Benen1and Jaap Visser1§ 1 Molecular Genetics of Industrial Microorganisms and2Food Chemistry, Wageningen University, Wageningen, The Netherlands The Aspergillus niger b-1,4-endogalactana
Trang 1The b-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
Ronald P de Vries1†, Lucie Parˇenicova´1‡, Sandra W A Hinz2, Harry C M Kester1, Gerrit Beldman2, Jacques A E Benen1and Jaap Visser1§
1
Molecular Genetics of Industrial Microorganisms and2Food Chemistry, Wageningen University, Wageningen, The Netherlands
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 andL-arabinose, suggesting that
galAis coregulated with both the pectinolytic genes as well as
the arabinanolytic genes The corresponding enzyme,
endogalactanase A (GALA), contains both active site
resi-dues 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
ara-binogalactans of potato, onion and soy with GALA resulted
initially in the release ofD-galactotriose andD -galactotetra-ose, whereas prolonged incubation resulted in D-galactose andD-galactobiose, predominantly MALDI-TOF analysis revealed the release of L-arabinose substituted D -galacto-oligosaccharides 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 towardsD-galacto-oligosaccharides that were substi-tuted withD-glucose at the reducing end
Keywords: Aspergillus niger; b-1,4-endogalactanase; galac-turonic acid; expression; galactan degradation
Endogalactanases are involved in the degradation of plant
cell wall polysaccharides, in particular pectin Two types of
arabinogalactan side chains are present in pectin Type I
consists of a chain of b-1,4 linked D-galactopyranose
linkages, while type II contains a backbone of b-1,3-linked
D-galactopyranose residues that can be substituted with
b-1,6-linkedD-galactopyranose residues [1] Both types can
be substituted with a)1-,3-linkedL-arabinofuranose chains
Type I arabinogalactan is degraded by
b-1,4-endogalacta-nase and b-galactosidase b-1,4-Endogalactab-1,4-endogalacta-nases cleave
within the galactan moiety of type I arabinogalactan,
releasing D-galacto-oligosaccharides Bacterial
b-1,4-endo-galactanases release mainly galactotriose and
galactotetra-ose [2–6], while some also release galactobigalactotetra-ose [3,5,7] Eukaryotic b-1,4-endogalactanases release predominantly galactobiose and galactose from galactan [8–10]
Genes encoding b-1,4-endogalactanase have been cloned from both bacteria and fungi [11–15] 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 Pseudo-monas fluorescens b-1,4-endogalactanase the catalytic resi-dues have been determined [11] Recently, the Aspergillus aculeatusb-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 arabinogalac-tans with a different degree ofL-arabinose substitution
M A T E R I A L S A N D M E T H O D S Strains and growth conditions
All A niger strains were derived from A niger N400 (CBS 120.49) and are described in Table 1 Escherichia coli DH5aF¢ was used for routine plasmid propagation E coli LE392 was used as a host for phage kEMBL3 Subcloning was performed using pBluescript SK+[18] and pGEM-T (Promega, Madison, WI, USA) The genomic library of
A nigerhas been described previously [19]
Minimal medium and complete medium were descri-bed before [20] Liquid cultures were inoculated with
Correspondence to R P de Vries, Microbiology, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands.
Fax: +31 302513655, Tel.: +31 302533016,
E-mail: r.p.devries@bio.uu.nl
Abbreviations: CREA, carbon catabolite repressor protein; HPAEC,
high performance anion exchange chromatography; GALA, the
A niger b-1,4-endogalactanase; galA, gene encoding the A niger
b-1,4-endogalactanase; Galp, galactopyranose; Glcp, glucopyranose;
LACA, A niger a-galactosidase; PACC, pH regulatory protein;
TOS, transgalactooligosaccharides.
Present address: Microbiology, Utrecht University, Padualaan 8,
3584 CH Utrecht, The Netherlands.
Present address: Dipartimento di Biologia, Universita degli studi di
Milano, Via Celoria 26, 20133 Milano, Italy.
