic digestibilities. Hot water pretreatment led to high oligosaccharide yields (18.2g/100g ATP at 190°C) while dilute acid pretreatment provided the highest monosaccharide yields (24.0g/100g ATP at 190°C) along with low concentrations of fermentation inhibitors. Glucose yields from enzymatic hydrolysis were strongly affected by both pretreatment type and pretreatment temperature. The highest temperature assayed for both hydrothermal and dilute sulphuric acid pretreatment maximized the glucose recovery (49.2% and 72.8%, respectively) while solids derived from alkaline peroxide treatment achieved maximal glucose concentrations (41.9g/L, 58.4% of potential yield).

Autohydrolysis of bamboo (Dendrocalamus giganteus Munro) culm for the production of xylo-oligosaccharides

Bamboo culm (Dendrocalamus giganteus Munro) was treated with hot water in non-isothermal conditions (140, 160, 180 and 200°C) for various reaction times (10, 30, 60 and 120 min) to generate xylo-oligosaccharides in a batch reactor at a solid to liquor ratio of 1:10 g/g. The influence of reaction conditions on yields, compositions and characteristics of the products were evaluated. The maximum yield of xylo-oligosaccharides of 47.49% was achieved at 180°C for 30 min with a relatively low level of xylose (4.73%) and other products (arabinose 0.20 g/L, galactose 0.19 g/L, HMF 0.15 g/L, furfural 0.36 g/L, acetic acid 0.64 g/L and formic acid 0.26 g/L). Higher temperature and/or longer reaction time reduced the yield of oligosaccharides, and enhanced concentrations of monosaccharides and byproducts. The result demonstrated that the yield of xylo-oligosaccharides obtained by autohydrolysis depends on the reaction temperature and time.

Direct saccharification of lignocellulosic biomass by hydrolysis with formic acid solution

An effective hydrolysis method for recovering oligosaccharides directly from lignocellulosic biomass has been investigated. Formic acid was selected as a hydrolysis reagent, due to its high acidity in organic acids. The degrees of cellulose hydrolysis and lignin elution were determined by the combination of the reaction temperature and formic acid aqueous solution concentration. We successfully obtained approximately 47 wt% of oligosaccharides, keeping the minimum lignin elution at 150°C and with 25 vol% formic acid aqueous solution concentration. Such a high yield of oligosaccharides was due to the relaxation of biomass structure. Furthermore, by diluting the formic acid aqueous solution concentration, or evaporating the solution, lignin can be separated from the oligosaccharides completely as deposits. Finally, two options for the efficient recovery of saccharides have been proposed.

Comprehensive Profiles of Human Milk Oligosaccharides Yield Highly Sensitive and Specific Markers for Determining Secretor Status in Lactating Mothers

Human milk oligosaccharides (HMOs), as an abundant and bioactive component of breast milk, work in many ways to promote the health of breast fed infants. The expression of HMOs has been shown to vary in accordance with Lewis blood type and secretor status, as women of different blood types differ in the expression of α1,2 fucosyltransferase (FUT2) and α1,3/4 fucosyltransferase (FUT3). In this study, HMOs were extracted from the milk of 60 women from The Gambia, Africa with various Lewis and secretor blood types. The HMOs were profiled using high resolution HPLC-Chip/TOF mass spectrometry. Notably, the amounts of fucosylation varied significantly between Le(a+b-) nonsecretors, Le(a-b+) and Le(a-b-) secretors, and Le(a-b-) nonsecretors. With higher frequency of expression of the recessive Lewis negative and nonsecretor phenotypes in West African populations, the HMO profiles of several milks from women of these phenotypes were examined, demonstrating decreased amounts of total oligosaccharide abundance and lower relative amounts of fucosylation. Also in this study, four specific fucosylated structures (2'FL, LNFP I, LDFT, and LNDFH I) were determined to be specific and sensitive glycan markers for rapidly determining secretor status without the need for serological testing.

Effect of Pretreatment Severity on Accumulation of Major Degradation Products from Dilute Acid Pretreated Corn Stover and Subsequent Inhibition of Enzymatic Hydrolysis of Cellulose

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R(o)), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26 mg/mL, while furfural increased from 1.04 to 1.33 mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23 mg/mL as formate rose from 0.62 to 1.83 mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.

