'ill IMII.II 1111 II. j

Indian Journal of Fibre & Textile Research

Vol. 21, December 1996, pp. 270-275

Degumming of silk with different protease enzymes

M L Gulrajani & Shailja Vaidya Gupta

Department ofTextile Technology, Indian Institute ofTechnology, New Delhi I IO 016

and

Abhilasha Gupta & Mona SuriLady Irwin College, Delhi 110 001

Received 16 February 1996; accepted 21 March 1996

Silk was degummed with eight different commercially available enzymes, viz. Degummase lOOOL,Protosol, Trypsin, AIcalase, Protease A, Protease N, Pepsin and Protease M. The degumming condi-tions with respect to concentration and time were optimised for each enzyme. Enzyme activity, animportant intrinsic property, and degumming efficiency were evaluated in terms of weight loss, ten-sile strength, handle and lustre. A weight loss of 24 ± 3% was observed for most of the enzymes atan optimum enzyme cone. of 15% except for Degummase lOOOLwhich gave this result at 25%cone. Trypsin and pepsin gave extremely poor results. The increase in treatment time at the opti-mum enzyme cone. showed no further significant weight lossThere was no significant strength lossin any of the degummed samples but a marked improvement in handle and lustre was observed.SEM showed that Protosol enzyme gave the best results both with respect to weight loss andsmoothness of the fabric.

Keywords: Degumming,Protease, Silk

1 Introduction

Degumming of silk has traditionally been carri-ed out with soap or alkali. These methods havesome major drawbacks, such as the degummedsilk obtained is not uniform, the strength loss ishigh and chemicals used cause environmental pol-lution. Enzymes are now being considered as alt-ernative degumming agents for the processing ofsilk. The action of the enzyme can be controlledto avoid strength loss but at the same time obtaina uniformly degummed silk. For this purpose,proteolytic enzymes or pro teases which attack theamide bonds of protein molecule are used. De-gumming of silk with protease enzymes has beenreviewed by Gulrajani 1.

Although, there are reports available wherecommercial preparations of' proteolytic enzymeshave been used to degum silk2•3, no study hasbeen carried out with respect to the enzyme activ-ity and degumming efficiency. In the presentstudy, it has been observed that the activity andthe specificity of the enzyme are important factorsfor degumming with a controlled weight loss ofsilk. For this purpose, eight different enzymeswhich .differ in their activity and the conditionsunder which they act, have been used. These en-

zymes belonged to three categories, viz. alkalineproteases, neutral proteases, and acidic proteases.Their efficiency with respect to weight loss,strength and overall improvement in the handleand lustre has been evaluated.

2 Materials and Methods2.1 Materials

Bivoltine silk yarn of 35.3 denier was obtainedfrom Bangalore, India. Alkaline protease Degum-mase lOOOL and Protosol were supplied by Adv-anced Biochemical Ltd., Bombay, India. Alcalasewas obtained from Novo Nordisk, Denmark.Neutral proteases Protease A 'Amano', Protease N'Amano', and acidic protease Protease M 'Amano'were given by Pfimex Pharmaceuticals Ltd" Hy-derabad, India. Trypsin and Pepsin were pur-chased from Central Drug House (CDH), Delhi,.India.

2.2 Methods2.2.1 Enzyme Activity

All the enzymes studied were assayed for activ-ity according to the Anson's method of analysis4•One unit of enzyme activity is defined as theamount of enzyme required to liberate 1 !Jogof

I I I II'

GULRAJANI et al. : DEGUMMING OF SILK 271

the tyrosine from casein at the required pH at45°C in 30 min.

2.2.2 Degumming of Silk

Raw silk yarn samples, weighing approximately1 g each, were conditioned and weighed. The de-gumming treatment was carried out in Atlas laun-derometer for 1 h, keeping the material-to-liquorratio at 1:50. The -samples were degummed to .aweight loss of about 25 ± 2% by the different pro-teases. The degummed samples were thenwashed, dried, conditioned and weighed.

The recipes used for degumming silk with var-ious enzymes are given in Table 1. In all thecases, 0.1% non-ionic surfactant was also added.

