Enzyme and Microbial Technology 40 (2006) 51–56

Bacterial biofilm removal using fungal enzymes

Belén Orgaz a , Juliana Kives a , Ana M. Pedregosa b , Inmaculada F. Monistrol b ,
Fernando Laborda b , Carmen SanJosé a,∗
a Department of Nutrition, Food Science and Technology, Fac. Veterinaria, University Complutense of Madrid, 28040 Madrid, Spain
b Department of Microbiology, Fac. Farmacia, University Alcala de Henares, 28801 Alcala, Spain

Received 18 March 2005; received in revised form 14 October 2005; accepted 17 October 2005

Abstract
Three fungal strains of Aspergillus niger, Trichoderma viride and Penicillium spp., were grown on four alternative plant polysaccharides as C-
sources, to induce enzymes able to degrade the bacterial biofilm matrix, for industrial cleaning purposes. Gum arabic and pectin were the C-sources
providing supernatants with better Pseudomonas fluorescens biofilm removal ability. Comparable efficiencies, however, could be achieved with
different enzymes, suggesting attacks to alternative points or structures of the biofilm matrix.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Biofilm removal; EPS degradation; Polysaccharidases; A. niger

1. Introduction be used as well to degrade bacterial biofilm matrices. There

When bacterial cells approach inert surfaces, they first get
bound to the substratum by relatively weak forces involving
their external structures (flagella, fimbriae and/or capsular com-
ponents). When the cells remain thus attached for some time,
they secrete sticky extracellular substances (EPS) forming a
matrix gel that embeds several layers of cells, as the biofilm
matures. This matrix is known to include mainly polysaccha-
rides, besides of proteins and nucleic acids; it may presumably
contain lipids, mineral ions and various cellular debris as well
[1]. Composition of those biofilm polysaccharides is still insuf-
ficiently known, but available data on planktonic EPS and a few
biofilm EPS, suggest that some of their monomers are identical
or similar to those in plant cell-wall materials [2]. In fact, some
bacterial exopolysaccharides have been reported to be accept-
able substrates for enzyme mixtures from non-bacterial sources
[3].
Many fungi can degrade complex plant cell-wall material,
by secreting a large variety of enzymes. This versatility makes
commercial polysaccharide-degrading enzyme mixtures to have
a widespread use in very different fields, such as fruit pro-
cessing [4] or wastewater treatment [5]. They could possibly
∗ Corresponding author. Tel.: +34 91 3943746; fax: +34 91 3943743.
E-mail address: serran@vet.ucm.es (C. SanJosé).
are some precedents on the use of commercial enzyme prepa-
rations for biofilm removal [6,7]. Preparations with different
polysaccharide-degrading activity profiles, can be obtained by
induction of different enzymes with appropriate culture sub-
strates [8], a practice that can increase those enzyme yields by
several fold [9–12]. The aim of this work was to obtain super-
natants from fungal cultures in which enzyme induction was
attempted with different plant polysaccharides, that could some-
what resemble those in biofilm EPS from Pseudomonas. Several
carbohydrate-degrading enzyme activities were determined in
those supernatants, to find out which of them were better asso-
ciated with biofilm removal efficiency.
2. Material and methods
2.1. Microorganisms and culture conditions
Aspergillus niger CECT 2574 and strains of Trichoderma viride and Peni-
cillium spp. H18, this one isolated from brown algae, belong to the collection
of the Department of Microbiology of the University of Alcalá de Henares,
Madrid, Spain, and were maintained in dextrose and potato agar, containing:
potato infusion (4 g/L), dextrose (20 g/L), agar (15 g/L), where they were incu-
bated at 28 ◦ C until adequate sporulation; filtered spores were stored at 5 ◦ C.
Pseudomonas fluorescens B52, originally isolated from refrigerated raw milk,
was kept at −25 ◦ C in Trypticase Soy Broth containing 15% glycerol.
For fungal growth, the following minimum medium [13] was used:
NaNO3 (6.0 g/L), KCl (0.52 g/L), KH2 PO4 (1.52 g/L) and MgSO4 ·7H2 O
(0.5 g/L), which was supplemented with a trace mineral solution [14]

