Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Structures of the xyloglucans in the monocotyledon family Araceae (aroids).

MAIN CONCLUSION: The xyloglucans of all aquatic Araceae species examined had unusual structures compared with those of other non-commelinid monocotyledon families previously examined. The aquatic Araceae species Lemna minor was earlier shown to have xyloglucans with a different structure from the fucogalactoxyloglucans of other non-commelinid monocotyledons. We investigated 26 Araceae species (including L. minor), from five of the seven subfamilies. All seven aquatic species examined had xyloglucans that were unusual in having one or two of three features: < 77% XXXG core motif [L. minor (Lemnoideae) and Orontium aquaticum (Orontioideae)]; no fucosylation [L. minor (Lemnoideae), Cryptocoryne aponogetonifolia, and Lagenandra ovata (Aroideae, Rheophytes clade)]; and > 14% oligosaccharide units with S or D side chains [Spirodela polyrhiza and Landoltia punctata (Lemnoideae) and Pistia stratiotes (Aroideae, Dracunculus clade)]. Orontioideae and Lemnoideae are the two most basal subfamilies, with all species being aquatic, and Aroideae is the most derived. Two terrestrial species [Dieffenbachia seguine and Spathicarpa hastifolia (Aroideae, Zantedeschia clade)] also had xyloglucans without fucose indicating this feature was not unique to aquatic species.

Brown Algae Carbohydrates: Structures, Pharmaceutical Properties, and Research Challenges.

Brown algae (Phaeophyceae) have been consumed by humans for hundreds of years. Current studies have shown that brown algae are rich sources of bioactive compounds with excellent nutritional value, and are considered functional foods with health benefits. Polysaccharides are the main constituents of brown algae; their diverse structures allow many unique physical and chemical properties that help to moderate a wide range of biological activities, including immunomodulation, antibacterial, antioxidant, prebiotic, antihypertensive, antidiabetic, antitumor, and anticoagulant activities. In this review, we focus on the major polysaccharide components in brown algae: the alginate, laminarin, and fucoidan. We explore how their structure leads to their health benefits, and their application prospects in functional foods and pharmaceuticals. Finally, we summarize the latest developments in applied research on brown algae polysaccharides.

The first bacterial ß-1,6-endoglucanase from Saccharophagus degradans 2-40T for the hydrolysis of pustulan and laminarin.

ß-1,6-glucan is a polysaccharide found in brown macroalgae and fungal cell walls. In this study, a ß-1,6-endoglucanase gene from Saccharophagus degradans 2-40T, gly30B, was cloned and overexpressed in Escherichia coli. Gly30B, which belongs to the glycoside hydrolase family 30 (GH30), was found to possess ß-1,6-endoglucanase activity by hydrolyzing ß-1,6-glycosidic linkages of pustulan (ß-1,6-glucan derived from fungal cell walls) and laminarin (ß-1,3-glucan with ß-1,6-branchings, derived from brown macroalgae) to produce gentiobiose and glucose as the final products. The optimal pH and temperature for Gly30B activity were found to be pH 7.0 and 40 °C, respectively. The kinetic constants of Gly30B, V max, K M, and k cat were determined to be 153.8 U/mg protein, 24.2 g/L, and 135.6 s-1 for pustulan and 32.8 U/mg protein, 100.8 g/L, and 28.9 s-1 for laminarin, respectively. To our knowledge, Gly30B is the first ß-1,6-endoglucanase characterized from bacteria. Gly30B can be used to hydrolyze ß-1,6-glucans of brown algae or fungal cell walls for producing gentiobiose as a high-value sugar and glucose as a fermentable sugar.

A Novel Glycoside Hydrolase Family 5 ß-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40T and Its Transglycosylase Activity.

UNLABELLED: In this study, we characterized Gly5M, originating from a marine bacterium, as a novel ß-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40(T) The gly5M gene was cloned and overexpressed in Escherichia coli Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry, Gly5M was identified as a novel ß-1,3-endoglucanase (EC 3.2.1.39) and bacterial ß-1,6-glucanase (EC 3.2.1.75) in GH5. The ß-1,3-endoglucanase and ß-1,6-endoglucanase activities were detected by using laminarin (a ß-1,3-glucan with ß-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a ß-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward ß-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes. IMPORTANCE: In this study, we have discovered a novel ß-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40(T) Gly5M was identified as the newly found ß-1,3-endoglucanase and bacterial ß-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both ß-1,3-glucans and ß-1,6-glucans. Gly5M also possesses a transglycosylase activity toward ß-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value ß-1,3- and/or ß-1,6-oligosaccharides.

Effective production of fermentable sugars from brown macroalgae biomass.