The first set of experiments was carried out tooptimise enzyme concentration. Samples weretreated with different concentrations of enzyme0.5%, 2.5%, 5%, 10% and 15% (in the case ofDeugummase and Trypsin, 25% and 30% cone.were also taken) at optimum pH and temperaturefor 1h. A control sample in buffer without en-zyme was also prepared. This procedure wasadopted for each enzyme. Optimum concentrationfor each enzyme was determined in terms ofweight loss. In the second set, experiments wereconducted to study the effect of time variation (lh,2h and 3h) at the optimum concentration deter-mined from the first set of experiments.

2.2.3 Weight Loss

Conditioned samples were weighed accuratelybefore and after enzyme treatment and the weightloss calculated as:

Wt.loss (%)= [(~ - "i)/~] x 100

where Wi and Wz are the weights of the fabric be-fore and after the enzyme treatment.

2.2.4 Tensile Strength

The samples obtained under the optimum en-zyme concentration were tested for strength using

lnstron 4202 Universal Materials Testing System.Peak values of load and elongation were notedand the mean of 20 readings of each was calculat-ed to obtain stress, % strain and Young's modulus('Y') using the followingformulae:

LoadStress=----

New denier

where,

New denier = Original denier - Denier loss

% Denier loss = % Wt.loss

Elongation x 100% Strain = Gauge length

Stress'Y'= Strain

2.2.5 Handle and Lustre

The untreated control sample and samplestreated under optimised conditions with each en-zyme were mounted on black chart paper andnumbered 1-9. Ten persons were asked to evalu-ate these and rank them (l-IX) on the basis ofhandle and lustre separately. Rank I was given 9points; II, 8 points and so on. Arithmetic meanwas computed for each sample and the sampleswere ranked.2.2.6 Degumming Efficiency

One yam sample was degummed with Mar-seilles soap using 25% (owf) soap at'boil for 90min. Material-to-liquor ratio was kept at 1:50.Taking this as standard 100% weight loss, de-gumming efficiency of the enzymes was calculatedusing the followingformula:

Degummingefficiency

% Wt.loss by enzyme treatment x 100% Wt. loss by soap treatment

Table I-Recipes used for deguming silk with various enzymes

Degummase

ProtosolTrypsinAlcalaseProteaseProteaseProteasePepsinlOOOL

ANM

Sodium carbonate

O.1MO.IM1MSodium bicarbonate

O.1MO.IM15% (owf) 15% (owf)Calcium chloride

--50mMDisodium hydrogen phosphate, gll

----9.469.46Dihydrogen sodium phosphate, gIl

----10.3910.39Acetic acid

------1M1MNaOH

------1MTemp., ·C

5050376050505060

pH

10.010.08.09.07.07.04.5-3.5

272 INDIAN J. FIBRE TEXT. RES., DECEMBER 1996

Enzyme

conc.,o/o _(x)

Nil

0.5

2.55.0

10.015.025.030.0

Fig. I-Effect of enzyme concentration on weight loss

Table 3- Regression equations for weight loss

Regression equation R RZ RZ SE ofestimate

ficieqcy of this enzyme was better than that ofthe Degummase enzyme. A high correlation co-efficient and low standard error of estimatemade the results predictable with a fair accura-cy .

• Alcalase (ALe), the third enzyme in the alkalinebuffer category, showed weight loss of 7.35% at

0.8251.0981.7641.4522.6811.105

0.995 0.990 0.9880.996 0.991 0.9860.990 0.981 0.9680.988 0.975 0.9590.960 0.923 0.8710.996 0.992 0.985

x LOSSPRo LOSSPN~LOSSPMo LOSSPA~LOSSDG

I-,..,-o LOSSALC -6 10152025

Concentration, %

oo

30

Yl = 1.901+ 0.836xYz= 2.599 + 2.784x- 0.088rY3 = 4.034 + 2.948x- 0.089rY4 =4.550+ 1.730x-0.037rYs= 5.753 + 3.069x- 0.136xzY6 = 3.776 + 4.092x- 0.198x2

20~ •..~52

~'0)3: 10

2.2:7 SEM Studies

Filaments of samples treated under optimisedconditions and those of the untreated sampl~were scanned using a Cambridge scanning elec-tron microscope.

