Pyrolysis gas-chromatography mass-spectrometry (Py-GC/MS) to identify compression wood in Pinus radiata saplings.
Brennan, M., McLean, J. P., Klingberg, A., Altaner, C. & Harris, P. J. (2014). Holzforschung, 68(5), 505-517.
The potential of pyrolysis followed by gas-chromatography and mass-spectrometry (Py-GC/MS) was investigated for identifying compression wood (CW) in saplings of radiata pine (Pinus radiata) by examining samples of CW and opposite wood (OW). Phenolic compounds and anhydrosugars were identified among the pyrolysis products that provided information about the cell-wall polymers. Sample preparation, such as coarse-milling, fine-milling, and fine-milling followed by calcium-chloride treatment was also investigated. Fine-milling typically decreased the total yield of phenolic compounds compared with coarse-milling. Fine-milling followed by calcium-chloride washing significantly increased the proportions of pyrolysis products from polysaccharides, specifically from (1→4)-β-D-galactans that were of interest in distinguishing CW from OW. Six pyrolysis products were identified that were unique to the CW samples examined, including derivatives of (1→4)-β-D-galactans and H-units of lignin. Other pyrolysis products were identified that had significantly different proportions between the two wood types, and sometimes among samples of the same wood type.
Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 β-galactosidases are arabinogalactan type I oligomer hydrolases.
Shipkowski, S. & Brenchley, J. E. (2006). Applied and Environmental Microbiology, 72(12), 7730-7738.
Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.
A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases.
Park, Y. B. & Cosgrove, D. J. (2012). Plant Physiology, 158(4), 1933-1943.
Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this “tethered network” model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.
Purification and characterization of Aspergillus β-D-galactanases acting on β-1,4- and β-1,3/6-linked arabinogalactans.
Luonteri, E., Laine, C., Uusitalo, S., Teleman, A., Siika-aho, M. & Tenkanen, M. (2003). Carbohydrate Polymers, 53(2), 155-168.
Arabinogalactan and arabinan fractions were isolated from kraft pulping black liquor. Both type I and type II arabinogalactans consisting of 1,4- and 1,3-linked β-D-galactose backbones, respectively, were found. Samples contained more arabino-1,3/6-galactan than arabino-1,4-galactan. Arabinan was mainly 1,5-linked slightly branched polysaccharide. Two enzymes acting on galactans, an endo-β-1,4-D-galactanase and a β-1,6-D-galactanase, were isolated from commercial pectinase preparations produced by Aspergillus aculeatus and A. niger, respectively. The purified enzymes showed molecular masses of 38 and 58 kDa, respectively. Based on its N-terminal amino acid sequence the endo-β-1,4-D-galactanase was the same as the previously studied GAL1 from A. aculeatus. It acted on β-1,4-linked galactan, producing a range of galacto-oligosaccharides. It was also able to liberate galactose from a lignin–carbohydrate complex isolated from softwood kraft pulp. No activity was detected towards β-1,3-liked galactan. The β-1,6-D-galactanase was active on arabino-1,3/6-galactan, liberating galactose and 1,6-β-D-galactobiose. It was found to be active only on β-1,6-linkages and no detectable hydrolysis of β-1,3-galactose linkages occurred. It also showed no activity on 1,4-β-D-galactan. However, β-1,6-D-galactanase was able to liberate arabinose from arabinan. Although chemical pulps contain only a minute quantity of galactans, both galactanases have recently been shown to enhance the bleachability of spruce kraft pulp.
Molecular cloning of endo-β-D-1,4-glucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae.
Moriya, T., Murashima, K., Nakane, A., Yanai, K., Sumida, N., Koga, J., Murakami, T. & Kono, T. (2003). Journal of Bacteriology, 185(5), 1749-1756.
Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.
Localization of pectic galactan in tomato cell walls using a monoclonal antibody specific to (1→4)-β-D-galactan.
Jones, L., Seymour, G. B. & Knox, J. P. (1997). Plant Physiology, 113(4), 1405-1412.