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.10.037
52 B. Orgaz et al. / Enzyme and Microbial Technology 40 (2006) 51–56

containing: FeSO2 ·7H2 O (1.0 mg/L), ZnSO4 ·H2 O (8.8 mg/L), CuSO4 ·5H2 O emission. Alanine was used as standard. One proteolytic unit produces 1 ␮mol
(0.4 mg/L), MnSO4 ·4H2 O (0.15 mg/L), NaB4 O7 ·10H2 O (0.1 mg/L) and of alanine/min.
(NH4 )6 Mo7 O24 ·4H2 O (0.05 mg/L). pH was adjusted to 6.5. Two C-sources
were added: 0.2% glucose as growth initiator and 0.8% of one of the follow- 2.3. P. fluorescens biofilm formation and removal
ing polysaccharides: pectin from apples, low viscosity sodium alginate from
brown algae, gum arabic from acacia tree and xylan from oat spelts, all from P. fluorescens B52 was cultured in mineral medium PMS7 -Ca, which
Sigma. A spore suspension to reach a final concentration of 106 spore/mL, contained: N,N-bis-(2-hydroxymetyl)-2-aminoethane sulphonic acid (BES)
was added to each 100 mL shake flask containing 20 mL of medium. Cultures (10.7 g/L); sodium pyruvate (11.00 g/L); dibasic potassium phosphate
were incubated at 28 ◦ C under shaking (200 rpm). Aliquot samples were with- (0.86 g/L); ammonium chloride (0.65 g/L); magnesium sulphate (0.20 g/L); this
drawn daily and the present mycelium was separated by filtration in Whatmant was adjusted to pH 7.0 and, after autoclaving, supplemented with 0.111 g/L of
paper no. 1; its weight, obtained by drying it at 90 ◦ C for 48 h, was used to filter-sterilised calcium chloride. Standard biofilms were obtained by attachment
measure growth. Supernatants were frozen until use. All cultures were run in of bacterial cells on sterile glass coupons (22 mm × 22 mm), clamped vertically
duplicate. to a Teflon carousel, which was placed inside a beaker almost filled with the
culture medium and covered by a lid. Washed cells to reach 107 CFU/mL were
2.2. Enzyme activities inoculated; after 24 h culture at 21 ◦ C, the coupons were withdrawn and dipped
into saline (0.9%, w/v) to discard loosely attached cells, before being used as
All enzymatic activities in culture supernatants were determined at 30 ◦ C. standard biofilm samples in removal tests.
All assays were run in triplicate, standard deviations being below 5%. To assay the biofilm removal efficiency, an standarized P. fluorescens biofilm
Pectin esterase (PE) activity was determined according to Ref. [15]. Reaction coupon was submerged into a Falcon tube with 12 mL of the corresponding
mixtures contained 2.95 mL of 0.1% (w/v) apple pectin solution and 0.068% fungal culture supernatant, to be incubated for 1 h at 30 ◦ C without shaking.
(w/v) bromocresol green, mixed in a proportion 10:1 with pH adjusted to The coupons were then soaked for 1 min in saline (0.9%, w/v), before being
5.1. The reaction was started with 50 ␮L of the enzyme source. After 30 min dried, fixed for 30 min with formaldehyde and stained for 2 min in Coomassie
incubation, absorption was measured at 617 nm; d-galacturonic acid (GA) Blue. Stained coupons were scanned in a Bio-Rad GS-690 densitometer, using
was used as a standard. One PE unit releases 1 ␮mol de GA/min from apple Molecular Analyst software (Bio-Rad Inc.) for image analysis. Biofilm biomass
pectin. removal assays were run in quadruplicate, standard deviations being below 10%.
For determination of polygalacturonase (PG) activity [16], samples were
diluted in 0.05 M sodium acetate buffer (pH 5.5). Reaction mixtures, consist-
ing of 500 ␮L enzyme source and 500 ␮L of 0.5% (w/v) polygalacturonic acid
solution, were incubated for 1 h. The hydrolysis product was quantified by the
increase in reducing end groups [17], using GA as standard. One PG releases
1 ␮mol of GA/min, from polygalacturonic acid.
Pectin lyase (PL) activity was determined according to Ref. [18], with 0.5%
apple pectin solution in phosphate-citrate buffer pH 5.2 (0.1 M dibasic potassium
phosphate and 0.1 M citric acid) as substrate. Reaction mixtures with 2 mL of
substrate solution and 0.1 mL of enzyme source were incubated and the absorp-
tion was monitored at 235 nm for 30 min. One PL unit produces an increase of
0.555 absorption units 235 nm/min.
To assay cellulase (CEL) activity, 1 g of carboximethyl cellulose dissolved
in 100 mL of 100 mM citrate buffer (pH 6.0) was used as substrate [19].
The reaction was started by mixing 0.2 mL of enzyme source with 0.3 mL
of substrate solution, and was stopped after 15 min, by addition of 1 mL of
a color reagent (0.1 M p-hydroxybenzoic acid hydrazide, 0.1 M sodium sul-
phite, 0.02 M calcium chloride, 0.05 M trisodium citrate and 0.5 M sodium
hydroxide, all from Sigma) and immersion of the test tubes in a boiling
water bath for 6 min. After cooling in tap water, absorption was measured at
420 nm, with glucose being used as standard. One CEL unit releases 1 ␮mol of
glucose/min.
Arabinase activity (ARA) was assayed according to Ref. [20], using 0.5%
arabinogalactane in 0.2 M acetate buffer pH 6.0, as substrate. Reaction mixtures,
contained 3.0 mL of the substrate solution, 1.0 mL of enzyme source and two
drops of toluene; the 30 min incubation was stopped by adding 4 mL of 0.2 M
NaOH. Hydrolysis products were quantified by the increase in reducing end
groups [17], using arabinose as standard. One ARA unit generates 1 ␮mol of
arabinose/min, from arabinogalactane.
For determination of alginate lyase (AL) activity, 2 mL of a substrate solu-
tion, prepared by dissolving 0.6 g of sodium alginate in 120 mL of 1 M Tris–HCl
buffer pH 7.5, were mixed with 3 mL of enzyme source [21]. Absorption at
230 nm was monitored every 5 min during incubation, for 30 min. One AL unit
produces an increase of 0.178 absorption units at 230 nm/min.
To determine proteinase activity, the fluorescamine assay was used [22].
One percent bovine seroalbumine in 0.1 M Tris–HCl buffer pH 7.6 was used as
a substrate. 2.3 mL of this solution was mixed with 200 ␮L of enzyme source.
After 20 min of incubation, the reaction was stopped by using trichloroacetic acid
(5%, v/v, in the final mixture). To complete the precipitation of TCA-insoluble
products, tubes were held for 1 h before being centrifuged for 10 min at 7500 rpm.
100 ␮L of the clear supernatant were mixed with 900 ␮L of the buffer and 50 ␮L
of the fluorescamine solution (3.4 mg/mL in dry acetone). Fluorescence intensity Fig. 1. Growth (a) and proteinase activity (b) in supernatant from Aspergillus
was immediately measured, with an excitation wavelength of 390 and 475 nm of niger cultured on: 䊉 gum arabic,  alginate,  xylan or  apple pectin.
 B. Orgaz et al. / Enzyme and Microbial Technology 40 (2006) 51–56 53

