Enzymatic Pre-Treatment of Cotton. Part 1. Desizing and Glucose Generation in Desizing Liquor

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Pervin Aniş, *Asim Davulcu, Enzymatic Pre-Treatment of Cotton. Hüseyin Aksel Eren Part 1. Desizing and Glucose Generation in Desizing Liquor

University of Uludag, Abstract Department of Textile Engineering, The objective of this study was to substitute cotton pretreatment chemicals with enzymes 16059, Bursa, Turkey. to create an environmentally friendly process for water and energy savings. In this study, E-mail: [email protected] enzyme selection and process optimisation was made in order to increase the glucose content of the desizing liquor of a starch-sized cotton fabric. Results indicated that *University of Erciyes, commercial desizing enzyme formulations of α-amylase enzymes were not appropriate to Department of Textile Engineering, produce a large quantity of glucose in the desizing bath; the glucose amounts obtained were 38039, Kayseri, Turkey. about 200 mg/l. However, the food market enzyme used, an amyloglucodidase/pullanase mixture (amyloglucosidase 186 Units/g, pullanase 395 Units/g), produced approximately 4000 mg/l glucose in the desizing bath after process optimisation. The desizing effect of the amyloglucodidase/pullanase enzyme mixture was compatible with those of commercial desizing enzymes. The results of peroxide generation from glucose and bleaching trials are reported in the second part of the study. Key words: desizing, starch, α-amylase, amyloglucosidase, glucose, enzymatic bleach.

n Introduction thus produce only glucose (glucoamylase cess of pure amyloglucosidase enzymes and α-glucosidase), maltose or dextrin reported in former studies [2, 11]. Starch is widely used as a sizing agent, (β-amylase). The third group of starch- being readily available, relatively cheap converting enzymes are the debranching and based on natural, sustainable raw ones that exclusively hydrolyse α,1-6 gly- n Experimental materials [1]. 75% of the sizing agents cosidic bonds: isoamylase and pullanase. Material used worldwide are starch and its deriva- The fourth group of starch-converting The fabric used was a plain weave 100% tives.[2] enzymes are transferases, which cleave raw cotton fabric with a mas per square the α,1-4 glycosidic bond of the donor meter of 175 g/m2 with equal weft and Using amylase enzymes for the removal molecule and transfer part of the donor to warp counts of 62.5 tex and densities of of starch sizes is one of the oldest enzyme a glycosidic acceptor with the formation 14 ends/cm. The fabric was sized with a applications.[1, 3-4] Amylases are en- of a new glycosidic bond [8]. zymes which hydrolyse starch molecules 100% starch sizing agent and 4% (owf: over the weight of the fabric) starch was to give diverse products, including dex- An enzymatic process is proposed to uti- present in the sized fabric. 9 conventional trins and progressively smaller polymers lise desizing baths for bleaching in which commercial desizing enzymes of classi- composed of glucose units [5]. These glucose oxidase (GOx) enzymes generate cal and thermo-stable types from 5 sup- partly degraded oligosaccarides can not hydrogen peroxide and gluconic acid us- be reused [2] and are usually discharged, ing glucose as a substrate [9 - 11]. Ad- pliers and 1 commercial enzyme used contributing large amounts of Chemical vantages of the process are reducing the in the food industry to produce glucose Oxygen Demand (COD) and Biological COD of the effluents by degrading glu- syrup from corn starch was used in the Chemical Oxygen Demand (BOD) to ef- cose units, and reducing the use of per- experiments (Table 1). fluent streams [6, 7]. 50-80% of the COD oxide stabilising agents with the help of in the effluents of textile finishing indus- gluconic acid, which is capable of com- Method tries is caused by sizing agents [2]. plexing catalysts as well as saving water Desizing trials were performed according and energy by using desizing liquor for to the recipes listed in Table 1 with fabric There are basically four groups of starch- bleach [2, 9 - 12]. However, starch has to specimens of 20 grams (approximately converting enzymes: (i) endoamylases; be degraded with glucose units in order 30 × 30 cm2) at a liquor ratio of 1:10 us- (ii) exoamylases; (iii) debranching en- to achieve process efficiency because of ing distilled water. The process time for zymes; and (iv) transferases. Endoamy- the high substrate selectivity of GOx en- the commercial desizing enzymes was lases are able to cleave α,1-4 glycosidic zyme. Conventional commercial desizing prolonged to 90 minutes (30-60-90 min- bonds present in the inner part of the am- enzymes do not seem appropriate for this utes) in order to investigate any further ylose or amylopectin chain. α-Amylase purpose since most include α-amylase in increase in the glucose content. Optimi- is a well-known endoamylase. The end formulations [1, 4, 13 - 14], whereas am- sation trials were performed for the amy- products of α-amylase action are oli- yloglucosidases are suitable amylase en- loglucosidase/pullanase mixture. gosaccharides. Enzymes belonging to zymes to degrade starch until it becomes the second group, the exoamylases, ei- glucose [2, 9 - 11]. Iodine tests were performed to determine ther exclusively cleave α,1-4 glycosidic the residual starch on the fabrics after de- bonds such as β-amylase, or cleave both The performance of an amyloglucosi- sizing. α,1-4 and α,1-6 glycosidic bonds like dase/pullanase mixture commercial en- amyloglucosidase or glucoamylase. Ex- zyme used in the food industry to pro- Glucose amounts of desizing liquor were oamylases act on the external glucose duce glucose syrup from corn starch was measured by use of a Thermo-Trace GOx residues of amylose or amylopectin and examined in this study, analysing the suc- solution. The composition of this solution

