Structural investigation of a thermostable 1,2-ß-mannobiose phosphorylase from Thermoanaerobacter sp. X-514.

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Enzyme-assisted Solvent Extraction of High-yield Paeonia suffruticosa Andr. Seed Oil and Fatty Acid Composition and Anti-Alzheimer's Disease Activity.

Enzyme-assisted solvent extraction (EASE) of Paeonia suffruticosa Andr. seed oil (PSO) was optimized by response surface methodology (RSM). The fatty acid composition and anti-Alzheimer's disease (AD) activity of PSO were analyzed. An enzyme mixture composed of cellulase and hemicellulase (1:1, w/w) was most effective in determining the extraction yield of PSO. The ideal extraction conditions were a pH value of 5.1, an enzymolysis time of 68 min, and a temperature of 50℃. The average extraction yield of PSO was 38.2 mL/100 g, 37.4% higher than that of untreated peony seed (27.8 mL/100 g). The fatty acid composition of PSO under optimal conditions for EASE was analyzed by gas chromatography-mass spectrometry (GC-MS). The predominant unsaturated fatty acids of PSO were determined to be more than 90.00%, including n-3 α-linolenic acid (43.33%), n-6 linoleic acid (23.40%) and oleic acid (23.59%). In this experiment, the anti-AD effect of PSO was also analyzed by performing learning and memory ability tests with Drosophila. PSO retarded the decrease in climbing ability in AD Drosophila. The 1% and 5% PSO groups were significantly different from the model group (b p < 0.05). The smell short-term memory ability test revealed the number of Drosophila in barrier and barrier-free centrifuge tubes in each group. PSO feeding improved learning and memory in AD Drosophila, with the highest number entering the barrierfree centrifuge tube. The performance index (PI) measured by the Pavlov olfactory avoidance conditioning test also demonstrated the effect of PSO on the learning and memory abilities of Drosophila. The PI of the PSO group was significantly increased compared to that of the model group. HE-stained brain tissue sections of AD Drosophila showed higher neurodegenerative changes, while PSO significantly reduced neurodegenerative damage. These results indicated that PSO can significantly improve the cognitive function of AD Drosophila and may help to prevent AD.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Elucidation of the microstructure of an immuno-stimulatory polysaccharide purified from Korean red ginseng using sequential hydrolysis.

The elucidation of the structural characteristics of polysaccharides from natural sources is generally difficult owing to their structural complexity and heterogeneity. In our previous study, an immuno-stimulatory polysaccharide (RGP-AP-I) was isolated from Korean red ginseng (Panax ginseng C.A. Meyer). The present study aims to elucidate the structural characteristics of RGP-AP-I. Sequential enzyme hydrolysis was performed using four specific glycosylases, and chemical cleavage via ß-elimination was carried out to determine the fine structure of RGP-AP-I. The degraded fragments were chemically identified using various chromatographic and spectrometric analyses, including HPLC-UVD, GC-MS, and tandem mass spectrometry. The results indicated that RGP-AP-I comprises a rhamnogalacturonan I (RG-I) backbone with repeating disaccharide units [→2)-Rhap-(1 â†’ 4)-GalAp-(1→] and three side chains substituted at the C(O)4 position of the rhamnose residue in the backbone. The three side chains were identified as a highly branched α-(1 â†’ 5)-arabinan, a branched ß-(1 â†’ 4)-galactan, and an arabino-ß-3,6-galactan. Our results represent the first findings regarding the fine structure of the immuno-stimulatory polysaccharide RG-AP-I isolated from red ginseng.

Thermosonication for the Production of Sulforaphane Rich Broccoli Ingredients.