3. Results and discussion

Growth data and enzyme activities assayed in the super-

natants of the daily aliquots taken along the 15 day cultures of
A. niger including as C-source either pectin, gum arabic, xylan
or sodium alginate, are shown in Figs. 1 and 2. The same, cor-
responding to T. viride cultures on pectin and Penicillium on
alginate, are in Figs. 3 and 4. Biofilm removal results are shown
in Table 1.

3.1. Proteinase

Proteinase activity did not change significantly along growth

in any of the four A. niger cultures, staying always below
13 U/mL. The highest values were detected at cultures on gum
arabic (Fig. 1b). This could be due to the small amounts of
protein included in gum arabic composition [23]. The lowest val-
ues of proteolytic activity were observed in cultures with apple
pectin as C-source, particularly the first days (Fig. 1). Low pro-
teinase may be one of the reasons for the high pectinesterase
activity registered in the same supernatants, which were very
efficient in biofilm removal (Table 1).

3.2. Cellulase

Cellulase was the activity showing the widest differences

in between cultures and also along growth (Figs. 2a and 4b).
No cellulase activity was observed in the pectin culture
supernants. Highest activity yields (140 U/mL) were observed
in the cultures on xylan, all along growth. Cellulose impurities
contained in the commercial oats xylan preparations (giving
rise, together with other ␤-glucans, to a maximum of 15%
glucose upon acid hydrolysis, according to the supplier) could
act both as substrates and inducers. Oats xylan was the C-source