Brown macroalgae are renewable and sustainable biomass resources for the production of biofuels and chemicals, owing to their high levels of carbohydrates and low levels of lignin. To increase the biological usage of brown macroalgae, it is necessary to depolymerize the polysaccharides that generate macroalgal monomeric sugars or sugar derivatives and to convert them into fermentable sugars for the production of biofuels and chemicals. In this review, we discuss the chemical and enzymatic saccharification of the major carbohydrates found in brown macroalgae and the use of the resulting constituents in the production of biofuels and chemicals, as well as high-value health-benefiting functional oligosaccharides and sugars. We also discuss recently reported experimental results, novel enzymes, and technological breakthroughs that are related to polysaccharide depolymerization, fermentable sugar production, and the biological conversion of non-favorable sugars for fermentation using industrial microorganisms. This review provides a comprehensive perspective of the efficient utilization of brown macroalgae as renewable resources for the production of biofuels and chemicals.

Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae.

This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-L-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides.

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2-4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

Recent advances in chromatography-mass spectrometry and electronic nose technology in food flavor analysis and detection.

Food flavor plays an important role in the consumption and acceptance of food, food production as well as food science research. Chromatography-mass spectrometry and electronic nose are the two most commonly used technologies in food flavor detection. Chromatography-mass has good qualitative and quantitative effect, wide detection range, and electronic nose is convenient and fast for practical application. In this paper, the principles, advantages and disadvantages, research progress and application in flavor fingerprinting of the two types of methods and their derived analytical techniques are reviewed. In particular, the application scenarios and advantages of different technologies combined are discussed in depth by summarizing studies that reflect the differences between different technologies. Finally, the current challenges and future directions of food flavor detection technology are discussed.

Current insights of factors interfering the stability of lytic polysaccharide monooxygenases.

Cellulose and chitin are two of the most abundant biopolymers in nature, but they cannot be effectively utilized in industry due to their recalcitrance. This limitation was overcome by the advent of lytic polysaccharide monooxygenases (LPMOs), which promote the disruption of biopolymers through oxidative mechanism and provide a breakthrough in the action of hydrolytic enzymes. In the application of LPMOs to biomass degradation, the key to consistent and effective functioning lies in their stability. The efficient transformation of biomass resources using LPMOs depends on factors that interfere with their stability. This review discussed three aspects that affect LPMO stability: general external factors, structural factors, and factors in the enzyme-substrate reaction. It explains how these factors impact LPMO stability, discusses the resulting effects, and finally presents relevant measures and considerations, including potential resolutions. The review also provides suggestions for the application of LPMOs in polysaccharide degradation.

Improving the thermostability of a novel PL-6 family alginate lyase by rational design engineering for industrial preparation of alginate oligosaccharides.

Alginate is degraded into alginate oligosaccharides with various biological activities by enzymes. However, the thermostability of the enzyme limits its industrial application. In this study, a novel PL-6 alginate lyase, AlyRm6A from Rhodothermus marinus 4252 was expressed and characterized. In addition, an efficient comprehensive strategy was proposed, including automatic design of heat-resistant mutants, multiple computer-aided ΔΔGfold value calculation, and conservative analysis of mutation sites. AlyRm6A has naturally high thermostability. Compared with the WT, T43I and Q216I kept their original activities, and their half-lives were increased from 3.68 h to 4.29 h and 4.54 h, melting point temperatures increased from 61.5 °C to 62.9 °C and 63.5 °C, respectively. The results of circular dichroism showed that both the mutants and the wild type had the characteristic peaks of ß-sheet at 195 nm and 216 nm, which indicated that there was no significant effect on the secondary structure of the protein. Molecular dynamics simulation (MD) analyses suggest that the enhancement of the hydrophobic interaction network, improvement of molecular rigidity, and denser structure could improve the stability of AlyRm6A. To the best of our knowledge, our findings indicate that AlyRm6A mutants exhibit the highest thermostability among the characterized PL-6 alginate lyases, making them potential candidates for industrial production of alginate oligosaccharides.

Controllable and complete conversion of agarose into oligosaccharides and monosaccharides by microwave-assisted hydrothermal and enzymatic hydrolysis and antibacterial activity of agaro-oligosaccharides.

