degumming of silk fabric with several proteases

Journal of Biotechnology 106 (2003) 101–112

Degumming of silk fabric with several proteases

Giuliano Freddia,∗, Raffaella Mossottib, Riccardo Innocentib

a Stazione Sperimentale per la Seta, via Giuseppe Colombo 83, 20133 Milano, Italyb C.N.R-ISMAC-Sezione di Biella, Corso Giuseppe Pella 16, 13900 Biella, Italy

Received 31 March 2003; received in revised form 11 August 2003; accepted 2 September 2003

Abstract

A crepe silk fabric was treated with different alkaline (3374-L, GC 897-H), neutral (3273-C), and acid (EC 3.4 23.18)proteases with the aim to study their effectiveness as degumming agents. Proteases were used under optimum conditions of pHand temperature, while enzyme dosage (0.05–2 U/g fabric) and treatment time (5–240 min) were changed in order to study thekinetics of sericin removal. Degumming loss with soap and alkali was 27 wt.%. The maximum amount of sericin removed in 1 hwas 17.6, 24, and 19 wt.% for 3374-L (2 U/g fabric), GC 897-H (1 U/g fabric), and 3273-C (0.1 U/g fabric), respectively. Underthe experimental conditions adopted, EC 3.4 23.18 was almost ineffective as a degumming agent. Degumming loss increased asa function of the treatment time, reaching a value of 25 wt.% with 1 U/g fabric of 3374-L. The morphological analysis showedthat sericin was completely removed from the warp yarns of the crepe fabric, while the highly twisted weft yarns still exhibitedthe presence of sericin deposits within the most internal parts of the close fibre texture. The chromatographic pattern of solublesericin peptides changed as a function of the kind of enzyme used, enzyme dosage, and treatment time. A mixture of peptidesfrom 5 to 20 kDa in weight, with a weight-average molecular weight of about 12 kDa was obtained.© 2003 Elsevier B.V. All rights reserved.

Keywords: Silk fabric; Sericin; Degumming; Proteases

1. Introduction

The silk filament spun by the silkwormBombyxmori is a composite material formed by two fibroinfilaments surrounded by a cementing layer of sericin.Both fibroin and sericin, which account for about 75and 25 wt.%, respectively, are proteins. Fibroin, thereal fibrous component, is a high molecular weightpolypeptide (∼=350 kDa), whose primary structureis rich in glycine, alanine, and serine amino acids(∼=85 mol%) in the molar ratio 3:2:1, which form typ-

∗ Corresponding author. Tel.:+39-02-2665990.E-mail address: [email protected] (G. Freddi).

ical -(ala–gly)n- repeating motifs (Lotz and ColonnaCesari, 1979; Zhou et al., 2000). In the fibre, fibroinchains are aligned along the fibre axis, held togetherby a close network of interchain hydrogen bonds,with adjacent -(ala–gly)n- sequences forming thewell known �-sheet crystals (Takahashi et al., 1991).Sericin, the silk gum glueing the fibroin filaments, is acomplex mixture of 5–6 polypeptides widely differingin size (40–400 kDa), chemical composition, structureand properties, such as: solubility, hydrophylicity, andstickiness (Gamo et al., 1977; Couble et al., 1987).Fine details of the primary structure of four sericinproteins have been reported (Garel et al., 1997).The amino acid composition is charaterized by an

0168-1656/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2003.09.006

102 G. Freddi et al. / Journal of Biotechnology 106 (2003) 101–112

extremely high concentration of serine, which rangesfrom 16 to 38 mol% in the different sericins. Theseproteins, which are synthesized, secreted, and storedin the middle silk gland, form a sheath around the fi-broin core during spinning of the silk filament. Theirphysiological function is to lower the shear stressand to absorb the water squeezed from the stretchedfibroin mass during the process of fibre formation.