3 Results and DiscussionThe effect of enzyme concentration on weight

loss of silk yam is shown in Table 2. In order tofind out the co-relation between weight loss andenzyme concentration, regression analysis wascarried out. The regression equations and correla-tion coefficients for all the enzymes investigatedare given in Table 3. With the help of the regres-sion equations, graphs between the two variableswere plotted (Fig. 1). Except in Degummase,which showed a linear relationship betweenweight loss and enzyme cone., in all other en-zymes the relationship was best represented byquadratic equation.

From the measured weight loss the followingobservations can be made:

• A weight loss of 2.3% at 0.5% Degummase con-centration was observed which increased to22'% at 25% enzyme concentration. The valueof standard error of estimate (0.825) was lowestfor Degummase enzyme. Also, the coefficient ofcorrelation was the highest(R= 0.988). Both thesecoefficient factors implied highest accuracy in pre-diction among the nine enzymes used. Degummase(DO) was the only enzyme that required a high-er enzyme concentration, i.e. 25%, to achieveover 20% weight loss. The enzyme was used inalkaline conditions and was the only enzyme forwhich linear correlation was observed betweenweight loss and enzyme cone.

• Protosol (PR), another protease functional underalkaline conditions, gave a weight loss of 25% at15% enzyme concentration. The degumming ef-

I I! 'II I II

GULRAJANI et al. : DEGUMMING OF SILK 273

Table 4-Effect of treatment time on weight loss of silk yarn

Enzyme Weight loss, %

33.9032.9030.7329.5031.7630.4220.5924.92

v

IVnIVillIXVIIVllI

10.5910.7211.52511.5211.1311.3014.6714.04

illnIVIVvnvnIX

3.413.433.463.253.343.382.9653.37

Control

Degummed withDegummaseProtosolAlca1aseProtease A

ProteaseNProteaseM

PepsinTrypsin

Table {;i~Effect of enzymatic degumming on, handle and lustre

Sample Rank

Handle Lustre

VI

Ih2h3h

Degufnmase (DG)

22.06 22.34 24.53

Protosol (PR)

24.86 27.26 30.93

Alcalase (ALe)

26.44 27.74 28.46

Protease A (PA)

22.45 25.93 27.57

Protease N (PN)

22.02 25.76 26.31

Protease M (PM)

25.07 27.9128.66

Control

Degummed withDegummase (25%)Protosol (15%)Alcalase (15%)ProteaseA(15%)Protease N (15%)Protease M (15%)Pepsin (15%)Trypsin (30%)

Table 5-Effect of enzymatic degumming on tensile strength

[Gauge length, 50 mm; Speed, 10 mmI s; Initial load < 1.00 g]

Sample Tensile strength Elongation Modulusglden % glden

3.43 12.82 27.16

where strength was concerned. These samples al-so showed a marked improvement in the handleand lustre as seen by the average ranking given inTable 6.

The scanning electron micrographs (Fig. 2) ofthese samples also substantiate the above results.The treatment with alkaline proteases results inthe complete and uniform ram()'\181of sericin.Amongst the alkaline proteases, Protosol enzymegave the best results both with respect to theweight loss and the smoothness of the fabric ob-tained (Figs 1 & 2b).

The neutral enzymes also gave fairly good re-sults although the removal of the sericin was notvery uniform (Figs 2f & 2g).. Micrographs ob-tained after treatment with acidic enzymes (Tryp-sin and Pepsin) showed as expected poorly de-

0.5% enzyme concentration which increased to26% at 15% enzyme concentration. This en-zyme was found to be the most effective in thecomplete removal of sericin. Also, the resultswere fairly predictable with high correlation co-efficient (R=0.968) and low s.tandard error ofestimate (1.674). When compared to the differ-ent enzymes used, the extent of sericin removalwith the alkaline buffer category enzymes wasfound to be maximum.

• Protease A 'Arnano' 2 (PA), a neutral buffer en-zyme, gave a good correlation value of 0.959and standard error of estimate, 1.452. A weightloss of 22% at 15% enzyme concentration wasobtained.

• The other neutral enzyme Protease N :.\mano'(PN) gave the lowest correlation coefficient andhighest standard error of estimate among the setof enzymes studied. The results of this neutralprotease could be predicted with least accuracy.A weight loss of 22.45% was obtained at 15%enzyme concentration. The enzymes in the neu-tral buffer category were not as effective de-gummingagents as compared to other enzymes.

