Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-β-D-galactanase.
Sørensen, S. O., Pauly, M., Bush, M., Skjøt, M., McCann, M. C., Borkhardt, B. & Ulvskov, P. (2000). Proceedings of the National Academy of Sciences, 97(13), 7639-7644.
Potato tuber pectin is rich in galactan (oligomer of β-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.
Expression and characterization of an endo-1,4-β-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans.
Michalak, M., Thomassen, L. V., Roytio, H., Ouwehand, A. C., Meyer, A. S. & Mikkelsen, J. D. (2012). Enzyme and Microbial Technology, 50(2), 121-129.
Potato pulp is a high-volume side-stream from industrial potato starch manufacturing. Enzymatically solubilized β-1,4-galactan-rich potato pulp polysaccharides of molecular weights >100 kDa (SPPP) are highly bifidogenic in human fecal sample fermentations in vitro. The objective of the present study was to use potato β-1,4-galactan and the SPPP as substrates for enzymatic production of potentially prebiotic compounds of lower and narrower molecular weight. A novel endo-1,4-β-galactanase from Emericella nidulans (anamorph Aspergillus nidulans), GH family 53, was produced in a recombinant Pichia pastoris strain. The enzyme was purified by Cu2+ affinity chromatography and its optimal reaction conditions were determined to pH 5 and 49°C via a statistical experimental design. The specific activity of the E. nidulans enzyme expressed in P. pastoris was similar to that of an endo-1,4-β-galactanase from Aspergillus niger used as benchmark. The E. nidulans enzyme expressed in P. pastoris generated a spectrum poly- and oligo-saccharides which were fractionated by membrane filtration. The potential growth promoting properties of each fraction were evaluated by growth of beneficial gut microbes and pathogenic bacteria. All the galactan- and SPPP-derived products promoted the growth of probiotic strains of Bifidobacterium longum and Lactobacillus acidophilus and generally did not support the propagation of Clostridium perfringens in single culture fermentations. Notably the growth of B. longum was significantly higher (p<0.05) or at least as good on galactan- and SPPP-derived products as fructooligosaccharides (FOS). Except in one case these products did not support the growth of the pathogen Cl. perfringens to any significant extent.
Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged.
Zykwinska, A., Thibault, J. F. & Ralet, M. C. (2007). Journal of Experimental Botany, 58(7), 1795-1802.
The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called ‘natural’ composites), rich in pectic neutral sugar side chains and cellulose, as well as ‘artificial’ composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.
Solubilization of galactosyltransferase that synthesizes 1,4‐β-galactan side chains in pectic rhamnogalacturonan I.
Geshi, N., Pauly, M. & Ulvskov, P. (2002). Physiologia Plantarum, 114(4), 540-548.
β-1,4-Galactan galactosyltransferase (GT) activity was solubilized from potato microsomal membranes in the presence of 78 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. The solubilized GT activity transferred 14[C]galactose from UDP-14[C]galactose onto the acceptor-substrates composed of rhamnogalacturonan (RG) with short galactan chains (RG-A, approximately 1.2 MDa, mol% Gal/Rha = 0.7; RG-B, approximately 21 kDa, mol% Gal/Rha = 1.2). However, shorter RG containing short galactan chains (approximately 2 kDa and 1.2 kDa), RG oligomers without galactosyl-residues, galactan, and galactooligomers did not act as acceptor-substrates. Optimal pH for 14[C] incorporation onto RG-A and RG-B was around 5.6 and 7.5, respectively. The 14[C]-labelled products synthesized upon RG-A and RG-B could be digested with a RG specific lyase into smaller RG fragments. 1,4-β-Endogalactanase could not digest the former product, whereas the latter product was digested to 14[C]galactobiose and 14[C]galactose. This demonstrates that at least two GT activities were solubilized from potato microsomal membranes. One had optimal pH around 5.6 to transfer galactosyl residues onto RG-A, whereas the other had optimal pH around 7.5 to transfer galactosyl residues onto RG-B. Both synthesized galactan attached to the RG backbone of RG-A and RG-B, and the galactan synthesized onto the RG-B acceptor was 1,4-β-linked.
In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I.