Silk processing from cocoons to the finished cloth-ing articles consists of a series of steps which include:reeling, weaving, degumming, dyeing or printing, andfinishing (Zahn, 1993). Degumming is a key processduring which sericin is totally removed and silk fibresgain the typical shiny aspect, soft handle, and elegantdrape highly appreciated by the consumers. The indus-trial process takes advantage of the different chemicaland physical properties of the two silk components, fi-broin and sericin. While the former is water-insolubleowing to its highly oriented and crystalline fibrousstructure, the latter is readily solubilized by boilingaqueous solutions containing soap, alkali, syntheticdetergents, or organic acids (Svilokos Bianchi andColonna, 1992; Freddi et al., 1996). Nowadays, batchdegumming of silk is mostly carried out in alkalinebaths containing soap and alkali. Soap is replacedby synthetic detergents in continuous degummingsystems, because it cannot compensate the acidityof sericin hydrolysis products accumulating in thebath, thus limiting the use of the degumming bath forweekly degumming cycles. The mechanism of sericinremoval in chemical degumming is a combinationof various effects such as: dispersion/solubilizationand hydrolysis of the different sericin polypeptides(Freddi et al., 1996). Hydrolysis prevails when strongalkaline compounds are added to the degumming bath.Therefore, suitable procedures for controlling processparameters, such as temperature, time, pH, and alka-linity must be implemented on an industrial scale inorder to attain effective sericin removal without trig-gering the hydrolytic degradation of fibres, which canbe easily induced by the presence of harsh chemicalsin the treatment bath. Fibre degradation often appearsas loss of aesthetic and physical properties, suchas dull appearance, surface fibrillation, poor handle,drop of tensile strength, as well as uneven dyestuffabsorption during subsequent dyeing and printing.

In recent years, various studies have dealt with theremoval of sericin by using proteolytic enzymes. Sev-

eral acidic, neutral, and alkaline proteases have beenused on silk yarn as degumming agents. Alkaline pro-teases performed better than acidic and neutral ones interms of complete and uniform sericin removal, reten-tion of tensile properties, and improvement of surfacesmoothness, handle, and lustre of silk (Gulrajani et al.,1996, 1998, 2000b). The combination of a lipase anda protease resulted in effective de-waxing and degum-ming, with positive effects on wettability of silk fibres(Gulrajani et al., 2000a). Enzyme degummed silk fab-ric displayed a higher degree of surface whiteness, buthigher shear and bending rigidity, lower fullness, andsoftness of handle than soap and alkali degummedfabric, owing to residual sericin remaining at the crossover points between warp and weft yarns (Chopraet al., 1996). To overcome these drawbacks and toenhance the effectiveness of the enzymatic process,the application of an ultrasonic field to an enzymaticdegumming bath has been proposed (Krasowski et al.,1999). However, the lower performance of enzymedegummed silk fabrics in terms of handle, as well asthe higher cost of enzymes compared to chemicalsand some concerns about the use of enzymes in con-tinuous degumming plants, have so far limited thedevelopment of industrial processes alternative to thetraditional degumming with chemical agents.

The increasing awareness of legislators and citizensfor the ecological sustainability of industrial pro-cesses has recently stimulated the interest of scientistsand technologists for the application of biotechnol-ogy to textile processing (Duran and Duran, 2000;Cavaco-Paulo, 1998; Gübitz and Cavaco-Paulo, 2001).Silk degumming is a high resource consuming processas far as water and energy are concerned. Moreover, itis ecologically questionable for the high environmen-tal impact of effluents. The development of an effectivedegumming process based on enzymes as active agentswould entail savings in terms of water, energy, chemi-cals, and effluent treatment. This could be made possi-ble by the milder treatment conditions, the recycling ofprocessing water, the recovery of valuable by-productssuch as sericin peptides, and the lower environmentalimpact of effluents. The present study focuses on theapplication of proteolytic enzymes characterized byhigh stability and efficiency to silk degumming. Theaim is to study the kinetics and mechanism of sericinremoval and to set up an experimental model that mayassist in developing enzyme-based degumming pro-

https://www.researchgate.net/publication/230237287_Degumming_of_silk_fabrics_with_tartaric_acid?el=1_x_8&enrichId=rgreq-46d31c4a-c94d-4fa4-961b-0498806f119f&enrichSource=Y292ZXJQYWdlOzg5OTE1Njk7QVM6MTE0OTg3ODM0MDIzOTM2QDE0MDQ0MjY2NDUzMTE=

G. Freddi et al. / Journal of Biotechnology 106 (2003) 101–112 103

cess competitive with the traditional one in terms ofeffectiveness, costs, and quality of the final product.

