Targeted differential display of abundantly expressed sequences ...

Curr Genet (1998) 33: 70–76 © Springer-Verlag 1998
ORIGINAL PAPER
Paul R. J. Birch
Targeted differential display of abundantly expressed sequences
from the basidiomycete Phanerochaete chrysosporium
which contain regions coding for fungal cellulose-binding domains
Received: 9 October / Accepted: 28 October 1997
Abstract Cellulose-binding domains (CBDs) are present
in the majority of fungal cellulases studied to-date. This
work describes the use of targeted differential display, employing
degenerate primers designed to anneal to variants
of a region conserved in fungal CBDs, each in combination
with an oligo-dT primer, to PCR-amplify cDNA sequences
containing regions coding for such domains from
Phanerochaete chrysosporium. After growth on either Avicel
or carboxymethyl cellulose (CMC), five distinct, abundantly
expressed cDNA sequences were obtained. Two of
these originated from transcripts of the previously characterised
cbhI.1 and cbhI.2 genes, whereas three were from
novel genes. One of the latter was isolated only after growth
on CMC. No such sequences were obtained after growth
on xylan, suggesting that the expression of sequences containing
such regions is down-regulated on this substrate.
The use of targeted differential display both for isolating
novel sequences and for studying the expression of known
genes within a family is discussed.
Key words Phanerochaete chrysosporium ·
Differential display · Cellulose-binding domain ·
Differential gene expression
Introduction
The polysaccharide cellulose is the most abundant organic
material on earth. It is thus our principle renewable resource
and, as such, is of significant importance as a source of food,
fuel and fibre. In Nature, it is intimately associated with the
polymers lignin and hemicellulose to form lignocellulose.
P. R. J. Birch
Fungal and Bacterial Plant Pathology Department,
Scottish Crop Research Institute,
Invergowrie, Dundee, DD2 5DA, UK
Fax: +44-1382-562426
e-mail: pbirch@scri.sari.ac.uk.
Communicated by H. N. Arst
Many microorganisms can de-polymerise cellulose; the
best studied of these is the deuteromycete fungus Trichoderma
reesei (Béguin and Aubert 1994). However, very few
can break down all three components of lignocellulose. Of
these, only the basidiomycete fungi have been studied in
detail and only Phanerochaete chrysosporium has been
well characterised (Gold and Alic 1993; Broda et al. 1996).
Two classes of hydrolytic enzymes are involved in the
de-polymerisation of crystalline cellulose, endoglucanases
(EGs) and cellobiohydrolases (CBHs). In both fungal and
bacterial systems such enzymes generally have a structure
comprising a catalytic domain, separated from a cellulosebinding
domain (CBD) by a linker region (Gilkes et al.
1991). Of these, the CBDs, although differing between
fungi and bacteria, are highly conserved in all species that
have been studied within each kingdom (Gilkes et al. 1991;
Béguin and Aubert 1994; Hoffrén et al. 1995).
Recently, two novel methods for isolating genes encoding
fungal cell-wall hydrolases have been reported.
Dalbøge and Heldt-Hansen (1994) isolated both cellulase
and xylanase genes from Humicola insolens by expression
of a cDNA library in yeast and subsequent screening for
appropriate enzyme activities. An alternative strategy was
employed by Sheppard et al. (1994) who designed degenerate
primers for PCR, based on intra-family sequence similarities,
exploiting the hydrophobic cluster analysis-derived
cellulase classification of Henrissat and Bairoch
(1993). Genomic DNA fragments were amplified and used
as probes to screen a cDNA library; five cDNAs coding
for distinct cellulase homologues were cloned and sequenced.
A drawback of both methods is the time and labour involved
in cDNA library construction, which requires microgram
amounts of mRNA and is normally made after
growth on a particular substrate. We have shown previously
that cellulase genes from P. chrysosporium are differentially
expressed, both temporally and according to
substrate (Tempelaars et al. 1994; Birch et al. 1995; Broda
et al. 1995), and thus a number of cDNA libraries would
be needed to screen all of the genes encoding this family
of enzymes.

A number of PCR-based methods for rapidly comparing
profiles of gene expression have been reported. One
such method, differential display (DDRT-PCR), involves
the random amplification of sequences from cDNA populations
of interest using short oligonucleotide primers in
combination with primers which anneal to the polyA tail
of the cDNA; amplification products are then directly compared
on polyacrylamide gels (Liang and Pardee 1992).