100 Aniş P., Davulcu A., Eren H. A.; Enzymatic Pre-Treatment of Cotton. Part 1. Desizing and Glucose Generation in Desizing Liquor. FIBRES & TEXTILES in Eastern Europe 2008, Vol. 16, No. 4 (69) pp. 100-103. is as follows: glucoseoxidase > 15,000 Table 1. Enzyme types and desizing recipes recommended by manufacturers; CD: Commer- U/l, peroxidase > 100 U/l, 4-aminoan- cial Desizing enzyme, a - Amyloglucosidase 186 Units/g, pullanase 395 Units/g. b - Com- tiprin – 0.5 mmol/l, 4-hydroxybenzoic mercial enzyme for food industry, no data available for desizing process. Temp. Time, acid - 10 mmol/l, phosphate - 119 mmol/l Enzyme Type pH Dosage Supplier and stabiliser. The parameters were: °C min. Temperature: CD1- α-amylase 6 - 7 0.25-1.3 g/l 70 30 Novozymes, Bagsvaerd, Denmark. 37 °C CD2- α-amylase 6 - 7 0.06-0.3 g/l 70 - 110 30 CD3- α-amylase 6 - 7 0.5-2 g/l 90 - 98 30 Primary wavelength: R-Duraner, Bursa, Turkey. 500 nm (460-560 nm) CD4- α-amylase 6 - 7 0.2-0.4 g/l 90 - 98 30 CD5- α-amylase 6.5 0.02-0.05 g/l 80 - 90 30 AB Chem., Bursa, Turkey. Secondary wavelength: CD6- α-amylase 7 - 7.5 0.05-0.2 % 90 - 95 10 - 20 600-660 nm Gemsan, Istanbul, Turkey. CD7- α-amylase 6.5 - 7 0.05-0.2 % 50 - 70 20 - 30 Desizing liquor : GOx solution ratio CD8- α-amylase 5.4 - 8 1.2 g/l 30 - 60 30 1:150 CHT, Istanbul, Turkey. CD8- α-amylase 5.4 - 8 0.5-2 g/l 60 - 100 30 Incubation time: AMG- Amylo-glucosidase/ Novozymes, Bagsvaerd, 4.1 - 4.3b - b 60 - 63b - b 5 minutes pullanase mixturea Denmark. Sensibility: 9-35 mmol/l (0-6300 mg/l) Table 2. Glucose generated in desizing liquor during the desizing process; CD: Commer- Reactions can be written as: cial Desizing enzyme, AMG: Amyloglucosidase/pullanase enzyme mixture. 1- Glucose + O2 + H2O → → Temp., Generated glucose, mg/l Iodine Gluconoic acid + H2O2 Enzyme pH Dosage °C 30 min. 60 min. 90 min. test 2- H2O2 + HBA + 4-AAP → CD1 6.5 1.3 g/l 70 200 210 205 7 - 8 → Quinoneimine dye + H2O HBA = 4-Hydrobenzonoic acid CD2 6.5 0.3 g/l 90 210 205 210 7 - 8 4-AAP = 4-aminoantiprin CD3 6.5 2.0 g/l 90 180 200 190 7 - 8 CD4 6.5 0.4 g/l 100 213 200 208 7 - 8 The absorbance of the solution was mea- CD5 6.5 0.05 g/l 85 180 200 190 7 - 8 sured using a spectrophotometer with a CD6 7.0 0.2% 90 180 190 185 7 - 8 460 - 560 nm interval; the darker the color, CD7 6.5 0.2% 60 190 185 200 6 - 7 the greater the glucose amount. The ab- CD8 6.5 2.0 g/l 50 205 220 208 6 - 7 sorbance of the desizing liquor was com- AMG 4.1 0.25% 62 3116 3920 3606 6 - 7 pared to the absorbance of standard glucose solution (5.55 mmol/l). The Glucose Amyloglucosidases, exoamylases and Despite the increase in glucose amount content of the desizing liquor was calcu- pullanases are debranching enzymes that while using the AMG enzyme, the glu- . lated by with formula Gd = (Ad/As) Gs, produce starch degradation until single cose amounts were still under the required where Gd and Ad are the glucose amount glucose units, as described before [8, 10]. level of approximately 4,000 mg/l [10]. (mg) and absorbance of the desizing li- The results reported in Table 2 for amy- Recommended process parameters for quor, and G and A are those of standard s s loglucosidase (AMG) enzyme conformed the AMG enzyme were not available for glucose solution. The calibration curve desizing, but pH 4.1 - 4.3 and tempera- this hypothesis, indicating a great in- obtained is shown in Figure 1. ture of 60 - 63 °C have been applied to crease in the amount of glucose: an aver- produce glucose syrup from corn starch age glucose generation of 199.8 mg/l for n Results and discussion in food industry. A set of trials were per- α-amylases and 3,544 mg/l for theamylo- formed to find optimum circumstances Table 2 compares the glucose genera- glucosidase/pullanase mixture. to generate maximum glucose during the tion and desizing effect of the enzymes desizing process. used. Results indicate an acceptable de- Iodine test results indicate a sufficient sizing effect but very low glucose gen- desizing performance for AMG enzyme Above all it has to be stated that the acid eration for α-amylases. The amount of comparable to that of α-amylases. consumption of untreated cotton fabrics hydrogen peroxide required to obtain a satisfactory whiteness is reported to be