2. Materials and methods

2.1. Silk fabric

The treatments were carried out on a 100% raw silkfabric (crepe). Construction parameters are listed inTable 1. Before degumming, the fabric was extractedwith a mixture of methanol and toluene (75/25 vol.%)to remove sizing agents.

2.2. Proteolytic enzymes

The commercial proteases used for enzymaticdegumming of the silk fabric are listed inTable 2.They represent different protease types generally usedin industrial laundry and food applications. 3374-L,GC 897-H, and 3273-C were kindly provided byGenencor Inc., USA. EC 3.4 23.18 was purchasedfrom Sigma–Aldrich.

2.3. Determination of enzymatic activity

The assay for proteases 3374-L and GC 897-H isbased on the ability of the enzyme to cleave a syntheticpeptide, N-succinyl-ala-ala-ala-p-nitroanilide (succ-

Table 1Construction properties of the silk fabric (crepe)

Warp yarn Weft yarn

Number of ends (cm−1) 120 40Counts (den) 29.2 23.3× 3Torsions (m−1) 2S (2400) 2Z (2500)

Fabric weight (gm−2) 79.6

Table 2Proteolytic enzymes used for silk degumming

Enzyme code Origin Characteristics pHa T (◦C)a Activitya

3374-L (A) Bacillus subtilis(genetically modified)

Oxidative-stable endopeptidase 7.5–12 (10) 20–60 (60) 55.9 MPU/g

GC 897-H (B) Bacillus lentus(genetically modified)

Bacterial high alkaline strain 7–12 (10) 40–65 (65) 44.7 GSU/ml

3273-C (C) Carica papaya Papain, thiol protease 3.5–9 (5–7) 65–78 (65) 58.349 FCCPU/gEC 3.4 23.18 (D) Aspergillus saitoi Aspergillus pepsin I 2.5–6.5 (2.5–3) 30–60 (50) 1 U/mg

a Optimum pH and temperature values in parentheses.

AAApNA), resulting in an increase in absorbance at405 nm. The activity was expressed in MPU and GSUfor 3374-L and GC 897-H, respectively.

The assay for protease 3273-C is based on the abilityof the enzyme to hydrolyze casein as a substrate at pH6 and 40◦C. FCCPU (FCC Papain Unit) is defined asthe quantity of enzyme which liberates the equivalentof 1�g of tyrosine per hour under the conditions ofthe assay.

Enzymatic assay for protease EC 3.4.23.6 was madeby using hemoglobin as a substrate at pH 2.8 and37◦C. One unit (U/mg) is defined as the quantity ofenzyme which hydrolyzes hemoglobin to produce acolor equivalent to 1�mol of tyrosine per minute.

2.4. Silk degumming

Standard degumming was carried out in an alka-line solution containing 10 g/l Marseille soap and1 g/l sodium carbonate, at 98◦C, for 1 h. Degummedsilk was thoroughly rinsed with warm distilled water,dried at room temperature, and then extracted withpetroleum ether to remove residual fatty acids.

The experimental conditions used for enzymaticdegumming are listed inTable 3. Silk fabric samplesof about 0.1 g were immersed in 20 ml of buffer solu-tion (material-to-liquor ratio 1:200) containing differ-ent amounts of enzyme. Blank samples were obtainedby treating silk with buffer alone, without enzyme. Op-timum pH and temperature for each enzyme were usedthroughout the tests. Enzyme dosage (0.05–2 Units/gfabric) and treatment time (5–240 min) were changed.Degumming tests were carried out in a thermostaticbath under gentle shaking. Inactivation of proteaseswas made at 85◦C for 10 min. At the end of the treat-ment, silk fabrics were rinsed with distilled water anddried at room temperature. All degumming tests wereperformed in duplicate.