Here, the DDRT-PCR approach has been modified to
screen specifically for sequences containing regions encoding
CBDs from P. chrysosporium, after growth on three
different carbon (C) sources. In this case, the short primers
used for DDRT-PCR are replaced by longer degenerate
primers designed to anneal to a conserved region of the
CBD-encoding sequence, allowing the annealing temperature
of the PCR reaction to be elevated, and thus to be
more specific. CBDs are present in the majority of fungal
cellulases studied to-date and have also been reported in a
xylanase from H. insolens (Dalbøge and Heldt-Hansen
1994), a β mannanase (Stålbrand et al. 1995) and an acetyl
xylan esterase (Margolles-Clark et al. 1996) from T. reesei.
Materials and methods
Organism and culture media. P. chrysosporium ME446 (ATCC
34541) was maintained on 2% (w/v) slopes malt-extract agar. The
culture medium was a modified Vogel’s medium as described previously
(Tempelaars et al. 1994), containing 0.2% (w/v) of a carbon
source from Avicel (microcrystalline cellulose), carboxymethyl cellulose
(CMC) (amorphous cellulose) or oatspelt arabinoxylan.
DNA manipulations. DNA was extracted from P. chrysosporium as
described by Raeder and Broda (1985). Southern blotting was onto
Hybond N membrane (Amersham) and hybridisation was performed
using the low-stringency conditions described in Sambrook et al.
(1989). All techniques referred to below were according to the
manufacturers’ recommendations. Colony hybridisations were made
on Hybond C extra membrane (Amersham). PCR-amplified DNA
fragments were purified for cloning using the Promega Wizard PCR
Preps kit. PCR-amplified DNA was cloned using the pGEM-T vector
cloning kit (Promega) and transformed into Stratagene Ultracompetent
Epicurian coli XL2-blue MRF′ cells. Plasmid preparations
were made using the Qiagen Plasmid Miniprep kit. Sequencing of
cloned PCR products was performed using the ABI PRISM Dye Terminator
cycle sequencing kit of Perkin Elmer. The 32 P-radiolabelled
probe DNA was prepared using the Random Primed Labelling kit of
Pharmacia.
RNA extraction and cDNA synthesis. P. chrysosporium was inoculated
into liquid culture as previously described and the mycelium
was harvested after 4-days stationary incubation at 37°C (Tempelaars
et al. 1994). The mycelium was ground under liquid nitrogen
and RNA extracted using the Qiagen RNeasy kit. Poly (A) + mRNA
was purified from this with a Dynal’s Dynabeads mRNA extraction
kit. cDNA was synthesised from mRNA with the Pharmacia First-
Strand cDNA synthesis kit, using the NotI primer supplied with the
kit.
Design of PCR primers and RT-PCR conditions for targeted differential
display. Figure 4 shows an alignment of the amino-acid sequences
of 30 previously published fungal CBD regions. From amino-acid
positions 2–8, 25 of these vary only at position 7, involving
sequences GQCGGI/N/QG. Three degenerate oligonucleotide primers
were designed to anneal to DNA sequences coding for these regions.
Primer 1 (5′-GGNCAGTGCGGNGGNATPyGG-3′) anneals
to sequences coding for GQCGGIG, primer 2 (5′-GGNCAGTGC
GGNGGNCAGGG-3′) anneals to sequences coding for GQCGGQG,
and primer 3 (5′-GGNCAGTGCGGNGGNAAPyGG-3′) anneals to
sequences coding for GQCGGNG. On the basis of codon usage in
known P. chrysosporium genes, the codon CAG was employed for
amino-acid Q. Each of these primers was used independently in
DDRT-PCR reactions with a primer which anneals to the poly-A tail
(5′-ATTCGCGGCCGCAGGAT 15 ), which is derived from the Pharmacia
NotI dT primer used for cDNA synthesis. Primer 4
(5′-GCACTGCGAGTAGTA-3′) was used in combination with primer
1 to PCR-amplify the region coding for the CBD from the cbhI.1
gene of P. chrysosporium, cloned in 3E2D (Sims et al. 1994). The
cbhII upstream primer, 5′-CCTCAGCCCTTACTACGC-3′, was as
used for RT-PCR in Tempelaars et al. (1994) with an annealing temperature
of 55°C. To prevent primers 1–3 acting as RAPD primers,
generating PCR products in the absence of any other primer, an annealing
temperature of 65°C was employed. The RT-PCR conditions
were: one cycle of 94°C for 1 min, 65°C for 1 min, 72°C for 2 min;
30 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 1.5 min, and a
cycle of 72°C for 10 min.