400 - 600 mg/l, requiring a glucose amount 0.06 of approximately 4,000 mg/l in the desizing bath for hydrogen peroxide genera- 0.05 tion of GOx [10]. The amounts of glucose 0.04 reported in Table 2 for α-amylases were m g not enough even for prolonged processing 0.03 Figure 1. Calibra-

times. The low glucose generation can be G l u c o s e , tion curve obtained 0.02 y = 0.005x + 0.005 attributed to the reaction mechanism of for a standard glu- R2 = 1 α-amylases, which are endoamylase en- cose solution to de- 0.01 zymes and do not involve the degradation termine the amount 0 of starch until single glucose units exists, of glucose in desizing liquor using a 0.0753 0.1106 0.1432 0.1722 0.2001 despite their well-known and satisfactory Thermo-Trace GOx Absorbance desizing effect [1 - 7]. kit.

FIBRES & TEXTILES in Eastern Europe 2008, Vol. 16, No. 4 (69) 101 was found to be a very important fac- utes, where a prolonged process time did Table 3. Acid consumption of the untreated tor and was considered during the trials not contribute to glucose generation. test fabrics. (Treatment: Blank dyeing at (Table 3). 45 °C for 10 minutes after acid addition). pH optimisation for AMG Acetic acid, ml/l Initial pH Final pH Table 3 shows the increase in liquor pH Although a pH of 4.1 - 4.3 was recom- 1.00 3.50 4.55 after 10 minutes of circulation with the mended as appropriate by the manufac- fabric in the liquid. According to these 1.25 3.45 4.45 turer of the enzyme, this pH was recom- 1.50 3.38 4.40 results, a process of ten minute’s circula- mended for glucose syrup production tion and re-adjustment of the pH before 1.75 3.28 4.25 from corn starch in food industry; how- 2.00 3.21 4.19 the addition of enzyme was applied in ever it lacked optimisation for desizing our experiments. 2.25 3.18 4.17 purposes. A set of trials were performed 2.50 3.16 4.14 to find the optimum pH to generate maxi- Enzyme dosage optimisation for AMG 2.75 3.16 4.10 mum glucose during the desizing pro- 3.00 3.15 4.03 Figure 2 illustrates the minimum nec- cess. 3.25 3.14 4.00 essary enzyme dosage - 0.5% (o.w.f.). 3.50 3.12 3.96 However, increasing the enzyme dos- Figure 4 illustrates that the optimum pH 3.75 3.10 3.98 age to 0.75% (o.w.f.) resulted in higher for glucose generation in desizing bath 4.00 3.08 3.94 glucose amounts than 0.5% (o.w.f.). An is pH 4.1, requiring an acetic amount of appropriate enzyme dosage of AMG en- 2.25 ml/l. zyme 0.75% (o.w.f.) was chosen from cose is observed. The linearized line with R2 = 0.9779 indicated a direct proportion these results. Optimum circumstances obtained for the AMG enzyme (amyloglucosidase/ pul- between the amount of starch and gener- Process time optimisation for AMG lanase mixture) as a result of the trials ated glucose. Since there were no data available con- made are: cerning the use of AMG enzyme in the n 0.75% (o.w.f.) enzyme, n Conclusions desizing process, the effect of the process n 2.25 ml/l of acetic acid (pH 4.1), time