Prunin Laurate Derived from Natural Substances Shows Antibacterial Activity against the Periodontal Pathogen Porphyromonas gingivalis.

Periodontal disease is an inflammatory disease caused by infection with periodontopathogenic bacteria. Oral care is essential to prevent and control periodontal disease, which affects oral and systemic health. However, many oral hygiene products currently on the market were developed as disinfectants, and their intense irritation makes their use difficult for young children and older people. This study investigated the antibacterial effects of prunin laurate (Pru-C12) and its analogs on periodontopathogenic bacteria, Porphyromonas gingivalis (P. gingivalis). Pru-C12 and its analogs inhibited in vitro bacterial growth at more than 10 µM and biofilm formation at 50 µM. Among its analogs, only Pru-C12 showed no cytotoxicity at 100 µM. Three of the most potent inhibitors also inhibited the formation of biofilms. Furthermore, Pru-C12 inhibited alveolar bone resorption in a mouse experimental periodontitis model by P. gingivalis infection. These findings may be helpful in the development of oral hygiene products for the prevention and control of periodontal disease and related disorders.

Fractionation of cassava pectins and their detailed structural analyses using various pectinolytic enzymes.

In this study, we analyzed the pectin structure within the pulp of cassava. Cassava pectin, derived from cassava pulp treatment at 120 °C for 90 min, was separated into four fractions (CP-P, CP-SD1, CP-SD2F, and CP-SD2R) based on variations in water solubility, electrical properties, and molecular weights. Sugar composition analysis demonstrated an abundance of homogalacturonan (HG) in CP-P and CP-SD2F, rhamnogalacturonan I (RG-I) in CP-SD2R, and neutral sugars in CP-SD1. Because RG-I possesses a complex structure, we analyzed CP-SD2R using various pectinolytic enzymes. Galactose was the major sugar in CP-SD2R accounting for 49 %, of which 65 % originated from arabinogalactan I, 9 % from galactose and galactooligosaccharides, 5 % from arabinogalactan II, and 11 % from galactoarabinan. Seventy-four percent of arabinose in CP-SD2R was present as galactoarabinan. The methylation (DM) and acetylation (DAc) degrees of cassava pectin were 11 and 15 %, respectively. The HG and RG-I regions exhibited DAc values of 5 and 44 %, respectively, signifying the high DAc of RG-I compared to HG. Information derived from the structural analysis of cassava pectin will enable efficient degradation of pectin and cellulose, leading to the use of cassava pulp as a raw material for biorefineries.

Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

Gelation of konjac glucomannan by acetylmannan esterases from Aspergillus oryzae.

Konjac glucomannan (KGM) is a principal component of the gelatinous food Konjac. Konjac production through alkali treatment releases an undesirable amine-odor. Two acetylesterases (AME1 and AME2) active against konjac glucomannan (polymer or oligomer) were purified from the supernatant of Aspergillus oryzae RIB40 culture. We cloned the genes encoding AME1 and AME2 based on the genomic information of A. oryzae, constructed their expression systems in A. oryzae, and obtained the recombinant enzymes (rAME1 and rAME2). rAME1 did not act on the KGM polymer but only on the KGM oligomer, releasing approximately 60% of the acetic acid in the substrate. However, rAME2 was active against both KGM substrates, releasing approximately 80% and 100% of acetic acid from the polymer and oligomer, respectively. Both enzymes were active against xylan and exhibited a trace activity on ethyl ferulate. The acetyl group position specificities of both enzymes were analyzed via heteronuclear single quantum correlation NMR using oligosaccharides of glucomannan prepared from Aloe vera (AGM), which has a higher acetyl group content than KGM. rAME1 acted specifically on single-substituted acetyl groups and not on double-substituted ones. In contrast, rAME2 appeared to act on all the acetyl groups in AGM. Treatment of 3% KGM with rAME2 followed by heating to 90 °C resulted in gel formation under weakly acidic conditions. This is the first study to induce gelation of KGM under these conditions. A comparison of the breaking and brittleness properties of gels formed by alkaline and enzymatic treatments revealed similar texture of the two gels. Furthermore, scanning electron microscopy of the surface structure of both gels revealed that both formed a fine mesh structure. Our findings on enzymatic gelation of KGM should lead to the development of new applications in food manufacturing industry.