Evaluation of Oligosaccharide Synthesis from Lactose and Lactulose Using β-Galactosidases from Kluyveromyces Isolated from Artisanal Cheeses

The β-galactosidase activity of 15 Kluyveromyces strains isolated from cheese belonging to Kluyveromyces lactis and Kluyveromyces marxianus species was tested for the production of oligosaccharides derived from lactose (GOS) and lactulose (OsLu). All Kluyveromyces crude cell extracts (CEEs) produced GOS, such as 6-galactobiose and 3'-, 4'-, and 6'-galactosyl-lactose. At 4 h of reaction, the main trisaccharide formed was 6'-galactosyl-lactose (20 g/100 g of total carbohydrates). The formation of OsLu was also observed by all CEEs tested, with 6-galactobiose, 6'-galactosyl-lactulose, and 1-galactosyl-lactulose being found in all of the reaction mixtures. The synthesis of trisaccharides predominated over other oligosaccharides. K. marxianus strain O3 produced the highest yields of GOS and OsLu after 4 h of reaction, reaching 42 g/100 g of total carbohydrates (corresponding to 80% lactose hydrolysis) and 45 g/100 g of total carbohydrates (corresponding to 87% lactulose hydrolysis), respectively. Therefore, the present study contributes to a better insight into dairy Kluyveromyces β-galactosidases and shows the feasibility of these enzymes to transglycosylate lactose and lactulose, producing high yields of prebiotic oligosaccharides.

Isolation and Characterization of Shigella flexneri G3, Capable of Effective Cellulosic Saccharification under Mesophilic Conditions

A novel Shigella strain (Shigella flexneri G3) showing high cellulolytic activity under mesophilic, anaerobic conditions was isolated and characterized. The bacterium is Gram negative, short rod shaped, and nonmotile and displays effective production of glucose, cellobiose, and other oligosaccharides from cellulose (Avicel PH-101) under optimal conditions (40°C and pH 6.5). Approximately 75% of the cellulose was hydrolyzed in modified ATCC 1191 medium containing 0.3% cellulose, and the oligosaccharide production yield and specific production rate reached 375 mg g Avicel(-1) and 6.25 mg g Avicel(-1) h(-1), respectively, after a 60-hour incubation. To our knowledge, this represents the highest oligosaccharide yield and specific rate from cellulose for mesophilic bacterial monocultures reported so far. The results demonstrate that S. flexneri G3 is capable of rapid conversion of cellulose to oligosaccharides, with potential biofuel applications under mesophilic conditions.

Chemical Production of Pectic Oligosaccharides from Orange Peel Wastes

Orange peel wastes (OPW) were extracted with water to remove soluble material, and the resulting solid phase (which was obtained at a yield of 48.8 kg/100 kg of OPW, oven-dry basis) was employed as a substrate for the manufacture of pectic oligosaccharides by nonisothermal processing with hot, compressed water (autohydrolysis or hydrothermal treatments). The effects of treatments on the composition of both liquors and spent solids were assessed in terms of the severity factor (Ro). Operating under selected conditions (maximum temperature of 160 °C, corresponding to Ro = 288 min), the oligosaccharide yield (including oligogalacturonides, arabinooligosaccharides, and galactooligosaccharides) accounted for 25.1 kg/100 kg of extracted OPW, whereas limited concentrations monosaccharides and nonsaccharide compounds were present in the reaction media.

Evolved β-Galactosidases from Geobacillus stearothermophilus with Improved Transgalactosylation Yield for Galacto-Oligosaccharide Production

A mutagenesis approach was applied to the beta-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved beta-galactosidases, a major trisaccharide was formed. Its structure was characterized as beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucopyranoside (3'-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3'-galactosyl-lactose production of 23%.

Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.

Glucosylation of raffinose via alternansucrase acceptor reactions

The glucansucrase known as alternansucrase [EC 2.4.1.140] can transfer glucosyl units from sucrose to raffinose to give good yields of oligosaccharides, which may serve as prebiotics. The main products were the tetrasaccharides α- d-Glc p-(1→3)-α- d-Gal p-(1→6)-α- d-Glc p-(1↔2)-β- d-Fru f and α- d-Glc p-(1→4)-α- d-Gal p-(1→6)-α- d-Glc p-(1↔2)-β- d-Fru f in ratios ranging from 4:1 to 9:1, along with lesser amounts of α- d-Glc p-(1→6)-α- d-Gal p-(1→6)-α- d-Glc p-(1↔2)-β- d-Fru f. Ten unusual pentasaccharide structures were isolated. Three of these arose from glucosylation of the major tetrasaccharide product, two each from the minor tetrasaccharides, and three were the result of glucosylations of the fructose acceptor product leucrose or isomaltulose. The major pentasaccharide product arose from glucosylation of the major tetrasaccharide at position 4 of the fructofuranosyl unit, to give a subunit structure analogous to that of maltulose. A number of hexasaccharides and higher oligosaccharides were also produced. Unlike alternansucrase, dextransucrase [EC 2.4.1.5] gave only a single tetrasaccharide product in low yield, and no significant amounts of higher oligosaccharides. The tetrasaccharide structure from dextransucrase was found to be α- d-Glc p-(1→4)-α- d-Gal p-(1→6)-α- d-Glc p-(1↔2)-β- d-Fru f, which is at odds with the previously published structure.

