A trapped covalent intermediate as a key catalytic element in the hydrolysis of a GH3 ß-glucosidase: An X-ray crystallographic and biochemical study.

Glycoside hydrolases (GHs) are industrially important enzymes that hydrolyze glycosidic bonds in glycoconjugates. In this study, we found a GH3 ß-glucosidase (CcBgl3B) from Cellulosimicrobium cellulans sp. 21 was able to selectively hydrolyze the ß-1,6-glucosidic bond linked glucose of ginsenosides. X-ray crystallographic studies of the ligand complex ginsenoside-specific ß-glucosidase provided a novel finding that support the catalytic mechanism of GH3. The substrate was clearly identified within the catalytic center of wild-type CcBgl3B, revealing that the C1 atom of the glucose was covalently bound to the Oδ1 group of the conserved catalytic nucleophile Asp264 as an enzyme-glycosyl intermediate. The glycosylated Asp264 could be identified by mass spectrometry. Through site-directed mutagenesis studies with Asp264, it was found that the covalent intermediate state formed by Asp264 and the substrate was critical for catalysis. In addition, Glu525 variants (E525A, E525Q and E525D) showed no or marginal activity against pNPßGlc; thus, this residue could supply a proton for the reaction. Overall, our study provides an insight into the catalytic mechanism of the GH3 enzyme CcBgl3B.

Ginseng-derived type I rhamnogalacturonan polysaccharide binds to galectin-8 and antagonizes its function.

Background: Panax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear. Methods: P. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis. Results: Our ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its ß-1,4-galactan side chains, with sub-micromolar KD values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function. Conclusion: P. ginseng RG-I pectin ß-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

Isomerization of proline-46 in the N-terminal tail of galectin-3 enhances T cell apoptosis via the ROS-ERK pathway.

Galectin-3 (Gal-3) is unique in the galectin family, due to the presence of a long N-terminal tail (NT) arising from its conserved carbohydrate recognition domain (CRD). Although functional significance of the NT has remained elusive, our previous studies demonstrated the importance of NT prolines to Gal-3 function. Here, we show that during the time Gal-3 stands in solution for three or more days, Gal-3 NT undergoes a slow, intra-molecular, time-dependent conformational/dynamical change associated with proline cis-trans isomerization. From initial dissolution of Gal-3 in buffer to three days in solution, Gal-3-mediated T cell apoptosis is enhanced from 23 % to 37 %. Western blotting and flow cytometry show that the enhancement occurs via the ROS-ERK pathway, and not by the PKC-ERK pathway. To assess which proline(s) is (are) responsible for this effect, we individually mutated all 14 NT prolines within the first 68 residues to alanines, and assessed their effect on ROS production. Our study shows that isomerization of P46 alone is responsible for the upregulation of ROS and T cell apoptosis. NMR studies show that this unique effect is mediated by a change in dynamic interactions between the NT and CRD F-face, which in turn leads to this change in Gal-3 function.

Preparation of ß-galacto-oligosaccharides using a novel endo-1,4-ß-galactanase from Penicillium oxalicum.

Endo-1,4-ß-galactanase is an indispensable tool for preparing prebiotic ß-galacto-oligosaccharides (ß-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-ß-galactanase (PoßGal53) belonging to glycoside hydrolase family 53 from Penicillium oxalicum sp. 68 was cloned and expressed in Pichia pastoris GS115. Upon purification by affinity chromatography, recombinant PoßGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoßGal53 showed optimal reaction conditions of pH 4.0, 40 °C, and was thermostable, retaining >80 % activity after incubating below 45 °C for 12 h. Significantly, PoßGal53 exhibited relatively conserved substrate specificity for (1 â†’ 4)-ß-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-ß-galactanase identified to date. By using PoßGal53, ß-GOS monomers were prepared from potato galactan and separated using medium pressure liquid chromatography. HPAEC-PAD, MALDI-TOF-MS and ESI-MS/MS analyses demonstrated that these ß-GOS species ranged from 1,4-ß-D-galactobiose to 1,4-ß-D-galactooctaose (DP 2-8) with high purity. This work provides not only a highly active tool for enzymatic degradation of pectic galactan, but an efficient protocol for preparing ß-GOS.