Fig. 2. Polysaccharide-degrading activities in the same A. niger cultures shown

in Fig. 1 (a) Cellulase (CEL), (b) pectin esterase (PE), (c) polygalacturonase Fig. 3. Growth in supernatant from T. viride cultured on apple pectin () and
(PG), (d) pectin lyase (PL), (e) alginate lyase (AL) and (f) arabinase (ARA). Penicillium H18 cultured on alginate ().
54 B. Orgaz et al. / Enzyme and Microbial Technology 40 (2006) 51–56

providing better A. niger growth (Fig. 1a), probably as a result

of the polysaccharide heterogeneity in the commercial product.
Xylans contain arabinose, d-glucuronic acid and acetylated
sugar units [27]. Due to its compositional complexity, a large
number of enzymes have been observed to have some effect
on xylan depolymerization [28], including endoxylanases,
glucuronidases and acetyl esterases.
The results with xylan could also involve shared regulation
of xylanase/cellulase expression, as observed by Aro et al. [24].
Constitutiveness of cellulase expression, which was suggested
by the low and decreasing, but present, CEL activities in the A.
niger cultures on alginate or gum arabic, was not supported by
the observed CEL absence in the cultures on pectin (Fig. 2a).
May be in this last case, constitutive CEL was repressed. It has
been reported that fungal growth on compounds such as glucose,
experience repression of cellulase production [8]. The glucose
that was added here as growth initiator could have had a negative
effect on CEL production. Trichoderma results seem to support
a constitutive and later repressed CEL production (Fig. 4b).

3.3. Pectin-degrading activities

Interestingly enough, pectin did not induce production of its

degrading enzymes in A. niger, or did it very briefly. Highest
levels of PE were observed on the first day of culture (up to
147 U/mL in Aspergillus and 100 U/mL in Trichoderma cul-
tures) but the activity practically disappeared after the second
day (Figs. 2b and 4c). May be PE is very sensitive to proteoly-
sis. Low polygalacturonase activity was detected in all cultures
(Figs. 2c and 4d); this may be related to the scarcity of PE activ-
ity, possibly not giving a chance for the pectin to be used as a
fungal substrate; growth on this C-source was slow (Fig. 1a).
Endopolygalacturonases in general, and also those from A.
niger, prefer pectins with a low degree of esterification as sub-
strates [25]. Unexpectedly, in our case, the best PG activity levels
provided by this strain of A. niger were with xylan as C-source.
Recently, Bai et al. [9] reported the use of sugar beet pulp (28.7%
pectin in dry weight) for induction of pectinases in cultures of
a selected strain of A. niger, obtaining maximum PE and PL
activities after 4 days culture.
As different agricultural waste products containing pectin
have been used as C-source for induction of pectinases in
fungi [26], it may be that complex substrates, possibly includ-
ing demethylated pectin and/or low proteinase levels, allow for
higher pectin-degrading enzyme production.

3.4. Alginate lyase

Alginate lyase was induced in this strain of A. niger by the

presence of alginate (Fig. 2e), but at very low levels, with a max-
imum value of 0.19 U/mL. When this Penicillium strain, isolated
from brown algae, was cultured on alginate (Fig. 4f), induced
levels of the lyase were only slightly higher (about 0.5 U/mL).

Fig. 4. Proteinase activity (a) and polysaccharide-degrading activities in Tricho- 3.5. Arabinase
derma viride cultured on pectin (), and Penicillium H18 cultured on alginate
(). (b) Cellulase (CEL), (c) pectin esterase (PE), (d) polygalacturonase (PG), This activity was only detected, and at rather low levels,
(e) pectin lyase (PL), (f) alginate lyase (AL). when A. niger was grown in gum arabic (Fig. 2f). This complex
 B. Orgaz et al. / Enzyme and Microbial Technology 40 (2006) 51–56 55

Table 1
Maximal biofilm removal efficiences with chosen fungal supernatants
Culture Culture day Maximum biofilm removal (%) PROT (U/mL) CEL (U/mL) PE (U/mL) PL (U/mL) AL (U/mL)

A. niger (gum arabic) 14 65 ± 5 13 19 nd nd nd

A. niger (alginate) 9 38 ± 7 8 8 nd nd nd
A. niger (xylan) 8 19 ± 6 6 89 nd 0.8 nd
A. niger (pectin) 1 60 ± 5 nd 0.5 147 38 nd
T. viride (pectin) 1 84 ± 2 nd 135 102 0.8 nd
Penicillium H18 (alginate) 8 45 ± 5 4 13 nd nd 0.04

Prot, proteinase; Cel, cellulase; PE, pectinesterase; PL, pectin lyase; AL, alginate lyase; nd, non detected.

exudate from Acacia trees, contains about 24% of arabinose
[23]. As in the case of pectin-degrading enzymes, A. niger pro-
duced more of this activity at the beginning of the culture. Fungal
cultures have been previously found to produce arabinase when
grown on arabinan and in the presence of inducers, such as ara-
binose or arabitol [29,30].
3.6. Use of fungal supernatants to remove biofilms
terial polysaccharides is still scarce. Gum arabic, for instance,
is known to contain 16 ± 5% glucuronic acid, also a compo-
nent of Pseudomonas biofilm EPS (results not shown). It might
thus occur that some enzyme activities induced in A. niger by
gum arabic, different from the ones assayed here, might attack
an structurally important part of the biofilm matrix, involving
glucuronic acid.

