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Applied and Environmental Microbiology, January 2000, p. 36-41, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Endo-Xylogalacturonan Hydrolase, a Novel
Pectinolytic Enzyme
C. J. B.
van der
Vlugt-Bergmans,1,*
P. J. A.
Meeuwsen,2
A. G. J.
Voragen,2 and
A.
J. J.
van Ooyen1
Industrial Microbiology
Group,1 and Food Chemistry
Group,2 Department of Food Technology and
Nutritional Sciences, Wageningen Agricultural University, NL-6700
EV Wageningen, The Netherlands
Received 1 March 1999/Accepted 1 October 1999
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ABSTRACT |
We screened an Aspergillus tubingensis expression
library constructed in the yeast Kluyveromyces lactis for
xylogalacturonan-hydrolyzing activity in microwell plates by using a
bicinchoninic acid assay. This assay detects reducing carbohydrate
groups when they are released from a carbohydrate by enzymatic
activity. Two K. lactis recombinants exhibiting
xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a
406-amino-acid protein, designated XghA, which was encoded by a
single-copy gene, xghA. A multiple-sequence alignment
revealed that XghA was similar to both polygalacturonases (PGs) and
rhamnogalacturonases. A detailed examination of conserved regions in
the sequences of these enzymes revealed that XghA resembled PGs more.
High-performance liquid chromatography and matrix-assisted laser
desorption ionization-time of flight mass spectrometry of the products
of degradation of xylogalacturonan and saponified modified hairy
regions of apple pectin by XghA demonstrated that this enzyme uses an
endo type of mechanism. XghA activity appeared to be specific for a
xylose-substituted galacturonic acid backbone.
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INTRODUCTION |
Pectin occurs as constituent of
higher-plant cell walls, where it is embedded in the cellulose fibrils.
The composition of pectin is different in different plant species and
also depends on the age and maturity of the plant part. Most pectin
polymers consist of smooth homogalacturonan regions and ramified hairy regions. The smooth regions consist of a linear homogalacturonan backbone. The hairy regions, as identified in apples (28),
consist of three different subunits; subunit I is xylogalacturonan
(xga) (a galacturonan backbone heavily substituted with xylose),
subunit II is a short section of a rhamnogalacturonan backbone that has many relatively long arabinan, galactan, and/or arabinogalactan side
chains (the hairs), and subunit III is rhamnogalacturonan composed of
alternating rhamnose and galacturonic acid residues. Some of the
rhamnose residues are substituted with single galactose residues. It is
thought that subunit III connects the other two subunits. Isolated
hairy regions are referred to as modified hairy regions (mhr), since
the isolation procedure may alter the sugar compositions and degrees of
methylation of the regions (27).
In industrial food processing (e.g., clarification of fruit juices), it
is important that pectins are degraded completely (36). The
smooth region (polygalacturonan) is readily degraded by pectinases,
such as endo-polygalacturonase (endo-PG), endo-pectate lyase,
endo-pectin lyase, and pectin methyl esterase. The corresponding genes
have been cloned from various plants, fungi, and bacteria, including
Aspergillus niger (5, 13, 15, 24). A. niger is of considerable economic importance, since it is employed
to produce extracellular enzymes used in the food industry.
The hairy regions of apple pectin can be degraded by
rhamnogalacturonases (RHGs), arabinases, and galactanases. Two enzymes, RHG-A and RHG-B, a hydrolase and a lyase, respectively (21, 26), which are able to split the rhamnogalacturonan backbone in
mhr, have been identified and isolated from A. niger
(32). In addition, workers have found two enzymes that
degrade the rhamnogalacturonan backbone in an exo fashion; a
rhamnohydrolase and a galacturonohydrolase catalyze the release of Rha
and GalA residues, respectively, from rhamnogalacturonans (20,
22). Also, rhamnogalacturonan acetyl esterases hydrolyze the
acetyl esters present in mhr (29). The side chains in the
hairy regions are hydrolyzed by arabinases and galactanases, and the
corresponding genes have also been cloned (6, 11).
