Residue Effect-Guided Design: Engineering of S. Solfataricus ß-Glycosidase to Enhance Its Thermostability and Bioproduction of Ginsenoside Compound K.

ß-Glycosidase from Sulfolobus solfataricus (SS-BGL) is a highly effective biocatalyst for the synthesis of compound K (CK) from glycosylated protopanaxadiol ginsenosides. In order to improve the thermal stability of SS-BGL, molecular dynamics simulations were used to determine the residue-level binding energetics of ginsenoside Rd in the SS-BGL-Rd docked complex and to identify the top ten critical contributors. Target sites for mutations were determined using dynamic cross-correlation mapping of residues via the Ohm server to identify networks of distal residues that interact with the key binding residues. Target mutations were determined rationally based on site characteristics. Single mutants and then recombination of top hits led to the two most promising variants SS-BGL-Q96E/N97D/N302D and SS-BGL-Q96E/N97D/N128D/N302D with 2.5-fold and 3.3-fold increased half-lives at 95 °C, respectively. The enzyme activities relative to those of wild-type for ginsenoside conversion were 161 and 116%, respectively..

Engineered Glycosidase for Significantly Improved Production of Naturally Rare Vina-Ginsenoside R7.

Ginsenosides are the main bioactive ingredients in plants of the genus Panax. Vina-ginsenoside R7 (VG-R7) is one of the rare high-value ginsenosides with health benefits. The only reported method for preparing VG-R7 involves inefficient and low-yield isolation from highly valuable natural resources. Notoginsenoside Fc (NG-Fc) isolated in the leaves and stems of Panax notoginseng is a suitable substrate for the preparation of VG-R7 via specific hydrolysis of the outside xylose at the C-20 position. Here, we first screened putative enzymes belonging to the glycoside hydrolase (GH) families 1, 3, and 43 and found that KfGH01 can specifically hydrolyze the ß-d-xylopyranosyl-(1 → 6)-ß-d-glucopyranoside linkage of NG-Fc to form VG-R7. The I248F/Y410R variant of KfGH01 obtained by protein engineering displayed a kcat/KM value (305.3 min-1 mM-1) for the reaction enhanced by approximately 270-fold compared with wild-type KfGH01. A change in the shape of the substrate binding pockets in the mutant allows the substrate to sit closer to the catalytic residues which may explain the enhanced catalytic efficiency of the engineered enzyme. This study identifies the first glycosidase for bioconversion of a ginsenoside with more than four sugar units, and it will inspire efforts to investigate other promising enzymes to obtain valuable natural products.

BST1 regulates nicotinamide riboside metabolism via its glycohydrolase and base-exchange activities.

Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.

Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases in the Prevention of Experimental Invasive Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.

Comparative evaluation for phytochemical composition and regulation of blood glucose, hepatic oxidative stress and insulin resistance in mice and HepG2 models of four typical Chinese dark teas.

BACKGROUND: Dark tea, comprising one of the six major teas, has many biological activities, which originate from their active substrates, such as polyphenols, polysaccharides, and so on. The hypoglycemic effect is one of its most prominent activities, although less is known about their evaluation and potential role in the hypoglycemic mechanism. RESULTS: In the present study, we separately analyzed the phytochemical composition, glycosidase inhibition and free radical scavenging activities, and hypoglycemic activity in type 2 diabetes mellitus mice, as well as the alleviation of insulin resistance in HepG2 cells of four dark tea aqueous extracts. The results showed that the phytochemical composition of dark tea aqueous extracts was significantly different, and they all had good glycosidase inhibition and free radical scavenging activities, in vivo hypoglycemic activity and alleviation of insulin resistance, and could also activate the phosphatidylinositol 3-kinase-Akt-perixisome proliferation-activated receptor cascade signaling pathway to regulate glucose and lipid metabolism, change the key enzyme activities related to glucose metabolism and antioxidant activity, and reduce oxidative stress and inflammatory factor levels. Among them, Liubao brick tea (LBT) and Pu-erh tea (PET) possessed better glycosidase inhibitory activity, in vivo hypoglycemic activity and improved insulin resistance activity, whereas Qingzhuan brick tea and Fuzhuan brick tea had better free radical scavenging activity, which may be explained by their distinct phytochemical compositions, such as tea proteins, polysaccharides, polyphenols, catechins, and tea pigments and some elements. CONCLUSION: Dark tea is a highly attractive candidate for developing antidiabetic food, LBT and PET may be good natural sources of agricultural products with anti-diabetic effects. © 2021 Society of Chemical Industry.