All fungal supernatants were tested as cleaning solutions 4. Conclusions

for P. fluorescens biofilms attached to glass surfaces. All the
cleaning incubations were made at 30 ◦ C, though optimum tem-
peratures for the enzymes involved ranged between 25 ◦ C for
the pectinesterase activity and 40 ◦ C for cellulase. Maximum
biofilm removal values and enzymatic activities found in the
corresponding supernatants, are shown in Table 1.
An straight forward conclusion on what enzyme(s) could bet-
ter remove biofilm, does not come up from those results, but
three options seem possible. One is the “high proteinase” alterna-
tive, suggested by old A. niger cultures on gum arabic (65 ± 5%
removal efficiency). The two other options are offered by the
young cultures on pectin, both rich in pectinesterase and poor
in proteinase. Best of those are the Trichoderma preparations,
which were besides rich in cellulase. Those of Aspergillus were
besides relatively rich in pectin lyase. One could conclude, there-
fore, that both proteolytic and carbohydrate-degrading activi-
ties are good for biofilm removal. The combination of both,
however, as it occurs naturally in the cultures of Penicillium
and Aspergillus on sodium alginate, does only achieve par-
tial removal, probably due to proteolytic attack on the other
enzymes. When pectin esterase is not itself degraded, it could
possibly deacetylate a polysaccharide such as alginate in the
biofilm matrix, making it softer and possibly more porous, thus
allowing other enzymes to penetrate the gel framework. Acety-
lation of bacterial polysaccharides is common, and the release
of those substituting groups is known to strongly affect some
physical properties, such as gel firmness [30]. As noted by
O’Toole and Kolter [31], bacterial protein-rich appendages play
an important role in the early steps of cell adhesion to surfaces.
Proteinases, reaching bottom layers of the biofilm, could there-
fore facilitate its detachment even without overall disintegration.
The possibility of removal not being actually (or not singly)
caused by these assayed enzymes cannot be discarded. Other
activities, induced or not, could be present in the tested culture
supernatants, and be active against the biofilm matrix. Knowl-
edge of similarities in components or structures of plant and bac-
On the whole, though the induction attempts failed to pro-
duce high enzyme yields, these fungal strains seem able to
produce enzyme activities (the ones assayed or others) that can
degrade bacterial biofilms to a significant extent. Of the plant
polysaccharides tested as C-sources, pectin provided the culture
supernatants (from T. viride) that removed biofilms from sur-
faces more efficiently. The complexity of the biofilm matrix
material probably allows different alternatives for enzymatic
removal strategy.
References
[1] Flemming HC, Wingender J. Relevance of microbial extracellular poly-
meric substances (EPSs)–Part I: structural and ecological aspects. Water
Sci Technol 2001;43(6):1–8.
[2] Sutherland IW. Microbial exopolysaccharides. In: Dumitriu S, editor.
Polysaccharides, structural diversity and functional versatility. New York:
Marcel Dekker; 2005. p. 431–57.
[3] Sutherland I. Biofilm exopolysaccharides: a strong and sticky framework.
Microbiology 2001;147:3–9.
[4] Lowe DA. Fungal enzymes. In: Arora DK, Elander RP, Mukerji KG, editors.
Handbook of applied micology, vol. 4. New York: Marcel Dekker; 1992.
p. 681–706.
[5] Raj HG, Saxena M, Aliameh A, Mujerki KG. Metabolism of foreign
coumpounds by fungi. In: Arora DK, Elander RP, Mukerji KG, editors.
Handbook of applied micology. New York: Marcel Dekker; 1992. p. 681–
706.
[6] Hahn Berg IC, Kalfas S, Malmsten M, Arnebrant T. Proteolytic degradation
of oral biofilms in vitro and in vivo: potencial of proteases originating from