Enzymic digestion of mhr with the enzymes that have been identified
thus far leaves xga as an inert carbohydrate. Only one xga-degrading
enzymes has been described to date; this exo-galacturonase is able to
remove a Xyl-GalA disaccharide from xga (1). However, by
analogy to the array of enzymes that degrade the smooth regions and
subunits II and III of the hairy regions of pectin, there should be a
set of enzymes that degrade xga. For example, in addition to an
endo-xylogalacturonase, there could be a lyaselike enzyme which splits
glycosidic linkages between methylated and xylosylated galacturonic
acid residues. In addition, there could be a xylosidase which
hydrolyzes the xylose residues from the galacturonic acid backbone,
releasing a backbone sensitive to PG or pectate lyase activities. Such
enzymes could be very valuable analytical tools for studying plant cell
wall structures and for comparing xga polysaccharides in pectins from
various plant sources.
The availability of sufficient amounts of purified xga from gum
tragacanth (19) and the expression cloning technique
(8) should allow cloning of an xga-hydrolyzing enzyme if it
is present in Aspergillus sp. In contrast to expression
cloning in Escherichia coli (34), the yeast
Kluyveromyces lactis is able to secrete extracellular
Aspergillus enzymes, and this allows screening for carbohydrolases in the supernatants of recombinant K. lactis
clones. This allows screening in microwell plates instead of the
commonly used plate assays. The bicinchoninic acid (BCA) assay
(12) has been used to screen for carbohydrolase activities
in microwell plates (18).
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MATERIALS AND METHODS |
Construction of the expression library in K. lactis.
The Aspergillus tubingensis expression library was
constructed from poly(A)+ RNA from mycelia cultivated for
10, 16, and 24 h in minimal medium (33) containing
0.5% soyoptim (Société Industriel Oléagineux, Saint
Laurent Blangy, France). The cDNA was ligated to the expression vector
pCVlacK (33). The primary library in E. coli
consisted of 7,400 colonies (33). After amplification, the
library was transferred to K. lactis by using the method of
Faber et al. (10).
Cultivation of K. lactis (i) In microwell
plates.
Individual K. lactis transformants were
transferred to 35 microwell plates (Nunc type 96F). They were
cultivated for 2 days at 30°C in 200 µl of minimal medium I
(adapted from the medium of Blondeau et al. [2])
containing mannitol as the sole carbon source and 80 ng of Geneticin
G418 sulfate (Gibco BRL) per ml. Subsequently, cultures were replica
plated on fresh microwell plates. Glycerol was added to the parental
plates to a final concentration of 15% (vol/vol), and the plates were
stored at 
80°C. The replicates were incubated for 2 days at 30°C.
The cells were pelleted by centrifugation at 3,000 rpm with a Hermle
model zk380 centrifuge, and the supernatant was used in the BCA assay.
(ii) In Erlenmeyer flasks.
K. lactis transformant 27E8
secreting xylogalacturonan hydrolase (XghA) was transferred from a
glycerol stock preparation in a microwell plate to a reagent tube
containing 2 ml of medium I supplemented with 80 µg of Geneticin G418
sulfate per ml. Wild-type K. lactis CBS2359 was cultivated
in a tube containing 2 ml of medium. Both tubes were incubated for 2 days at 30°C, and the cultures were then used to inoculate 500-ml
portions of the corresponding media in 1-liter Erlenmeyer flasks. After
2 days of incubation, the cells were pelleted, and each supernatant was
dialyzed extensively against 50 mM sodium acetate buffer (pH 5.0) and
then used for enzyme studies.
Microwell plate screening.
The substrate xga, which was
isolated from gum tragacanth (18), consisted of 2%
rhamnose, 1% arabinose, 23% xylose, 5% galactose, 3% glucose, and
66% galacturonic acid (molar percentages). The composition was
determined by using a Carlo Erba model 4200 gas-liquid chromatography
system and a J&W DB225 column after hydrolysis with 2 M trifluoroacetic
acid (1 h, 121°C) and conversion of the monomers to alditol acetates
(9).
