Biotechnological modification of polyester surfaces

V.A. Nierstrasz, Aachen-Dresden Int. Textile Conf., 26-27 Nov. 2009Ghent University, Faculty of Engineering, Department of Textiles

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Biotechnological modification of polyester surfacesV.A. Nierstrasz1, I. Donelli2, G. Freddi2, P.F. Smet3, D. Poelman3,L. Van Langenhove1, P. Kiekens1

1 Department of Textiles, Ghent University,Technologiepark 907, 9052 Zwijnaarde (Gent)Belgium

2 Stazione Sperimentale per la Seta, Milano, Italy3 Dept. of Solid State Sciences, Ghent University, Gent, Belgium


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Global market info White Biotechnology

Market shares (2007) industrial enzymes:

Food & feed industry ~49% Technical enzymes (detergents, textile, paper en pulp, …) ~51 %.

Enzymes for industrial applications in textiles ~10 % of the industrial enzymesEnzymes for detergents approx. 34 % of the industrial enzymes

75% produced in Europe.

Novozymes and Genencor/Danisco world leaders industrial enzymes.


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Textile and Polymer Biotechnology

• Vast potential in the industrial production‣ ~200 million euros enzymes in textile processing

today mainly amylases, cellulases increasing importance and potential and new developments

• pectinases, catalases, proteases, cutinases, … • chemo-enzymatic approaches

‣ Biopolymers PLA, PHA, chitosan, …

‣ Advanced applications biosensors, medical applications, …

‣ Novel technology and processes

‣ BIOTEX roadmap (EuropaBio, Euratex)


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Markets & applications

Industry Enzyme class Application Detergent (laundry and dish wash) Protease Protein stain removal

Amylase Starch stain removal Lipase Lipid stain removal Cellulase Cleaning, color clarification, anti-redeposition (cotton) Mannanase Mannanan stain removal (reappearing stains)

Starch and fuel Amylase Starch liquefaction and saccharification Amyloglucosidase Saccharification Pullulanase Saccharification Glucose isomerase Glucose to fructose conversion Cyclodextrin-glycosyl Transferase Cyclodextrin production Xylanase Viscosity reduction (fuel and starch) Protease Protease (yeast nutrition – fuel)

Food (including dairy) Protease Milk clotting, infant formulas (low allergenic), flavor Lipase Cheese flavor Lactase Lactose removal (milk) Pectin methyl esterase Firming fruit-based products Pectinase Fruit-based products Transglutaminase Modify visco-elastic properties

Baking Amylase Bread softness and volume, flour adjustment Xylanase Dough conditioning Lipase Dough stability and conditioning (in situ emulsifier) Phospholipase Dough stability and conditioning (in situ emulsifier) Glucose oxidase Dough strengthening Lipoxygenase Dough strengthening, bread whitening Protease Biscuits, cookies Transglutaminase Laminated dough strengths

Animal feed Phytase Phytate digestibility – phosphorus release Xylanase Digestibility β-Glucanase Digestibility

Beverage Pectinase De-pectinization, mashing Amylase Juice treatment, low calorie beer β-Glucanase Mashing Acetolactate decarboxylase Maturation (beer) Laccase Clarification (juice), flavor (beer), cork stopper treatment

Textile Cellulase Denim finishing, cotton softening Amylase De-sizing Pectate lyase Scouring Catalase Bleach termination Laccase Bleaching Peroxidase Excess dye removal Protease Degumming of silk, sand washing of silk, wool finishing Hemicellulase Flax retting Pectinase Flax retting Xylanase Flax retting

Pulp and paper Lipase Pitch control, contaminant control Protease Biofilm removal Amylase Starch-coating, de-inking, drainage improvement Xylanase Bleach boosting Cellulase De-inking, drainage improvement, fiber modification

Fats and oils Lipase Transesterification Phospholipase De-gumming, lyso-lecithin production

Organic synthesis Lipase Resolution of chiral alcohols and amides Acylase Synthesis of semisynthetic penicillin Nitrilase Synthesis of enantiopure carboxylic acids

Leather Protease Unhearing, bating Lipase De-pickling

Personal care Amyloglucosidase Antimicrobial (combined with glucose oxidase) Glucose oxidase Bleaching, antimicrobial Peroxidase Antimicrobial

Textile Cellulase Denim finishingCotton softening

Amylase De-sizingPectate lyase ScouringCatalase Bleach terminationLaccase BleachingPeroxidase Excess dye removalProtease Degumming of silk

Sand washing of silkWool finishing

Hemicellulase Flax rettingPectinase Flax rettingXylanase Flax retting


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Synthetic fibers form an important part of the textile industry: Global annual production (2008) of fibers and yarns was estimated (Oerlikon) to be:• 30.3 million tons of polyester• 3.6 million tons of polyamide• 1.9 million tons of acrylics• 23.6 million tons of cotton

The production volume of polyester fibers and yarns justifies

research into effective production.

Global market info synthetic fibers


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Textile and Polymer BiotechnologyThe importance and potential of biotechnology in textiles has been assessed in the last 10-20 years.