Results
Isolation of cDNA sequences which hybridise
to the CBD-encoding region of cbhI.1
from P. chrysosporium ME446
71
P. chrysosporium was grown for 4 days at 37°C in medium
containing either Avicel, CMC, or oatspelt arabinoxylan.
The rationale for choosing these C sources is that Avicel
is commonly regarded as an exocellulase substrate, CMC
as an endocellulase substrate, while xylan is the major component
of hemicellulose; the expression of different components
of the lignocellulolytic system has been shown to
occur after growth on each (Broda et al. 1995). cDNA was
synthesised from poly (A) + mRNA prepared from mycelia
grown on each medium and, in each case, 50 ng was
used in PCR reactions containing either CBD primer 1, 2
or 3, each in combination with an oligo-dT primer which
anneals specifically to the poly-A tail of cDNA.
Initially, however, to test whether, under all conditions,
intact mRNA had been extracted and converted to cDNA,
RT-PCR was performed using a primer which anneals to
the cbhII gene of P. chrysosporium in combination with
the oligo-dT primer. This cellulase gene is expressed after
growth on Avicel, CMC, and xylan (Tempelaars et al. 1994)
and thus cDNA derived from it should be detected in each
sample. This proved to be the case, and a PCR product of
a size expected from cbhII cDNA using these primers, approximately
1.4 kb, was generated from each cDNA sample,
whereas no such product was generated from genomic
DNA (Fig. 1).
To test whether the products amplified, using CBD
primers 1, 2 or 3 with the oligo-dT primer, contained sequences
which share homology with a CBD-encoding region,
each population of PCR products was separated by
gel electrophoresis. These were then Southern blotted and
hybridised, using low-stringency conditions, to a probe derived
from the P. chrysosporium cbhI.1 gene. To prepare

72
Fig. 1 PCR amplification with a primer which anneals to the cbhII
gene of P. chrysosporium, in combination with the oligo-dT primer,
using cDNA prepared after growth on Avicel (2), CMC (3) or xylan
(4), or genomic DNA (1) as the template. The size of the cbhII cDNAamplification
product in base pairs is given to the right of the
panel
Fig. 2A,B PCR amplification products (the left half of each panel),
generated using either CBD-specific primer 1 (panel A) or primer
2 (panel B) each in combination with the oligo-dT primer. Templates
were either genomic DNA from P. chrysosporium (lane 1), or
cDNA prepared after growth on either Avicel (2), CMC (3) or xylan
(4), or no template (negative control; 5). The right half of each panel
shows Southern hybridisation, under low-stringency conditions,
of DNA transferred from each of these agarose gels to a probe derived
from the CBD-encoding region of cbhI.1 from P. chrysosporium.
Size markers are given in base pairs to the right of each panel
this probe, primers 1 and 4 were used to amplify from position
1452 to 1548, containing the CBD-encoding region
of the cbhI.1 cDNA sequence cloned into plasmid 3E2D
(Sims et al. 1994). Amplification products obtained from
cDNA prepared after growth on either Avicel or CMC, using
either primer 1 or primer 2, strongly hybridised to sequences
of between 200 and 400 bp (Fig. 2). However,
only weak hybridisation was observed to xylan-derived
PCR products. In all cases, amplification products were
obtained which failed to hybridise to the probe (Fig. 2).
Only very weak hybridisation was observed to amplification
products obtained using primer 3, and these products
were thus excluded from further analyses (data not shown).
To isolate sequences which hybridise to the CBD probe,
each population of amplification products, obtained after
amplification with either primer 1 or 2 from cDNA derived
from P. chrysosporium grown on Avicel, CMC or xylan,
was shotgun-cloned into the vector pGEM-T. One-hundred
white colonies from each cloning event were screened by
low-stringency hybridisation to the CBD probe. Several
strongly hybridising clones were obtained from cDNAs
amplified after growth on either Avicel or CMC. However,
only weakly hybridising clones were obtained from
cDNAs amplified after growth on xylan. Plasmid DNA was
prepared for sequence analysis from both strongly and
weakly hybridising colonies.