After incubation of the K. lactis expression library in
microwell plates, 25 µl of supernatant was pipetted into a new
microwell plate. Next, 25 µl of a 0.2% xga solution in 100 mM sodium
acetate buffer (pH 5.0) was added. After incubation overnight at
30°C, the increase in the reducing carbohydrate content was measured by using a modified BCA assay (18). Ten microliters of the
enzyme incubation preparation was mixed with 90 µl of water and 100 µl of BCA reagent in a microwell plate. The BCA reagent was freshly prepared by mixing stock solutions A and B (1:1, vol/vol). Solution A
contained (per liter of distilled water) 54.3 g of
Na2CO3, 24.2 g of NaHCO3, and
1.9 g of Na2BCA. Solution B contained (per liter of
distilled water) 1.25 g of CuSO4 · 5H2O and 1.26 g of L-lysine.
Following incubation for 1 h at 80°C in a water bath, the plate
was cooled on ice, and the absorbance at 550 nm was determined
with a
microwell plate reader. Transformants that produced absorbance
which
was 0.1 U higher than the absorbance of the blank were
retested.
General cloning and sequencing techniques.
Standard methods
were used for DNA manipulation and Southern blotting (25).
The blots were hybridized by using a digoxigenin-labelled probe and
chemiluminescence detection as recommended by the manufacturer (Boehringer Mannheim). The pCVlacK expression plasmid from K. lactis was isolated by using the glass bead method of Sobanski and
Dickinson (30). Sequencing was performed with a
Taq DYE primer cycle sequencing kit (Applied Biosystems).
The nucleotide sequences of both strands were determined. Sequence
analysis and multiple alignment were performed by using the Wisconsin
Package, version 9.0 (Genetics Computer Group, Madison, Wis.).
HPLC analysis of xga and mhr-s degradation.
Dialyzed culture
supernatant (250 µl) was incubated with 500 µl of substrate (1%
xga or 1% saponified mhr of apple pectin [mhr-s]
[27] in 50 mM sodium acetate [pH 5.0]) and 250 µl
of 50 mM sodium acetate (pH 5.0) for 18 h at 30°C. The samples
were heated in a boiling water bath for 5 min before the degradation products were analyzed by high-performance liquid chromatography (HPLC). A polygalacturonan digest was prepared in a similar way by
incubating polygalacturonan (ICN Biomedicals) with endo-PG (EC
3.2.1.15) from Kluyveromyces fragilis purified in our
laboratory (23).
High-performance anion-exchange chromatography (HPAEC) was performed by
using a Dionex system (Dionex, Sunnyvale, Calif.)
which included a
model BioLC quaternary gradient pump, a model
EDM (He) deventilating
unit, a Carbopac PA-1 column (4 by 250
mm) with a matching guard
column, and a pulsed electrochemical
detector operated in pulsed
amperometric detection mode. A linear
gradient of sodium acetate in 100 mM NaOH at a flow rate of 1
ml/min was used as follows: 0 to 50 min, 0 to 620 mM; 50 to 55
min, 620 to 1000 mM; and 55 to 65 min, 1,000
mM.
High-performance size exclusion chromatography (HPSEC) was performed by
using a model SP8800 HPLC (Spectra Physics) equipped
with three Bio-Gel
TSK columns (each 300 by 7.8 mm) in series
(40XL, 30XL, and 20XL;
Bio-Rad Laboratories) in combination with
a Bio-Gel TSK XL guard column
(40 by 6 mm). The temperature of
the columns was 30°C. The columns
were eluted with 0.4 M sodium
acetate buffer (pH 3.0) at a flow rate of
0.8 ml/min. During elution
the refractive index was monitored with a
Shodex model SE-61 refractive
index
detector.