Novel acid trehalase belonging to glycoside hydrolase family 37 from Pleurotus sp.: cloning, expression and characterization.

The N-terminal amino acid sequence of the Pleurotus sp. 90 kDa protein was in good agreement with the corresponding sequence of the glycoside hydrolase (GH) family 37 protein (trehalase) from P. ostreatus PC 15 v2.0. The length of the Pleurotus sp. trehalase gene was 2247 bp, encoding a protein of 749 amino acids with a predicted molecular mass of 81.2 kDa. The molecular mass of the recombinant enzyme was estimated to be about 117 kDa by SDS-PAGE. We found that the recombinant enzyme comprised an N-glycosylated sugar chain and that its optimum pH and temperature were 4.5 and 40 ºC, respectively. Moreover, this enzyme exhibited high activity against trehalose exclusively. We found that the enzyme is novel acid trehalase belonging to GH family 37.

Effects of an environmental endocrine disruptor, para-nonylphenol on the cell growth of Euglena gracilis: association with the cellular oxidative stress.

Effects of an environmental endocrine disruptor, para-nonylphenol (NP) on the cell growth of a photosynthetic eukaryotic microorganism, Euglena gracilis were analysed under different cell culture conditions. Although NP did not show significant inhibitory effects on the cell growth of E. gracilis (Z and SM strains) under light culture condition, NP exhibited significant suppressive effects under dark culture condition. Exogenous supplementation with lipophilic antioxidants (α-tocopherol, ß-carotene or 6-O-palmitoyl-ascorbic acid) to E. gracilis caused strong preventive effects against NP-induced cell growth inhibition under dark culture condition, but hydrophilic antioxidants [ascorbic acid, glutathione and epigallocatechin gallate (EGCG)] did not show significant preventive effects. NP caused significant generation of reactive oxygen species (ROS) in E. gracilis under dark culture condition, but E. gracilis under light culture condition did not show significant increase in ROS generation. Supplementation with lipophilic antioxidants to E. gracilis caused significant suppressive effects against NP-induced cellular ROS generation under dark culture condition, but hydrophilic antioxidants did not show significant suppressive effects. Furthermore, the productivities of typical cellular antioxidants (α-tocopherol, ß-carotene and ascorbic acid) in E. gracilis under light culture conditions were much higher than those under dark culture conditions.

NADPH-to-NADH conversion by mitochondrial transhydrogenase is indispensable for sustaining anaerobic metabolism in Euglena gracilis.

Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization.

We identified the raw-starch-digesting α-amylase genes a earthworm Eisenia fetid α amylase I and II (Ef-Amy I and Ef-Amy II). Each gene consists of 1,530 base pairs (bp) that encode proteins of 510 amino acids, as indicated by the corresponding mRNA sequences. Ef-Amy I and II showed an 89% amino acid identity. The amino acid sequences of Ef-Amy I and II were similar to those of the α-amylases from porcine pancreas, human pancreas, Tenebrio molitor, Oryctolagus cuniculus, and Xenopus (Silurana) tropicalis. Each gene encoding mature Ef-Amy I and II was expressed in the GS115 strain of Pichia pastoris. The molecular masses of the recombinant Ef-Amy I and II were 57 kDa each, and catalytically important residues of α-amylases of the GH family 13 were conserved in both proteins. These amylases exhibited raw-starch-digesting activity at 4 °C. The substrate specificities of rEf-Amy I and II were dissimilar. rEf-Amy I and II were shown to be active even in 40% ethanol, 4 M NaCl, and 4 M KCl.

Homogalacturonan and xylogalacturonan region specificity of self-cloning vector-expressed pectin methylesterases (AoPME1-3) in Aspergillus oryzae.

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family.

Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the ß-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1→4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1→4)-d-glucuronic acid -bound FoRham1 were determined, and the active site was identified on the anterior side of the ß-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.

Biochemical and structural characterization of a novel 4-O-α-l-rhamnosyl-ß-d-glucuronidase from Fusarium oxysporum.