OPTIMIZATION OF CONDITIONS OFLEUCONOSTOC MESENTEROIDESNRRL B-640 FOR PRODUCTION OF A DEXTRANSUCRASE AND ITS ASSAY

ABSTRACT The optimum culture conditions for dextransucrase production from Leuconostoc mesenteroides NRRL B-640 were investigated. The dextransucrase production was studied in the temperature range, 22 to 30C under shaking and static flask conditions. The optimum temperature for dextransucrase production was 25C. The shaking flask culture gave higher enzyme activity by 20% than the static flask culture. The dextransucrase activity of L. mesenteroides NRRL B-640 was compared with L. mesenteroides NRRL B-512F. The results showed that the maximum dextransucrase activity achieved by L. mesenteroides NRRL B-640 at 25C under shaking flask culture, was 15% higher than that the enzyme activity given by L. mesenteroides NRRL B-512F grown at 23C under the static flask culture. The dextransucrase elaborated by L. mesenteroides NRRL B-640 displayed a maximum enzyme activity when assayed in the temperature range of 30 to 35C and at pH of 5.4. The enzyme was stable with in the 10–500 mM concentrations of sodium acetate buffer. PRACTICAL APPLICATIONS Dextransucrase synthesizes dextran that has several commercial applications in both its native and modified forms. Flocculation agent in waste-water treatment, blood volume expander, stabilizer in ice cream and a cryo-protective agent for cells are a few of dextran applications in its native form. It can serve as stabilizer in photographic, cosmetics and agriculture-based industries with generation of charged derivatives of dextran. Dextransucrases are reported to generate varieties of gluco-oligosaccharides that serve as prebiotics. Dextransucrases are reported in the glycoconjugation of several peptides for the development of vaccines, contraceptives and antibiotics. Dextran conjugates with iron and fluorophores are used in the clinical applications. Dextran hydrogels are used in pharmaceutical and biomedical applications such as contact lenses and in drug delivery. Looking at the wide applications of dextransucrase and growing demand there is a need for exploration of new strains for production of efficient and pure dextransucrase, producing better quality and yield of oligosaccharides and polysaccharides. The results of optimized conditions attain importance for large-scale production of dextransucrase.

Separation of galacto-oligosaccharides mixture by nanofiltration

Four commercial 1812 spiral wound nanofiltration membranes were firstly applied to examine the separation performance of sugar solutions in total recycle mode of operation. Membrane NF-3 (supplied by Sepro Co. (USA)) with molecular weight cut-off of 800–1000 Da was selected for separation of a commercial galactose-oligosaccharide (GOS) mixture with low content of oligosaccharides in constant volume diafiltration (CVD) process at 50 °C and 6 bar pressure. The choosing of membrane and operation conditions for CVD process was based on a compromise between the permeate fluxes and apparent rejections of the sugar solution. In CVD course, the concentration of various sugars and the relationship between the yield and purity of oligosaccharides predicted well with mathematical models for the following situation: when the volume of every removing batch of permeate is equal to the volume of tank, the increased rate of sugar rejections is less than 8% and the decreased rate of tank concentrations is less than 15%. 90.5% monosaccharide and 52.5% lactose in the mixture were removed with NF-3 membrane in this study, and the oligosaccharides purity of 54.5% was achieved (1.5 times of the raw material), obtaining oligosaccharides yield of 70.0%.