Effect of anti-skin disorders of ginsenosides- A Systematic Review.

Ginsenosides are bioactive components of Panax ginseng with many functions such as anti-aging, anti-oxidation, anti-inflammatory, anti-fatigue, and anti-tumor. Ginsenosides are categorized into dammarane, oleanene, and ocotillol type tricyclic triterpenoids based on the aglycon structure. Based on the sugar moiety linked to C-3, C-20, and C-6, C-20, dammarane type was divided into protopanaxadiol (PPD) and protopanaxatriol (PPT). The effects of ginsenosides on skin disorders are noteworthy. They play anti-aging roles by enhancing immune function, resisting melanin formation, inhibiting oxidation, and elevating the concentration of collagen and hyaluronic acid. Thus, ginsenosides have previously been widely used to resist skin diseases and aging. This review details the role of ginsenosides in the anti-skin aging process from mechanisms and experimental research.

Preparation and structural analysis of fucomannogalactan and ß-1,6-glucan from Grifola frondosa mycelium.

Introduction: Polysaccharides, key components present in Grifola frondosa, can be divided into those derived from fruiting bodies, mycelium, and fermentation broth based on their source. The structure of G. frondosa fruiting body-derived polysaccharides has been fully characterized. However, the structure of G. frondosa mycelium-derived polysaccharides remains to be elucidated. Methods: In this study, we obtained mycelia from G. frondosa by liquid fermentation and extracted them with water and alkaline solution. Then, the mycelia were isolated and purified to obtain homogeneity and systematically characterized by methylation and FT infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. Results and discussion: Structural analysis showed that two neutral fractions (WGFP-N-a and AGFP-N-a1) have a common backbone composed of α-1,6-D-Me-Galp and α-1,6-D-Galp that were substituted at O-2 by 1,2-Manp, α-1,3-L-Fucp, and α-T-D-Manp and thus are identified as fucomannogalactans. WGFP-A-a, AGFP-A-b, and AGFP-A-c are ß-1,6-glucans with different molecular weights and are branched with ß-1,3-D-Glcp and T-D-Glcp at the O-3 of Glc. Our results provide important structural information about G. frondosa mycelium-derived polysaccharides and provide the basis for their further development and application.

Schisandra chinensis (Turcz.) Baill. polysaccharide inhibits influenza A virus in vitro and in vivo.

Influenza virus is prone to seasonal spread and widespread outbreaks, which pose important challenges to public health security. Therefore, it is important to effectively prevent and treat influenza virus infection. Schisandra polysaccharide (SPJ) is a polysaccharide derived from the fruit of Schisandra chinensis (Turcz.) Baill. In this study, we evaluated the antiviral activity of SPJ in vitro and in vivo, especially against influenza A virus (IAV) infection. By analyzing SPJ structure and monosaccharide composition, the molecular weight of SPJ was determined to be 115.5 KD, and it is composed of galacturonic acid (89.4%), rhamnose (0.8%), galactose (4.4%), arabinose (3.8%), and glucose (1.7%). Immunofluorescence analysis showed that SPJ treatment reduced the positive rate of viral nucleoproteins in cells, indicating that the compound had an inhibitory effect on influenza virus replication. Furthermore, SPJ therapy improved the survival of infected mice. Lung virus titer assays indicated that SPJ treatment significantly reduced viral loading in the lung tissue of infected mice and alleviated the pathological damage caused by influenza virus infection. Moreover, SPJ reduced cytokine expression during influenza virus challenge. In conclusion, SPJ has anti-influenza virus effects and may have potential as an anti-influenza drug candidate in further clinical studies.

An efficient protocol for preparing linear ß-manno-oligosaccharides.

Linear ß-manno-oligosaccharides (l-ß-MOS) are widely used to investigate oligo- and poly-saccharide structures and mannanolytic enzyme activities. l-ß-MOS are also being used as prebiotic agents with potential bio-active properties. In this study, we developed an efficient protocol to prepare a series of l-ß-MOS by hydrolyzing cassia gum (CG) using mannanolytic enzymes (endo-1,4-ß-mannanase, α-galactosidases and ß-glucosidases). By using medium pressure liquid chromatography (MPLC), we purified l-ß-MOS with different degrees of polymerization (DPs). HPAEC-PAD, MALDI-TOF-MS and NMR studies confirmed that these l-ß-MOS species ranged from 1,4-ß-d-mannobiose to 1,4-ß-d-mannononaose (DP 2-9) with >95% purity. Our results provide a robust approach to preparing l-ß-MOS, thus enabling l-ß-MOS to be further used in the fields of chemistry, life science, and nutritional food.