MALDI-TOF mass spectrometry of the degradation products of
xga.
xga was incubated with XghA as described above. The molecular
masses of the resulting oligomers were determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass
spectrometry. Each sample was analyzed with a Perspective Biosystems
Inc. Voyager-DE RP instrument equipped with a nitrogen laser
(wavelength, 337 nm; pulse width, 3 ns). The mass spectrometer was
operated in the positive ion mode with a delayed extraction time of 200 ns. The ions were accelerated to an energy of 12 kV before they entered the time of flight mass spectrometer. The minimum laser power for
obtaining a good spectrum was used, and 20 to 50 spectra were obtained
for each analysis. Approximately 0.35 g of Dowex 50WX8 (50-100 mesh; Fluka Chemika-BioChemika, Buchs, Switzerland) was added to the
mixture of oligomers. The sample was thoroughly mixed and centrifuged
to pellet the Dowex material. One microliter of the clear supernatant
was applied to a matrix-assisted laser desorption ionization plate and
mixed on the plate with 1 µl of matrix suspension, and then the
preparation was dried with a gentle stream of air at room temperature.
The matrix suspension was prepared by dissolving 9 mg of
2,5-dihydroxybenzoic acid and 3 mg of isocarbostyril in 1 ml of a
water-acetonitrile mixture (7:3). The mass spectrometer was calibrated
externally with a polygalacturonic acid endo-PG digest (7).
Nucleotide sequence accession number.
The nucleotide
sequence of the xghA gene has been deposited in the EMBL
database under accession no. AJ 249460.
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RESULTS |
Screening of the expression library.
An A. tubingensis expression library constructed in K. lactis
was initially designed to screen for arabinogalactan-modifying enzymes
(33). The shuttle vector pCVlacK used for expression of the
cDNA library in K. lactis also facilitated isolation and analysis of the cDNA insert in E. coli.
Screening of expression libraries on plates by using chromogenic
substrates or overlays and Congo red staining has been used
extensively. Here we describe screening of a library in microwell
plates by using a modified BCA assay (
18). This assay is
based
on reduction of Cu(II) to Cu(I) by reducing carbohydrate mono-
and oligomers. A purple complex consisting of BCA and Cu(I) is
formed,
and this complex can be measured spectrophotometrically.
Screening 35 microwell plates (about 3,400
K. lactis transformants)
by
using xga as the substrate yielded two transformants (27E8
and 42B4)
with xga-hydrolyzing activity. The pCVlacK plasmids
were isolated from
both
K. lactis recombinants. After transformation
and
propagation of the plasmids in
E. coli, the cDNA inserts
were
excised from pCVlacK by
HindIII-
XhoI
digestion. This digestion
released 1.0- and 0.4-kb fragments due to an
internal
HindIII
site, as determined from the nucleotide
sequence. The plasmid
inserts of the transformants were identical, as
determined by
restriction
analysis.
Structural analysis of XgaA.
The DNA sequences of both strands
of the cDNA insert derived from K. lactis transformant 27E8
were determined by using 5'- and 3'-specific primers flanking the cDNA
insert and specific primers based on the cDNA sequence obtained with
the former primers. The cDNA sequence, designated xghA,
contained a 1,218-bp open reading frame that began with an ATG start
codon and ended with a TAA stop codon (Fig.
1). It encoded a 406-residue polypeptide designated XghA that had a calculated molecular mass of 42,070 Da. The
open reading frame was preceded by a 20-bp 5' noncoding region and was
followed by a 130-bp 3' noncoding region and a poly(A) tail. The
TCATCATGGC sequence covering the ATG start codon closely
resembled the consensus sequence for initiation of translation in
higher eukaryotes (17). The xghA cDNA encoded an
apparent signal sequence consisting of 18 amino acids at the
NH2 terminus, and there was a typical signal peptidase
cleavage site between Ala-18 and Ala-19 (35). Two
potential N-glycosylation sites were present at Asn-278-Ser-Thr
and Asn-301-Val-Thr (Fig. 1).
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FIG. 1.
Nucleotide sequence of the cDNA encoding XghA
(xghA) and the deduced amino acid sequence of the encoded
protein (XghA). The arrow indicates the potential cleavage site of the
signal sequence. Two potential glycosylation sites are underlined.
