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Comparison of the hydrolysis of polyethylene terephthalate fibers by a

hydrolase from Fusarium oxysporum LCH I and Fusarium solani f. sp. pisi

Article  in  Biotechnology Journal · March 2007

DOI: 10.1002/biot.200600095 · Source: PubMed

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Biotechnol. J. 2007, 2, 361–364 DOI 10.1002/biot.200600095 www.biotechnology-journal.com

Short Communication

Comparison of the hydrolysis of polyethylene terephthalate

fibers by a hydrolase from Fusarium oxysporum LCH I and
Fusarium solani f. sp. pisi

Thidarat Nimchua1,2, Hunsa Punnapayak3 and Wolfgang Zimmermann1

1Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Leipzig, Germany
2BiologicalScience Ph.D. Program, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
3Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok,

Thailand

The hydrolysis of polyethylene terephthalate (PET) fibers by two fungal hydrolases was investigat- Received 28 June 2006
ed. The hydrolase from a newly isolated Fusarium oxysporum strain (LCH 1) was more efficient in Revised 9 August 2006
releasing terephthalic acid from PET fibers compared to the enzyme from F. solani f. sp. pisi DSM Accepted 5 September 2006
62420 when equal amounts of p-nitrophenyl butyrate-hydrolyzing activity were employed. PET fab-
rics treated under the same conditions with the enzyme from F. oxysporum LCH 1 also showed a
considerably higher increase in hydrophilicity compared to fabrics treated with the enzyme from
F. solani f. sp. pisi DSM 62420.

Keywords: Cutinase · Enzymatic hydrolysis · Hydrolase · Polyethylene terephthalate fibers

Synthetic fibers made of polyethylene terephthalate (PET)
are important textile materials with excellent strength
properties and resistance to abrasion, shrinking and wrin-
kling. However, their high hydrophobicity and limited
flexibility results in disadvantages in processing and
wearing comfort. Strong alkaline solutions are presently
used to increase the hydrophilicity of PET fibers. Howev-
er, an alkaline hydrolysis treatment can result in severe
weight losses of the fibers [1]. The use of hydrolytic en-
zymes for the modification of synthetic fibers, resulting in
their improved hydrophilicity and water absorbency, has
been reported recently [2, 3]. The activity of the enzymes
is limited to the fiber surfaces and the textile treatment
can be performed under mild and compliant conditions.
Besides esterases and lipases, hydrolytic enzymes acting
on plant polyesters such as cutin and suberin have a po-
Correspondence: Professor Wolfgang Zimmermann, Department of
Microbiology and Bioprocess Technology, Institute of Biochemistry,
University of Leipzig, 04103 Leipzig, Germany
E-mail: wolfgang.zimmermann@uni-leipzig.de
Fax: +49-341-97-36798
Abbreviation: PET, polyethylene terephthalate
tential application in biocatalytic fiber modification reac-
tions [4, 5]. The hydrolysis of PET by a fungal cutinase
from Fusarium solani f. sp. pisi and a bacterial hydrolase
from Thermobifida fusca has been demonstrated recently
[6, 7].
However, the reported treatment times of PET with
these enzymes are still too long for an application of this
technology in the textile industry. The identification of
novel enzymes with improved properties will therefore be
necessary. In this contribution, the hydrolysis of PET by a
hydrolase from Fusarium oxysporum LCH 1 obtained by
screening of microbial sources is reported, and the per-
formance of the enzyme is compared with the cutinase
from F. solani f. sp. pisi.
For the isolation of micro-organisms producing PET-
hydrolyzing enzymes, plant samples including leafs, flow-
ers, underground storage organs, and fruits as well as soil
samples were collected at different locations in North-
Thailand. F. solani f. sp. pisi DSM 62420 was obtained from
the German National Resource Center for Biological Ma-
terial, Braunschweig, Germany.
The soil samples were appropriately diluted and
aliquots were spread on agar plates containing mineral
medium supplemented with polycaprolactone (500 mg/L,

