Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans.
Hinz, S. W. A., Pastink, M. I., van den Broek, L. A. M., Vincken, J. P. & Voragen, A. G. J. (2005). Applied and Environmental Microbiology, 71(9), 5501-5510.
A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and Vmax for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.
Characterization of the Erwinia chrysanthemi gan locus, involved in galactan catabolism.
Delangle, A., Prouvost, A. F., Cogez, V., Bohin, J. P., Lacroix, J. M. & Cotte-Pattat, N. H. (2007). Journal of Bacteriology, 189(19), 7053-7061.
β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.
Large-scale extraction of rhamnogalacturonan I from industrial potato waste.
Byg, I., Diaz, J., Øgendal, L. H., Harholt, J., Jørgensen, B., Rolin, C., Rolin, C., Svava, R. & Ulvskov, P. (2012). Food Chemistry, 131(4), 1207-1216.
Potato pulp is rich in dietary fibres and is an underutilised material produced in large quantities by the potato starch factories. Potato fibres are especially rich in rhamnogalacturonan I (RG I). RG I is a pectic polysaccharide with a high degree of branching and until now undegraded RG I has only been extracted in small amounts limiting the application possibilities for RG I. The present paper describes a large-scale extraction process providing large quantities of undegraded RG I readily available. The extraction process includes enzymatic starch removal using purified Termamyl, enzymatic RG I solubilisation using a highly purified polygalacturonase, and finally purification using depth filtration and ultrafiltration. The extracted RG I has a high molecular weight and a monosaccharide composition comparable to RG I extracted by analytical extraction procedures. The large amount of RG I available by the presented method allows for thorough structure–function analyses and tailoring of RG I to specific functionalities.
Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus.
Jensen, M. H., Otten, H., Christensen, U., Borchert, T. V., Christensen, L. L. H., Larsen, S. & Leggio, L. L. (2010). Journal of Molecular Biology, 404(1), 100-111.
We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the − 3/+ 3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the β-elimination on [-,2)-α-L-rhamno-(1,4)-α-D-galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.
Simultaneous in vivo truncation of pectic side chains.
Øbro, J., Borkhardt, B., Harholt, J., Skjøt, M., Willats, W. G. T. & Ulvskov, P. (2009). Transgenic Research, 18(6), 961-969.
Despite the wide occurrence of pectin in nature only a few source materials have been used to produce commercial pectins. One of the reasons for this is that many plant species contain pectins with high levels of neutral sugar side chains or that are highly substituted with acetyl or other groups. These modifications often prevent gelation, which has been a major functional requirement of commercial pectins until recently. We have previously shown that modification of pectin is possible through heterologous expression of pectin degrading enzymes in planta. To test the effect of simultaneous modification of the two main neutral pectic side chains in pectic rhamnogalacturonan I (RGI), we constitutively expressed two different enzymes in Arabidopsis thaliana that would either modify the galactan or the arabinan side chains, or both side chains simultaneously. Our analysis showed that the simultaneous truncation of arabinan and galactan side chains is achievable and does not severely affect the growth of Arabidopsis thaliana.
The β-1,4‐endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions.
de Vries, R. P., Pařenicová, L., Hinz, S. W. A., Kester, H. C. M., Beldman, G., Benen, J. A. E. & Visser, J. (2002). European Journal of Biochemistry, 269(20), 4985-4993.
The Aspergillus niger β-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, D-galacturonic acid and L-arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescens β-1,4-endogalactanase. The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of D-galactotriose and D-galactotetraose, whereas prolonged incubation resulted in D-galactose and D-galactobiose, predominantly. MALDI-TOF analysis revealed the release of L-arabinose substituted D-galactooligosaccharides from soy arabinogalactan. This is the first report of the ability of a β-1,4-endogalactanase to release substituted D-galacto-oligosaccharides. GALA was not active towards D-galacto-oligosaccharides that were substituted with D-glucose at the reducing end.
Investigating the binding of β-1, 4‐galactan to Bacillus licheniformis β-1, 4‐galactanase by crystallography and computational modeling.
Le Nours, J., De Maria, L., Welner, D., Jørgensen, C. T., Christensen, L. L. H., Borchert, T. V., Larsen, S. & Lo Leggio, L. (2009). Proteins: Structure, Function, and Bioinformatics, 75(4), 977-989.
Microbial β-1,4-galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH-A of glycoside hydrolases, which cover many different poly- and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformi β-1,4-galactanase and its inactive nucleophile mutant have been obtained with methyl-β(1→4)-galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the β-1,4-galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a β(1→4)-galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite −4 to +5. In particular, this analysis newly identified a conserved β-turn, which contributes to subsites −2 to +3. This β-turn is unique to family 53 β-1,4-galactanases among all clan GH-A families that have been structurally characterized and thus might be a structural signature for endo-β-1,4-galactanase specificity.
The Structure of endo-β-1, 4-galactanase from Bacillus licheniformis in Complex with Two Oligosaccharide Products.
Ryttersgaard, C., Le Nours, J., Lo Leggio, L., Jørgensen, C. T., Christensen, L. L. H., Bjørnvad, M. & Larsen, S. (2004). Journal of Molecular Biology, 341(1), 107-117.