was also examined to a large scale n at 62 °C, for 45 minutes. The performance of a commercial en- in order to find optimum circumstances. zyme (an amyloglucosidase/ pullanase Trials were performed for 90 minutes The optimum recipe attained was tested mixture) was tested and process optimi- with 15 minute intervals. by directly adding starch to the liquor, sation trials were performed. Optimum the results of which are illustrated in circumstances obtained were: 0.75% The results illustrated in Figure 3 indicat- Figure 5 where a linear relation between (o.w.f.) enzyme, pH 4.1, 62 °C and a pro- ed an optimum process time of 45 min- the amount of starch and generated glu- cess time of 45 minutes. Acid consump-

4200 4400 l l 4200 / 4000 / g g m m 4000 , 3800 , e e s s 3800 c o 3600 c o u u l l 3600 G G 3400 3400 3200 3200 0.25 0.50 0.75 1.00 1.25 1.50 15 30 45 60 75 90 Enzyme dosage, % Process time, minutes

Figure 2. Enzyme dosage dependency of glucose generation by the Figure 3. Process time dependency of glucose generation by the amyloglucosidase/pullanase enzyme mixture; Process parameters: amyloglucosidase/pullanase enzyme mixture; Process parameters: pH 4.1 (acetic acid), 62 °C, 30 minutes. 0.75% enzyme, pH 4.1 (acetic acid), 62 °C.

8000 Initial pH Final pH Generated glucose, mg/l 4.80 4400 y = 895.97x + 1747.6 7000 2 4 . 6 0

4 . 5 R = 0.9779 4 . 5

4.60 4200 l l / / 4 . g g 4 . 0 4 . 3 9 6000 4 . 3 5 m

4.40 4000 m 4 . 2 8 , , e e H s 4 . 1 5

s 5000 4.20 4 . 1 2 3800 p 4 . 1 0 o 4 . 0 8 c 4 . 0 3 c o 4 . 0 3 . 9 8 u 3 . 9 6 u l 4.00 3600 l 4000 G G 3.80 3400 3000 3.60 3200 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 2000 Acetic acid, ml/l 0.8 1.0 1.2 1.4 1.8 2.0 Starch, %

Figure 4. pH dependency of glucose generation by the amyloglu- Figure 5. Glucose generation by the amyloglucosidase/pullanase cosidase/pullanase enzyme mixture; Process parameters: 1% en- enzyme mixture after starch addition. (Process parameters: 0.75% zyme, 62 °C, 30 minutes. enzyme, pH 4.1(acetic acid), 62 °C, 45 minutes).

102 FIBRES & TEXTILES in Eastern Europe 2008, Vol. 16, No. 4 (69) tion of the raw cotton fabric played an important role in pH adjustments.

Similar results were reported using pure amyloglucosidase enzymes [10]; however, storage and usage properties as well as the availability of pure enzymes may cause problems when compared to The 8th International Symposium commercial ones. A commercial enzyme readily used in the food industry was uti- lized in this study.

The sufficiency of the generated glucose to utilise the desizing liquor for bleaching was tested by using glucose oxidase EL-TEX 2008 enzyme to produce hydrogen peroxide from this glucose. Bleaching trials were Electrostatic and Electromagnetic Fields performed with the glucose generated. Compatible whiteness degrees were at- New Materials and Technologies tained. Results are reported in the second will be held on 26-27 November 2008 in Łódź, Poland. part of the study.