Hydrolysis of agar or agarose can yield two types of oligosaccharides: agaro-oligosaccharides (AOS) and neoagaro-oligosaccharides (NAOS). These oligosaccharides have various biological activities and promising applications in the future food industry and pharmaceuticals. In this study, we prepared AOS from agarose by microwave-assisted hydrothermal hydrolysis and then used a commercial ß-galactosidase to treat AOS for producing NAOS. A complete conversion from agarose to AOS or NAOS can be achieved by microwave hydrothermal treatment and one-step enzyme reaction, and the production process was completely green. In addition, we combined ß-galactosidase and α-neoagarobiose hydrolase from Saccharophagus degradans 2-40 (SdNABH) to treat AOS, and AOS was completely converted into monosaccharides. Then the results of the inhibitory activity of AOS on the growth of Streptococcus mutans showed that AOS might be a good potential sugar substitute for dental caries prevention. This study provides an efficient approach for the production of multiple mixed degrees of polymerization (DP) of pure AOS and NAOS without requiring acid catalyst and agarases while simplifying the production processes and reducing costs.

Characterization of degradation patterns and enzymatic properties of a novel alkali-resistant alginate lyase AlyRm1 from Rubrivirga marina.

Alginate lyase is essential for the production of alginate oligosaccharides (AOSs), which exhibit diverse bioactivities and have numerous applications in the food and pharmaceutical industries. The creation of recombinant alginate lyase by genetic engineering lays a crucial foundation for the commercialization of alginate lyase. This study cloned and expressed the polysaccharide lyase family 6 (PL6) alginate lyase gene alyrm1 from Rubrivirga marina.The optimum temperature and pH for recombinant AlyRm1 are 30 °C and 10.0, respectively. AlyRm1 shows good alkaline stability, for it remained over 80% of the enzyme activity after being incubated at pH 10.0 for 24 h AlyRm1 preferentially degrades PolyM into AOSs with degrees of polymerization (DP) 2-5 and monosaccharides as an endolytic bifunctional lyase. In addition, the analysis of degradation products toward oligosaccharides revealed that the minimal substrate of AlyRm1 is trisaccharide and clarified the degradation patterns.

Coating peanut shell lignin nanospheres with gelatin via non-covalent adsorption: Key parameters, consequences, and underlying interactions.

In the present work, lignin nanospheres (LNS, average diameter 166.43 nm) were prepared and the affecting parameters, the absorbed types, and mechanisms of their interactions with type-A gelatin (AG) were explored. The findings demonstrated that upon AG coating, the ζ-potential of LNS sharply decreased and concluded a negative-to-positive shift, while the average diameter and polydispersity index increased significantly. AG presented the highest coating capacity (0.32 mg/mg, db) onto LNS (0.5 mg/mL) at an optimum pH of 4.0 and an AG concentration of 1.0 mg/mL. The adsorption of AG onto LNS could be well described by the Hill model (R2 = 0.9895), which was characterized as positive synergistic adsorption by the Hill coefficient (1.32) and physical adsorption by the free energy (3.70 kJ/mg). The spectral analysis revealed that the interactions between AG and LNS were mainly driven by electrostatic forces (ΔG < 0, ΔH < 0, and ΔS > 0) together with the assistance of hydrogen bonds and hydrophobic interactions, which companied a decrease of α-helix (4.04 %) and ß-turn (0.60 %) and an increase of ß-sheet (3.10 %) and random coil (1.53 %) of the secondary structure of AG. The results herein certainly favored the hydrophilic/hydrophobic change of LNS/AG and the quality control of a binary system consisting of lignin and gelatin.

Depolymerization of alginate into a monomeric sugar acid using Alg17C, an exo-oligoalginate lyase cloned from Saccharophagus degradans 2-40.

Macroalgae are considered to be promising biomass for fuels and chemicals production. To utilize brown macroalgae as biomass, the degradation of alginate, which is the main carbohydrate of brown macroalgae, into monomeric units is a critical prerequisite step. Saccharophagus degradans 2-40 is capable of degrading more than ten different polysaccharides including alginate, and its genome sequence demonstrated that this bacterium contains several putative alginate lyase genes including alg17C. The gene for Alg17C, which is classified into the PL-17 family, was cloned and overexpressed in Escherichia coli. The recombinant Alg17C was found to preferentially act on oligoalginates with degrees of polymerization higher than 2 to produce the alginate monomer, 4-deoxy-L: -erythro-5-hexoseulose uronic acid. The optimal pH and temperature for Alg17C were found to be 6 and 40 °C, respectively. The K (M) and V (max) of Alg17C were 35.2 mg/ml and 41.7 U/mg, respectively. Based on the results of this study, Alg17C could be used as the key enzyme to produce alginate monomers in the process of utilizing alginate for biofuels and chemicals production.

Complete conversion of agarose into water soluble agaro-oligosaccharides by microwave assisted hydrothermal hydrolysis.