Effects of nutrient restriction and melatonin supplementation from mid-to-late gestation on maternal and fetal small intestinal carbohydrase activities in sheep.

The objective of this experiment was to evaluate the effects of nutrient restriction and melatonin supplementation during mid-to-late gestation on maternal and fetal small intestinal carbohydrase activities in sheep. Ewes were randomly assigned to one of 4 dietary treatments arranged in a 2 × 2 factorial design. Ewes were fed to provide 100% (adequate; ADQ) or 60% (restricted; RES) of nutrient recommendations, and diets were supplemented with either no melatonin (control; CON) or 5 mg melatonin/d (melatonin; MEL). This resulted in 4 treatment groups: CON-ADQ (n = 7), CON-RES (n = 8), MEL-ADQ (n = 8), MEL-RES (n = 8). Treatments began on day 50 of gestation, and ewes were euthanized on day 130 for tissue collection. The maternal and fetal small intestine were collected and assayed for small intestinal carbohydrase activities. Data were analyzed using the GLM procedure of SAS with fetal sex, melatonin, nutrition, and the melatonin by nutrition interaction included in the model statement. There were no melatonin by nutrition interactions for maternal or fetal small intestinal protein concentration or carbohydrase activities (P ≥ 0.11). Dietary melatonin supplementation decreased (P = 0.03) maternal small intestinal protein concentration by 22.7% and increased (P = 0.03) maternal small intestinal glucoamylase, isomaltase, and maltase activity per gram protein by 45.5%, 41.3%, and 40.6%, respectively. Nutrient restriction from mid-to-late gestation did not influence (P ≥ 0.46) maternal small intestinal protein concentration, or maltase, isomaltase, and lactase activity. Maternal glucoamylase activity per gram intestine increased (P = 0.05) with nutrient restriction by 49.1%. Melatonin supplementation and maternal nutrient restriction did not influence (P ≥ 0.15) fetal small intestinal protein concentration, or glucoamylase, isomaltase, and lactase activity. Maternal nutrient restriction from mid-to-late gestation decreased (P = 0.05) fetal maltase activity per gram intestine by 20.5% but did not influence fetal maltase activity per gram protein. These data indicate that some maternal and fetal carbohydrases are influenced by nutrient restriction and melatonin supplementation in sheep. More information is needed to understand how nutritional and hormonal factors regulate digestive enzyme activity in ruminants to design improved maternal nutrition programs to optimize fetal growth and development while maintaining maternal productivity.

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.

Therapeutic Activity of Type 3 Streptococcus pneumoniae Capsule Degrading Enzyme Pn3Pase.

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.

Characterization of two extracellular arabinanases in Lactobacillus crispatus.

