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.

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.

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).

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.

Ferulic acid and its water-soluble derivatives inhibit nitric oxide production and inducible nitric oxide synthase expression in rat primary astrocytes.

We recently reported that two water-soluble derivatives of ferulic acid (1-feruloyl glycerol, 1-feruloyl diglycerol) previously developed by our group exhibited protective effects against amyloid-ß-induced neurodegeneration in vitro and in vivo. In the current study, we aimed to further understand this process by examining the derivatives' ability to suppress abnormal activation of astrocytes, the key event of neurodegeneration. We investigated the effects of ferulic acid (FA) derivatives on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The results showed that these compounds inhibited NO production and iNOS expression in a concentration-dependent manner and that the mechanism underlying these effects was the suppression of the nuclear factor-κB pathway. This evidence suggests that FA and its derivatives may be effective neuroprotective agents and could be useful in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions.

In Euglena gracilis, pyruvate:NADP+ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP+ oxidoreductase. In contrast, pyruvate:NADP+ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.

Purification, characterization, and overexpression of an endo-1,4-ß-mannanase from thermotolerant Bacillus sp. SWU60.

Endo-ß-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the ß-1,4-linkage in the mannan backbone and release short ß-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60 °C, respectively. The enzyme was stable up to 60 °C for 1 h and at pH 5-9 at 4 °C for 16 h. Its enzyme activity was inhibited by Hg2+. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.

Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages.

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2-C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

Water-soluble ferulic acid derivatives improve amyloid-ß-induced neuronal cell death and dysmnesia through inhibition of amyloid-ß aggregation.

Ferulic acid (FA) has been reported to exhibit protective effects against amyloid-ß (Aß)-induced neurodegeneration in vitro and in vivo. Recently, we developed two water-soluble FA derivatives: 1-feruloyl glycerol and 1-feruloyl diglycerol. In this study, we examined the neuroprotective effects of these water-soluble FA derivatives on Aß-induced neurodegeneration both in vitro and in vivo. FA and water-soluble FA derivatives inhibited Aß aggregation and destabilized pre-aggregated Aß to a similar extent. Furthermore, water-soluble FA derivatives, as well as FA, inhibited Aß-induced neuronal cell death in cultured neuronal cells. In in vivo experiments, oral administration of water-soluble FA derivatives to mice improved Aß-induced dysmnesia assessed by contextual fear conditioning test and protected hippocampal neurons against Aß-induced neurotoxicity. This study provides useful evidence suggesting that water-soluble FA derivatives are expected to be effective neuroprotective agents.

Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

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.

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.

The preventive effect of ß-carotene on denervation-induced soleus muscle atrophy in mice.

Muscle atrophy increases the production of reactive oxygen species and the expression of atrophy-related genes, which are involved in the ubiquitin-proteasome system. In the present study, we investigated the effects of ß-carotene on oxidative stress (100 µM-H2O2)-induced muscle atrophy in murine C2C12 myotubes. ß-Carotene (10 µM) restored the H2O2-induced decreased levels of myosin heavy chain and tropomyosin (P< 0·05, n 3) and decreased the H2O2-induced increased levels of ubiquitin conjugates. ß-Carotene reduced the H2O2-induced increased expression levels of E3 ubiquitin ligases (Atrogin-1 and MuRF1) and deubiquitinating enzymes (USP14 and USP19) (P< 0·05, n 3) and attenuated the H2O2-induced nuclear localisation of FOXO3a. Furthermore, we determined the effects of ß-carotene on denervation-induced muscle atrophy. Male ddY mice (8 weeks old, n 30) were divided into two groups and orally pre-administered micelle with or without ß-carotene (0·5 mg once daily) for 2 weeks, followed by denervation in the right hindlimb. ß-Carotene was further administered once daily until the end of the experiment. At day 3 after denervation, the ratio of soleus muscle mass in the denervated leg to that in the sham leg was significantly higher in ß-carotene-administered mice than in control vehicle-administered ones (P< 0·05, n 5). In the denervated soleus muscle, ß-carotene administration significantly decreased the expression levels of Atrogin-1, MuRF1, USP14 and USP19 (P< 0·05, n 5) and the levels of ubiquitin conjugates. These results indicate that ß-carotene attenuates soleus muscle loss, perhaps by repressing the expressions of Atrogin-1, MuRF1, USP14 and USP19, at the early stage of soleus muscle atrophy.

Peculiarities and applications of galactanolytic enzymes that act on type I and II arabinogalactans.