Structural characterization and antioxidant activity of pectic polysaccharides from Veronica peregrina L.

Background: Pectins are a class of acidic polysaccharides with complex structures. Different pectin molecules are composed of different domains, which have an important impact on their biological activity. Objective: This study aimed to determine the structural features and the antioxidant activities of the pectic polysaccharides isolated from Veronica peregrina L. Methods: The polysaccharide was isolated from Veronica peregrina L by water extraction and fractionated by ion exchange chromatography and gel permeation chromatography. The structure features of the pectic polysaccharides were determined by Fourier transforminfrared spectroscopy (FT-IR) and Nuclear magnetic resonance (NMR). The antioxidant activities was evaluated by the DPPH, OH and ABTS radical scavenging ability. Results: WVPP-A2b and WVPP-A3b, with molecular weights of 48.7 × 104 and 77.6 × 104 kDa, respectively, contained homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) domains with a mass ratio of 2.08:2.64:1.00 and 3.87:4.65:1:00, respectively. The RG-I domain contained an arabinogalactan II backbone and arabinans consisting of t-Araf, (1→5)-α-Araf, and (1→3,5)-α-Araf. WVPP-A3b also contained short chains consisting of the [t-Araf-(1→5)-α-Araf-(1→] structural unit. WVPP-A3b showed stronger ability to scavenge DPPH, hydroxyl, and ABTS radicals, which was potentially associated with its high content of galacturonic acid and presence of the HG domain. Conclusion: The results provide information for enhancing knowledge of the structureactivity relationship of pectic polysaccharides from V. peregrina and their potential application in the healthcare food field.

The interrelation of galectins and autophagy.

Autophagy is a vital physiological process that maintains intracellular homeostasis by removing damaged organelles and senescent or misfolded molecules. However, excessive autophagy results in cell death and apoptosis, which will lead to a variety of diseases. Galectins are a type of animal lectin that binds to ß-galactosides and can bind to the cell surface or extracellular matrix glycans, affecting a variety of immune processes in vivo and being linked to the development of many diseases. In many cases, galectins and autophagy both play important regulatory roles in the cellular life course, yet our understanding of the relationship between them is still incomplete. Galectins and autophagy may share common etiological cofactors for some diseases. Hence, we summarize the relationship between galectins and autophagy, aiming to draw attention to the existence of multiple associations between galectins and autophagy in a variety of physiological and pathological processes, which provide new ideas for etiological diagnosis, drug development, and therapeutic targets for related diseases.

Purification and identification of oligosaccharides from Cimicifuga heracleifolia Kom. rhizomes.

Even though Cimicifuga sp. is widely used in functional foods around the world, the content and structure of its oligosaccharides remain unclear. Here, we isolated a mixture of oligosaccharides from Cimicifuga heracleifolia Kom. rhizomes with a yield of 9.5% w/w. Twenty-six oligosaccharide monomers from the mixture were purified using optimized SEC and HILIC techniques. The oligosaccharides were identified as belonging to two groups by using HPAEC-PAD, MALDI-TOF-MS, NMR and GC-MS methylation analyses. One group belongs to sucrose and inulin type fructo-oligosaccharides (FOS) {ß-d-Fruf-(2 â†’ 1)-[ß-d-Fruf-(2 ↔ 1)]n=1-12-α-d-Glcp} with a 3-14 degree of polymerization (DP). Oligosaccharides in the other group belong to the inulo-n-ose type FOS {ß-d-Fruf-(2 â†’ 1)-[ß-d-Fruf-(2 â†’ 1)]m=0-12-ß-d-Frup} with a DP of 2-14. This appears to be the first time that these oligosaccharides have been purified from Cimicifuga heracleifolia Kom., thus providing useful information concerning the utilization of Cimicifuga heracleifolia Kom. in functional foods.

The model polysaccharide potato galactan is actually a mixture of different polysaccharides.

