Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae ß-galactosidase, followed by purification using silica gel.

The aim of this study was to optimize and compare the production of galactooligosaccharides (GOSs) by free and cotton cloth-immobilized Aspergillus oryzae ß-galactosidase, and perform economical evaluation of production of GOSs (100%) between them. Using the response surface method, the optimal reaction time (3.9 h), initial lactose concentration (57.13%), and enzyme to lactose ratio (44.81 U/g) were obtained for the free enzyme, which provided a GOSs yield of 32.62%. For the immobilized enzyme, the optimal yield of GOSs (32.48%) was obtained under reaction time (3.09 h), initial lactose concentration (52.74%), and temperature (50.0 ℃). And it showed desirable reusability during five successive enzymatic reactions. The recovery rate of GOSs (100%) is 65% using silica gel filtration chromatography. The economical evaluation showed almost no difference in the manufacturing cost for the GOSs (100%) between these two systems, and that the recovery rate had a great impact on the cost.

Extraction and Characterization of Hemicellulose from Eucalyptus By-product: Assessment of Enzymatic Hydrolysis to Produce Xylooligosaccharides.

Eucalyptus wood is the primary source of fibers to produce paper and cellulose in South American countries. The major by-product generated in the cellulose industry is sawdust derived from chip wood production, which is designated as Eucalyptus by-product (EB). The xylooligosaccharides (XOS) are xylose-based oligomers with proven effects over maintenance and stimulation of beneficial human gut bacteria. This study reported the EB extraction and characterization along with an assessment of hemicellulose hydrolysis using commercial xylanases to produce XOS. Hemicellulose derived from extracted and NaClO2 pretreated (HEEBPT) presented xylan content of 55%, which was similar to 58.5% found in commercial Birchwood hemicellulose (CBH). The enzymatic hydrolysis of HEEBPT and CBH presented 30% as maximum conversion of xylan into XOS without significant difference among the enzymatic extracts evaluated. The XOS production from EB was proven as a technically feasible alternative to recover a value-added product from hemicellulosic fraction generated in the cellulose industry. However, lignin removal with NaClO2 from EB affects the feasibility of an industrial process because they generate toxic compounds in the pretreatment step. Thus, further studies with alternative reagents, such as ionic liquids, are required to asses selectively lignin removal from EB. Graphical Abstract.

Oligosaccharide biotechnology: an approach of prebiotic revolution on the industry.

Oligosaccharides are polymers with two to ten monosaccharide residues which have sweetener functions and sensory characteristics, in addition to exerting physiological effects on human health. The ones called nondigestible exhibit a prebiotic behavior being fermented by colonic microflora or stimulating the growth of beneficial bacteria, playing roles in the immune system, protecting against cancer, and preventing cardiovascular and metabolic issues. The global prebiotics market is expected to grow around 12.7% in the next 8 years, so manufacturers are developing new alternatives to obtain sustainable and efficient processes for application on a large scale. Most studied examples of biotechnological processes involve the development of new strategies for fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, and mannanooligosaccharide synthesis. Among these, the use of whole cells in fermentation, synthesis of microbial enzymes (ß-fructofuranosidases, ß-galactosidases, xylanases, and ß-mannanases), and enzymatic process development (permeabilization, immobilization, gene expression) can be highlighted, especially if the production costs are reduced by the use of agro-industrial residues or by-products such as molasses, milk whey, cotton stalks, corncobs, wheat straw, poplar wood, sugarcane bagasse, and copra meal. This review comprises recent studies to demonstrate the potential for biotechnological production of oligosaccharides, and also aspects that need more investigation for future applications in a large scale.

Characteristics of Raw Starch-Digesting α-Amylase of Streptomyces badius DB-1 with Transglycosylation Activity and Its Applications.