A large proportion of broccoli biomass is lost during primary production, distribution, processing, and consumption. This biomass is rich in polyphenols and glucosinolates and can be used for the production of bioactive rich ingredients for food and nutraceutical applications. This study evaluated thermosonication (TS) (18 kHz, 0.6 W/g, 40-60 °C, 3-7 min) for the pre-treatment of broccoli florets to enhance enzymatic conversion of glucoraphanin into the bioactive sulforaphane. TS significantly increased sulforaphane yield, despite a decrease in myrosinase activity with increasing treatment intensity. The highest sulforaphane yield of ~2.9 times that of untreated broccoli was observed for broccoli thermosonicated for 7 min at 60 °C, which was 15.8% higher than the corresponding yield for thermal processing without sonication (TP) at the same condition. This was accompanied by increase in the residual level of glucoraphanin (~1.8 and 2.3 time respectively after TP and TS at 60 °C for 7 min compared to control samples) indicating that treatment-induced release of bound glucoraphanin from the cell wall matrix and improved accessibility could be at least partially responsible for the enhanced sulforaphane yield. The result indicates the potential of TS for the conversion of broccoli biomass into high sulforaphane broccoli-based ingredients.

Development of pullulanase mutants to enhance starch substrate utilization for efficient production of ß-CD.

The inhibitory effect of ß-CD on pullulanase which hydrolyzes α-1,6 glycosidic bond in starch to release more available linear substrates, limited substrate utilization thus influencing the yield of ß-CD. Here, an aspartic acid residue (D465) which interacted with cyclodextrin ligand by hydrogen bond, was mutated to explore its contribution to bind inhibitors and obtain mutants with lower affinity to ß-CD. Enzyme activity results showed that mutants D465E and D465N retained higher activity than wild-type pullulanase in presence of 10 mM ß-CD. Circular dichroism spectra and fluorescence spectra results showed that D465 was related to structure stability. Chain length distribution results confirmed the improvement of substrate utilization by the addition of D465E. The conversion rate from potato starch, cassava starch, and corn starch into ß-CD, increased to 56.9%, 55.4% and 54.7%, respectively, when synchronous using ß-CGTase and D465E in the production process.

Generation of short-chained granular corn starch by maltogenic α-amylase and transglucosidase treatment.

We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.

Thermostable and alkalistable exopolygalacturonase of Bacillus pumilus dcsr1: Characteristics and applicability.

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL-1, 116 µmole mg-1 min-1, 74.73 KJmol-1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high ß-sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in ß-sheet, and a significant increase in α-helix, suggesting a key role of ß-sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.

An Atomic Force Microscopy Study on the Effect of ß-Galactosidase, α-L-Rhamnosidase and α-L-Arabinofuranosidase on the Structure of Pectin Extracted from Apple Fruit Using Sodium Carbonate.

The enzyme driven changes in plant cell wall structure during fruit ripening result in debranching, depolymerization and solubilization of pectin polysaccharides, which has an effect in terms of the postharvest quality losses in fruit. Atomic force microscopy (AFM) has revealed that diluted alkali soluble pectins (DASP) from fruit and vegetables have an interesting tendency to self-assemble into regular structures. However, the mechanism is not yet fully understood. The current study is aimed at investigating the role of neutral sugars, namely galactose, rhamnose and arabinose in the formation of the branched structure of DASP. ß-galactosidase, α-L-rhamnosidase and α-L-arabinofuranosidase enzymes were used for the treatment of DASP extracted from Golden Delicious apple flesh (Malus domestica cv. Golden Delicious). The effects of the selective degradation of pectic polysaccharides after 15, 30, 60, 90 and 120 min of incubation were observed using AFM. The α-L-rhamnosidase enzyme activity on pectin extracted with Na2CO3 did not cause any visible or measurable degradation of the molecular structure. The moderate effects of ß-galactosidase enzymatic treatment suggested the possible role of galactose in the branching of DASP molecules deposited on mica. Data obtained for α-L-arabinofuranosidase indicated the crucial role of arabinose in the formation and preservation of the highly branched structure of the DASP fraction.

Determination of chemical structure of pea pectin by using pectinolytic enzymes.