• Biocatalysis has already proven to be very profitable in industrialtextile pre-treatment processes of natural fibers.

• Application of enzymes is not limited to biological materials, relativelyrecently it has been demonstrated enzymes are able to modify thesurfaces of synthetic textile materials as well (PET, PA, ..).

• Enzymes are specific towards a certain reaction


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Textile and Polymer Biotechnology

• Biotechnology in textile and fiber engineering

‣ Biotechnological surface modification and functionalisation of textilematerials synthetic, biopolymers, natural

surface engineering characteristics

material

functionalityBiotechnology


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Textile and polymer biotechnology

Objective and aimBiotechnologically functionalised (bio-inspired) textile materials

Open possibility for design of novel biotechnological production processes for textiles that exhibit the desired functionalities

Focus• Enzymatic of functionalisation of textile fibers/surfaces.• Specific enzymatic surface modification to obtain

functional structured surfaces.


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Biotechnological surface modification and functionalisation

Motivation• Manipulation of surface characteristics is of fundamental importance in the production of functional textiles.

• Research efforts often focus on chemical or physical modification or structuring of the surfaces.

• The introduction of functionalities using biotechnology is arelatively unexplored and modern scientific area. The advantageof enzymes over other technologies is their high specificitytowards a certain substrate.

Need for a concerted multi-disciplinary approach.


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Biotechnological surface modification and functionalisation Challenges

• Control of the enzymatic reaction at the specific time and length scales

• Innovative enzymatic processes to functionalize textile surfaces

• Characterization of structured surfaces/functional material at alltime and length scales (e.g. AFM, SEM, XPS, confocal microscopy, FT-IR, …).

• Functionalities have to be related to the material properties, surface characteristics and materials engineering. Methodologies will be developed to establish the interrelationships between parameters.

prerequisite to achieve thea structuring effect orcomplex functionalities


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Biotechnological surface modification and functionalisation: Topics• Selection of enzymes and synthetic materials for modification/functionalisation.

• New enzymes via (research) cooperation's.

• Control of enzymatic reaction at correct time and length scales.

• Characterization of (nano-)structured textile surfaces/functional material at alltime and length scales (e.g. AFM, XPS, confocal microscopy, FT-IR, …).

• Development suitable technology for enzymatic modification/functionalisation.

• Development and application of suitable minimal application techniquese.g. inkjet technology.


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Enzymatic modification / functionalisation of PETSynthetic materials have generally been considered resistantto biological degradation: cutinase, lipase, laccase

Model substrates, films, fabrics, yarns, fibers, oligomers

Research on enzymatic PET hydrolysis in by various research groups, mainly in EU

Industry• Inotex• Genencor• Novozymes


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Cutinase in modification of PET

• hydrolysis of oligomers • degradation (recycling)• surface modification to improve (yarns, films, ….)

• hydrophilicity (wetting and absorbency)electrostatic chargedyeabilitywashabilitywear comfort

• improved functionalisationcoatingintroduction functional groups

• not to change the bulk properties


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Achieving hydrophilicity

Incorporate hydrophilic groups Co-polymerization Co-crystallisation

Generate hydrophilic groups Plasma treatments Alkali treatment Enzymes

– strength, pitting corrosion– hydroxide– temperature+ durable+ incubation time+ mild reaction conditions+ not affecting bulk properties+ durable– longer incubation time

– affecting bulk properties


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Enzymatic hydrolysis of PET

CO

OCH2CH2OCO

CO

O CO

OCH2CH2OCO

cutinasepolyesteraselipase

1 2 3 4

2 3

1 4

1 3 2 4

TPA (terephthalic acid )

BHET (bis(2 hydroxyethyl) terephthalate)or MHET (mono(2 hydroxyethyl) terephthalate)


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PET hydrolysis

Tota

l con

cent

ratio

n of

sol

uble

reac

tion

prod

ucts

(mm

ol/l)

Incubation time (hrs)Total main peak areas of all cutinase and lipase treatments in time;

▲:APET-cutinase, ♦:GPET-cutinase, •:OPET-cutinase, ■:APET-lipase, +:GPET-lipase, ×: OPET-lipase

Crystallinity:APET 4.1%OPET 48.2%GPET 13.7%

0

500

1000

0 50 100

Vertommen, M.A.M.E., Nierstrasz, V.A., Veer, M.V.D.,and Warmoeskerken, M.M.C.G., J. Biotechnol. 120(4), 376-386, 2005.


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MutantsDirected evolutionEnzyme engineering

• Serine hydrolase• 45/30/30 Å• Ser 120, Asp175, His188• No interfacial activation• Absence of flap

Cutinase in PET modification Improving cutinase (stability and reaction rate)


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Cutinase in PET modification, so far ….

Araújo et al. J. Biotechnology 128, 849-857, 2007

Improving cutinase (reaction rate)

Increased activity

• Enlarged active site viasite directed mutagenesis

• L182A: Ala instead of Val• more open, less restrained active site• model compound betteraccommodated

• 5 fold increase (TPA release fromPET in 24 hours)


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Enzymes or NaOH

untreated cutinase 20 U/mg 2 h, 40°C NaOH, 1M 2 h, 40°C

untreated cutinase 4 U/mg 74 h, 30°C

Billig, s., Agrawal, P.B.., Birkemeyer, C.,Nierstrasz, V.A., Warmoeskerken,M.M.C.G., and Zimmermann, W., in prep.