• Among the acidic proteases studied, Protease M'Amano' (PM) showed a high weight loss of25% at 10% enzyme concentration. Moreover,correlation coefficient was 0.985 and standarderror of estimate was 1.105, which implied fairlyhigh accuracy in terms of prediction.Trypsin (TRY) and Pepsin (PEP) enzymes

showed exceptionally poor results of 4% weightloss at 30% and 15% enzyme concentration re-spectively.

In general, as can be observed from Table 2and Fig. 1, an increase in the enzyme concentra-tion shows a marked increase in weight loss. Aweight loss of 24 ± 3% was observed for most ofthe enzymes at an enzyme concentration of 15%except for Degummase which gave the general re-sult at 25% concentration (Table 2). Two excep-tions to the general trend were the enzymes tryp-sin and pepsin which gaveextremelypoor results.

After having optimised the concentration of theenzymes at a constant time of lh, the time wasvaried at the constant optimised concentration.,The increase in the treatment time from Ih to 3hshowed no further significant weight loss (Table4).

The silk yam with a weight loss of 24 ± 3% wastested for strength loss, handle and lustre.' Therewas no significant strength loss in any of the de-gummed samples (Table 5). However, in general,the alkaline proteases gave more consistent results

~-"

~.;;;

~~

zo>=z~'Tj

53:::0tT1

@><:-l

~Y'otT1(")tT1

~I:;l:ltT1:::0•.....\0\00-.

Fig. 2-Scanning electron micrographs of silk yarn degummed with Degummase (a), Protosol (b), Alcalase (c), Protease A'Amano'(d), Protease N 'Amano' (e), Protease M 'Amano' (f), Pepsin (g), Trypsin (h), and Control (i)

~ ~ ~ A...

GULRAJANI et al. : DEGUMMING OF SILK

~~

275

Table 8-Degumnring efficiency of enzymes

Degumming agent Wt.lQSS Degumming°/~ efficiency, %

References1 Gulrajani M L, Rev Prog Color, 22 (1992) 79-89.2 Chopra S, Garg S & Gulrajani M L, Korean J Sene Se~

36(1 )(1994) 44.3 Chopra S & Gulrajani M L, Asian Text J, January (1994)

39.

4 Anson M L, J Gen Physio~22 (1939) 79-89.

these results, enzymes having activity of morethan 3400 AU/mg were the most significant de-gumming agents, giving degumming efficiency ofmore than 80% when compared with the 100% jdegumming obtained with Marseilles soap (Table!8~ '

Interestingly, Protease 'M' which has. comparat-ively low amopnt of enzyme activity of 745 AU/mg (Table 7) showed a good degumming efficien-cy of 92.96% (Table 8). One probable reason forthis aberration could be due to the specificity ofthe enzyme. This can cause the ~me to attackonly certain specific regions of the sericin, result-ing in high weight loss but non-lUliform removalof the sericin. This observation was further sup-ported by the fact that the highest coefficient ofvariation of strength measurements (0.267) wasobserved for this enzyme. This is an indicator ofuneven degumming and the presence of thick andthin sections in the.degummed sample.

Thus, to use an enzyme as an effective de-gumming agent it is important that the activity ofthe enzyme should be taken into considerationbefore evolvinga protocol for the use of enzyme.

100.0081.7992.1896.9283.2481.6592.9616.4215.16

26.9722.0624.8626.1422.4522.0225.07

4.434.09

Table 7-Activity of enzymes

Enzyme ActivityAU/mg·3400

3836393645913891

745

DegummaseProtosolAlcalaseProtease AProteaseNProteaseM

Marseilles soapDegummase (25%)Protosol (15%)Alcalase (15%)Protease A (15%)Protease N (15%)Protease M (15%)Pepsin (15%)Trypsin (30%)

gummed samples with considerable amount ofsericin still remaining on the surface (Figs 2h &2i).

To analyse the degumming efficacy of the dif-ferent enzymes under similar conditions of en-zyme concentration (w/v), it was important to de-termine the activity of the enzymes. The activityof the individual enzymes as obtained by the An-son's method is given in Table 7. According to