In this study, we have isolated the novel enzyme 4-O-α-l-rhamnosyl-ß-d-glucuronidase (FoBGlcA), which releases α-l-rhamnosyl (1→4) glucuronic acid from gum arabic (GA), from Fusarium oxysporum 12S culture supernatant, and for the first time report an enzyme with such catalytic activity. The gene encoding FoBGlcA was cloned and expressed in Pichia pastoris. When GA was subjected to the recombinant enzyme, > 95% of the l-rhamnose (Rha) and d-glucuronic acid in the substrate were released, which indicates that almost all Rha binds to the glucuronic acid at the end of the GA side chains. The crystal structure of FoBGlcA was determined using a single-wavelength anomalous dispersion at 1.51 Å resolution. FoBGlcA consisted of an N-terminal (ß/α)8 -barrel domain and a C-terminal antiparallel ß-sheet domain. This configuration is characteristic of glycoside hydrolase (GH) family 79 proteins. A structural similarity search showed that FoBGlcA mostly resembled GH79 ß-d-glucuronidase (AcGlcA79A) of Acidobacterium capsulatum; however, the root-mean-square deviation value was 3.2 Å, indicating that FoBGlcA has a high structural divergence. FoBGlcA had a low sequence identity with AcGlcA79A (19%) and differed from other GH79 ß-glucuronidases. The structures of FoBGlcA and AcGlcA79A also differed in terms of the loop structure location near subsite -2 of their catalytic sites, which may account for the unique substrate specificity of FoBGlcA. The amino acid residues involved in the catalytic activity of this enzyme were determined by evaluating the activity levels of various mutant enzymes based on the crystal structure analysis of the FoBGlcA reaction product complex. DATABASE: Atomic coordinates and structure factors (codes 7DFQ and 7DFS) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Exopolysaccharides from a Scandinavian fermented milk viili increase butyric acid and Muribaculum members in the mouse gut.

Starter culture of viili contains lactic acid bacteria belonging to Lactococcus lactis. These bacteria secrete large polysaccharides (EPSs) into milk, resulting in a ropy texture of viili. In mouse experiments, a large dose of EPS (5-140 mg/day) has been shown to alleviate severity of artificially induced illness through modulation of the gut microbiota. The present study investigated whether supplementary amounts of EPS affects the gut microbiota of normal mouse. EPS with high glucosamine content (VEPS) was isolated from home-made viili. C57BL/6J male mice fed ordinary diet took 49 ± 1 µg VEPS/day for 28 days by drinking ad libitum tap water containing 8 µg/mL VEPS. The relative abundance of Muribaculum increased significantly by VEPS supplementation. The relative abundance of fecal butyric acid decreased in control mice, and VEPS prevented this decrease. These findings indicated that the gut microbiota can be modulated by a small dose of VEPS.

Substrate-recognition mechanism of tomato ß-galactosidase 4 using X-ray crystallography and docking simulation.

MAIN CONCLUSION: TBG4 recognize multiple linkage types substrates due to having a spatially wide subsite + 1. This feature allows the degradation of AGI, AGII, and AGP leading to the fruit ripening. ß-galactosidase (EC 3. 2. 1. 23) catalyzes the hydrolysis of ß-galactan and release of D-galactose. Tomato has at least 17 ß-galactosidases (TBGs), of which, TBG 4 is responsible for fruit ripening. TBG4 hydrolyzes not only ß-1,4-bound galactans, but also ß-1,3- and ß-1,6-galactans. In this study, we compared each enzyme-substrate complex using X-ray crystallography, ensemble refinement, and docking simulation to understand the broad substrate-specificity of TBG4. In subsite - 1, most interactions were conserved across each linkage type of galactobioses; however, some differences were seen in subsite + 1, owing to the huge volume of catalytic pocket. In addition to this, docking simulation indicated TBG4 to possibly have more positive subsites to recognize and hydrolyze longer galactans. Taken together, our results indicated that during tomato fruit ripening, TBG4 plays an important role by degrading arabinogalactan I (AGI), arabinogalactan II (AGII), and the carbohydrate moiety of arabinogalactan protein (AGP).

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.

Characterization of three GH35 ß-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Identification and characterization of ferulic acid esterase from Penicillium chrysogenum 31B: de-esterification of ferulic acid decorated with l-arabinofuranoses and d-galactopyranoses in sugar beet pectin.

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg2+, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-ß-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-ß-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both ß-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.