Table 3Experimental conditions used for enzymatic degumming of silk

Enzyme code Buffer pH T (◦C) Enzyme concentration (Units/g fabric) Time (min)

3374-L Tris–HCl 0.1 M 10 60 0.05–2 5–240GC 897-H Tris–HCl 0.1 M 10 65 0.05–2 5–2403273-C Citric acid–Na phosphate, 0.1 M 6 65 0.05–2 5–240EC 3.4 23.18 Citric acid–Na phosphate 0.2 M 3 50 0.05–60 5–240

2.5. Degumming loss

Degumming loss, which represents a quantitativeevaluation of the degumming efficiency, indicates theweight loss of the fabric (expressed as a percentageof the initial weight) after standard or enzymaticdegumming. Before weight measurement, sampleswere conditioned at 20◦C and 65% relative humidityfor 24 h.

2.6. Scanning electronic microscopy (SEM)

Morphological characterization of silk fabrics wasperformed by means of scanning electron microscopy(SEM) (Stereoscan 440, LEO Electron Microscopy

Fig. 1. Degumming kinetics of the crepe silk fabric at different enzyme dosage (0.05–2 U/g fabric). 3374-L and GC 897-H: alkalineproteases. 3273-C: neutral protease. Time: 1 h. Other parameters as inTable 3. The dotted line shows the level of degumming loss obtainedwith soap and alkali (27 wt.%). Results are the average of duplicate tests.

Ltd.). Samples were observed at 10 kV accelerationvoltage, after gold sputtering.

2.7. High performance-size exclusionchromatography (HP-SEC) of sericin peptides

HP-SEC runs were performed with a Waters Cor-porate (USA) chromatographic system controlled bya Maxima 820 Workstation, which included a GPCdata handling module (Freddi et al., 2000). At the endof degumming, the silk fabric was removed and thesolution containing sericin peptides was pooled withwashing waters and immediately freeze-dried. For HP-SEC analysis, freeze-dried samples were dissolved ina fixed volume of 50 mM sodium phosphate buffer, pH


7.2, containing 0.15 M KCl, filtered, and analyzed witha protein Pak-60 column (Waters Corporate, USA).Injection volumes ranged from 50 to 100�l, flow ratewas 0.5 ml/min. Eluate was detected at 254 nm. Molec-ular weight calibration was performed by using theLMW gel filtration calibration kit (Amersham Bio-sciences, Sweden).

0

10

20

30

5 10 30 60 120 180 240

Time (min)

Deg

um

min

g lo

ss (

%)

Inactivation

3374-L

0

10

20

30

5 10 30 60 120 180 240

Time (min)

Deg

um

min

g lo

ss (

%)

Inactivation

GC897-H

(a)

(b)

Fig. 2. Time dependence of the enzymatic degumming of the crepe silk fabric (5–240 min). Enzyme dosage: 3374-L 2 U/g fabric; GC 897-H1 U/g fabric; 3273-C 0.1 U/g fabric. Other parameters as inTable 3. Dark + light bars: total degumming loss. Light bars: contribution ofthe inactivation step to the degumming loss.

3. Results and discussion

3.1. Study of the degumming kinetics: effect ofenzyme dosage and time

The effect of enzyme dosage on the extent of sericinremoval was studied by treating silk fabric samples for


0

10

20

30

5 10 30 60 120 180 240

Time (min)

Deg

um

min

g lo

ss (

%) 3273-C

(C)

Fig. 2. (Continued ).

1 h with different amounts of proteases.Fig. 1 showsthe results obtained with the alkaline proteases A andB, and with the neutral protease C, (Table 2for en-zyme codes), whose amount ranged from 0.05 to 2 U/gfabric. It is worth noting that the acid protease D dis-played a very low degumming efficiency (7.6 wt.%degumming loss after 3 h treatment with 60 U/g fab-ric). For these reasons, this protease was not includedin Fig. 1and was not used for further tests. The dottedline drawn in the graph at about 27 wt.% degummingloss indicates the target value for complete degum-ming, as obtained under standard conditions with soapand alkali. Without enzymes, the degumming loss wasnegligible (2–3 wt.%), owing to the low treatment tem-perature. In fact, it is well known that sericin can beremoved by using water alone, but high temperatureis needed to attain complete degumming (110–120◦C,under pressure).