Commercially-supplied potato galactan (PG) is widely used as a model polysaccharide in various bioactivity studies. However, results using this galactan are not always consistent with the stated composition. Here, we assessed its composition by fractionating this commercial PG and purified its primary components: PG-A, PG-B and PG-Cp with weight-averaged molecular weights of 430, 93, and 11.3 kDa, respectively. PG-Cp consists of free ß-1,4-galactan chains, whereas PG-A and PG-B are type I rhamnogalacturonans with long ß-1,4-galactan side chains of up to 80 Gal residues and short ß-1,4-galactan side chains of 0 to 3 Gal residues that display a "trees in lawn" pattern. Structures of these polysaccharides correlate well with their activities in terms of galectin-3 binding and gut bacterial growth assays. Our study clarifies the confusion related to commercial PG, with purified fractions serving as better model polysaccharides in bioactivity investigations.

MHC class I links with severe pathogenicity in C57BL/6N mice infected with SARS-CoV-2/BMA8.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes non-symptomatic infection, mild influenza-like symptoms to pneumonia, severe acute respiratory distress syndrome, and even death, reflecting different clinical symptoms of viral infection. However, the mechanism of its pathogenicity remains unclear. Host-specific traits have a breakthrough significance for studying the pathogenicity of SARS-CoV-2. We previously reported SARS-CoV-2/BMA8, a mouse-adapted strain, was lethal to aged BALB/c mice but not to aged C57BL/6N mice. Here, we further investigate the differences in pathogenicity of BMA8 strain against wild-type aged C57BL/6N and BALB/c mice. METHODS: Whole blood and tissues were collected from mice before and after BMA8 strain infection. Viral replication and infectivity were assessed by detection of viral RNA copies and viral titers; the degree of inflammation in mice was tested by whole blood cell count, ELISA and RT-qPCR assays; the pathogenicity of SARS-CoV-2/BMA8 in mice was measured by Histopathology and Immunohistochemistry; and the immune level of mice was evaluated by flow cytometry to detect the number of CD8+ T cells. RESULTS: Our results suggest that SARS-CoV-2/BMA8 strain caused lower pathogenicity and inflammation level in C57BL/6N mice than in BALB/c mice. Interestingly, BALB/c mice whose MHC class I haplotype is H-2Kd showed more severe pathogenicity after infection with BMA8 strain, while blockade of H-2Kb in C57BL/6N mice was also able to cause this phenomenon. Furthermore, H-2Kb inhibition increased the expression of cytokines/chemokines and accelerated the decrease of CD8+ T cells caused by SARS-CoV-2/BMA8 infection. CONCLUSIONS: Taken together, our work shows that host MHC molecules play a crucial role in the pathogenicity differences of SARS-CoV-2/BMA8 infection. This provides a more profound insight into the pathogenesis of SARS-CoV-2, and contributes enlightenment and guidance for controlling the virus spread.

A ginseng-derived rhamnogalacturonan I (RG-I) pectin promotes longevity via TOR signalling in Caenorhabditis elegans.

Panax ginseng C. A. Meyer (ginseng), a traditional Chinese herb, is usually used to improve health and increase anti-aging activity for human. Polysaccharides are bioactive components of ginseng. Herein, using Caenorhabditis elegans as a model, we discovered a ginseng-derived rhamnogalacturonan I (RG-I) pectin WGPA-1-RG promoted longevity via TOR signalling pathway with transcription factors FOXO/DAF-16 and Nrf2/SKN-1 accumulated in the nucleus, where they activated target genes. And the WGPA-1-RG-mediated lifespan extension was dependent on endocytosis, rather than a bacterial metabolic process. Glycosidic linkage analyses combined with arabinose- and galactose-releasing enzyme hydrolyses identified the RG-I backbone of WGPA-1-RG was primarily substituted with α-1,5-linked arabinan, ß-1,4-linked galactan and arabinogalactan II (AG-II) side chains. Feeding worms with the WGPA-1-RG-derived fractions which lost distinct structural elements by enzymatic digestions, we found the arabinan side chains prominently contributed to the longevity-promoting activity of WGPA-1-RG. These findings provide a novel ginseng-derived nutrient that potentially increases human longevity.