§Present address: FGT Consultancy, PO Box 396, 6700 AJ
Wagenin-gen, The Netherlands.
(Received 27 March 2002, revised 1 July 2002,
accepted 21 August 2002)
Trang 2106 sporesÆmL)1, and incubated at 30C in an orbital
shaker at 250 r.p.m For the growth of strains with
auxotrophic mutations, the necessary supplements were
added to the medium
Transfer experiments were performed by pregrowing the
strains for 16 h in complete medium containing 2% (w/v)
fructose as carbon source, after which the mycelium was
harvested and washed with minimal medium without
carbon source Aliquots (1.5 g) of wet mycelium were then
transferred to 50 mLminimal medium containing carbon
sources as indicated in the text After 4 h of incubation in a
rotary shaker at 250 r.p.m and 30C mycelium was
harvested, dried between tissue paper, frozen in liquid
nitrogen and stored at)70 C
For purification of the b-1,4-endogalactanase, A niger
transformant NW290:: pIM3982.77 was grown in minimal
medium supplemented with 0.003% (w/v) yeast extract and
3% (w/v) fructose The pH was adjusted to 3.8 Cultivation
was performed at 30C in a 5-litre jacketed stirred tank
reactor (Applikon) Air-saturated culture medium was
inoculated with 106 spores per litre The spores were
allowed to germinate for 6 h at low stirring speed
(400 r.p.m) after which the cultivation was continued for
18 h at 750 r.p.m Culture pH was maintained at 3.8 by the
addition of a 5-Msodium hydroxide solution The cultures
were sparged with air (2 v.v.m) and 0.5 mL30% (v/v)
polypropylene glycol in ethanol was added per litre of
medium as antifoam agent
Materials
D-Xylose, D-glucose, D-fructose, D-galactose, D-mannose,
and lactose were obtained from Merck (Darmstadt,
Germany) D-Glucuronic and D-galacturonic acid were
from Fluka (Buchs, Switzerland) Mellibiose, raffinose,
stachyose, L-arabinose, gum arabic, gum karaya, locust
bean gum, and beechwood xylan were from Sigma (St Louis,
Mo.) Potato pectic galactan was from Megazyme
Interna-tional (Bray, Ireland) Taq polymerase was from Gibco BRL
(Breda, The Netherlands) All other standard chemicals
were either obtained from Sigma or Merck Potato
arabi-nogalactan and onion arabiarabi-nogalactan were obtained as
described previously (Fractions F44) [21] Soy
arabinoga-lactan was kindly provided by NOVO Nordisk (Dittingen,
Switzerland) Sugar compositions were determined as
des-cribed [21] Borculo Whey Products (Borculo, The
Nether-lands) kindly provided transgalactooligosaccharides (TOS)
PCR cloning of a specific fragment ofgalA
Two oligonucleotides were designed based on the sequence
of the A aculeatus b-1,4-endogalactanase encoding gene
(5¢-CTCTTCTCTCTTGCTCTTG-3¢ and 5¢-GTTCGTCT
CCACAACCAC-3¢, respectively) and used in PCRs under the following conditions: 1 min denaturing at 95C, 1 min annealing at 50C and 2 min amplification at 72 C, 30 cycles Chromosomal DNA of A niger N402 was used as a template This resulted in a fragment of approximately
700 bp which was cloned in pGEM-T easy (Promega) Sequence analysis was performed as described below Cloning and characterization ofgalA
Plaque hybridizations were performed as previously des-cribed [22] Hybridizations were performed overnight at
65C by using the galA PCR fragment as a probe Filters were washed to 0.2· NaCl/Cit (1 · NaCl/Cit is 0.15M
NaCl plus 0.015M Na3-citrate, pH 7.6)-0.5% (mass/vol) SDS Positive plaques, identified on duplicate replicas after autoradiography, were recovered from the original plates and purified by rescreening at low plaque density Standard methods were used for other DNA manipulations, such as Southern analysis, subcloning, DNA digestions, and lambda phage and plasmid DNA isolations [23] Chromosomal DNA was isolated as previously described [24] Sequence analysis was performed on both strands of DNA by using the Cy5 AutoCycle Sequencing kit (Pharmacia Biotech, Uppsala, Sweden) The reactions were analyzed with an ALFred DNA Sequencer (Pharmacia Biotech) Nucleotide sequences were analyzed with computer programs based on those of Devereux et al [25] RT-PCRs were performed using the Enhanced Avian RT-PCR kit (Sigma) according