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A comparison of the XghA amino acid sequence with sequences in the EMBL
data library revealed homology to PG (EC 3.2.1.15)
sequences of
prokaryotes, fungi, and plants and to RHG-A (EC 3.2.1.-)
and RHG-B (EC
4.2.2.-) sequences of
Aspergillus aculeatus (
16,
31) and
A. niger (
32). Many PGs have been
cloned from members
of the genus
Aspergillus, but only the
A. niger and
A. tubingensis sequences (Fig.
2) were used for a detailed sequence
comparison.
XghA exhibited 31 to 39% similarity to the endo-PGs and
44% similarity
to the exo-PG of
A. tubingensis; these
values are higher than
the levels of similarity between the endo-PGs
and the exo-PG (26
to 33%). The levels similarity of XghA to the two
RHG-As were
30% (
A. niger RHG-A) and 32% (
A. aculeatus RHG-A), whereas the
level of similarity to RHG-B of
A. niger was very low (for the
whole sequence).
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FIG. 2.
Part of a multiple alignment of PG, XghA, and RHG amino
acid sequences. Amino acids identical to the amino acids in the XghA
sequence are indicated by dots, and gaps that were introduced to obtain
the optimal alignment are indicated by dashes. Amino acids conserved in
all plant, fungal, and prokaryotic PGs are shaded. An HG sequence
(domain III in PG and XghA) is underlined in the sequences of A. aculeatus RHG-A and A. niger RHG-B. Abbreviations:
A. tubingensis PGAII Atub-PgaII, (EMBL accession no. P19805
[3]); Anig-PgaII, A. niger PGAII (P26214
[3]); Anig-PgaI, A. niger PGAI (P26213
[4]); Anig-PgaC, A. niger PGAC (X64356
[5]); Atub-PgaX, A. tubingensis exo-PG
(X99795 [14]); Aac-RhgA, A. aculeatus RHG-A
(A544425 [16] and S80208 [31]);
Anig-RhgA, A. niger RHG-A (X94220 [32]);
Anig-RhgB, A. niger RHG-B (X94221 [32]);
Atub-XghA, A. tubingensis XghA. The A. niger PG
sequence (24) deposited under EMBL accession no. X54146 is
identical to the A. tubingensis PGAII sequence
(3).
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Based on the values given above, the levels of similarity of XghA to
the PGs and the RHG-As seem to be comparable. However,
an analysis of a
multiple alignment of the enzymes produced different
results. Figure
2
shows part of a multiple alignment of four domains
of conserved amino
acids which were first described for PGs of
plant, fungal, and
bacterial origin (
3). When all of the PGs
recovered from a
database search were aligned, only the following
four short stretches
of amino acids were conserved: NXD, DD, HG,
and RXK (
14)
(Fig.
2). Possible candidates for the essential
amino acids involved in
the hydrolysis reaction were the three
aspartic acid residues of
domains I and II and the histidine residue
of domain III
(
3). These domains were completely conserved
in XghA. The
fourth domain contained amino acids that may be involved
in substrate
binding. The arginine residue of this domain was
replaced by a glycine
residue in XghA. The domains were less conserved
in the RHG sequences.
Only two of the three aspartic acid residues
were conserved, and the
histidine in domain III was replaced by
glycine. It has been suggested
that the HG sequence is present
further along the sequence
(
31) (Fig.
2), but it is not present
in the RHG-A sequence
of
A. niger.
Southern blot analysis.
The copy number of the xghA
gene was determined by performing a Southern blot analysis of the
genomic DNA of A. tubingensis digested with several enzymes
(Fig. 3). Hybridization under stringent conditions (65°C, 0.2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) and less stringent conditions (60°C, 1× SSC) with
a 1.0-kb HindIII fragment of xghA clearly
revealed single hybridizing fragments (Fig. 3). This demonstrated that
a single copy of the xghA gene is present in the A. tubingensis genome.
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FIG. 3.