Supercritical hydrolysis of cellulose for oligosaccharide production in combined technology

A combined supercritical/subcritical technology was used as a pre-treatment and hydrolysis method for ethanol production from cellulose/lignocelluloses. In a batch study for supercritical hydrolysis, which is the primary step of the combined technology, 60 mg of microcrystalline cellulose in 2.5 ml deionized water was loaded into each reactor and heated in a salt bath at a selected temperature for a specified reaction time. Cellulose was quickly hydrolyzed to oligosaccharides, hexoses and other small molecular products at temperatures above the critical point of water. Temperature and reaction time were the two key parameters that determined the products of cellulose hydrolysis. The highest yield of oligosaccharides (approximately 40%) was obtained at optimum conditions of 380 °C and a reaction time of 16 s. The corresponding yield of hexoses was 24%, giving a maximum yield of hydrolysis products of approximately 63%. A complete decomposition of hydrolysis products occurred at higher temperatures and/or longer reaction times. A kinetic analysis was performed to explain the reaction of cellulose in supercritical water. The results presented here provide a rigid framework for the use of combined supercritical/subcritical technology in subsequent research.

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.

Enzyme‐Catalyzed Synthesis of a Hybrid N‐Linked Oligosaccharide using N‐Acetylglucosaminyltransferase I

AbstractThe soluble catalytic domain of human N‐acetylglucosaminyltransferase I was purified from Escherichia coli and utilized in the enzyme‐catalyzed conversion of high mannose N‐linked oligosaccharide 1 into the rare hybrid oligosaccharide 2. Analysis of the reaction showed that the conversion of high mannose 1 into hybrid oligosaccharide 2 proceeded to 100% completion as assessed by MALDI‐TOF‐MS. Purification of the large polar oligosaccharide by gel filtration and silica gel chromatography afforded a 42% isolated yield of oligosaccharide 2. This enzyme‐catalyzed reaction can be utilized to produce rare hybrid oligosaccharides for biochemical and structural studies.

Response surface methodology: Synthesis of inulooligosaccharides with an endoinulinase from Aspergillus niger

Oligosaccharides of chain length up to degree of polymerisation (DP) 5 have been synthesised using a purified endoinulinase and response surface methodology with central composite experimental design for the optimisation of the product yield. The endoinulinase was purified through a three-step procedure in a yield of 1.11% and 3.5-fold and its molecular weight was 68.1 kDa by SDS-PAGE. It had optimum pH and temperature values of 6 and 60 °C, respectively with only 10% loss in activity over 60 min under these conditions. From the kinetic studies K m and V max were estimated at 3.53 mM and 666.7 μmol min −1 ml −1, respectively. Using the purified endoinulinase preparation isolated from Aspergillus niger, at a temperature (60 °C), inulin concentration (150 mg ml −1), incubation time (48 h), pH (6.0) and enzyme dosage (60 U ml −1) produced high yields of inulotrioses (70.3 mM), inulotetraoses (38.8 mM), and inulopentaoses (3.5 mM). Response surface regression predicted similar product levels under the same conditions. A comparative analysis of the yields of individual oligosaccharides produced from both the crude and purified enzyme is also presented.

Synthesis of a Series of Fructooligosaccharides with Sucrose and Cycloinulohexaose Extending over Ten Degrees of Polymerization Using Cycloinulooligosaccharide Fructanotransferase fromBacillus circulansOKUMZ 31B

Prolonged incubation with sucrose as an acceptor and cycloinulohexaose as a donor at a high concentration of cycloinulooligosaccharide fructanotransferase afforded a series of fructooligosaccharides. Their chain-length distribution extended over 10 degrees of polymerization when the donor concentration was increased to 120 mM. Increasing the acceptor concentration proved effective in improving the yield of inulin-type oligosaccharides because hydrolysis was suppressed.

Coproduction of Oligosaccharides and Glucose from Corncobs by Hydrothermal Processing and Enzymatic Hydrolysis

Corncobs were subjected to nonisothermal reaction in aqueous conditions to reach temperatures in the range 202−216 °C. The liquors were assayed for composition to assess the conversion of xylan into oligosaccharides and reaction byproducts, whereas the spent solids from treatments were subjected to enzymatic hydrolysis. Enzymatic assays were carried out according to an incomplete, factorial, centered design of experiments, in which the following independent variables were considered: liquor to solid ratio (in the range 6−16 g/g), enzyme to substrate ratio (in the range 6−28 filter paper units/g), and β-glucosidase activity/cellulase activity ratio (in the range 1−9 IU/FPU). The concentration/time data were fitted to an equation selected from the literature, and the model parameters were expressed as a function of the operational variables considered in the experimental design. Under selected conditions, high oligosaccharide yields and near quantitative cellulose conversion into glucose were obtained.

Glucuronylation in Escherichia coli for the bacterial synthesis of the carbohydrate moiety of nonsulfated HNK-1

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.