The chemical structure of pea pectin was delineated using pectin-degrading enzymes and biochemical methods. The molecular weight of the pea pectin preparation was 488,000, with 50 % arabinose content, and neutral sugar side chains attached to approximately 60 % of the rhamnose residues in rhamnogalacturonan-I (RG-I). Arabinan, an RG-I side chain, was highly branched, and the main chain was comprised of α-1,5-l-arabinan. Galactose and galactooligosaccharides were attached to approximately 35 % of the rhamnose residues in RG-I. Long chain ß-1,4-galactan was also present. The xylose substitution rate in xylogalacturonan (XGA) was 63 %. The molar ratio of RG-I/homogalacturonan (HG)/XGA in the backbone of the pea pectin was approximately 3:3:4. When considering neutral sugar side chain content (arabinose, galactose, and xylose), the molar ratio of RG-I/HG/XGA regions in the pea pectin was 7:1:2. These data will help understand the properties of pea pectin.

Quantification of Mushroom-Derived Soluble ß-1,6-Glucan Using the Function-Modified Recombinant ß-1,6-Glucanase.

Mushroom is one of the major sources of ß-glucan used in medical applications and traditional therapies. Thus, structure analysis and quantification of ß-glucan content is crucial to evaluate medicinal mushrooms. Most studies concerning mushroom-derived ß-glucan have been focused on ß-1,3-glucans. However, recent investigations suggest that ß-1,6 glucans have important roles for immunomodulating activity. Therefore, to elucidate the fine structure of various mushroom-derived ß-glucans, we recently developed a novel ß-1,6 glucan detection system using the function-modified recombinant ß-1,6-glucanase. In this study, we performed an ELISA-like assay using modified ß-1,6-glucanase and soluble dectin-1-Fc as the probes for ß-1,6-glucan and ß-1,3-glucan, respectively. Reactivity of ELISA to crude hot water extracts of edible mushrooms (Grifola frondosa, Agaricus bisporus, Pleurotus tuoliensis, P. eryngii, P. ostreatus, Hypsizygus marmoreus, and Lentinus edodes) was compared and L. edodes showed the strongest reactivity among them. An additional 19 different products of fresh L. edodes (shiitake mushroom) commercially available in Japan were also analyzed. This revealed limited differences in amounts of ß-1,6-glucan and ß-1,3-glucan in each shiitake mushroom. Furthermore, structural analysis of some purified ß-glucans derived from medicinal mushrooms was performed, and their action for inducing tumor necrosis factor-α production from the murine bone marrow-derived dendritic cells was investigated. We found relation between reactivity to modified ß-1,6-glucanase and its cytokine inducing activity. This assay could be useful for evaluating the strains of edible or medicinal mushrooms, which may be used as alternative medicines.

Phytochemical content, antioxidant activity, and enzyme inhibition effect of Salvia eriophora Boiss. & Kotschy against acetylcholinesterase, α-amylase, butyrylcholinesterase, and α-glycosidase enzymes.

Many taxa of Salvia genus have been used in herbal beverages, food flavoring, cosmetics, and pharmaceutical industry. In this paper, chemical compounds of Salvia eriophora (S. eriophora) leaves were determined by LC-MS/MS (Liquid Chromatography tandem Mass Spectrometry). Salvigenin (158.64 ± 10.8 mg/kg), fumaric acid (123.09 ± 8.54 mg/kg), and quercetagetin-3.6-dimethylether (37.85 ± 7.09 mg/kg) were detected as major compounds in the ethanol extract, whereas fumaric acid (555.96 ± 38.56 mg/kg), caffeic acid (103.62 ± 20.51 mg/kg), and epicatechin (83.19 ± 8.43 mg/kg) were detected as major compounds in the water extract. Furthermore, enzyme inhibition of S. eriophora against acetylcholinesterase (AChE), α-amylase (AM), butyrylcholinesterase (BChE), and α-glycosidase (AG) enzymes were detected. AChE, BChE, AG, and AM enzymes were very strongly inhibited by S. eriophora water extract (WES) and S. eriophora methanol extract (MES). Additionally, antioxidant potential of S. eriophora was determined by in vitro analytical methods. IC50 values of WES and MES were performed for radicals. PRACTICAL APPLICATIONS: Metabolic enzymes have crucial functions on living systems due to inhibition or activation of them mainly attributed with some health disorders. AChE, BChE, AM, and AG enzymes have important roles on carbohydrate metabolism or cholinergic pathways. The relation between enzyme inhibition effect and phenolic compounds or antioxidant activity need to be confirmed. Thus, many studies tested to clarify this relation for pure samples or plant extracts. To the best of our knowledge, this is the first report about inhibition effects of Salvia eriophora extracts against AChE, BChE, AM, and AG enzymes as well as their phenolic contents and antioxidant activities.