Autoradiographs of Southern blots containing genomic DNA
of A. tubingensis digested with four restriction enzymes
(lanes H, HindIII; lanes I, EcoRI; lanes V,
EcoRV; lanes P, PstI). The positions of molecular
size markers (in kilobases) are indicated on the left. (A) Blot
hybridized under low-stringency conditions (60°C, 1× SSC). (B) Blot
hybridized under high-stringency conditions (65°C, 0.2× SSC). A
1.0-kb HindIII fragment of xghA was used as a
probe.
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Characterization of the enzymatic activity of XghA.
Degradation of xga or mhr-s by XghA was monitored by HPAEC and HPSEC.
Figure 4 shows HPAEC elution profiles for
xga and mhr-s incubated with a wild-type K. lactis culture
supernatant (Fig. 4A) or with an XghA-containing supernatant of
transformant 27E8 (Fig. 4B). A polygalacturonan digest (Fig. 4C) was
included as a reference (7). Incubation with the wild-type
K. lactis culture supernatant did not result in breakdown of
xga or mhr-s, whereas incubation with the XghA-containing supernatant
yielded oligomers with both substrates. When the HPAEC elution times of
these products were compared with the elution times of the
polygalacturonic acid standards (Fig. 4C), it was obvious that the xga
and mhr-s products eluted at different times, indicating that none of
the degradation products was a galacturonic acid oligomer. Considering
the sugar composition of the substrates, we concluded that xga
oligomers were produced. The molecular masses of the oligomers released from xga after incubation with XghA were determined by
MALDI-TOF mass spectrometry. The molecular weights of the
resulting oligomers corresponded to the molecular weights of sodium
adducts of oligomers consisting of xylose and galacturonic acid
monomers. The compositions of these oligomers as related to the
detected masses are shown in Table 1.
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FIG. 4.
HPAEC elution profiles of digests of xga and mhr-s
incubated with K. lactis wild-type (wt) culture supernatant
(A) or XghA-containing supernatant of K. lactis transformant
27E8 (B). (C) Polygalacturonic acid (pga) digested with PG. The
subscript numbers indicate the form of galacturonic acid (1, monomer;
2, dimer; etc.) (7). Pad, pulsed amperometric detection.
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TABLE 1.
Molecular weights and compositions of the oligomers
released from xga after incubation with XghA, as determined by
MALDI-TOF mass spectrometry
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Only two products were formed when mhr-s was incubated with XghA. The
same two products plus some additional products whose
elution times
were greater than the elution time of the galacturonic
acid trimer
standard were formed when xga was degraded by XghA.
These larger
oligomers appeared even after a short incubation
time (results not
shown), indicating that the mechanism of the
enzyme was an endo type of
mechanism.
XghA activity has also been tested with other substrates, such as
polygalacturonic acid, linear arabinan, soy galactan, and
xylan from
oat spelt. No breakdown was detected (data not shown).
Apparently, XghA
activity requires a galacturonic acid backbone
substituted with
xylose.
The HPSEC results obtained show that there was a dramatic decrease in
the molecular weight of xga after incubation with XghA
(Fig.
5A). This decrease appeared after
incubation for 1 h (results
not shown), and developed rapidly,
again indicating the endo nature
of the enzyme. Degradation of mhr-s
(Fig.
5B) resulted in a shift
of the high-molecular-weight fraction to
a lower molecular weight.
However, this shift was less prominent, which
could be explained
if the xga were present at the ends of the mhr-s
chains.
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FIG. 5.
Elution profiles of digests of xga (A) and mhr-s (B)
incubated with XghA-containing supernatant of K. lactis
transformant 27E8 (dark lines). The light lines indicate the results
obtained with substrate incubated with water.
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DISCUSSION |
xga is an important constituent of the hairy regions of certain
pectins. Although it has been suggested that an array of enzymes which
are able to hydrolyze xga exist in nature, only an exogalacturonase which is able to remove a Xyl-GalA disaccharide from xga has been identified (1). Because of the availability of xga
(19) as a very specific substrate and the availability of an
A. tubingensis expression library in K. lactis,
we performed the work described in this paper, which was aimed at
finding enzymes that degrade xga in an endo fashion.
