Purification and Properties of Endo-<i>β</i>-1,4-<scp>D</scp>-galactanase from<i>Aspergillus niger</i>

Yamaguchi, Fumihide; Inoue, Shinichi; Hatanaka, Chitoshi

doi:10.1271/bbb.59.1742

Cited by 20 publications

(6 citation statements)

References 2 publications

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“…This suggests that the putative N‐glycosylation site identified in the sequence is indeed functional. The determined molecular mass and pH optimum (pH 4–4.5) of GALA are similar to those reported previously for Aspergillus β‐1,4‐endogalactanases [10,12,36–40].…”

Section: Discussionsupporting

confidence: 86%

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

Vries¹,

Pařenicová²,

Hinz³

et al. 2002

European Journal of Biochemistry

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The Aspergillus niger b-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 b-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 b-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. Genes encoding b-1,4-endogalactanase have been cloned from both bacteria and fungi [11][12][13][14][15]. Based on their derived amino acid sequence, the corresponding enzymes have been assigned to family 53 of the glycosyl hydrolases [16]. These enzymes have a retaining mechanism and for the Pseudomonas fluorescens b-1,4-endogalactanase the catalytic residues have been determined [11]. Recently, the Aspergillus aculeatus b-1,4-endogalactanase has been expressed in vivo in potato resulting in a 30% reduction of the galactosyl content of the pectin fraction of the cell walls [17].In this paper we describe the cloning, characterization and expression analysis of the A. niger galA gene, encoding b-1,4-endogalactanase. This is the first paper that describes the expression of this gene in detail and that compares the activity of the gene product, GALA, against arabinogalactans with a different degree of L-arabinose substitution.

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Section: Discussionsupporting

confidence: 86%

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

Vries¹,

Pařenicová²,

Hinz³

et al. 2002

European Journal of Biochemistry

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“…A comparable reaction may also be occurring in some fungi. In comparison to bacterial galactanases, most fungal galactanases (5,11,39,45,52,68) yield ␤-1-4-galactobiose and some fungi have ␤-galactosidases with reasonable K m values (4.5 to 19.4 mM) on galactobiose (46). The association between GHF 53 and GHF 2 genes in a few bacteria also supports possible GHF 53-GHF 2 synergy.…”

Section: Vol 72 2006 Functions For Ghf 42 ␤-Galactosidases 7735mentioning

confidence: 85%

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Shipkowski

Brenchley

2006

Appl Environ Microbiol

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Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the ␤-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 ␤-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 ␤-galactosidases. Similar functions for other ␤-galactosidases in both GHFs 2 and 42 are suggested by genomic data.␤-Galactosidases, such as the LacZ ␤-galactosidase of Escherichia coli, are used as molecular biology tools, industrial enzymes, and models for exploring structure-function relationships. However, these uses do not often yield insight into the physiological roles of these enzymes. Although ␤-galactosidases (EC 3.2.1.23) are also found in glycoside hydrolase families (GHFs) 1, 2, and 35 (groups differing in secondary structure and other attributes [20,21]), we were especially curious about the in vivo function of GHF 42 ␤-galactosidases because they occur in organisms isolated from diverse habitats where lactose would not be present, e.g., hot springs (33, 47, 65), hypersaline environments (27, 54), and soil (Bacillus and Streptomyces spp.).Over a dozen GHF 42 enzymes have been characterized, and most are ␤-galactosidases (as shown by maximal activity with the) with less than 10% relative activity on nongalactosidic chromogens (with two exceptions [27,34]). However, experimental data supporting lactose hydrolysis by GHF 42 ␤-galactosidases are absent (34, 44, 48) or weak. Several prokaryotes possessing a GHF 42 gene are unable to grow on lactose as a sole carbon source (1, 9, 27), and at least two GHF 42 ␤-galactosidases do not cleave lactose in vitro (27,64). The determination of growth on lactose can be complicated by the presence of multiple ␤-galactosidases (7,16,25) because not all of the ␤-galactosidases may be participating equally (or at all) as...

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“…b-D-Galactanase (GNase, speciˆc for b-1,4-galactan) was puriˆed according to the method of Yamaguchi et al 14) The puriˆed AFase and ANase samples had no other hemicellulase activity and could respectively hydrolyze a-1,3-and a-1,5-arabinan to give the arabinose monomer, and a-1,5-arabinan to give arabinooligosaccharide. 15,16) a-L-Rhamnosidase (RPase) and rhamnogalacturonase (RGase) were puriˆed from the commercial enzyme, Pectinex Ultra SP-L, from Aspergillus aculeatus (kindly presented by Novo Nordisk A W S, Denmark) by the method of Mutter et al 17) and Schols et al 18) Among the four kinds of RPases found in the commercial enzyme preparation, an enzyme of pI 5.4 could only hydrolyze rhamnose residues, which were present at the non-reducing end of RG and linked to galacturonic acid, to give the rhamnose monomer.…”

Section: )mentioning

confidence: 99%

Structural Studies by Stepwise Enzymatic Degradation of the Main Backbone of Soybean Soluble Polysaccharides Consisting of Galacturonan and Rhamnogalacturonan

Nakamura¹,

Furuta²,

Maeda³

et al. 2002

Bioscience, Biotechnology, and Biochemistry

154

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Purification and Properties of Endo-β-1,4-D-galactanase fromAspergillus niger

Cited by 20 publications

References 2 publications

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

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

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Structural Studies by Stepwise Enzymatic Degradation of the Main Backbone of Soybean Soluble Polysaccharides Consisting of Galacturonan and Rhamnogalacturonan

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