Degumming loss increased linearly as the amountof protease B increased until 2 U/g fabric, attaininga value of 24 wt.%. On the other hand, proteasesA and C reached a plateau at 1 and 0.1 U/g fabric,corresponding to 17.6 and 19 wt.% degumming loss,respectively. The values of maximum turnover at sat-uration, expressed as degumming loss, were 0.29 and

3.22 wt.% for protease A and C, respectively. Enzymeconcentration at half maximum turnover was 0.15 U/gfabric for the alkaline protease A and 0.04 U/g fabricfor the neutral protease B. These preliminary valuesare of chief interest in view of characterizing the ac-tivity of different proteases. However, a deeper kineticinvestigation of the proteolytic degradation of sericinis needed because the enzyme-substrate system underconsideration is quite complicated. In fact, as it willbe discussed later in more detail, several factors maysignificantly affect the protease activity, such as fabrictexture, accessibility of the cleavage sites, and treat-ment conditions (agitation). Moreover, competitionof already solubilized sericin peptides for the activesites of the enzyme cannot be excluded.

To study the effect of the treatment time on theextent of sericin removal, the amount of enzyme waskept constant, while the time was changed in therange 5–240 min. The enzyme dosage was 2, 1, and0.1 U/g fabric for proteases A, B, and C, respectively.These values were chosen on the basis of the resultsreported inFig. 1, because they resulted in almostsimilar levels of degumming loss (18–19 wt.%).Fig. 2shows the time dependence of sericin removal for thethree proteases. The total bar height corresponds to


the degumming loss obtained in a complete degum-ming cycle, in which the enzymatic treatment wasfollowed by the inactivation step at 85◦C for 10 min.The amount of sericin removed reached 15–20 wt.%after 5–10 min, and then tended to increase further asthe treatment time increased. Alkaline proteases per-formed better than the neutral one. In particular, pro-tease A attained a degumming loss of about 25 wt.%,very close to the target value. It is worth noting thesignificant contribution of the inactivation step to the

Fig. 3. Crepe silk fabric degummed under standard (a) and enzymatic (b) conditions (GC 897-H, 2 U/g fabric, 60 min).

final value of the degumming loss, especially at shorttreatment times. This behavior can be attributed tovarious factors. The temperature rise from 60–65◦Cto 85◦C probably contributed to enhance the solu-bility of partially hydrolyzed sericin fractions stilladhering to the fibrous core of the silk. Moreover,the extension of the treatment time probably exposedsericin to the action of a residual enzymatic activity.

None of the proteases used in this study allowedthe attainment of the target degumming loss (27 wt.%)


under the experimental conditions adopted. However,this result is only apparently negative, because thecrepe fabric is one of the most difficult substrates totreat, owing to the presence of highly twisted weftyarns. In fact, higher amounts of alkalis are needed toachieve complete sericin removal also during standardchemical degumming. It is likely that other factors be-side the lack of hydrolytic power of proteases haveplayed a role in lowering the extent of degumming.The morphological features of the samples which gotvery close to the target degumming loss seem to con-firm this hypothesis.

3.2. Morphological characterization

The surface morphology of the silk fabric after stan-dard and enzymatic degumming is shown inFig. 3.Removal of sericin resulted in the separation of theindividual silk filaments, which were glued togetherby sericin and sizing agents in the raw fabric. The

Fig. 4. Details ofFig. 3b. (a) Warp yarn. (b–d) Weft yarn.

volume of the yarns increased, especially for the un-twisted warp yarn, while the weft yarn shrank due tothe high number of twists. This conferred on the fab-ric the denser texture and the rough surface typical oflight weight crepe silk fabrics. The dull appearanceand stiff handle of the raw fabric disappeared, and thedegummed fabric became shiny, soft, and scroopy.

By comparing the fabrics degummed with the stan-dard and enzymatic methods, it is possible to observethat the latter exhibited a flatter surface, mostly at-tributable to incomplete shrinkage of the weft yarns,which appeared straighter and thinner than in the ref-erence fabric. The closer SEM examination of thewarp yarn showed that the individual silk filamentssplit off and their surface was clean and free of sericin(Fig. 4a). On the other hand, weft yarns still showedthe presence of sericin residues (Fig. 4b–d). It is in-teresting to note that the deposits were mainly locatedat the cross over points between warp and weft yarnsand/or in the more internal parts of the weft yarns.