to the suppliers instructions using galA-specific oligonucleo-tides (5¢-GATGATCTACCCTCTGCTTC-3¢ and 5¢-GTC ACGGACGGACTGGGT-3¢) Northern analysis was per-formed as described previously [20] Per sample, 5 lg of total RNA was loaded on the gel
The A niger galA accession number is AJ305303 Sequence alignments
Nucleic acid and amino acid sequence alignments were performed by using the Blast programs [26] at the server
of the National Center for Biotechnology Information (Bethesda, Md., USA)
Purification of b-1,4-endogalactanase Culture fluid was collected by filtration through cheesecloth and diluted twofold with distilled water, after which the pH was adjusted to 6.5 by the addition of sodium hydroxide Proteins were collected by batchwise adsorption to Stream-line Q XL(Amersham Pharmacia Biotech, Sweden) For this 40 mLof the matrix was added to the culture fluid and stirred for 2 h Bound protein was eluted by a pulse of
10 mMpiperazine/HCl pH 6.0, 1MNaCl After extensive dialysis against 10 mMpiperazine/HCl pH 6.0, the protein was loaded onto a Source 30 Q column (Amersham Pharmacia Biotech, Sweden, 16 mL) followed by elution with a 300-mLlinear NaCl gradient (0–1M) Fractions (10 mL) were collected and assayed for b-1,4-endogalac-tanase activity b-1,4-Endogalacb-1,4-endogalac-tanase containing fractions were pooled, diluted fivefold with buffer and reapplied to the same column Elution was performed with a 300-mL linear NaCl gradient (0–0.5M) The purity of b-1,4-endo-galactanase was determined by SDS/PAGE
Table 1 Strains used in this study.
Strain Genotype Reference
NW200 cspA1, bioA1, creAd4,
pyrA13/pyrA+, areA1/areA+
[43]
NW290 cspA1, fwnA12, DargB/argB + , pyrA6,
prtF28, DpgaA, DpgaB, goxC17
[44]
Trang 3Enzyme assays
Transformants producing elevated levels of
b-1,4-endoga-lactanase were selected using AZCL-galactan (Megazyme,
Bray, Ireland) according to the supplier’s instructions
During the purification endogalactanase activity was
moni-tored using azo-galactan (Megazyme) as the substrate Two
hundred microlitres of a solution of 1% (w/v) azo-galactan
in water was mixed with 50 lLof a 50-mMsodium acetate
buffer (pH 4.2) followed by the addition of 50 lLenzyme
solution Incubations were performed for 20 min at 40C
Reactions were stopped by the addition of 650 lLethanol
The precipitated substrate was removed by centrifugation
and the supernatant was used to measure absorbance at
590 nm The pH optimum of GALA was determined using
azo-galactan as described above with a pH range of 3.5–7.5
The specific activity of GALA was determined using
GALACTAZYME tablets (Megazyme) with conditions as
indicated by the supplier and using 0, 10, 25, 50, 100, 200
and 500 lLof the GALA preparation Incubations were
performed in duplicate
The hydrolysis activity of GALA with different
arabino-galactans was measured in duplicate by HPAEC and
MALDI-TOF MS after incubation of 1 mgÆmL)1 of
potato, onion and soy arabinogalactan with 1.33 lgÆmL)1
GALA at 30C, pH 4.5 (McIlvain buffer) for 15 min,
30 min, 2 h and 20 h, respectively The incubation was
stopped by heating the samples for 5 min at 100C
Transgalactooligosaccharides (TOS) were incubated in
duplicate with GALA to analyse the activity of the enzyme
towards these oligosaccharides The oligosaccharides used
all contained a glucose residue at the reducing end and were:
b-D-Galp-1,4-D-Glcp (b-D-Galp-1,4-)2D-Glcp
(b-D-Galp-1,4-)3D-Glcp (b-D-Galp-1,4-)4D-Glcp (b-D-Galp-1,4-)5
D-Glcp Incubations were performed in duplicate For this,
0.5 mgÆmL)1 of TOS was incubated with 1.33 lgÆmL)1
GALA at 30C, pH 4.5 (McIlvain buffer) for 15 min,
30 min, 2 h and 20 h, respectively The incubation was
stopped by heating the samples for 5 min at 100C
a-L-1,3-Arabinofuranosidase activity in the GALA
pre-paration was measured as described before [27], using 10
and 100 lLof the enzyme preparation (in duplicate) and
incubation times of 1 and 4 h
Analytical methods
SDS/PAGE Electrophoresis of proteins was performed
under denaturing conditions on 10% (w/v) gels using the