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mol. wt. 10 000) as the sole source of carbon [8]. From the
plant materials, small pieces were cut and placed on agar
plates containing mineral medium and polycaprolactone
as described above. The plates were incubated at 30°C for
several days and examined for growth and the formation
of clear zones around colonies indicating the hydrolysis of
polycaprolactone. The isolates producing a clearing zone
on polycaprolactone plates and F. solani f. sp. pisi were
cultivated in the mineral medium described above sup-
plemented with 2 g/L PET yarn (small pieces of untreated
pre-washed PET yarn, a kind gift of Dr. T. Böhme KG,
Geretsried, Germany). The pH was adjusted to 7.0. The
cultures were incubated at 30°C for 21 days. Samples of
culture supernatant from F. oxysporum LCH 1 and F. solani
f. sp. pisi were collected by centrifugation and partially
purified enzyme preparations were obtained by ammoni-
um sulfate precipitation (80% saturation) followed by re-
moval of the salt by dialysis.
Esterase activity in the culture supernatant and en-
zyme preparations was determined with p-nitrophenyl
butyrate as substrate as described previously [4]. One unit
(U) of enzyme activity was defined as the amount of en-
zyme releasing 1 µmol p-nitrophenol per minute.
The release of terephthalic acid from PET was esti-
mated by incubating pieces of PET yarn (2.0 g/L) with
crude enzyme preparations from F. oxysporum LCH 1 and
F. solani f. sp. pisi (20, 40, and 80 U p-nitrophenyl butyrate-
hydrolyzing activity) in 50 mL 0.1 M phosphate buffer,
pH 7.0 at 30°C for up to 168 h. Periodically, aliquots were
removed and the terephthalic acid released was quanti-
20
18
Terephthalic acid released (µg/ml)
16
14
12
10
0
0 24 48
Figure 1. Time course of the release of terephthalic acid from PET yarn by reaction with enzyme preparations (80 U p-nitrophenyl butyrate-hydrolyzing activ-
ity) from F. oxysporum LCH 1 (solid line) and F. solani f. sp. pisi (dotted line).
362 © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Biotechnol. J. 2007, 2, 361–364
fied by fluorimetric determination at 425 nm of hydroxy-
terephthalic acid formed by the reaction of terephthalic
with hydrogen peroxide [9].
Changes in the water adsorption ability of enzyme-
treated PET fabrics were estimated by incubating pre-
washed swatches of PET fabrics (20 × 2 cm, a kind gift of
Amtex Ltd., Barcelona, Spain) with crude enzyme prepa-
rations from F. oxysporum LCH 1 and F. solani f. sp. pisi
(20, 40, and 80 U p-nitrophenyl butyrate-hydrolyzing ac-
tivity) in 50 mL 0.1 M phosphate buffer, pH 7.0 at 30°C for
up to 168 h. The samples were washed in 2 g/L sodium
carbonate solution at 70°C for 30 min to remove adsorbed
protein and the rising height of water in the treated fab-
ric swatches and controls was determined as described
previously [10]. All experiments were carried out in dupli-
cate.
Twenty-two of the fungal isolates hydrolyzed poly-
caprolactone, a synthetic polyester, as indicated by clear-
ing zones detected around colonies growing on agar
plates containing polycaprolactone after 3–5 days of incu-
bation. Polycaprolactone is also depolymerized by the
phytopathogenic fungus F. solani f. sp. pisi, which pro-
duces cutinase, a serine esterase. This enzyme degrades
cutin, a polyester located in the plant cuticle and is also
able to hydrolyze PET [6, 11]. We therefore used poly-
caprolactone hydrolysis as a first screen to identify fungal
isolates producing enzymes that may also be active on
aromatic synthetic polyesters such as PET. When culti-
vated in a liquid medium containing PET as the sole
source of carbon, only 1 of the 22 isolates (LCH 1) and F.
72 96 120 144 168
Reaction time (h)
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20