This study aims to produce oligosaccharides by microwave-assisted hydrothermal hydrolysis of agarose, which is a more environmentally friendly and green economical approach than acid or enzymatic hydrolysis. Under the optimized condition of 160 °C and 25 min, total liquefaction of agarose was achieved, the dominant products in the hydrolysate are agaro-oligosaccharides (AOs), with only a small portion of d-galactose and 5- hydroxymethylfurfural (5-HMF). This approach can produce even and odd number AOs simultaneously, which is due to the random hydrolysis of α-1,3 glycosidic linkage at first and subsequent hydrolysis of the ß-1,4 glycosidic linkage at the reducing end. The yield of AOs of a low degree of polymerization (DP) can reach about 57% of the theoretical maximum, while the total yield of AOs is over 90%. In conclusion, microwave-assisted hydrothermal hydrolysis is a way of producing oligosaccharides from agarose with extra-high efficiency and practical significance.

Recent advances in biotransformation, extraction and green production of D-mannose.

D-mannose is a natural and biologically active monosaccharide. It is the C-2 epimer of glucose and a component of a variety of polysaccharides in plants. In addition, D-mannose also naturally exists in some cells of the human body and participates in the immune regulation of cells as a prebiotic. Its good physiological benefits to human health and wide application in the food and pharmaceutical industries have attracted widespread attention. Therefore, in-depth research on preparation methods of D-mannose has been widely developed. This article summarizes the main production methods of D-mannose in recent years, especially the in-depth excavation from biomass raw materials such as coffee grounds, konjac flour, acai berry, etc., to provide new ideas for the green manufacture of D-mannose.

Novel Two-Step Process in Cellulose Depolymerization: Hematite-Mediated Photocatalysis by Lytic Polysaccharide Monooxygenase and Fenton Reaction.

To transform cellulose from biomass into fermentable sugars for biofuel production requires efficient enzymatic degradation of cellulosic feedstocks. The recently discovered family of oxidative enzymes, lytic polysaccharide monooxygenase (LPMO), has a high potential for industrial biorefinery, but its energy efficiency and scalability still have room for improvement. Hematite (α-Fe2O3) can act as a photocatalyst by providing electrons to LPMO-catalyzed reactions, is low cost, and is found abundantly on the Earth's surface. Here, we designed a composite enzymatic photocatalysis-Fenton reaction system based on nano-α-Fe2O3. The feasibility of using α-Fe2O3 nanoparticles as a composite catalyst to facilitate LPMO-catalyzed cellulose oxidative degradation in water was tested. Furthermore, a light-induced Fenton reaction was integrated to increase the liquefaction yield of cellulose. The innovative approach finalized the cellulose degradation process with a total liquefaction yield of 93%. Nevertheless, the complex chemical reactions and products involved in this system require further investigation.

Recent Advances in Bioutilization of Marine Macroalgae Carbohydrates: Degradation, Metabolism, and Fermentation.

Marine macroalgae are considered renewable natural resources due to their high carbohydrate content, which gives better utilization value in biorefineries and higher value conversion than first- and second-generation biomass. However, due to the diverse composition, complex structure, and rare metabolic pathways of macroalgae polysaccharides, their bioavailability needs to be improved. In recent years, enzymes and pathways related to the degradation and metabolism of macroalgae polysaccharides have been continuously developed, and new microbial fermentation platforms have emerged. Aiming at the bioutilization and transformation of macroalgae resources, this review describes the latest research results from the direction of green degradation, biorefining, and metabolic pathway design, including summarizing the the latest biorefining technology and the fermentation platform design of agarose, alginate, and other polysaccharides. This information will provide new research directions and solutions for the biotransformation and utilization of marine macroalgae.

Structural compositions and biological activities of cell wall polysaccharides in the rhizome, stem, and leaf of Polygonatum odoratum (Mill.) Druce.

Polygonatum odoratum is a perennial rhizomatous medicinal plant and different plant parts have been used in the treatment of various ailments. Herein, we have investigated the structural compositions of rhizome, leaf, and stem cell walls. We found 30-44% of polysaccharides in these wall preparations were cyclohexanediaminetetraacetic acid (CDTA) extractable, the proportion of heteromannans (HMs) in the rhizome is nearly three-fold compared to that of the leave and stem. The pectic polysaccharides of the rhizome are also structurally more diverse, with arabinans and type I and type II arabinogalactans being richest as shown by linkage study of the sodium carbonate (Na2CO3) extract. In addition, the 2-linked Araf was rhizome-specific, suggesting the cell walls in the rhizome had adapted to a more complex structure compared to that of the leaf and stem. Water-soluble polysaccharide fractions were also investigated, high proportion of Man as in 4-linked Manp indicated high proportion of HMs. The 21.4 kDa pectic polysaccharides and HMs derived from rhizome cell walls induced specific immune response in mice macrophage cells producing IL-1α and hematopoietic growth factors GM-CSF and G-CSF in vitro.