Expression libraries are most commonly screened by performing plate
assays. In this paper we describe a BCA assay for detecting carbohydrolase activity in the supernatants of K. lactis
recombinants in which microwell plates were used. The advantage of this
method is that less substrate is required than the amount required for plate assays. Furthermore, it is very sensitive and fast.
Two identical K. lactis recombinants from the approximately
3,400 colonies screened exhibited hydrolyzing activity with xga. An
analysis of the cDNA insert demonstrated that it codes for a
406-residue polypeptide with a signal sequence consisting of 18 amino
acids. A single copy of the gene is present in the A. tubingensis genome. HPLC analysis and MALDI-TOF mass spectrometry of xga degradation by XghA-containing supernatant of the K. lactis recombinant clearly showed that the mechanism of the enzyme
was an endo type of mechanism. To our knowledge, this is the first report of a true endo-xylogalacturonase. Since XghA exhibits no activity with polygalacturonic acid, linear araban, soy galactan, or
xylan from oat spelt, we concluded that XghA requires a galacturonic acid backbone substituted with xylose.
When mhr-s was incubated with XghA, only two major products were
produced, whereas several peaks were formed when xga was incubated with
XghA (Fig. 4). The difference could have been the result of the
different Xyl/GalA ratios; the Xyl/GalA ratio was two to three times
higher in mhr-s than in xga obtained from gum tragacanth. Steric
hindrance by other subunits of mhr-s could also have been the reason
for the difference. Also, HPSEC analysis of mhr-s incubated with XghA
revealed a shift to the lower-molecular-weight fraction, which was less
significant than it was in the elution profile of xga with XghA. A
possible explanation for this is the presence of xga at the ends of the
mhr-s chains. The mhr-s degradation data obtained in the HPAEC and
HPSEC analyses show that XghA is valuable for determining the
structures of complex polysaccharide fractions, like mhr-s.
A search of the EMBL data library for sequences homologous to the XghA
sequence revealed that the levels of similarity of XghA to PGs and RHGs
were low. The percentages of similarity were approximately the same for
both groups of enzymes. However, an analysis of a multiple alignment of
conserved domains in these proteins showed that XghA resembled PGs more
than RHGs. The putative active site residues aspartate and histidine
(present in domains I to III [Fig. 2]) are conserved in the PGs and
XghA. In RHGs, however, the histidine is replaced by a glycine, and
only two aspartic acid residues are conserved. This suggests that the
catalytic mechanism of RHGs may be different from the catalytic
mechanisms of the PGs and XghA and that PGs and XghA might have similar
catalytic mechanisms. This could also correspond to the cleavage sites
in the backbones of the respective substrates. RHGs cleave a
rhamnosyl-galacturonic acid linkage, whereas the PGs and XghA cleave a
galacturonic acid-galacturonic acid linkage.
The positively charged sequence Arg-Ile-Lys present in domain IV of
PGs, postulated to play a role in substrate binding, is not fully
conserved in XghA and RHGs. This arginine residue is not conserved in
either XghA or RHGs, but the substrate for these enzymes is different
from the substrate for PGs, which may account for the discrepancy
(14). Although the substrate backbone for both PGs and XghA
consists of GalA, XghA requires a substrate with a xylose-substituted backbone.
In conclusion, XghA is the first enzyme that has been reported to
hydrolyze xga in an endo fashion. Based on sequence similarities of the
active site residues, we propose that the catalytic mechanism of XghA
is similar to that of PGs. This possibility must be investigated further, however. XghA should be a useful tool that allows workers to
study plant cell wall structures. In practice, XghA can be a valuable
component of tailor-made enzyme preparations that are used in fruit
juice manufacturing.
| |
ACKNOWLEDGMENT |
This work was supported by Gist-brocades, Delft, The Netherlands.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Industrial
Microbiology Group, Department of Food Technology and Nutritional
Sciences, Wageningen Agricultural University, P.O. Box 8129, NL-6700 EV Wageningen, The Netherlands. Phone: 31 317 484980. Fax: 31 317 484978. E-mail: cecile.vandervlugt.imb.ftns.wau.nl.
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Abstract
Introduction