Extracellular glycosyl hydrolases are uncommon in lactobacilli and include amylases and fructosidases mediating starch and fructan utilization, respectively. Extracellular arabinanases have not been described in lactobacilli. This study is aimed at identifying the function of an arabinan utilization operon in Lactobacillus crispatus DSM29598 and at characterizing two putative extracellular arabinanases that are located on that operon. The arabinan utilization operon of L. crispatus DSM29598 encodes enzymes for degradation of arabinan, α-galactosidases, ß-galactosidases, and enzymes and for utilization of arabinose including phosphoketolase. The two putative extracellular arabinanases, AbnA and AbnB, are homologous to family GH43 endo-arabinanases. In Lactobacillaceae, homologs of these enzymes were identified exclusively in vertebrate-adapted species of the genus Lactobacillus. L. crispatus grew with arabinan from sugar beet pectin as sole carbon source, indicating extracellular arabinanase activity, and produced lactate and acetate, indicating metabolism via the phosphoketolase pathway. The two arabinanases AbnA and AbnB were heterologously expressed and purified by affinity chromatography. AbnA hydrolyzed linear and branched arabinan, while AbnB hydrolyzed only linear arabinan. The optimum pH for AbnA and AbnB was 6 and 7.5, respectively; 40 °C was the optimum temperature for both enzymes. The application of arabinan degrading L. crispatus as probiotic or as synbiotic with pectins may improve the production of short-chain fatty acids from pectin to benefit host health. KEY POINTS: • An arabinan utilization operon in L. crispatus encodes two extracellular arabinanases. • The same operon also encodes metabolic genes for arabinose conversion. • In Lactobacillaceae, extracellular arabinanases are exclusive to Lactobacillus species.

Three novel rhamnogalacturonan I- pectins degrading enzymes from Aspergillus aculeatinus: Biochemical characterization and application potential.

Rhamnogalaturonans I (RGI) pectins, which are a major component of the plant primary cell wall, can be recalcitrant to digestion by commercial enzymatic cocktails, in particular during fruit juice clarification process. To overcome these problems and get better insights into RGI degradation, three RGI degrading enzymes (RHG: Endo-rhamnogalacturonase; ABF: α-Arabinofuranosidases; GAN: Endo-ß-1,4-galactanase) from Aspergillus aculeatinus were expressed in Pichia pastoris, purified and fully biochemically characterized. All three enzymes showed acidic pH optimum, and temperature optima between 40-50 °C. The Km values were 0.5 mg.ml-1, 1.64 mg.ml-1 and 3.72 mg.ml-1 for RHG, ABF, GAN, respectively. NMR analysis confirmed an endo-acting mode of action for RHG and GAN, and exo-acting mode for ABF. The application potential of these enzymes was assessed by measuring changes in viscosity of RGI-rich camelina mucilage, showing that RHG-GAN enzymes induced a decrease in viscosity by altering the structures of the RGI backbone and sidechains.

Influence of dietary fructose supplementation on visceral organ mass, carbohydrase activity, and mRNA expression of genes involved in small intestinal carbohydrate assimilation in neonatal calves.

The hypothesis of this experiment was that dietary fructose would influence visceral organ mass, carbohydrase activity, and mRNA expression of carbohydrases and nutrient transporters in the small intestine in neonatal calves. Therefore, our objective was to use the neonatal calf as a model to evaluate the effects of postruminal fructose supply on small intestinal carbohydrate assimilation. Ten calves (<7 d of age; 41.2 ± 1.46 kg of body weight) were fed milk replacer at 2.0% of body weight daily (816 ± 90.5 g/d; 272 ± 30.1 g/L; dry-matter basis) in 2 equal portions and assigned to the following dietary treatment groups: (1) milk replacer (control; n = 6) or (2) milk replacer + 2.2 g of fructose/kg of body weight (fructose; n = 4). Calves were fed dietary treatments for 28 d, with jugular blood sampled every 7 d before and after the morning feeding. Calves were slaughtered, and visceral weights were recorded. Postruminal carbohydrase activities were assayed. Quantitative real-time PCR was conducted for small intestinal mRNA expression of nutrient transporters [solute carrier family 2 member 5 (GLUT5), solute carrier family 2 member 2 (GLUT2), and solute carrier family 5 member 1 (SGLT1)], carbohydrases (lactase, maltase-glucoamylase, and sucrase-isomaltase), and ketohexokinase (KHK). Data were analyzed using MIXED procedures in SAS version 9.4 (SAS Institute Inc, Cary, NC). Dietary fructose supplementation decreased serum glucose concentration. Small intestinal mass was greater in calves supplemented with fructose. Dietary fructose supplementation did not influence pancreatic α-amylase, small intestinal isomaltase, or maltase activities. Sucrase activity was undetected in the small intestine. Dietary fructose supplementation increased small intestinal glucoamylase activity per gram of tissue by 30% and increased maltase-glucoamylase mRNA expression by 6.8-fold. Dietary fructose supplementation did not influence mRNA expression of GLUT5, SGLT1, GLUT2, or KHK. Dietary fructose supplementation increased small intestinal lactase mRNA expression by 3.1-fold. Sucrase-isomaltase mRNA expression in the small intestine decreased 5.1-fold with dietary fructose supplementation. Dietary fructose supplementation does not induce sucrase activity in neonatal calves; however, sucrase-isomaltase may be transcriptionally regulated by dietary fructose in neonatal calves. More research is needed to compare glucose and fructose at isocaloric intakes to examine effects of dietary fructose at equal metabolizable energy intake.