Geshi, N., Jørgensen, B., Scheller, H. V. & Ulvskov, P. (2000). Planta, 210(4), 622-629.
The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[14C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [14C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [14C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the 14C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [14C]galactose and [14C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn2+. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [14C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [14C]galactose or [14C]galactobiose but also as larger fragments. The larger fragments were likely the [14C]galactose or [14C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [14C]galactose. The data indicate that the majority of the [14C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I.
A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A.
Boraston, A. B, Tomme, P., Amandoron, E. A. & Kilburn, D. G. (2000). Biochem. J, 350, 933-941.
The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of 1×102 M-1–1×103 M-1. It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2±0.6)×103/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the α, β and γ sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only ≈ (0.5±0.1)×103/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail.
Characterization of the bga1-encoded glycoside hydrolase family 35 β-galactosidase of Hypocrea jecorina with galacto-β-D-galactanase activity.
Gamauf, C., Marchetti, M., Kallio, J., Puranen, T., Vehmaanperä, J., Allmaier, G., Kubicek, C. P. & Seiboth, B. (2007). FEBS Journal, 274(7), 1691-1700.
The extracellular bga1-encoded β-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-β-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-β-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is 5.0, and the optimum temperature was found to be 60°C. Bga1 is also capable of releasing D-galactose from β-galactans and is thus actually a galacto-β-D-galactanase. β-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.
Pectin biosynthesis: GALS1 in Arabidopsis thaliana is a β-1,4-galactan β-1,4-galactosyltransferase.
Liwanag, A. J. M., Ebert, B., Verhertbruggen, Y., Rennie, E. A., Rautengarten, C., Oikawa, A., Andersen, M. C. F., Clausen, M. H. & Scheller, H. V. (2012). The Plant Cell, 24(12), 5024-5036.
β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.
Glycation of lysozyme with galactose, galactooligosaccharides and potato galactan through the Maillard reaction and optimization of the production of prebiotic glycoproteins.
Seo, S., Karboune, S., Yaylayan, V. & L’Hocine, L. (2012). Process Biochemistry, 47(2), 297-304.
The production of glycated lysozyme (LZM), with galactose, galactooligosaccharides (GOSs) and potato galactan through the Maillard reaction, was investigated. The percent blocked lysine, estimated from the furosine content, reached a maximum value of 11.2% for LZM:galactan conjugates after 1 day incubation at a aw of 0.65. A maximum percent blocked lysine of 7.0 and 13.5% were obtained for LZM:galactose/GOS conjugates at a lower aw of 0.45 after 3 and 7 days, respectively. However, the low percent blocked lysine and the high protein aggregation index of LZM:galactose/GOS conjugates at aw 0.79 and 0.65 revealed the prevalence of the degradation of the Amadori compounds and the protein cross-linking. Mass spectrometry of LZM conjugates revealed the formation of different glycoforms. Glycated LZMs containing up to seven galactose moieties were formed; while only mono- and diglycated LZMs with GOSs were detected. 2–3 mol of galactan were conjugated to 1 mol of LZM. Response surface methodology, based on a 5-level and 3-factor central composite design, revealed that molar ratio and temperature were the most significant variables for the glycation of LZM with GOSs. The optimal conditions leading to a high percent blocked lysine (16.11%) with a low protein aggregation index (0.11) were identified: temperature of 49.5°C, LZM:GOS molar ratio of 1:9 and aw of 0.65. To the best of our knowledge, this is the first study on the optimization of LZM glycation with GOSs.
Subcellular localization and topology of β(1→4) galactosyltransferase that elongates β(1→4) galactan side chains in rhamnogalacturonan I in potato.
Geshi, N., Jørgensen, B. & Ulvskov, P. (2004). Planta, 218(5), 862-868.
The subcellular localization and topology of rhamnogalacturonan I (RG-I) β(1→4)galactosyltransferase(s) (β[1→4]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[14C]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180–2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and β(1,4) endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires β(1→4)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing β(1→4)galactans producing RG-I side chains in total longer than a trimer. Thus, the β(1→4)GalT activity that elongates β(1→4)-linked galactan on RG-I was located in the Golgi apparatus. This β(1→4)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the β(1→4)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I β(1→4)GalT that elongates β(1→4)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.