method of Laemmli [28] in a Mini-V system (Life
Technologies B.V., Breda, The Netherlands)
HPAEC High performance anion exchange
chromato-graphy (HPAEC) was performed using a SpectraSystem
P4000 (Thermo Separation Products) equipped with a
Dio-nex CarboPac PA-1 (4· 250 mm) column and a Dionex
ED40 Electrochemical Detector in de Pulsed Amperometric
Detection mode The samples were analyzed using a linear
gradient of 0–400 mM sodium acetate in 100 mM sodium
hydroxide for 40 min D-galactose, D-galactobiose and
D-galactotetraose were used as standards to identify the
D-galactose andD-galacto-oligosaccharides The calculated
areas forD-galactose andD-galacto-oligosaccharides were
expressed as percentages of the area of 1 m -galactose
MALDI-TOF MS MALDI-TOF MS was performed with a Voyager-DE RP Biospectrometry Workstation (PerSeptive Biosystems, Framingham, VS) in the positive mode The laser intensity was 2300, the pulsed delay time was 200 nsec, the accelerating voltage was 12 000 V, the grid voltage was 7200 V and the guide wire voltage was 9.6 V The instrument was used in the reflector mode The mass spectrometer was calibrated with maltodextrins
R E S U L T S Cloning and characterization ofgalA Based on the sequence of the Aspergillus aculeatus b-1,4-endogalactanase encoding gene, two oligonucleotides were designed and used in PCRs, resulting in a specific fragment of A niger galA, as described in Material and methods Screening of a genomic library of A niger using this fragment as a probe resulted in the isolation of two galA-containing phage k clones From one of these clones
a 4.5-kb EcoRI fragment was cloned into pBluescript
SK+, resulting in plasmid pIM3980 Double stranded sequence was determined for a region of this construct containing galA and some of the flanking regions, resulting in the genomic sequence of A niger galA The presence of one putative intron was confirmed by RT-PCR using total RNA of A niger mycelium (transferred
to minimal medium containing 15 mM D-galacturonic acid) as a template The galA gene has a length of
1122 bp, is interrupted by one intron of 72 bp, and encodes a protein of 350 amino acids Computer analysis predicted a eukaryotic signal sequence of 16 amino acids The putative mature enzyme has a calculated pI of 3.67, a calculated molecular mass of 37 053.9 Da and contains one potential N-glycosylation site BLAST analysis of the deduced amino acid sequence of GALA revealed very high similarity to the endogalactanases of A tubingensis (95.7% amino acid sequence identity) and A aculeatus (78.9% amino acid sequence identity) and lower similarity
to bacterial endogalactanases (between 21% and 28% amino acid sequence identity) This is illustrated by a CLUSTALW analysis [29] of the deduced amino acid sequences of GALA and b-1,4-endogalactanases of Aspergillus tubingensis [15], A aculeatus [12], Bacillus circulans (Acc No P48843), Yersinia pestis [14], Bacillus subtilis (Acc No O07013), Clostridium acetobutylicum [13], and Pseudomonas fluorescens [11] (Fig 1) Both catalytic residues identified in P fluorescens (E161, E270) [11] are present in the eukaryotic b-1,4-endogalactanases Sequence analysis of the promoter of galA revealed the presence of several sequences possibly involved in the regulation of galA expression Putative CREA binding sites [30] were detected at position)204, )218, 299, )482, and )580 from the start codon In addition, one putative PACC site ()549) [31] and two CCAAT sites ()280, )300) were detected
Expression analysis ofgalA The expression pattern of galA on a selection of mono-saccharides, oligosaccharides and polysaccharides was com-pared to the previously reported expression pattern of lacA (encoding b-galactosidase) (Fig 2) [32] For this, mycelium
Trang 4was pregrown as described in Material and methods, and
transferred for 4 h to minimal medium containing carbon
sources as indicated in Fig 2 Expression of galA was only
detected in the presence of sugar beet pectin, whereas
expression of lacA was detected in the presence ofL -arabi-nose,D-xylose, sugar beet pectin and xylan (Fig 2)