These sericin residues still sticking the silk filamentstogether hampered the weft yarns from reaching theexpected degree of shrinkage.

The morphological observations confirm that pro-teases failed to completely remove sericin from thehighly twisted weft yarns. Since the ability of these en-zymes to hydrolyze sericin is unquestionable, the ob-served behavior can be attributed to other factors, suchas the lack of an effective mechanical agitation in thelab-scale degumming system used for this study. Thisprobably limited penetration of enzymes into the closetexture of weft yarns. The influence of the mechanicalagitation on the enzymatic treatment of cellulose-based textiles has been stressed by various authors(Cavaco-Paulo et al., 1996; Morgado et al., 2000;Radhakrishnaiah et al., 1999; Traore and Buschle-Diller, 1999). It has been shown that not only the con-

15 20 25Time (min)

A 2

54 n

m (

a.u

.)

18.0

15.8

12.9

11.3

9.5

7.7 5.9 4.9

Buffer

GC897-H

3374-L

3273-C

Fig. 5. HP-SEC profiles of soluble sericin peptides obtained by enzymatic degumming with 3374-L, GC 897-H, and 3273-C, for 1 h.Enzyme dosage: 2 U/g fabric. Other parameters as inTable 3.

tact of the enzyme with the substrate was drasticallyimproved by applying suitable mechanical agitation,but also the kinetics and mechanism of the enzymeaction was influenced. Moreover, the mechanical agi-tation during enzyme treatment has proved to changethe aesthetic, tactile, thermal, and comfort propertiesof the fabric. The surface of silk fabrics is very del-icate and sensitive to mechanical stresses during wettreatments. These may result in severe faults knownas chafe marks, that is, surface fibrillation at creases,which impart a typical dull appearance on the fabricand irremediably impair its final quality. Therefore,the degumming system must be optimized with theimplementation of a suitable bath and/or materialmovement that assists the hydrolytic action of the en-zyme and allows to obtain completely degummed silkfabrics without causing significant damages to the


10

12

14

16

18

20

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2

Enzyme (units/g fabric)

Ave

rag

e M

W (

kDa)

3374-LGC897-H3273-C

10

12

14

16

18

20

0 50 100 150 200 250

Time (min)

Ave

rag

e M

W (

kDa)

3374-LGC897-H3273-C

(b)

(a)

Fig. 6. Enzyme dosage (a) and time (b) dependence of the weight-average molecular weight of soluble sericin peptides obtained byenzymatic degumming.


textile goods. Lab-scale degumming tests focusing onthese aspects are in progress.

3.3. HP-SEC screening of sericin peptides

Fig. 5shows the HP-SEC curves of sericin peptidesobtained by degumming the silk fabric with alkalineand neutral proteases. A common feature of the threechromatographic profiles is that the whole peaks fallin the low molecular weight range, from about 5 to20 kDa. It is worth noting that native sericin proteinscover a much wider molecular weight range, from 40to 400 kDa (Garel et al., 1997). The narrower molecu-lar weight distribution of sericin by-products is mostlydependent on the specific mechanism of action of pro-teases, that is, the selective cleavage of target peptidebonds along the protein chains. This resulted in mainchain fission and formation of a range of small sizesoluble peptides.

The two alkaline proteases A and B displayed arather similar chromatographic pattern of the hy-drolyzed sericin peptides, with a major peak centeredat about 12.9 kDa. The neutral protease C resulted intwo main peaks at 18.0 and 15.8 kDa. This featurecan be attributed to the different substrate specificityof the proteases, that is, the chemical structure ofthe target cleavage site. Bacterial proteases are usu-ally characterized by a low degree of specificity, andthis may explain the similarity in the pattern of theresulting sericin peptides. The neutral protease (pa-pain) hydrolyzes the “X–Y” peptide bonds where Xis arginine, lysine, or phenylalanine (Mathews andvan Holde, 1994), and therefore resulted in a differentpattern of soluble sericin peptides.