Sequence analysis of cloned cDNAs which hybridise
to the CBD probe
Sequences derived from weakly hybridising clones contained
no open reading frames (ORFs) which shared identity
with known fungal CBDs (data not shown). However,
such sequences were obtained from all strongly hybridising
colonies. Using primer 1 with the oligo-dT primer,
three different CBD-encoding sequences were identified
(Fig. 3). Two of these contain sequences matching the previously
published cbhI.1 and cbhI.2 genes (Fig. 3 A and B
respectively). However, the third sequence obtained is
novel (Fig. 3 C). The lengths of the sequences varied, from
220 bp in the case of the region amplified from cbhI.1, to
approximately 370 bp in the case of ac1 (Fig. 3 C). This
was expected, as the CBD probe had hybridised to fragments
of 200–400 bp (see above; Fig. 2 A). Variation in
sequence length was most pronounced in amplification
products derived from cbhI.1; four sites of polyadenylation
were observed (Fig. 3 A), resulting in fragments from
220- to 260-bp long.
Two different classes of sequences containing CBD-encoding
ORFs were obtained using primer 2 with the oligodT
primer. Each of these represents a novel sequence
(Fig. 3 D and E). Clones containing sequence ac2 were approximately
210-bp long, whereas those containing cmc1
were approximately 240-bp long. Again, these sizes are in
agreement with the hybridisation reported in Fig. 2 B.
Table 1 shows the number of each class of sequence obtained
as a percentage of the colonies screened by hybridisation.
Sequences matching cbhI.1 and cbhI.2, and novel
sequences ac1 and ac2, were all amplified from cDNAs
prepared after growth on both Avicel and CMC. In contrast,
sequence cmc1 was only detected in cDNAs prepared
after growth on CMC. With the exception of cmc1, the sequence
differences between clones suggested that both alleles
of each sequence were identified (Fig. 3). To allow
for differences arising from PCR, such allelic differences
were based on consistent nucleotide differences being observed
in two or more clones of each class of transcript for
Table 1 Number of clones (%) isolated of each CBD-encoding sequence
Sequence Number of clones sequenced (%)
Avicel CMC Xylan
cbhI.1 26 21 0
cbhI.2 16 19 0
ac1 2 3 0
ac2 10 7 0
cmc1 0 12 0

73
Fig. 3 A–E DNA sequences
of each of the five classes of
amplified cDNA fragments
containing CBD-encoding regions
(A–E), where A = cbhI.1,
B = cbhI.2, C = ac1, D = ac2
and E = cmc1. Differences
between alleles are indicated by
underlining and the alternative
base is given beneath. Underlining
of a base with a star (*)
beneath indicates alternative
positions at which the poly-A
tail is added. The predicted
amino-acid sequence is shown
above the corresponding coding
sequence (capital letters). The
sequence in bold represents a
sequence present in one allele
of the cbhI.2 gene (B) which is
absent in the other. Alternative
stop codons in differentially
spliced transcripts of each allelic
variant of cbhI.2 (B) are indicated
in both bold and italics

from P. chrysosporium, or from unspliced transcripts of
cbhI.1 and cbhI.2. Firstly, more abundant classes of transcript
may out-compete rarer classes in the PCR. Such competition
may explain the failure to detect unspliced transcripts
of cbhI.1 and cbhI.2, as the fully spliced transcripts
are more abundantly synthesised after growth on these substrates
and may thus have out-competed them (Sims et al.