Donelli, I., Taddei, P., Smet, P.F., Poelman, D.Nierstrasz, V.A. and Freddi, G., Biotechnologyand Bioengineering, 103(5), 845-856, 2009.


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Enzymes or NaOH

Untreated ~75

Cutinase ~58 endo mechanism introduction new groups

NaOH ~45 hydrolysis end groups little or no introduction new groups


Contact angle(water/PET amorphous)


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Functionalisation with 2-(bromomethyl)naphthalene (BrNP)

0

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100000

150000

200000

250000

300000

350000

400000

450000

500000

350 400 450 500 550 600 650 700

Emission wavelength (nm)

Emis

sion

inte

nsit

y (a

.u.)

A

B

C

D

E

F

The emission spectra of the 6 samples (the spectra are corrected detector sensitivity). A= PET-Cr + BrNP, B=PET-Cr + Enzyme + BrNP, C=PET-Am + Enzyme + BrNP,D=PET-Cr + Enzyme + BrNP, E= PET-Am + Enzyme + BrNP and F=PET-Cr + NaOH + BrNP.

Opacity of films increased



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Functionalisation with BrNP

mono-chromator

Light source

Opticalfiber

spectro-meter

mono-chromator

Light source

Opticalfiber

spectro-meter

0

20

40

60

80

100

A B C D E F

Tota

l lum

ines

cenc

e in

tens

ity (a

.u.)

A= PET-Cr + BrNP, B=PET-Cr + Enzyme + BrNP,C=PET-Am + Enzyme + BrNP, D=PET-Cr + Enzyme + BrNP,E= PET-Am + Enzyme + BrNP and F=PET-Cr + NaOH + BrNP.


To evaluate the effect of increased opacity and scattering effects of the amorphous PET films the total photoluminescence intensity was measured using an integrating sphere.


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Cutinase in PET modification, so far ….

• Cutinase exhibits significant hydrolytic activity towards amorphous regions. (ratio TPA, MHET, BHET is function of enzyme concentration and substrate)

• Model substrates vs ‘real’ substrates• Introduction of carboxyl and hydroxyl groups in PET surface

• pHopt ~ 8 -8.5 • Topt ~25°C• Long incubation times / high enzyme concentration• Enzyme stability (surfactants, temperature)• Enzyme adsorption inhibits analysis of induced chemical surface changes

and increase in hydrophilicity, wetting characteristics are difficult to assessdenaturated enzymeswashing procedureprotease


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Inkjet

Specific enzymaticsurface modification

Building3D structures/devices

Enzymatic synthesisfunctional groups

Research inkjet application e.g.Digitex project (functionalities), VTT (electronics), Setti et al. 2007 (enzymes), Nishioka et al. 2004 (enzymes), Di Risio et al. 2007 (enzymes)


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Development ‘bio-ink’A piezo-based inkjet system requires the design of a jettable ink: ‣ viscosity 2-30 mPa s‣ surface tension 30 mN/m

Specific challenges in developing a biological ‘ink’.compatibility of surfactantspH and ionic strengthshear (enzyme inactivation)high enzyme concentrations can cause to ‘ink’ to become non-Newtonian

difficulties or even a non-jetable liquid.

In addition we need to consider evaporation in the nozzle as well as on the substrate‣ evaporation in the nozzle results in clogging,‣ evaporation at the substrate will result in lack of water and thus reduced hydrolysis


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InkTwo jetable ‘bio-inks’ developed compatible with cutinase• carboxymethylcellulose, buffer, non-ionic surfactant, glycerol• polyvinyl pyrrolidone, buffer, non-ionic surfactant, glycerolOther inks in development.

Task -> characterisation of ‘bio-inks’• enzyme stability / inactivation• enzyme activity in ‘bio-ink’• optimal enzyme concentration for surface modification

Both inks allow printing on PET films (amorphous, crystallinity ~ 4%).

Ink based on PVP allows printing with more detail


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Functional surface• Bioresponsive/sensor• UV protection• …..

PVCAdhesive layer &binding agent

PET

Guebitz and co-workers., 2008

Potential applications

Tarpaulin (PVC on PET)reduction adhesive: from 6% to 0,5 / 1%

moisturemanagement

sustainableprocesses

tires

enzymaticallytreated PET or PA


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Biotechnologically functionalised materials

Challenges

Today’s challenge is to make the enormous potential of modernbiotechnology for production and synthesis of materials with advanced functionalities an opportunity for industry.

• Novel processes for textiles exhibiting the desired functionalities.• Novel enzyme technology for structuring and functionalisation ofsurfaces.

To contribute to the transition towards a biobased economy


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• Inotex and Novozymes• Cost Action 868• Work is supported by a grant of the European Commission

FP7 Grant Agreement Number PIEF-GA-2008-219665

Acknowledgements

Documents

Biotechnological modification of polyester surfaces