The Symposium is organised by the Textile Research Institute (IW) in Łódź, the Institute of Acknowledgment Telecommunication, Teleinformatics and Acoustics (ITT&A) of the Technical University The authors would like to thank TUBITAK- of Wrocław, and the Polish Committee of Electrostatics (SEP), Warsaw. TAM for their financial support. (Project no. TAM-2003-08). El-Tex Symposium has been organised by the Textile Research Institute since 1994. On the 1st of July 2007 the Textile Research Institute was enlarged by the inclusion of the following formerly independent institutions: References n Institute of Textile Architecture (Instytut Architektury Tekstyliów – IAT) n Institute of Textile Materials Engineering 1. Lange N. K.; Textile Chemist and Colorist, (Instytut Inżynierii Materiałów Włókienniczych) 1997, 29, pp. 23-26. n 2. Opwis K., Knittel D., Kele A., Scholl- TRICOTEXTIL Institute of Knitting Techniques and Technologies meyer E.; Starch/Starke, 1999, 51, pp. (Instytut Technik i Technologii Dziewiarskich Tricotextil) 348-353. 3. Lange N. K., Liu J., Husain P. A., Condon The main aim of El-Tex 2008 is the survey and promotion of scientific, technological and B.; Novo Nordisk Enzyme Business Ar- applicational developments and the exchange of experiences between experts representing ticle A 6648a-GB, 1999, pp. 1-12. different scientific disciplines. 4. Ciechańska D., Kazimierczak J.; Fibres & Textiles in Eastern Europe, 2006, 14, The program will cover a selection of issues in the scope of: No 1(55), pp. 92-95. n the effects of electromagnetic fields (EMF) onto human health, 5. Gupta R., Gigras P., Mohapatra H., n eliminating or restricting the hazards caused by the occurrence of static electricity, Goswami V.K., Chauhan B.; Process n environmental aspects of EMF, Biochemistry, 2003, 38, pp. 1599-1616. n production methods and application of textile shielding materials, 6. Laursen S. E., Hansen J., Andersen T. A., n electrostatic evaluation of textile fabrics, Knudsen, H. H.; Danish experience. BAT n the metrology of electrostatic and electromagnetic fields. - in the clothing and textile industry, www. mst.dk, (accessed 20 April 2006). Deadlines: 7. EPA Sector Notebook Textile, www.epa. n abstract submission in English (2 pages A4) 25.04.2008 gov, (accessed 10 November 2005). n paper/poster acceptance 31.05.2008 8. Maarel M. J. E. C., Veen B., Uitdehaag n manuscripts ready for printing 30.06.2008 J. C. M., Leemhuis H., Dijkhuisen L.; n Journal of Biotechnology, 2002, 94, pp. submission of presentations in electronic form 30.06.2008 137-155. n payment of registration fee 30.09.2008 9. Tzanov T., Calafell M., Guebitz G. M., Cavaco-Paulo A.; Enzyme and Microbial Registration fee: authors 200 EURO, other participants 250 EURO Technology, 2001, 29, pp. 357–362. 10. Buschle-Diller G., Yang X.D.; Textile Re- The fee covers: participation in sessions, CD with abstracts and presentations, boarding, search Journal, 2001, 71, pp. 388-394. and social event. Hotel costs are not included. 11. Tzanov T., Costa S. A., Gubitz G. M., Ca- Recommended accommodation and Symposium venue: Hotel AMBASADOR, vaco-Paulo A.; Journal of Biotechnology, address: ul. Kosynierów Gdyńskich 8, 93-320 Łódź, Poland tel.+4842 646 49 04 2002, 93, pp. 87-94. 12. Lebiedowski M., Fibres & Textiles in Eastern Europe, 2005, 13, No 1(49), Information: pp. 75-79. Textile Research Institute (IW), Brzezińska 5/15, 92-103 Łódź, Poland, 13. Kirk O., Borchert V., Fuglsang C. C.; fax: +4842 6792638 Current Opinion in Biotechnology, 2002, 13, pp. 345-351. Contact persons: 14. Product Data Sheets, (Gemsan, Novozy- Katarzyna Grzywacz tel. (+4842) 6163 195, e-mail: [email protected] mes, CHT, Rudolph-Duraner). Joanna Koprowska tel (+4842) 6163 116, e-mail: [email protected]

Received 20.02.2007 Reviewed 29.02.2008

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