1994; Birch et al. 1995). Such a bias of DDRT-PCR towards
high copy number cDNAs has been demonstrated
previously (Bertioli et al. 1995). Secondly, there may be
insufficient identity between the degenerate primers and
the CBD-encoding regions within the cellulase genes. This
is the case for the cbh1-2 gene, which contains a region
coding for SQCGGLG; this differs from equivalent sequences
chosen for the design of primers 1, 2 and 3 and,
indeed, is different to analogous regions in any other
known fungal CBD (Fig. 4). Thirdly, the CBD-encoding
regions of all sequences isolated in this study are located
at the 3′ ends of the ORF. In the case of cbhII, this region
is present at the 5′ end of the ORF. Use of a specific primer
for cbhII confirmed that cDNAs derived from this gene
were present in samples prepared after growth on Avicel,
CMC, and xylan. However, when using CBD-specific
primer 1, cbhII cDNA templates were out-competed in all
PCR reactions by templates yielding smaller amplification
products, including those from cbhI.1 and cbhI.2 in the
cases of Avicel and CMC. Although such competition may
be due simply to different levels of gene expression, the
abundance of cbhII templates relative to those from cbhI.1
and cbhI.2 may also be effected by the efficiency of cDNA
synthesis, which is primed from the 3′ end of mRNA. In
addition, the larger PCR product predicted from cbhII
cDNA may be amplified less efficiently than the small PCR
products isolated in this study. Both of these factors should
be considered when designing degenerate primers to target
a family of genes. It is thus recommended that conserved
sequences should be sought which are present at
only one location in all known genes in the family and that
the location should be as near to the 3′ end as possible.
Interestingly, partial protein sequence from one of the
EGs produced by P. chrysosporium, EG1, has revealed an
N-terminal CBD containing the sequence GQCGGIG
(Uzcategui et al. 1991 b), identical to those encoded by
equivalent nucleotides in cbhI.1, cbhI.2 and cbhII, from
which primer 1 was designed. As was the case for cbhII,
however, cDNAs coding for this gene were also not detected.
Further work will be required to identify the genes from
which the three novel cDNAs containing CBD-encoding
regions are derived. However, they may originate from
transcripts of the partially characterised cbh1-5 and
cbh1-6 (Covert et al. 1992 b) genes, or from the gene that
is predicted to code for both EG38 and EG36 (Uzcategui
et al. 1991 a). Indeed, although Northern analysis would
be required to confirm patterns of regulation for these
genes, one sequence isolated in this study, cmc1, was amplified
only from cDNA prepared after growth on CMC,
which is commonly regarded as an EG substrate. In addition,
a cellulose-binding β-glucosidase has been purified
from cellulose-degrading cultures of P. chrysosporium and
this too may contain a CBD (Lymar et al. 1995). However,
not all cellulolytic enzymes that bind to microcrystalline
cellulose contain a conventional fungal CBD; this is the
case for cellobiose dehydrogenase from P. chrysosporium
(Li et al. 1996).
In conclusion, the targeted DDRT-PCR approach described
here has been successfully applied to the isolation
of novel, abundant cDNA sequences containing regions
coding for CBDs and has also confirmed previous observations
on the regulation of known genes containing such
regions. Although not a comprehensive screen, the speed
of analysis of this method makes it attractive for an initial
study of more highly expressed genes from fungi which
contain such regions. The requirement for only small quantities
of mRNA means that a broad number of growth conditions
can easily be studied in parallel. In addition, this
approach could readily be adapted to the investigation of
other gene families, provided sufficient homology exists
between related sequences for a general probe to be developed
for the hybridisation-screening of cloned cDNAs.
Failing this, additional conserved regions should be sought
within the amplified region which can be used to design
degenerate primers for screening by nested PCR.
Acknowledgements This work was supported by grant RO361
from the Scottish Office Agriculture, Environment and Fisheries Department.
References
75
Béguin P, Aubert J-P (1994) The biological degradation of cellulose.
FEMS Microbiol Rev 13:25–58
Bertioli DJ, Schlichter UHA, Adams MJ, Burrows PR, Steinbiß
H-H, Antoniw JF (1995) An analysis of differential display shows
a strong bias towards high copy number mRNAs. Nucl Acids Res
23:4520–4523
Birch PRJ, Sims PFG, Broda P (1995) Substrate-dependent differential
splicing of introns in the regions encoding the cellulose-binding
domains of two exocellobiohydrolase I-like genes in P. chrysosporium.