To determine which of the monosaccharides present in sugar beet pectin is the actual inducer of galA expression a
Fig 1 Alignment of the derived amino acid sequences of fungal and bacterial b-(1,4)-endogalactanase encoding genes In the consensus sequence the amino acids indicated are those present in at least four sequences Amino acids present in all sequences are in bold The eukaryotic signal sequence
of A niger GALA is depicted in lower case letters The putative N-glycosylation site of A niger GALA is in bold, italics, underlined and indicated above the sequence (") The two catalytic residues identified in the P fluorescens endogalactanase [11] are indicated above the alignment (^) and are
in white against a grey background.
Trang 5second experiment with identical experimental setup was
performed in which the expression of galA was studied in
two strains, a wild type (N402) and a mutant with a
derepressed phenotype for CREA repression (NW200)
Five of the carbon sources used in this experiment (D-xylose,
L-arabinose,D-galactose,L-rhamnose,D-galacturonic acid,
Fig 3) have been identified as components of sugar beet
pectin [1], whereas the others served as controls Expression
of galA in the wild type strain was only detected in
the presence ofD-galacturonic acid (Fig 3) However, in the
CREA mutant expression of galA was detected in the
presence ofD-galacturonic acid andL-arabinose
The expression of galA is very low on the inducing
compounds Using approximately 50 ng of the PCR
product for labelling, the radiation level of the probe after
purification was 42.6 kBq It was necessary to expose the
autoradiogram for two weeks in order to visualize the
hybridizing bands
Purification and characterization of GALA
To obtain an A niger transformant that produces increased
levels of b-1,4-endogalactanase, a construct was made in
which galA was fused to the promoter of the A niger pkiA
gene, encoding pyruvate kinase [24] This gene has a high
constitutive expression level and has been used previously
for the production of high levels of proteins in A niger [33]
The fusion construct (pIM3982) was made by introducing
an NsiI site at the translation start point of galA and by then
cloning a fragment, starting at this NsiI site and containing
the galA gene and approximately 700 bp 3¢-flanking region,
in pIM4700 (containing the pkiA promoter) A niger
NW290 was transformed with pIM3982 and transformants were analyzed for b-1,4-endogalactanase production using azo-galactan Transformant NW290::pIM3982.77 was selected as the highest b-1,4-endogalactanase producing transformant and was used for the purification of GALA Growth of this transformant and subsequent purification of the enzyme were performed as described in Materials and methods Purity of the preparation was checked by SDS/ PAGE (Fig 4) The purification resulted in 10 mLof the GALA preparation with a concentration of 31.6 lgÆmL)1 Using galactazyme tablets a specific activity of 12.3 UÆmg)1 was determined for GALA
The purified protein had a molecular mass of 48.5 kDa as determined by SDS/PAGE (Fig 4) The pH optimum was between 4 and 4.5
No activity of GALA could be detected against any of the transgalactooligosaccharides listed in Materials and methods
Hydrolysis of arabinogalactans The sugar composition of the arabinogalactans from potato, onion and soy was determined as described [21] Onion arabinogalactan consists of 99% D-galactose and 0.3% L-arabinose and is predominantly linear Potato arabinogalactan consists of 86% D-galactose and 6.6%
L-arabinose, while soy arabinogalactan consists of 57%
D-galactose and 38% L-arabinose Methylation analysis demonstrated that a substantial amount of theL-arabinose residues (14%) in soy arabinogalactan is present as terminal residues [21], suggesting that of these arabinogalactans, soy arabinogalactan is the most highly branched substrate
Fig 2 Comparison of the expression of the
A niger b-(1,4)-endogalactanase (galA) and
b-galactosidase (lacA) encoding gene A
frag-ment of the 18S rRNA gene [45] was used as
an RNA loading control.