To further characterize the properties of the sericinpeptides produced by the proteases used for silkdegumming, the following quantitative parametersof the molecular weight distribution were calculatedfrom the raw chromatographic data: weight-average(Mw) and number-average (Mn) molecular weights,which provide a measure of the average chain weightand length; peak molecular weight (Pmw), which givesthe weight of the most abundant polypeptide frac-tion in the sample; polydispersity index (Mw/Mn),which indicates the breadth of the molecular weightdistribution. As shown inTable 4, the average size ofthe sericin peptides (Mw) was in the following order:A > B > C. However, the difference among the sam-

Table 4Molecular weight distribution parameters of sericin by-products

Samplea Mw (kDa) Mn (kDa) Pmw (kDa) Mw/Mn

3374-L 12.9 11.8 12.9 1.092GC 897-H 11.9 11.1 12.9 1.0783273-C 11.4 10.5 15.8 1.087

a Reaction conditions: 2 U/g fabric, 1 h.

ples was very low, despite the above noticed changesin peak distribution and intensity. Accordingly, thevalues of the polydispersity indexes were similar.

Fig. 6shows the behavior of weight-average molec-ular weight as a function of the enzyme dosage andthe treatment time. The plot ofMw versus enzymedosage (Fig. 6a) shows that the size of sericin peptidesdecreased sharply with increasing the amount of theproteases A, B, and C until 0.5 U/g fabric, and thenremained constant. The plotMw versus treatment time(Fig. 6b) indicates that the size of the soluble sericinpeptides remained essentially unchanged regardless ofthe degumming time. These results suggest that thesoluble sericin peptides tended to reach a minimumsize, which was probably dependent on the number ofsites available for enzymatic cleavage, and that the ex-tent of the proteolytic attack towards the peptide frac-tions in solution was negligible.

4. Conclusions

Alkaline and neutral proteases effectively degum-med silk fabrics. Almost complete sericin removal wasobtained by using a crepe silk fabric as the substrate.Owing to the presence of the highly twisted weft yarns,this fabric is one of the most difficult substrates todegum, even by using the standard chemical degum-ming method.

Hydrolytic degradation of sericin took place by se-lective peptide bond cleavage. Degumming kineticswere dependent on enzyme dosage and treatment time.Moreover, chemical properties of soluble sericin pep-tides changed as a function of the kind of enzyme used.A mixture of peptides ranging from 5 to 20 kDa, witha weight-average molecular weight of about 12 kDa,was obtained with the alkaline and neutral proteasesused in this study. Recovery from waste water andreuse of these peptides as additives for cosmetic prod-


ucts appears feasible, due to the lack of contaminantssuch as alkalis and detergents.

Further optimization of the degumming conditionsis needed, with particular reference to the study ofthe mechanical agitation effect on the degummingefficiency of proteases. This parameter is likely toenhance enzyme penetration within the closest partsof the fabric texture and to make sericin removalmore effective. As a consequence of complete sericinremoval, the quality of silk goods in terms of han-dle, appearance and tensile properties is expected toincrease, due to the lower extent of chemical andphysical stresses to which silk is subjected to duringenzymatic processing, as compared to the traditionalchemical process. To this purpose, tests on pilot plantsare in progress, with the aim to scale-up the processfor its implementation on industrial equipments.

Acknowledgements

Authors wish to express their thanks to Dr. MeeYoung Yoon of Genencor Inc. (USA) for providingalkaline (3374-L, GC 897-H), and neutral (3273-C)proteases, for the determination of enzymatic activi-ties, as well as for scientific and technical informationand support.

References

Cavaco-Paulo, A., 1998. Processing textile fibres with enzymes:an overview. In: Eriksson, K.E.L., Cavaco-Paulo, A. (Eds.),Enzyme Applications in Fibre Processing, ACS SymposiumSeries 687, American Chemical Society, Washington, DC,pp. 180–189.

Cavaco-Paulo, A., Almeida, L., Bishop, D., 1996. Effects ofagitation and endoglucanase pre-treatment on the hydrolysisof cotton fabrics by a total cellulose. Textile Res. J. 66, 287–294.

Chopra, S., Chattopadhyay, R., Gulrajani, M.L., 1996. Low stressmechanical properties of silk fabric degummed by differentmethods. J. Textile Inst. 87, 542–553.