The gut microbiome stability is altered by probiotic ingestion and improved by the continuous supplementation of galactooligosaccharide.

The stable gut microbiome plays a key role in sustaining host health, while the instability of gut microbiome also has been found to be a risk factor of various metabolic diseases. At the ecological and evolutionary scales, the inevitable competition between the ingested probiotic and indigenous gut microbiome can lead to an increase in the instability. It remains largely unclear if and how exogenous prebiotic can improve the overall gut microbiome stability in probiotic consumption. In this study, we used Lactobacillus plantarum HNU082 (Lp082) as a model probiotic to examine the impact of the continuous or pulsed supplementation of galactooligosaccharide (GOS) on the gut microbiome stability in mice using shotgun metagenomic sequencing. Only continuous GOS supplement promoted the growth of probiotic and decreased its single-nucleotide polymorphisms (SNPs) mutation under competitive conditions. Besides, persistent GOS supplementation increased the overall stability, reshaped the probiotic competitive interactions with Bacteroides species in the indigenous microbiome, which was also evident by over-abundance of carbohydrate-active enzymes (CAZymes) accordingly. Also, we identified a total of 793 SNPs arisen in probiotic administration in the indigenous microbiome. Over 90% of them derived from Bacteroides species, which involved genes encoding transposase, CAZymes, and membrane proteins. However, neither GOS supplementation here de-escalated the overall adaptive mutations within the indigenous microbes during probiotic intake. Collectively, our study demonstrated the beneficial effect of continuous prebiotic supplementation on the ecological and genetic stability of gut microbiomes.

Enhancement of Production of D-Glucosamine in Escherichia coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc.

D-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that D-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The D-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of D-glucosamine by a factor of 2.16. Moreover, for further increasing D-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of D-glucosamine in E. coli BL21 glmU-Δgpa', E. coli BL21ΔmurQ', and E. coli BL21ΔnanE' were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.

Zearalenone Removal from Corn Oil by an Enzymatic Strategy.

The estrogen-like mycotoxin zearalenone (ZEN) is one of the most widely distributed contaminants especially in maize and its commodities, such as corn oil. ZEN degrading enzymes possess the potential for counteracting the negative effect of ZEN and its associated high safety risk in corn oil. Herein, we targeted enhancing the secretion of ZEN degrading enzyme by Pichia pastoris through constructing an expression plasmid containing three optimized expression cassettes of zlhy-6 codon and signal peptides. Further, we explored various parameters of enzymatic detoxification in neutralized oil and analyzed tocopherols and sterols losses in the corn oil. In addition, the distribution of degraded products was demonstrated as well by Agilent 6510 Quadrupole Time-of-Flight mass spectrometry. P. pastoris GSZ with the glucoamylase signal was observed with the highest ZLHY-6 secretion yield of 0.39 mg/mL. During the refining of corn oil, ZEN in the crude oil was reduced from 1257.3 to 13 µg/kg (3.69% residual) after neutralization and enzymatic detoxification. Compared with the neutralized oil, no significant difference in the total tocopherols and sterols contents was detected after enzymatic detoxification. Finally, the degraded products were found to be entirely eliminated by washing. This study presents an enzymatic strategy for efficient and safe ZEN removal with relatively low nutrient loss, which provides an important basis for further application of enzymatic ZEN elimination in the industrial process of corn oil production.