Fig 3 Expression of galA in the presence of
different monomeric carbon sources in the wild
type and CREA derepressed A niger strains.
A fragment of the 18S rRNA gene [45] was
used as an RNA loading control.
Trang 6The three arabinogalactans were incubated with GALA
for 15 min, 30 min, 2 h and 20 h to determine the substrate
specificity of the enzyme The degradation of the polymer
and subsequent oligosaccharide formation was analyzed by HPAEC and MALDI-TOF MS HPAEC analysis demon-strated that already after 15 min the formation of tetramers, trimers, dimers and monomers ofD-galactose was visible for all three substrates (Fig 5) Using potato and onion arabinogalactan as substrate, a small increase in all four products is observed during the incubation The product formation using soy arabinogalactan as a substrate is clearly different (Fig 5) After initial release of trimers and tetramers, these products disappear during prolonged incubation A relatively higher amount of monomers and dimers is released from soy arabinogalactan, which increased upon prolonged incubation
MALDI-TOF MS analysis allowed for a more detailed analysis of the products formed during the incubations, although the method used cannot detect monomers and dimers GALA initially releasedD-galactotriose,D -galacto-tetraose and D-galactopentaose from potato and onion arabinogalactan, while after prolonged incubations pre-dominantlyD-galactotriose andD-galactotetraose could be detected (Fig 6) Using soy arabinogalactan as a substrate, not onlyD-galactotriose, D-galactotetraose andD -galacto-pentaose were detected but also L-arabinose substituted forms of these three oligosaccharides (Fig 6) After pro-longed incubations (2 h), only D-galactotriose could be detected However, HPAEC demonstrated that after even longer incubations (20 h)D-galactotriose also disappears
To determine whether a small amount of a-L -1,3-arabino-furanosidase was responsible for the disappearance of
Fig 4 SDS/PAGE-of the purified b-(1,4)-endogalactanase
prepar-ation 1, 2: purified GALA preparation; 3: molecular mass marker
proteins.
Fig 5 HPAEC analysis of the hydrolysis of arabinogalactans by GALA Three different arabinogalactans were used: potato (r), onion (j) and soy (m) The release of monomers (A), dimers (B), trimers (C) and tetramers (D) was studied during the incubations The areas calculated for
D -galactose and D -galacto-oligosaccharides were expressed as percentages of the area of 1 m M D -galactose The values used for the figure are the average of duplicate incubations.