Arabinogalactans (AGs) are branched galactans to which arabinose residues are bound as side chains and are widely distributed in plant cell walls. They can be grouped into two types based on the structures of their backbones. Type I AGs have ß-1,4-galactan backbones and are often covalently linked to the rhamnogalacturonan-I region of pectins. Type II AGs have ß-1,3-galactan backbones and are often covalently linked to proteins. The main enzymes involved in the degradation of AGs are endo-ß-galactanases, exo-ß-galactanases, and ß-galactosidases, although other enzymes such as α-L-arabinofuranosidases, ß-L-arabinopyranosidases, and ß-D-glucuronidases are required to remove the side chains for efficient degradation of the polysaccharides. Galactanolytic enzymes have a wide variety of potential uses, including the bioconversion of AGs to fermentable sugars for production of commodity chemicals like ethanol, biobleaching of cellulose pulp, modulation of pectin properties, improving animal feed, and determining the chemical structure of AGs. This review summarizes our current knowledge about the biochemical properties and potential applications of AG-degrading enzymes.

Identification of an exo-ß-1,3-D-galactanase from Fusarium oxysporum and the synergistic effect with related enzymes on degradation of type II arabinogalactan.

An exo-ß-1,3-D-galactanase (Fo/1,3Gal) was purified from the culture filtrate of Fusarium oxysporum 12S. A cDNA encoding Fo/1,3Gal was isolated by in vitro cloning. Module sequence analysis revealed a "GH43_6" domain and a "CBM35_galactosidase-like" domain in Fo/1,3Gal. The recombinant enzyme (rFo/1,3Gal) expressed in Pichia pastoris degraded ß-1,3-galactan and ß-1,3-galactobiose (Gal2), and released only galactose (Gal). In contrast, the enzyme did not hydrolyze p-nitrophenyl ß-D-galactopyranoside, ß-1,4-Gal2, or ß-1,6-Gal2. The enzyme also showed low activity towards native type II arabinogalactans such as larchwood arabinogalactan (LWAG) and gum arabic. Using LWAG as substrate, rFo/1,3Gal released Gal, ß-1,6-Gal2, ß-1,6-galactotriose (Gal3), and ß-1,6-Gal3 substituted with a single arabinofuranose residue accompanied with unidentified oligosaccharides, indicating that the enzyme can by-pass the branching points of ß-1,3-galactan backbones. A time course analysis of products released by rFo/1,3Gal on LWAG revealed that ß-1,6-Gal2 is the main side chain in LWAG and that the activity of rFo/1,3Gal was decreased when degrees of polymerization of side chains increase. rFo/1,3Gal worked synergistically with three other recombinant F. oxysporum enzymes (ß-1,6-galactanase, ß-L-arabinopyranosidase, and α-L-arabinofuranosidase) that degrade side chains, on the degradation of LWAG. However, the synergism was much lower than anticipated, probably because LWAG have longer side chains than the three enzymes used are able to remove or ß-1,3-galactan main chain is interrupted with glycosidic linkages that are different from the ß-1,3-galactosyl linkage. Affinity gel electrophoresis revealed that rFo/1,3Gal specifically bound to ß-1,3-galactan.

Biochemical characterization of a GH53 endo-ß-1,4-galactanase and a GH35 exo-ß-1,4-galactanase from Penicillium chrysogenum.

An endo-ß-1,4-galactanase (PcGAL1) and an exo-ß-1,4-galactanase (PcGALX35C) were purified from the culture filtrate of Penicillium chrysogenum 31B. Pcgal1 and Pcgalx35C cDNAs encoding PcGAL1 and PcGALX35C were isolated by in vitro cloning. The deduced amino acid sequences of PcGAL1 and PcGALX35C are highly similar to a putative endo-ß-1,4-galactanase of Aspergillus terreus (70% amino acid identity) and a putative ß-galactosidase of Neosartorya fischeri (72%), respectively. Pfam analysis revealed a "Glyco_hydro_53" domain in PcGAL1. PcGALX35C is composed of five distinct domains including "Glyco_hydro_35," "BetaGal_dom2," "BetaGal_dom3," and two "BetaGal_dom4_5" domains. Recombinant enzymes (rPcGAL1 and rPcGALX35C) expressed in Escherichia coli and Pichia pastoris, respectively, were active against lupin galactan. The reaction products of lupin galactan revealed that rPcGAL1 cleaved the substrate in an endo manner. The enzyme accumulated galactose and galactobiose as the main products. The smallest substrate for rPcGAL1 was ß-1,4-galactotriose. On the other hand, rPcGALX35C released only galactose from lupin galactan throughout the reaction, indicating that it is an exo-ß-1,4-galactanase. rPcGALX35C was active on both ß-1,4-galactobiose and triose, but not on lactose, ß-1,3- or ß-1,6-galactooligosaccharides even after 24 h of incubation. To our knowledge, this is the first report of a gene encoding a microbial exo-ß-1,4-galactanase. rPcGAL1 and rPcGALX35C acted synergistically in the degradation of lupin galactan and soybean arabinogalactan. Lupin galactan was almost completely degraded to galactose by the combined actions of rPcGAL1 and rPcGALX35C. Surprisingly, neither rPcGAL1 nor rPcGALX35C released any galactose from sugar beet pectin.