18
Terephthalic acid released (µg/ml)

16

14

12

10

0
0 20 40 80
Enzyme concentration (U)
Figure 2. Terephthalic acid released from PET yarn after a reaction time of 168 h by different concentrations of enzymes from F. oxysporum LCH 1 (solid
line) and F. solani f. sp. pisi (dotted line).

solani f. sp. pisi were able to grow in this medium. The iso- samples (Table 1). Treatments with inactivated enzymes
late LCH 1 was identified as F. oxysporum based on its did also not result in increased rising heights of water in
growth conditions and spore morphology [12]. The extra- the fabrics. Therefore, the observed changes in water ad-
cellular fluid of the cultures contained 60 mU/mL esterase sorption of the enzyme-treated fabrics cannot be attrib-
activity, determined using p-nitrophenyl butyrate as the uted to protein adsorbed to the fibers and indicate an in-
substrate after 21 days of cultivation. crease in the hydrophilicity of the PET fabric caused by
For comparison of the aromatic polyester-hydrolyzing the hydrolytic action of the enzymes on PET. Compared to
activity of the hydrolases from F. oxysporum LCH 1 and F. F. solani f. sp. pisi, treatments with the enzyme from F.
solani f. sp. pisi, PET fibers were incubated with 80 U oxysporum LCH 1 resulted in considerably stronger in-
p-nitrophenyl butyrate-hydrolyzing activity for 168 h creases in hydrophilicity. These results are in agreement
(Fig. 1). Determination of the amount of terephthalic acid with previous reports on the increase in hydrophilicity of
released showed a considerablely higher activity of the
enzyme from F. oxysporum LCH 1 on PET compared to F.
solani f. sp. pisi. An enzyme concentration dependence of Table 1. Water adsorption of PET fabrics treated with different amounts of
the activity of the hydrolases on PET was observed when hydrolases from F. oxysporum LCH 1 and F. solani f. sp. pisi
the fibers were incubated with different amounts of en-
Treatment Rising height of
zymes from F. oxysporum LCH 1 and F. solani f. sp. pisi
water (mm/10 min)
(Fig. 2). The results of the hydrolysis of PET by the two
fungal enzymes suggest that the amount of terephthalic Water (control) 11
acid liberated depends on the type of hydrolase employed. Enzyme preparation from F. oxysporum LCH 1
Since crude enzyme preparations were used, it is still pos- Inactivated enzyme 14
sible that the hydrolase from F. oxysporum LCH 1 was not 20 U 24
as effective on p-nitrophenyl butyrate as the F. solani en- 40 U 31
zyme and a higher dose based on the amount of protein of 80 U 57
Enzyme preparation from F. solani f. sp. pisi
the F. oxysporum LCH 1 preparation caused the higher ac-
Inactivated enzyme 14
tivity in the PET hydrolysis experiments.
20 U 14
The water adsorption ability of PET fabrics treated
40 U 22
with enzyme preparations from F. oxysporum LCH1 and F.
80 U 36
solani f. sp. pisi was increased compared to untreated

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PET fabrics following treatment with enzymes active on
PET substrates [10, 13].
Aromatic polyesters have been considered previously
as very resistant to degradation by hydrolytic enzymes
[14]. However, the results obtained with esterases and
cutinases from various fungi and bacteria [3–7, 13, 15]
provide accumulating evidence for the hydrolysis of ester
bonds in PET catalyzed by microbial hydrolases.
The results obtained in this study show that efficient
novel enzymes can be identified for the modification and
hydrolysis of aromatic synthetic polyesters with potential
use in biocatalytic textile finishing processes.
The authors thank the Thailand Research Fund (Royal
Golden Jubilee Program) for financial support to T. N. Dr.
P. Otto, Institute of Biology I, University of Leipzig, is kind-
ly acknowledged for the identification of F. oxysporum
LCH 1.
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