Anaerobic respiration coupled with mitochondrial fatty acid synthesis in wax ester fermentation by Euglena gracilis.

In Euglena gracilis, wax ester fermentation produces ATP during anaerobiosis. Here, we report that anaerobic wax ester production is suppressed when the mitochondrial electron transport chain complex I is inhibited by rotenone, whereas it is increased by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ADP/ATP ratio in anaerobic cells is elevated by treatment with either rotenone or CCCP. Gene silencing experiments indicate that acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) participate in wax ester production. These results suggest that fatty acids are synthesized in mitochondria by the reversal of ß-oxidation, where trans-2-enoyl-CoA is reduced mainly by acyl-CoA dehydrogenase using the electrons provided by NADH via the electron transport chain complex I, RQ, and ETF, and that ATP production is highly supported by anaerobic respiration utilizing trans-2-enoyl-CoA as a terminal electron acceptor.

Gene cloning, expression, and X-ray crystallographic analysis of a ß-mannanase from Eisenia fetida.

The endo-1,4-ß-mannanases (Ef-Man) gene from Eisenia fetida was determined to consist of 1131 bp and encode a 377 amino acid protein. The amino acid sequence showed similarity with the endo-1,4-ß-mannanases of Daphnia pulex (62%), Cryptopygus antarcticus (64%), Crassostrea gigas (61%), Mytilus edulis (60%), and Aplysia kurodai (58%). The gene encoding mature Ef-Man was expressed in Pichia pastoris (GS115 strain). Based on SDS-PAGE analysis, the molecular mass of the purified recombinant Ef-Man (rEf-Man) was estimated to be 39 kDa. All catalytically important residues of endo-1,4-ß-mannanases in the glycoside hydrolase (GH) family 5 were conserved in Ef-Man. The optimal temperature for rEf-Man was identified as 60 °C. HPLC and HPAEC analyses suggest that Ef-Man requires at least six subsites for efficient hydrolysis and is capable of performing transglycosylation reactions. The overall structure of rEf-Man is similar to those of GH5 family proteins, and tertiary structures around the active site are conserved among endo-1,4-ß-mannanase families. X-ray crystallographic analysis supports the hydrolysis and transglycosylation reaction mechanism determined by HPLC and HPAEC analyses.

Secretion of Legumain Increases in Conditioned Medium from DJ-1-Knockout Cells and in Serum from DJ-1-Knockout Mice.

BACKGROUND: Asparaginyl endopeptidase, also known as legumain (EC 3.4.22.34) shows strong activity in the mouse kidney. Legumain is also highly expressed in tumors. DJ-1/PARK7 is a Parkinson's disease- and cancer-associated protein. DJ-1 is a coactivator of various transcription factors. Recently, we reported that transcription of the legumain gene is regulated by p53 through DJ-1. METHODS: We measured the secretion levels of legumain in a conditioned medium of DJ-1 knockout cells and in serum from DJ-1 knockout mice using Western blotting and ELISA. We performed immunocytochemical staining of legumain to examine the localization of legumain in DJ-1-knockout cells. RESULTS: We found that the secretion levels of legumain were increased in the conditioned medium of DJ-1-knockout cells and in serum from DJ-1-knockout mice. Dot structures of legumain were also increased in DJ-1-knockout cells. CONCLUSION: The results suggest that legumain secretion from DJ-1-knockout cells and in mice increases through its increased expression and accumulation in membrane-associated vesicles.

Identification and characterization of GH62 bacterial α-l-arabinofuranosidase from thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched l-arabinofuranoses in wheat arabinoxylan.

We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity at pH 5.0 and 50 °C, and more than 70% of its initial enzymatic activity remained after incubation at pH 4.1-10.5, while SWUAbf62A lost all activity after 1 h at 60 °C. SWUAbf62A activity was stimulated by Ba2+, Ca2+, and Mn2+ and decreased by Ag+, Cu2+, Fe2+, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-l-arabinofuranoside or p-nitrophenyl-ß-d-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg, and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using 1H-NMR analysis, SWUAbf62A was found to release l-arabinofuranoses singly linked to O-3 of wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase (XynSW3) and α-l-arabinofuranosidase, which releases arabinose linked to O-3 of double-substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and can be applied in various industrial biotechnologies.