The β-1, 4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of β-1, 4-galactan in the hairy regions of pectin. The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of β-1, 4-galactan. As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (βα)8-barrel architecture. However, BLGAL βα-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously. The structure of BLGAL additionally shows a calcium ion linking the long βα-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases. Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, −3 and −4. A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent. This difference in substrate preference can be explained structurally by the presence of the extra subsites −3 and −4 in BLGAL.
Mining Dictyoglomus turgidum for enzymatically active carbohydrases.
Brumm, P., Hermanson, S., Hochstein, B., Boyum, J., Hermersmann, N., Gowda, K. & Mead, D. (2011). Applied Biochemistry and Biotechnology, 163(2), 205-214.
The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes. Screening a random clone library generated from D. turgidum resulted in the discovery of five novel biomass-degrading enzymes with low homology to known molecules. Whole genome sequencing of the organism followed by bioinformatics-directed amplification of selected genes resulted in the recovery of seven additional novel enzyme molecules. Based on the analysis of the genome, D. turgidumdoes not appear to degrade cellulose using either conventional soluble enzymes or a cellulosomal degradation system. The types and quantities of glycosyl hydrolases and carbohydrate-binding modules present in the genome suggest that D. turgidum degrades cellulose via a mechanism similar to that used by Cytophaga hutchinsonii and Fibrobacter succinogenes.
Expression cloning in Kluyveromyces lactis.
van der Vlugt-Bergmans, C. J. B. & van Ooyen, A. J. J. (1999). Biotechnology Techniques, 13(1), 87-92.
Kluyveromyces lactis was used as host for an Aspergillus tubingensis expression library. A new episomal vector was constructed to direct the expression of the A. tubingensis cDNAs and to allow subsequent analysis in Escherichia coli. Using three different plate assays, 18000 K. lactis recombinants were screened, yielding 60 galactanase-, 26 polygalacturonase- and 16 cellulase-secreting colonies. The galactanase-secreting recombinants were analysed in detail: they are transcripts of the same galactanase gene with similarity to an A. aculeatus β-1,4-galactanase gene. The results of the K. lactis system compare favourably to those obtained by Saccharomyces cerevisiae.
Structural and functional characterization of a novel family GH115 4-O-methyl-α-glucuronidase with specificity for decorated arabinogalactans.
Aalbers, F., Turkenburg, J. P., Davies, G. J., Dijkhuizen, L. & van Bueren, A. L. (2015). Journal of Molecular Biology, 427(24), 3935-3946.
Glycoside hydrolases are clustered into families based on amino acid sequence similarities, and belonging to a particular family can infer biological activity of an enzyme. Family GH115 contains α-glucuronidases where several members have been shown to hydrolyze terminal α-1,2-linked glucuronic acid and 4-O-methylated glucuronic acid from the plant cell wall polysaccharide glucuronoxylan. Other GH115 enzymes show no activity on glucuronoxylan, and therefore, it has been proposed that family GH115 may be a poly-specific family. In this study, we reveal that a putative periplasmic GH115 from the human gut symbiont Bacteroides thetaiotaomicron, BtGH115A, hydrolyzes terminal 4-O-methyl-glucuronic acid residues from decorated arabinogalactan isolated from acacia tree. The three-dimensional structure of BtGH115A reveals that BtGH115A has the same domain architecture as the other structurally characterized member of this family, BoAgu115A; however the position of the C-terminal module is altered with respect to each individual enzyme. Phylogenetic analysis of GH115 amino sequences divides the family into distinct clades that may distinguish different substrate specificities. Finally, we show that BtGH115A α-glucuronidase activity is necessary for the sequential digestion of branched galactans from acacia gum by a galactan-β-1,3-galactosidase from family GH43; however, while B. thetaiotaomicron grows on larch wood arabinogalactan, the bacterium is not able to metabolize acacia gum arabinogalactan, suggesting that BtGH115A is involved in degradation of arabinogalactan fragments liberated by other microbial species in the gastrointestinal tract.
Effect of mutations on the thermostability of Aspergillus aculeatus β-1,4-galactanase.
Torpenholt, S., De Maria, L., Olsson, M. H., Christensen, L. H., Skjøt, M., Westh, P., Jensen, J. H. & Leggio, L. L. (2015). Computational and Structural Biotechnology Journal, 13, 256-264.
New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions were experimentally validated by measurements of the melting temperature (Tm) by differential scanning calorimetry. We found that the Tm is elevated by 1.1°C for G306A, slightly increased (in the range of 0.34 to 0.65°C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability. Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also investigated for the wild-type and three variants and found to be biphasic. The half-lives of thermal inactivation were approximately doubled in G306A, unchanged for D182N and, disappointingly, a lot lower for D58N. In conclusion, this study tests a new method for estimating Tm changes for mutants, adds to the available data on the effect of substitutions on protein thermostability and identifies an interesting thermostabilizing mutation, which may be beneficial also in other galactanases.
Aspergillus hancockii sp. nov., a biosynthetically talented fungus endemic to southeastern Australian soils.
Pitt, J. I., Lange, L., Lacey, A. E., Vuong, D., Midgley, D. J., Greenfield, P., Bradbury, M. I., Lacey, E., Busk, P. K., Pilgaard, B., Chooi, Y. H. & Piggott, A. M. (2017). PloS One, 12(4), e0170254.
Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.
Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter.
Huang, Y., Yi, Z., Jin, Y., Huang, M., He, K., Liu, D., Luo, H., Zhao, D., He, H., Fang, Y. & Zhao, H. (2017). Frontiers in Microbiology, 8, 1747.
Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.