Taurolidine improved protection against highly pathogenetic avian influenza H5N1 virus lethal-infection in mouse model by regulating the NF-κB signaling pathway.

Taurolidine (TRD), a derivative of taurine, has anti-bacterial and anti-tumor effects by chemically reacting with cell-walls, endotoxins and exotoxins to inhibit the adhesion of microorganisms. However, its application in antiviral therapy is seldom reported. Here, we reported that TRD significantly inhibited the replication of influenza virus H5N1 in MDCK cells with the half-maximal inhibitory concentration (EC50) of 34.45 â€‹µg/mL. Furthermore, the drug inhibited the amplification of the cytokine storm effect and improved the survival rate of mice lethal challenged with H5N1 (protection rate was 86%). Moreover, TRD attenuated virus-induced lung damage and reduced virus titers in mice lungs. Administration of TRD reduced the number of neutrophils and increased the number of lymphocytes in the blood of H5N1 virus-infected mice. Importantly, the drug regulated the NF-κB signaling pathway by inhibiting the separation of NF-κB and IκBa, thereby reducing the expression of inflammatory factors. In conclusion, our findings suggested that TRD could act as a potential anti-influenza drug candidate in further clinical studies.

Heterogalactan WPEP-N-b from Pleurotus eryngii enhances immunity in immunocompromised mice.

This study reports on in vivo immunomodulatory activities mediated by WPEP-N-b, a heterogalactan from Pleurotus eryngii. Using cyclophosphamide (CTX)-induced immunosuppressed mice, we demonstrate here that WPEP-N-b enhances immunity as determined by the immune organ index, peripheral blood immune cell content, splenocyte proliferation, NK cell activity and T lymphocyte subpopulations. WPEP-N-b prevented apoptosis of bone marrow cells induced by CTX. The level of cytokines (i.e. TNF-α, IL-6 and IL-1ß) and macrophage activity in these immunocompromised mice were restored upon treated with WPEP-N-b. Mechanistically, it appears that WPEP-N-b enhances splenocyte proliferation and NK cell activity might through the Toll-like receptor 4 (TLR4)-PKC signaling axis, and increases macrophage activity by activating JNK, p38 and NF-κB signaling pathways and Toll-like receptor 2 (TLR2) is the possible receptor of WPEP-N-b in macrophages. Our findings indicate that WPEP-N-b may function as a natural immune stimulant.

Structural characterization and anti-oxidation activity evaluation of pectin from Lonicera japonica Thunb.

Pectins are nutrient components of plants and are widely used in the food industry. In this study, one major pectin fraction (WLJP-A0.2b) with Mw of 40.6 kDa was purified from Lonicera japonica Thunb. The structural feature and antioxidant activity of it was investigated. Monosaccharide composition, Fourier transform infrared (FT-IR) spectra, enzymatic hydrolysis, and nuclear magnetic resonance (NMR) spectra analysis indicated that WLJP-A0.2b consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and homogalacturonan (HG) domains, with mass ratio of 0.4:1.0:2.1. The RG-I domain contained highly branched α-L-1,5-arabinan, ß-D-1,4-galactan and type II arabinogalactan (AG-II) side chains. The HG domain was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with degree of polymerization 1-8 after degradation by endo-polygalacturonase. Radical scavenging assays indicated that WLJP-A0.2b exhibited antioxidant activity through the synergistic effects of different pectin domains. Oligogalacturonides, especially de-esterified oligogalacturonides, showed better antioxidant activities than RG-II and RG-I domains. Moreover, de-esterified oligogalacturonides remarkably reduced H2O2-induced reactive oxygen species production in HEK-293T cells. These results provide useful information for screening of natural antioxidants from Lonicera japonica Thunb. and application of pectin in functional food field.

Dual crosslinking of folic acid-modified pectin nanoparticles for enhanced oral insulin delivery.