A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

Myrosinase: insights on structural, catalytic, regulatory, and environmental interactions.

Glucosinolate-myrosinase is a substrate-enzyme defense mechanism present in Brassica crops. This binary system provides the plant with an efficient system against herbivores and pathogens. For humans, it is well known for its anti-carcinogenic, anti-inflammatory, immunomodulatory, anti-bacterial, cardio-protective, and central nervous system protective activities. Glucosinolate and myrosinase are spatially present in different cells that upon tissue disruption come together and result in the formation of a variety of hydrolysis products with diverse physicochemical and biological properties. The myrosinase-catalyzed reaction starts with cleavage of the thioglucosidic linkage resulting in release of a D-glucose and an unstable thiohydroximate-O-sulfate. The outcome of this thiohydroximate-O-sulfate has been shown to depend on the structure of the glucosinolate side chain, the presence of supplementary proteins known as specifier proteins and/or on the physiochemical condition. Myrosinase was first reported in mustard seed during 1939 as a protein responsible for release of essential oil. Until this date, myrosinases have been characterized from more than 20 species of Brassica, cabbage aphid, and many bacteria residing in the human intestine. All the plant myrosinases are reported to be activated by ascorbic acid while aphid and bacterial myrosinases are found to be either neutral or inhibited. Myrosinase catalyzes hydrolysis of the S-glycosyl bond, O-ß glycosyl bond, and O-glycosyl bond. This review summarizes information on myrosinase, an essential component of this binary system, including its structural and molecular properties, mechanism of action, and its regulation and will be beneficial for the research going on the understanding and betterment of the glucosinolate-myrosinase system from an ecological and nutraceutical perspective.

Enhanced inulinase production by Fusarium solani JALPK from invasive weed using response surface methodology.

The present study is the first report of utilizing Tithonia rotundifolia weed as a substrate for inulinase production from Fusarium solani JALPK. It also deals with the statistical optimization of culture conditions to enhance the enzyme yield. Amongst the 11 variables screened by Plackett- Burman design, Inulin in combination with Agave sisalana extract, Tithonia rotundifolia extract and NaNO3 had a significant influence on inulinase production and their concentrations were further optimized employing Box Behnken design. An enhancement of inulinase production from 970 EU/mL to 3261.011 EU/mL was gained after media optimization. Amongst the screened carbon sources Tithonia rotundifolia was found to be very effective in stimulating elevated inulinase synthesis. The Tithonia rotundifolia weed extract was treated with inulinase from Fusarium solani JALPK to form fructose which was estimated spectrophotometrically. This liberated fructose was also confirmed by osazone formation test and FTIR. HPTLC analysis of product revealed the exoinulinase nature of the enzyme produced by Fusarium solani JALPK since fructose was the only end product after hydrolysis of inulin rich weed in fermented broth. Thus the elevated extracellular inulinase yielding novel property of Fusarium solani JALPK (KY914560) contributes in considering it as a potential candidate with food, pharmaceutical and bioremediation applications.

Detailed Mode of Action of Arabinan-Debranching α-L-Arabinofuranosidase GH51 from Bacillus velezensis.