Euphasia superba for plaque control. Eur J Oral Sci 2001;109:316–24.
[7] Johansen C, Falholt P, Gram L. Enzymatic removal and disinfection of
bacterial biofilm. Appl Environ Microbiol 1997;63:3724–8.
[8] Priest FG. Extracellular enzymes. Aspects of microbiology, vol. 9. Wok-
ingham: Van Nostrand Reinhold; 1984.
[9] Bai ZH, Zhang HX, Qi HY, Peng XW, Li BJ. Pectinase production by A.
niger using wastewater in solid state fermentation for eliciting plant disease
resistance. Bioresour Technol 2004;95:49–52.
[10] Eriksson KEL, Blanchette RA, Ander P. Microbial and enzymatic degra-
dation of wood and wood components. Berlin: Springer Verlag; 1990.
56 B. Orgaz et al. / Enzyme and Microbial Technology 40 (2006) 51–56
[11] Jorgensen H, Morkeberg A, Krogh KBR, Olsson L. Growth and enzyme
production by three Penicillium species on monosaccharides. J Biotechnol
2004;109:295–9.
[12] Olsson L, Christensen T, Hansen KP, Palmqvist EA. Influence of the car-
bon source on production of cellulases, hemicellulases and pectinases by
Trichoderma reesei Rut C-30. Enzyme Microb Technol 2003;33:612–9.
[13] Pontecorvo G, Roper JA, Hemmons LM, Mc Donald KD, Bufón AVJ. The
genetics of Aspergillus nidulans. Adv Genet 1953;5:141–238.
[14] Armita S, McCullogh W, Roberts CF. Analysis of acetate non-utilizing
(acu) mutans in Aspergillus nidulans. J Gen Microbiol 1976;92:
263–82.
[15] Vilariño C, Del Giorgio JF, Hours RA, Cascone O. Spectrophotometric
method for fungal pectinesterase activity determination. Lebensm Wiss
u-Technol 1993;26:107–10.
[16] Blanco P, Sieiro C, Dı́az A, Villa TG. Production and partial characteriza-
tion of an endopolygalacturonase from Saccharomyces cerevisiae. Can J
Microbiol 1994;40:974–7.
[17] Nelson NJ. Colorimetric analysis of sugars. Methods Enzymol 1957;3:
85–6.
[18] Albersheim P. Pectin lyase from fungi. Methods Enzymol 1966;8:
628–31.
[19] Lever M. Colorimetric and fluorometric carbohydrate determination with
p-hydroxybenzoic acid hydrazide. Biochem Med 1973;7:274–81.
[20] Kaji A, Saheki T. Endo-arabinase from Bacillus subtilis F-11. Biochim
Biophys Acta 1975;410:354–60.
[21] Boyd A, Chakrabarty AM. Role of alginate lyase in cell detachment of
Pseudomonas aeruginosa. Appl Environ Microbiol 1994;60:2355–9.
[22] Sarath G, De la Motte R, Wagner F. In: Beynon RJ, Bond JS, editors.
Proteolytic enzymes: a practical approach. New York: Oxford University
Press; 1990. p. 25.
[23] Imeson A. Thickening and gelling agents for food. London: Blackie Aca-
demic and Professional; 1992.
[24] Aro N, Saloheimo A, Ilmén MY, Penttı̈la M. ACEII, a novel transcrip-
tional activator involved in regulation of celullase and xylanase genes of
Trichoderma reesei. J Biol Chem 2001;276:24309–14.
[25] Parenicovà L, Benen JAE, Kester HCM, Visser J. pgaA and pgaB encode
constitutively expressed members of the A. niger endopolygalacturonase
gene family. Biochem J 2000;345:637–44.
[26] Said S, Fonseca MJV, Siéssere V. Pectinase production by Penicillium fre-
quentans. World J Microbiol Biotechnol 1991;7:607–8.
[27] Burchard W. Light scattering from polysaccharides. In: Dumitriu S, editor.
Polysaccharides, structural diversity and functional versatility. New York:
Marcel Dekker; 2005. p. 89–236.
[28] Thompson JA. Molecular biology of xylan degradation. FEMS Microbiol
Rev 1993;104:65–82.
[29] Roche N, Berna P, Desgranges CY, Durand A. Substrate use and produc-
tion of ␣-L-arabinofuranosidase during solid-state culture of Trichoderma
reesei on sugar beet pulp. Enzyme Microb Technol 1995;17:935–41.
[30] Sutherland I. Polysaccharases for microbial exopolysaccharides. Carbo-
hydr Polym 1999;38:319–28.
[31] O’Toole GA, Kolter R. The initiation of biofilm formation in Pseudomonas
aeruginosa WCS365 proceeds via multiple, convergent signalling path-
ways: a genetic analysis. Mol Microbiol 1998;28:449–61.