Streptomyces badius DB-1 produces α-amylase extracellularly, and its production was enhanced 5.1-fold (from 9.47 ± 0.51 to 48.23 ± 1.45 U mL-1) due to optimization by one-variable-at-a-time and statistical approaches. Soluble starch emerged as the most influential factor that strongly affected enzyme production. The purified enzyme is a monomer with a molecular mass of ~57 kDa and optimally active at 50 °C and pH 6.0. The enzyme hydrolyzes soluble as well as raw starches into simpler sugars with a high proportion (>40.0 %) of maltotetraose. It is optimally active at moderate temperature and generates maltooligosaccharides from starch, thus, useful as an antistale in bread making. It also plays a role in increasing the formation of maltooligosaccharides due to transglycosylation activity, thus, finds application in functional foods. This is the first report on the production of raw starch-digesting α-amylase by S. badius with transglycosylation activity.

Endoinulinase production by a new endoinulinase producer Aspergillus tritici BGPUP6 using a low cost substrate.

Endoinulinase is an inulolytic enzyme which is used for the production of fructooligosaccharides from inulin. A new endoinulinase producing fungal strain BGPUP6 was identified as Aspergillus tritici on the basis of its molecular characterization. Response surface methodology was used to optimize the endoinulinase production at shake-flask level from Aspergillus tritici BGPUP6 using raw Asparagus inulin as carbon source. Four independent variables (raw inulin, 2-4%; peptone, 0.3-0.7%; (NH4)H2PO4, 0.3-0.7% and pH, 4.5-6.5) were selected for the study based on the CCRD model of RSM. The other media supplements (FeSO4·7H2O, 0.001%; MgSO4·7H2O, 0.05% and KCl, 0.02%) were kept constant in the production medium, throughout the study. Endoinulinase production and biomass yield were selected as dependent responses. The optimal combination of media ingredients obtained from the study was 3% raw inulin, 0.5% peptone, 0.5% (NH4)H2PO4 and pH 5.5. Using the optimized media constituents, maximum endoinulinase production (25.01 IU/mL) and biomass yield (0.514g dry weight/50 mL) obtained were in good agreement with the predicted values. Crude enzyme produced was also used for the hydrolysis of inulin. The hydrolysate showed the presence of a mixture of fructooligosaccharides with varied degree of polymerization. This is the first report on the production of an endoinulinase from Aspergillus tritici.

Assessment of suitability of vine shoots for hemicellulosic oligosaccharides production through aqueous processing.

Vine shoots were subjected to non-isothermal aqueous processing. A range of severities (S0) from 3.20 to 4.65 was assayed and their effects in terms of solubilization, composition, molar mass distribution, structural characterization and thermal stability of the liquors were studied using HPLC, HPSEC, TGA and FTIR. The spent solids were characterized by HPLC and FTIR. When autohydrolysis was carried out at S0=4.01, the substrate solubilization achieved a 38.7% of the raw material and 83.1% of the initial xylan was converted into xylooligosaccharides (XOS). The amount of TOS (total oligosaccharides) in the hydrolysates was 28.4g/L while the other non volatile compounds (ONVC) were 0.08g/g NVC. The spent solid from the treatment at S0=4.01 was composed about 90% of cellulose and lignin. Therefore, it can be concluded that autohydrolysis is a suitable pretreatment of vine shoots such as a first stage of a biomass refinery.

Economic analysis and environmental impact assessment of three different fermentation processes for fructooligosaccharides production.

Three different fermentation processes for the production of fructooligosaccharides (FOS) were evaluated and compared in terms of economic aspects and environmental impact. The processes included: submerged fermentation of sucrose solution by Aspergillus japonicus using free cells or using the cells immobilized in corn cobs, and solid-state fermentation (SSF) using coffee silverskin as support material and nutrient source. The scale-up was designed using data obtained at laboratory scale and considering an annual productivity goal of 200 t. SSF was the most attractive process in both economic and environmental aspects since it is able to generate FOS with higher annual productivity (232.6 t) and purity (98.6%) than the other processes; reaches the highest annual profit (6.55 M€); presents the lowest payback time (2.27 years); and is more favourable environmentally causing a lower carbon footprint (0.728 kg/kg, expressed in mass of CO2 equivalent per mass of FOS) and the lowest wastewater generation.

Microbial degradation of chitin waste for production of chitosanase and food related bioactive compounds.