Appl Environ Microbiol 61:3741–3744
Broda P, Birch PRJ, Brooks PR, Sims PFG (1995) PCR-mediated
analysis of lignocellulolytic gene transcription by Phanerochaete
chrysosporium: substrate-dependent differential expression
within gene families. Appl Environ Microbiol 61:2358–2364
Broda P, Birch PRJ, Brooks PR, Sims PFG (1996) Lignocellulose degradation
by Phanerochaete chrysosporium: gene families and gene
expression for a complex process. Mol Microbiol 19: 923–932
Covert SF, Vanden Wymelenburg A, Cullen D (1992 a) Structure,
organisation and transcription of a cellobiohydrolase gene cluster
from Phanerochaete chrysosporium. Appl Environ Microbiol
58:2168–2175
Covert SF, Bolduc J, Cullen D (1992 b) Genomic organisation of a
cellulase gene family in Phanerochaete chrysosporium. Curr
Genet 22:407–413
Dalbøge H, Heldt-Hansen HP (1994) A novel method for efficient
expression cloning of fungal enzyme genes. Mol Gen Genet
243:253–260
Gilkes NR, Henrissat B, Kilburn DG, Miller RC, Warren RAJ (1991)
Domains in microbial β-1,4-glycanases: sequence conservation,
function and enzyme families. Microbiol Rev 55:303–315
Gold M H, Alic M (1993) Molecular biology of the lignin-degrading
basidiomycete Phanerochaete chrysosporium. Microbiol
Rev 57:605–622

76
Henrissat B, Bairoch A (1993) New families in the classification of
glycosyl hydrolases based on amino-acid sequence similarities.
Biochem J 293:781–788
Hoffrén A-M, Teeri TT, Teleman O (1995) Molecular dynamics simulation
of fungal cellulose-binding domains: differences in molecular
rigidity but a preserved cellulose-binding surface. Protein
Eng 8:443–450
Li B, Nagalla SR, Renganathan V (1996) Cloning of a cDNA encoding
cellobiose dehydrogenase from Phanerochaete chrysosporium.
Appl Environ Microbiol 62:1329–1335
Liang P, Pardee AB (1992) Differential display of eukaryotic messenger
RNA by means of the polymerase chain reaction. Science
257:967–970
Lymar ES, Li B, Renganathan V (1995) Purification and characterisation
of a cellulose-binding β-glucosidase from cellulose-degrading
cultures of Phanerochaete chrysosporium. Appl Environ
Microbiol 61:2976–2980
Margolles-Clark E, Tenkanen M, Soderlund H, Pentilla M (1996)
Acetyl xylan esterase from Trichoderma reesei contains an active-site
serine residue and a cellulose-binding domain. Eur J Biochem
237:553–560
Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous
fungi. Lett Appl Microbiol1:17–20
Rasmussen G, Mikkelson J, Schülein M, Hagen F, Hjort C, Hastrup
S (1991) A cellulase preparation comprising an endoglucanase
enzyme. World Pat Appl WO 9117243
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a
laboratory manual. Cold Spring Harbour Laboratory, Cold Spring
Harbour, New York
Sheppard PO, Grant FJ, Oort PJ, Sprecher CA, Foster DC, Hagen
FS, Upshall A, Mcknight GL, O’Hara PJ (1994) The use of conserved
cellulase family specific sequences to clone cellulase homologue
cDNAs from Fusarium oxysporum. Gene 150:163–167
Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S, Myambo
K, Innis M (1983) Molecular cloning of exocellobiohydrolase
I derived from Trichoderma reesei strain L27. Bio/Technol
1:691–696
Sims PFG, Soares-Felipe MS, Wang Q, Gent ME, Tempelaars CAM,
Broda P (1994) Differential expression of multiple exocellobiohydrolase
I-like genes in the lignin-degrading fungus Phanerochaete
chrysosporium. Mol Microbiol 12:209–216
Ståhlbrand H, Saloheimo A, Vehmaanpera J, Henrissat B, Pentilla
M (1995) Cloning and expression in Saccharomyces cerevisiae
of a Trichoderma reesei β-mannanase gene containing a cellulose-binding
domain. Appl Environ Microbiol 61:1090–1097
Tempelaars CAM, Birch PRJ, Sims PFG, Broda P (1994) Isolation,
characterisation and analysis of the expression of the cbhII gene
of Phanerochaete chrysosporium. Appl Environ Microbiol
60:4387–4393
Uzcategui E, Johansson G, Ek B, Pettersson G (1991 a) The 1,4-β-
D-glucan glucanohydrolases from Phanerochaete chrysosporium.
Reassessment of their significance in cellulose degradation
mechanisms. J Biotechnol 21:143–160
Uzcategui E, Ruiz A, Montesino R, Johansson G, Pettersson G
(1991 b) The 1,4-β-D-glucan cellobiohydrolases from Phanerochaete
chrysosporium: a system of synergistically acting enzymes
homologous to Trichoderma reesei. J Biotechnol 19:
271–286