Pectin-based drug delivery systems hold great potential for oral insulin delivery, since they possess excellent gelling property, good mucoadhesion and high stability in the gastrointestinal (GI) tract. However, lack of enterocyte targeting ability and premature drug release in the upper GI tract of the susceptible ionic-crosslinked pectin matrices are two major problems to be solved. To address these issues, we developed folic acid (FA)-modified pectin nanoparticles (INS/DFAN) as insulin delivery vehicles by a dual-crosslinking method using calcium ions and adipic dihydrazide (ADH) as crosslinkers. In vitro studies indicated insulin release behaviors of INS/DFAN depended on COOH/ADH molar ratio in the dual-crosslinking process. INS/DFAN effectively prevented premature insulin release in simulated GI fluids compared to ionic-crosslinked nanoparticles (INS/FAN). At an optimized COOH/ADH molar ratio, INS/DFAN with FA graft ratio of 18.2% exhibited a relatively small particle size, high encapsulation efficiency and excellent stability. Cellular uptake of INS/DFAN was FA graft ratio dependent when it was at/below 18.2%. Uptake mechanism and intestinal distribution studies demonstrated the enhanced insulin transepithelial transport by INS/DFAN via FA carrier-mediated transport pathway. In vivo studies revealed that orally-administered INS/DFAN produced a significant reduction in blood glucose levels and further improved insulin bioavailability in type I diabetic rats compared to INS/FAN. Taken together, the combination of dual crosslinking and FA modification is an effective strategy to develop pectin nano-vehicles for enhanced oral insulin delivery.

Determining Methyl-Esterification Patterns in Plant-Derived Homogalacturonan Pectins.

Homogalacturonan (HG)-type pectins are nutrient components in plants and are widely used in the food industry. The methyl-esterification pattern is a crucial structural parameter used to assess HG pectins in terms of their nutraceutical activity. To better understand the methyl-esterification pattern of natural HG pectins from different plants, we purified twenty HG pectin-rich fractions from twelve plants and classified them by their monosaccharide composition, Fourier transform-infrared spectroscopy (FT-IR) signatures, and NMR analysis. FT-IR shows that these HG pectins are all minimally esterified, with the degree of methyl-esterification (DM) being 5 to 40%. To examine their methyl-esterification pattern by enzymatic fingerprinting, we hydrolyzed the HG pectins using endo-polygalacturonase. Hydrolyzed oligomers were derivatized with 2-aminobenzamide and subjected to liquid chromatography-fluorescence-tandem mass spectrometry (HILIC-FLR-MSn). Twenty-one types of mono-/oligo-galacturonides having DP values of 1-10 were found to contain nonesterified monomers, dimers, and trimers, as well as oligomers with 1 to 6 methyl-ester groups. In these oligo-galacturonides, MSn analysis demonstrated that the number of methyl-ester groups in the continuous sequence was 2 to 5. Mono- and di-esterified oligomers had higher percentages in total methyl-esterified groups, suggesting that these are a random methyl-esterification pattern in these HG pectins. Our study analyzes the characteristics of the methyl-esterification pattern in naturally occurring plant-derived HG pectins and findings that will be useful for further studying HG structure-function relationships.

Cloning, Expression, and Characterization of Endo-ß-1,6-galactanase PoGal30 from Penicillium oxalicum.

Because ß-1,6-galactans are significant components in arabinogalactans from plant cell walls, identifying selective endo-ß-1,6-galactanases is crucial to degrading these polysaccharides and to analyzing and modifying their structures. Here, we cloned and expressed in E. coli a novel endo-ß-1,6-galactanase in the glycosidic hydrolase family 30 (GH30) from Penicillium oxalicum. Our recombinant PoGal30 hydrolase (1464 bp gene) that contains an N-terminal His-tag for purification by nickel affinity chromatography has a specific activity of 3.8 U/mg on the substrate de-arabinosylated gum Arabic (dGA) polysaccharide. The enzyme has 487 residues with a molecular mass of 60 kDa, an isoelectric point of 6, and functional pH and temperature optima of pH 2.5 to pH 5.0 and 40 °C, respectively. While the activity of PoGal30 is activated by Mg2+ (5 or 50 mmol/L), it is completely inhibited by Cu2+ and Fe3+ (50 mmol/L) and partially inhibited by Hg2+, EDTA, and SDS (50 mmol/L). The enzyme demonstrates high specificity towards ß-1,6-galactosidic linkages in dGA, but is inactive against aryl-glycosides and galactobioses with different linkages. Using PoGal30 is, therefore, an effective approach to analyzing the fine structure of polysaccharides and preparing bioactive oligosaccharides.