Couble, P., Michaille, J.-J., Garel, A., Couble, M.-L., Prudhomme,J.-C., 1987. Developmental switches of sericin mRNA splicingin individual cells ofBombyx mori silkgland. Dev. Biol. 124,431–440.

Duran, N., Duran, M., 2000. Enzyme applications in the textileindustry. Rev. Prog. Coloration 30, 41–44.

Freddi, G., Allara, G., Candiani, G., 1996. Degumming of silkwith tartaric acid. JSDC 112, 191–195.

Freddi, G., Berlin, A., Tsukada, M., Dubini Paglia, E., 2000. Useof HP-size exclusion chromatography to study the degree ofpolymerization of silk (Bombyx mori) fibroin fibres. Sericologia40, 363–373.

Gamo, T., Inokuchi, T., Laufer, H., 1977. Polypeptides offibroin and sericin secreted from the different sections ofthe silk gland in Bombyx mori. Insect Biochem. 7, 285–295.

Garel, A., Deleage, G., Prudhomme, J.-C., 1997. Structure andorganization of theBombyx mori sericin 1 gene and of thesericins 1 deduced from the sequence of the Ser 1B cDNA.Insect Biochem. Mol. Biol. 27, 469–477.

Gübitz, G.M., Cavaco-Paulo, A., 2001. Editorial: Biotechnologyin the textile industry – perspectives for the new millennium.J. Biotechnol. 89, 89–90.

Gulrajani, M.L., Gupta, S.V., Gupta, A., Suri, M., 1996.Degumming of silk with different protease enzymes. Indian J.Fibre Textile Res. 21, 270–275.

Gulrajani, M.L., Sen, S., Soria, A., Suri, M., 1998. Efficacy ofproteases on degumming of dupion silk. Indian J. Fibre TextileRes. 23, 52–58.

Gulrajani, M.L., Agarwal, R., Grover, A., Suri, M., 2000a.Degumming of silk with lipase and protease. Indian J. FibreTextile Res. 25, 69–74.

Gulrajani, M.L., Agarwal, R., Chand, S., 2000b. Degumming ofsilk with fungal protease. Indian J. Fibre Textile Res. 25, 138–142.

Krasowski, A., Müller, B., Föhles, J., Höcker, H., 1999.Degumming of silk in an ultrasonic field. MelliandTextilberichte 80, 543–545.

Lotz, B., Colonna Cesari, F., 1979. The chemical structure and thecrystalline structures ofBombyx mori silk fibroin. Biochimie61, 205–214.

Mathews, C.K., van Holde, K.E., 1994. Biochemistry, C.E.A.,Milano, p. 146.

Morgado, J., Cavaco-Paulo, A., Rousselle, M.-L., 2000. Enzymatictreatment of lyocell: clarification of depilling mechanisms.Textile Res. J. 70, 696–699.

Radhakrishnaiah, P., Meng, X., Huang, G., Buschle-Diller, G.,Walsh, W.K., 1999. Mechanical agitation of cotton fabricsduring enzyme treatment and its effect on tactile properties.Textile Res. J. 69, 708–713.

Svilokos Bianchi, A., Colonna, G.M., 1992. Developments in thedegumming of silk. Melliand Textilberichte 73, 68–75.

Takahashi, Y., Gehoh, M., Yuzuriha, K., 1991. Crystal structure ofsilk (Bombyx mori). J. Polym. Sci., Part B:Polym. Phys. 29,889–891.

Traore, M.K., Buschle-Diller, G., 1999. Influence of wettingagents and agitation on enzymatic hydrolysis of cotton. TextileChemist and Colourist and American Dyestuff Reporter 1, 51–56.

Zahn, H., 1993. Silk. Ullmann’s Encyclopedia of IndustrialChemistry, VCH Publisher Inc., vol. A24, pp. 95–106.

Zhou, C.Z., Confalonieri, F., Medina, N., Zivanovic, Y., Esnault,C., Yang, T., Jacquet, M., Janin, J., Duguet, M., Perasso, R., Li,Z.G., 2000. Fine organization ofBombyx mori fibroin heavychain gene. Nucleic Acids Res. 28, 2413–2419.

degumming of silk fabric with several proteases

Documents