Ecological samples rich in microbial diversity like cow dung, legume rhizosphere, fish waste and garden soil were used for isolation of chitosan-degrading microorganisms. Selected isolates were used for production of chitosanase and food related bioactive compounds by conversion of biowaste. Production of glucosamine (Gln), N-acetylglucosamine (NAG), chitooligosaccharides (COS), antioxidants, antibacterial compounds and prebiotics was carried out by microbial fermentation of biowaste. The highest chitosanase activity (8 U/mL) was observed in Aspergillus sp. isolated from fish market waste and it could produce Gln and NAG while Streptomyces sp. isolated from garden soil was able to produce COS along with Gln and NAG. Radical scavenging activity was observed in culture supernatants of 35% of studied isolates, and 20% isolates secreted compounds which showed positive effect on growth of Bifidobacterium. Antibacterial compounds were produced by 40% of selected isolates and culture supernatants of two microbial isolates, Streptomyces zaomyceticus C6 and one of garden soil isolates, were effective against both gram positive and negative bacteria.

Coupling reactions of trehalose synthase from Pyrococcus horikoshii: cost-effective synthesis and anti-adhesive activity of α-galactosyl oligosaccharides using a one-pot three-enzyme system with trehalose.

A new sugar nucleotide cycling (SNC) process was established in a one-pot three enzyme-coupled reaction using disaccharide trehalose. Trehalose synthase from Pyrococcus horikoshii could be applied to the SNC process for the synthesis of functional α-galactosyl oligosaccharides, α-galactose (Gal) epitopes and globotriose, using the effective regeneration of UDP-Gal. The α-Gal epitopes and globotriose were found to attach to the cell-surface of enteropathogenic Escherichia coli O127 (EPEC) which were bound to human Caco-2 cells. These α-galactosyl oligosaccharides were able to prohibit the attachment of EPEC, which could have resulted in colonization and disease. The α-Gal epitope III with a lactulose acceptor showed the most inhibitory activity of anti-adhesion. The results suggest that the α-galactosyl oligosaccharides may be alternative anti-adhesion molecules that overcome antibiotic resistance.

Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides.

We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan.

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.

Beet sugar syrup and molasses as low-cost feedstock for the enzymatic production of fructo-oligosaccharides.

Sugar syrup and molasses from beet processing containing 620 and 570 mg/mL sucrose, respectively, were assayed as low-cost and available substrates for the enzymatic synthesis of fructo-oligosaccharides (FOSs). A commercial pectinase (Pectinex Ultra SP-L, from Aspergillus aculeatus) characterized by the presence of a transfructosylating activity was used as a biocatalyst. The FOS production increased when lowering the initial pH value of syrup (7.5) and molasses (8.9) to 5.5. Sugar syrup and molasses were diluted in order to reduce substrate viscosity; interestingly, the percentage of FOS with regards to total sugars remained almost constant, which indicated a high transferase-to-hydrolase ratio for this enzyme. Kinetics of FOS production was analyzed. Using approximately 10 U transfructosylating activity per g sucrose, the FOS concentration reached a maximum of 388 mg/mL after 30 h using syrup and 235 mg/mL in 65 h with molasses. These values corresponded to approximately 56 and 49% (w/w), respectively, of the total amount of carbohydrates in the mixture. The enzyme was also covalently immobilized on an epoxy-activated polymethacrylate-based polymer (Sepabeads EC-EP5). We found that immobilized Pectinex Ultra SP-L can be efficiently applied to the synthesis of FOS using syrup and molasses as substrates.

Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli.

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.

Production of fructo-oligosaccharides from molasses by Aureobasidium pullulans cells.

Three different strains of Aureobasidium pullulans were grown in batch cultures to compare their abilities for the production of fructo-oligosaccharides. Specific intracellular enzyme activity was the highest with strain KCCM 12017 and enzyme production was closely coupled to growth. Using A. pullulans cells, 166 g/l fructo-oligosaccharides was produced from 360 g/l molasses sugar as sucrose equivalent at 55 degrees C and pH 5.5 after 24 h incubation.

Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex.

A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.