To develop antibody probes for the neutral side chains of pectins, antisera were generated to a pectic galactan isolated from tomato (Lycopersicon esculentum) pericarp cell walls and to a (1→4)-β-galactotetraose-bovine serum albumin neoglycoprotein. The use of these two antisera in immunochemical assays and immunolocalization studies indicated that they had very similar specificities. A monoclonal antibody (LM5) was isolated and characterized subsequent to immunization with the neoglycoprotein. Hapten inhibition studies revealed that the antibody specifically recognized more than three contiguous units of (1→4)-β-galactosyl residues. The antigalactan antibody was used to immunolocalize the galactan side chains of pectin in tomato fruit pericarp and tomato petiole cell walls. Although the LM5 epitope occurs in most cell walls of the tomato fruit, it was absent from both the locular gel and the epidermal and subepidermal cells. Furthermore, in contrast to other anti-pectin antibodies, LM5 did not label the cell wall thickenings of tomato petiole collenchyma.
Expression cloning, purification and characterization of a β-1,4-glactanase from Aspergillus aculeatus.
Christgau, S., Sandal, T., Kofod, L. V. & Dalbøge, H. (1995). Current Genetics, 27(2), 135-141.
Expression cloning has been used to isolate a cDNA encoding β-1,4-galactanase from the filamentous fungus Aspergillus aculeatus. A cDNA library was prepared from mycelia, inserted in a yeast expression vector and transformed into Saccharomyces cerevisiae. Thirteen clones secreting galactanase activity were identified from a screening of approximately 2.5×104 yeast colonies. All clones expressed transcripts of the same galactanase gene. The cDNA was re-cloned in an Aspergillus expression vector and transformed into Aspergillus oryzae. The recombinant enzyme had a molecular weight of 44 000 Da, an isoelectric point of pH 2.85, a pH optimum of pH 4.0–4.5, and a temperature optimum of 45–65°C, which is similar to values obtained for a β-1,4-galactanase purified from A. aculeatus. The enzyme degraded unsubstituted galactan to galactose and galactobiose. The deduced primary sequence of the enzyme showed no apparent homology to any known enzyme, in accordance with this being the first reported β-1,4-galactanase cDNA. However, the deduced aminoacid sequence of a Bacillus circulans DNA sequence containing an open reading frame (ORF) with no known function, showed 36% identity and 60% similarity to the galactanase amino-acid sequence.
Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893.
Ichinose, H., Kotake, T., Tsumuraya, Y. & Kaneko, S. (2008). Applied and Environmental Microbiology, 74(8), 2379-2383.
The putative endo-β-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote.
Family 6 carbohydrate‐binding modules display multiple β1,3‐linked glucan‐specific binding interfaces.
Correia, M. A. S., Pires, V. M. R., Gilbert, H. J., Bolam, D. N., Fernandes, V. O., Alves, V. D., Prates, J. A. M., Ferreira, L. M. A. & Fontes, C. M. G. (2009). FEMS Microbiology Letters, 300(1), 48-57.
Noncatalytic carbohydrate-binding modules (CBMs), which are found in a variety of carbohydrate-degrading enzymes, have been grouped into sequence-based families. CBMs, by recruiting their appended enzymes onto the surface of the target substrate, potentiate catalysis particularly against insoluble substrates. Family 6 CBMs (CBM6s) display unusual properties in that they present two potential ligand-binding sites termed clefts A and B, respectively. Cleft B is located on the concave surface of the β-sandwich fold while cleft A, the more common binding site, is formed by the loops that connect the inner and the outer β-sheets. Here, we report the biochemical properties of CBM6-1 from Cellvibrio mixtus CmCel5A. The data reveal that CBM6-1 specifically recognizes β1,3-glucans through residues located both in cleft A and in cleft B. In contrast, a previous report showed that a CBM6 derived from a Bacillus halodurans laminarinase binds to β1,3-glucans only in cleft A. These studies reveal a different mechanism by which a highly conserved protein platform can recognize β1,3-glucans.
Cellulose microfibril angles and cell-wall polymers in different wood types of Pinus radiate.
Brennan, M., McLean, J. P., Altaner, C. M., Ralph, J. & Harris, P. J. (2012). Cellulose, 19(4), 1385-1404.