The gene encoding an α-L-arabinofuranosidase (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at 45°C. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively L-arabinose. BvAF could cleave α-(1,2)- and/or α-(1,3)-L-arabinofuranosidic linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate L-arabinose from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting α-L-arabinofuranosidase possessing high debranching activity towards α-(1,2)- and/or α-(1,3)-linked branches of arabinan, which can facilitate the successive degradation of arabinan by endo-α-(1,5)-L-arabinanase.

Demonstration of allelopathy of horseradish root extract on lettuce seed.

Allelopathy plays crucial roles in invasive plant viability and agricultural production systems. However, there is no well-established hands-on learning activity to teach the concept of allelopathy. Nor is there an activity which allows students to gain knowledge about glucosinolates and their corresponding enzyme, myrosinase, which are present in almost all Brassica crops. Lettuce germination was counted by the students from three different treatments including water treated with Parafilm sealing, horseradish treated with Parafilm sealing, and horseradish treated without Parafilm sealing. Additionally, lettuce root length was measured by students using ImageJ software from each treatment using pictures captured by students' smartphones. Students took an identical quiz as a pre-laboratory and a post-laboratory assignment. Their average scores on the pre-laboratory and post-laboratory quizzes were 3.14 and 6.56 out of 10, respectively, indicating the lab activity significantly improved students' understanding of allelopathy and glucosinolate-myrosinase system. In addition, students (n = 76) completed a survey post-laboratory to assess their self-efficacy. This simple and cost-effective laboratory activity improved students' knowledge and skill development as it made learning more inviting, meaningful, and fun. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):333-340, 2019.

Enzymatic fingerprinting of isomalto/malto-polysaccharides.

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

Structural characterization of rhamnogalacturonan domains from Panax ginseng C. A. Meyer.

Rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains were isolated from ginseng pectin by alkali saponification and endo-polygalacturonase hydrolysis, then purified by anion-exchange and size-exclusion chromatography. Monoclonal antibody detection indicated that ginseng RG-I contained →4)-α-GalpA-(1→2)-α-Rhap-(1→ repeating units as backbone, with arabinan, galactan and type II arabinogalactan (AG-II) as side chains. The use of galactose- and arabinose-releasing enzymes, mass spectrometry analysis of the oligosaccharides generated by rhamnogalacturonan hydrolase, and glycosidic linkage analyses provided evidence that ginseng RG-I contains both single galactose-branched subunits and highly branched subunits with arabinan and AG-II side chains. RG-II was analyzed by sequential acid hydrolysis followed by mass spectrometry. Ginseng RG-II contains 9 galacturonic acid units as backbone. Side chain A is an octasaccharide with 0 ∼ 1 methyl ether group. Side chain B is a nonasaccharide with 0 ∼ 1 acetyl group. These results provide useful information for further investigation of structure-activity relationship of ginseng pectin.

Efficient Hydrolysis of Raw Microalgae Starch by an α-Amylase (AmyP) of Glycoside Hydrolase Subfamily GH13_37.

Microalgae starch is receiving increasing attention as a renewable feedstock for biofuel production. Raw microalgae starch from Tetraselmis subcordiformis was proven to be very efficiently hydrolyzed by an α-amylase (AmyP) of glycoside hydrolase subfamily GH13_37 below the temperature of gelatinization (40 °C). The hydrolysis degree reached 74.4 ± 2.2% for 4% raw microalgae starch and 53.2 ± 1.7% for 8% raw microalgae starch after only 2 h. The hydrolysis efficiency was significantly stimulated by calcium ions. The enzyme catalysis of AmyP and its mutants (Q306A and E347A) suggested that calcium ions contributed to the hydrolysis of cyclic structures in raw microalgae starch by a distinctive calcium-binding site Ca2 of AmyP. The study explored raw microalgae starch as a new resource for cold enzymatic hydrolysis and extended our knowledge on the function of calcium in amylolytic enzyme.