Substrate specificity and gene expression of two Penicillium chrysogenum α-L-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54.

We previously isolated two α-L-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L-arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.

Synthesis of highly water-soluble feruloyl diglycerols by esterification of an Aspergillus niger feruloyl esterase.

Ferulic acid (FA) is a component of plant cell walls that has applications in food, cosmetic, and health products, but its applications are limited by its high insolubility. We synthesized water-soluble FA derivatives by esterification of FA with diglycerol (DG) using feruloyl esterase purified from a commercial enzyme preparation produced by Aspergillus niger. The major reaction product, FA-DG1, was determined to be γ-feruloyl-α,α'-DG by NMR and electrospray ionization mass spectrometry analyses. FA-DG1 is a sticky liquid whose water solubility (>980 mg/ml) is dramatically higher than that of FA (0.69 mg/ml). Suitable conditions for esterification of FA with DG were 100 mg of FA in the presence of 1 g of DG and 0.1 ml of 1 M phosphate buffer (pH 6.0) at 50 °C under reduced pressure. Under these conditions, 168 mg of feruloyl DGs (FA-DG1, 2, and 3) was obtained, corresponding to a 95 % conversion rate of FA. We also developed a batch method which resulted in synthesis of 729 mg of feruloyl DGs and 168 mg of diferuloyl DGs from 600 mg of FA and 1 g of DG (corresponding to conversion of 69 % of the FA to feruloyl DGs and 21 % of the FA to diferuloyl DGs). As an anti-oxidant, feruloyl DGs were essentially equal to FA and butyl hydroxytoluene in scavenging 1,1-diphenyl-2-picrylhydrazyl radicals. In contrast, the scavenging abilities of diferuloyl DGs were twice those of feruloyl DGs.

Biochemical characterization and gene expression of two endo-arabinanases from Penicillium chrysogenum 31B.

We previously described five arabinanolytic enzymes secreted by Penicillium chrysogenum 31B into the culture medium. Here, we describe a sixth such enzyme, termed AbnS1. Analysis of the reaction products of debranched arabinan revealed that AbnS1 cleaved the substrate in an endo manner. The optimum temperature of AbnS1 was 60°C, which was much higher than that of a cold-adapted endo-arabinanase (Abnc) produced by this strain. The abns1 cDNA gene encoding AbnS1 was isolated by in vitro cloning. The deduced amino acid sequence of AbnS1 had 70% identity with that of Abnc. Pfam analysis revealed a Glyco_hydro_43 domain at positions 28 to 318 of AbnS1. Semi-quantitative reverse transcription-polymerase chain reaction analysis indicated that the abns1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was only slightly induced with arabinose and arabinan. In contrast, expression of the abnc gene encoding Abnc was strongly induced by arabinose, arabinitol, and arabinan. Using debranched arabinan as substrate, recombinant AbnS1 (rAbnS1) accumulated arabinobiose and arabinotriose as the major products. Recombinant Abnc (rAbnc) released mainly arabinotriose and lesser amounts of arabinose and arabinobiose than did rAbnS1. Branched arabinan was completely degraded to arabinose by the action of rAbnS1 or rAbnc in combination with α-L: -arabinofuranosidase.

Induction of apoptosis in MCF-7 cells by ß-1,3-xylooligosaccharides prepared from Caulerpa lentillifera.

ß-1,3-Xylan was prepared from the green alga, Caulerpa lentillifera, and hydrolyzed to oligosaccharides by a mild acid treatment. The average degree of polymerization was about 5. The oligosaccharides reduced the number of viable human breast cancer MCF-7 cells in a dose-dependent manner, and induced chromatin condensation and degradation of poly ADP-ribose polymerase, indicating that they induced apoptosis in MCF-7 cells.