Discovery and mechanism of intestinal bacteria in enzymatic cleavage of C-C glycosidic bonds.

C-Glycosides, a special type of glycoside, are frequently distributed in many kinds of medicinal plants, such as puerarin and mangiferin, showing various and significant bioactivities. C-Glycosides are usually characterized by the C-C bond that forms between the anomeric carbon of sugar moieties and the carbon atom of aglycon, which is usually resistant against acidic hydrolysis and enzymatic treatments. Interestingly, C-glycosides could be cleaved by several intestinal bacteria, but whether the enzymatic cleavage of C-C glycosidic bond is reduction or hydrolysis has been controversial; furthermore, whether existence of a "C-glycosidase" directly catalyzing the cleavage is not clear. Here we review research advances about the discovery and mechanism of intestinal bacteria in enzymatic cleavage of C-C glycosidic bond with an emphasis on the identification of enzymes manipulation the deglycosylation. Finally, we give a brief conclusion about the mechanism of C-glycoside deglycosylation and perspectives for future study in this field.

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.

Production of thermostable endo-1,5-α-L-arabinanase in Pichia pastoris for enzymatically releasing functional oligosaccharides from sugar beet pulp.

Sugar beet pulp is an agricultural processing residue that is a rich source of the cell wall polysaccharide arabinan. Functional oligosaccharides, specifically feruloylated arabino-oligosaccharides (FAOs), can be isolated from sugar beet pulp through selective action by endo-arabinanase (glycoside hydrolase family 43). This study aimed to develop yeast (Pichia pastoris) as an efficient, eukaryotic platform to produce a thermophilic endo-1,5-α-L-arabinanase (TS-ABN) for extracting FAOs from sugar beet pulp. Recombinant TS-ABN was secreted into yeast culture medium at a yield of ~ 80 mg/L, and the protein exhibited specific enzyme activity, pH and temperature optimum, and thermostability comparable to those of the native enzyme. Treatment of sugar beet pulp with Pichia-secreted TS-ABN released FAOs recovered by hydrophobic chromatography at 1.52% (w/w). The isolated FAOs averaged seven arabinose residues per ferulic acid, and treatment of T84 human colon epithelial cells significantly increased expression of two key tight junction-related proteins-zonula occludens-1 and occludin-in a dose-dependent manner. This research establishes a biochemical platform for utilizing sugar beet pulp to produce value-added bioproducts with potential nutraceutical applications.

Development of a DNA marker for variety discrimination specific to 'Manten-Kirari' based on an NGS-RNA sequence in Tartary buckwheat (Fagopyrum tataricum).

To discriminate the trace-rutinosidase variety of Tartary buckwheat 'Manten-Kirari', we developed DNA markers based on RNA polymorphism. Specifically, we mapped 17.76 GB RNA sequences, obtained using HiSeq2000, to create 11,358 large contigs constructed de novo from 'Manten-Kirari' RNA derived from GS-FLX+ titanium. From these, we developed eight DNA markers corresponding to single- to four-nucleotide polymorphisms between 'Manten-Kirari' and 'Hokkai T8', which is representative of normal rutinosidase content varieties in Japan. Using these markers, 'Manten-Kirari' was discriminated from 'Hokkai T8' by eight markers, from major Tartary buckwheat varieties by three markers, and from common buckwheats by two markers. We also performed direct PCR from flour and dried noodle made with 'Manten-Kirari' and 'Hokkai T8'. Based on the results, the DNA markers developed are promising for discriminating 'Manten-Kirari'. This is the first study to develop a DNA marker to discriminate varieties in the Polygonaceae family including buckwheat species.

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.