Four corewood types were examined from sapling trees of two clones of Pinus radiata grown in a glasshouse. Trees were grown either straight to produce normal corewood, tilted at 45° from the vertical to produce opposite corewood and compression corewood, or rocked to produce flexure corewood. Mean cellulose microfibril angle of tracheid walls was estimated by X-ray diffraction and longitudinal swelling measured between an oven dry and moisture saturated state. Lignin and acetyl contents of the woods were measured and the monosaccharide compositions of the cell-wall polysaccharides determined. Finely milled wood was analysed using solution-state 2D NMR spectroscopy of gels from finely milled wood in DMSO-d6/pyridine-d5. Although there was no significant difference in cellulose microfibril angle among the corewood types, compression corewood had the highest longitudinal swelling. A lignin content >32% and a galactosyl residue content >6% clearly divided severe compression corewood from the other corewood types. Relationships could be drawn between lignin content and longitudinal swelling, and between galactosyl residue content and longitudinal swelling. The 2D NMR spectra showed that the presence of H-units in lignin was exclusive to compression corewood, which also had a higher (1→4)-β-D-galactan content, defining a unique composition for that corewood type.
A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain.
Maruta, A., Yamane, M., Matsubara, M., Suzuki, S., Nakazawa, M., Ueda, M. & Sakamoto, T. (2017). Enzyme and Microbial Technology, 103, 25-33.
We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with α-D-galactopyranosidase (GPase) and β-L-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from α-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with α-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of α-galactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and α-L-arabinofuranosidase released four times more arabinose than α-L-arabinofuranosidase acting alone. This suggested that a large number of α-L-arabinofuranosyl residues is capped by α-galactosyl residues. 1H NMR experiments revealed that rFoGP1 hydrolyzed the α-1,3-galactosidic linkage within the side chain structure of [α-D-Galp-(1 → 3)-α-L-Araf-(1 → ] in gum arabic. In conclusion, rFoGP1 is highly active toward α-1,3-galactosyl linkages but negligibly or not active toward α-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent.
Heterologous expression and characterization of an Arabidopsis &beta-L-arabinopyranosidase and &alpha-D-galactosidases acting on &beta-L-arabinopyranosyl residues.
Imaizumi, C., Tomatsu, H., Kitazawa, K., Yoshimi, Y., Shibano, S., Kikuchi, K., Yamaguchi, M., Kaneko, S., Tsumuraya, Y. & Kotake, T. (2017). Journal of Experimental Botany, 68(16), 4651-4661.
The major plant sugar L-arabinose (L-Ara) has two different ring forms, L-arabinofuranose (L-Araf) and L-arabinopyranose (L-Arap). Although L-Ara mainly appears in the form of α-L-Araf residues in cell wall components, such as pectic α-1,3:1,5-arabinan, arabinoxylan, and arabinogalactan-proteins (AGPs), lesser amounts of it can also be found as β-L-Araf residues of AGPs. Even though AGPs are known to be rapidly metabolized, the enzymes acting on the β-L-Araf residues remain to be identified. In the present study, four enzymes, which we call β-L-ARAPASE (APSE) and α-GALACTOSIDASE 1 (AGAL1), AGAL2, and AGAL3, are identified as those enzymes that are likely to be responsible for the hydrolysis of the β-L-Araf residues in Arabidopsis thaliana. An Arabidopsis apse-1 mutant showed significant reduction in β-L-arabinopyranosidase activity, and an apse-1 agal3-1 double-mutant exhibited even less activity. The apse-1 and the double-mutants both had more β-L-Araf residues in the cell walls than wild-type plants. Recombinant APSE expressed in the yeast Pichia pastoris specifically hydrolyzed β-L-Araf residues and released L-Ara from gum arabic and larch arabinogalactan. The recombinant AGAL3 also showed weak β-L-arabinopyranosidase activity beside its strong α-galactosidase activity. It appears that the β-L-Araf residues of AGPs are hydrolysed mainly by APSE and partially by AGALs in Arabidopsis.