Trang 7the L-arabinose-substituted oligosaccharides the GALA
preparation was assayed for this activity However, no
a-L-1,3-arabinofuranosidase activity could be detected in
the preparation (data not shown)
This paper demonstrates the important role of GALA in
the degradation of both linear andL-arabinose-substituted
galactan side chains of pectin This is in agreement with a
previous study in which the synergy of enzymes degrading
the pectin side chains was studied [34] GALA had a positive
effect on the activity of the other enzymes involved in the
degradation of these side chains
D I S C U S S I O N
A niger galAis highly similar to the b 1,4-endogalactanase
encoding genes from A tubingensis and A aculeatus The
similarity of the galA genes is higher between the two
biseriate species, A niger and A tubingensis, than between
the monoseriate A aculeatus galA gene and either of the
other two Aspergillus genes A similar observation has
previously been reported for pectin lyase encoding genes of
these species [35] Significant similarity was also detected to
a number of bacterial b-1,4-endogalactanases The highest
similarity was detected in the region around the first
catalytic residue identified for the P fluorescens
b-1,4-endogalactanase [11] The similarity around the second
catalytic residue is lower
The determined molecular mass for A niger GALA
(48.5 kDa) is higher than the molecular mass based on the
amino acid sequence (37 kDa) This suggests that the
puta-tive N-glycosylation site identified in the sequence is indeed
functional The determined molecular mass and pH
opti-mum (pH 4–4.5) of GALA are similar to those reported
pre-viously for Aspergillus b-1,4-endogalactanases [10,12,36–40]
Expression of galA was observed in the presence of sugar
beet pectin,D-galacturonic acid andL-arabinose As A niger
is not able to import pectin, it is likely that the latter two
compounds, or metabolites derived from them, are the true
inducers of galA expression This indicates that galA is coexpressed with both the pectinolytic genes encoding main chain cleaving enzymes as well as with the arabinanolytic genes [1] The absence of expression of galA in the presence
of pectic galactan indicates that galactose is not an inducer
of galA expression This is in agreement with a role for
L-arabinose and D-galacturonic acid as inducers as men-tioned above Expression of lacA is also detected in the presence of these compounds, but in the presence ofD-xylose and xylan as well This can be explained by the structure of the polysaccharides Xylan contains single b-1,4-linked
D-galactose residues but no b-1,4-linked galactan chains Therefore it only requires an exo-acting galactose releasing enzyme (LACA) Previously it was shown that lacA is under the control of the xylanolytic activator protein [32,41] The increase in expression levels of galA in the CREA mutant, indicates that at least one of the detected putative CREA binding sites in the promoter of galA is functional
Hydrolysis of arabinogalactans with a different degree of
L-arabinose substitution by GALA results in different products Arabinogalactans with a low degree of L -arabi-nose substitution (potato, onion) as substrates result in the liberation of monomers, dimers, trimers and tetramers of
D-galactose Using arabinogalactans with a high degree of
L-arabinose substitution (soy), higher oligomers appear initially, but these disappear if the incubation is continued Another difference is that only with the latter substrate arabinose-substituted galacto-oligosaccharides are detected These disappear in time, suggesting the presence of an arabinose-releasing activity in the GALA preparation However, no a-1-,3-L-arabinofuranosidase activity could
be detected using PNP-a-L-arabinofuranoside as a sub-strate This most likely means that the removal of the
L-arabinose residues from the galactomannan oligosaccha-rides is caused by traces of an enzyme in the b-1,4-endogalactanase preparation that is not active against the PNP-substrate Galacto-oligosaccharides that were sub-stituted with -glucose at the reducing end were not
Fig 6 Maldi-TOF MS analysis of the hydrolysis of arabinogalactans by GALA Three different arabinogalactans were used: potato (A), onion (B), and soy (C) Spectra were taken after 15 min (1) and 2 h (2) of incubation with GALA.
Trang 8hydrolyzed by GALA So, heterosugar oligosaccharides
with the structures Gal–Gal–Glc and Gal–Gal–Gal–Glc can
not be hydrolyzed, while their corresponding homosugar
oligosaccharides Gal3 and Gal4 are degradable (Fig 5,
panels C and D) This indicates that the substrate binding
site in GALA that binds the reducing-end sugar is very
important in enzyme substrate interaction Previous studies
using an Aspergillus b-1,4-endogalactanases reported release
of D-galactose and D-galactobiose from arabinogalactan,
after initial release ofD-galactotriose andD-galactotetraose
[10,12,36,39] However, none of these papers reported the
release ofL-arabinose substituted galacto-oligosaccharides
A C K N O W L E D G E M E N T S
The authors thank Simon Flitter for the identification and isolation
of the galA containing phage clones and Matthew Illsley for the
analysis of a- L -1,5-arabinofuranosidase activity in the endogalactanase
preparation.
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