Indian Journal of Fibre & Textile Research Vol. 25, March 2000, pp. 69-74

Degumming of silk with lipase and protease

M L Gulrajani & Ritu Agarwal

Department of Textile Technology, Indian Institute of Technology, New Delhi 110016, India

and

Amrit Grover & Mona Suri

Lady Irwin College, New Delhi 11000 I, Indi a

Received J 8 Jam'([lI), J 999; accepted J 3 April J 999

A lipase enzyme in combinati on with a protease enzyme has been used for dewaxing and degumming of silk and the combined effect of dewaxing and degumming on weight loss, wettability , dye uptake, yellowing, microscopic struc ture, handle and lustre has been studied. It is observed that the combined enzyme treated samples show the same weight lo ss, cleaner longitudinal su rface and better wettability when compared with the Marseill es' soap treated samples .

Keywords: Dewaxing, Degumming, Lipase, Protease, Silk

1 Introduction The raw si lk does not merely consist of an inner

filament fibroin , sheathed in natural size sericin , but is also contaminated with small amounts of non­proteinaceous impurities like dust, minerals, pigments and waxy matter'. Along with sericin, these waxes playa role in obscuring the brilliance and softness of the silk . Only a few studies have been conducted on waxes and oils present in si lk, their nature, composi­tion, properties, exact quantities and methods of re­moval. Apart from naturally occurring waxes and oils, some additional waxes are applied to the yarns while weaving them into fabric which do not get re­moved even after degumming. These waxes hamper the processi ng of si lk due to poor wetting properties. Hence, complete elimi nation of these waxy sub­stances is very essential. Due to the ambiguity about the true nature and composition of these waxes, no method has been successfully de vised to remove them completely. It is believed that a fraction of these waxes is saponifiable and therefore can be removed with alkali or soap treatment, but these methods are believed to be harmful to fibroin . The use of enzymes in the silk industry is relatively unexplored and has generated a lot of interest and much research is being carried out internationally. For this purpose, a special class of enzymes called lipases, which are specialised in hydrolysing waxes into fatty acids and alcohols, seem to be aR appropriate answer.

The present work was therefore undertaken to study the effect of lipase treatment on silk wax and the combined effect of dewaxing and degumming on weight loss, wettability, dye uptake, microscopic structure, handle and lustre.

2 Materials and Methods

2.1 Materials

Two varieties of raw si lk fabric with plain weave (Silk A) and crepe weave (S ilk B) were used in this study.

Lipase enzyme Amano AK 20, from Pseu­domonas sp., with activity of 9.98 AU/g was obtained from th e Department of Biochemical Engineering and Biotechn ology, lIT-Delhi. Pro­tease enzyme Alcalase 2,4 LFG with activity of 2.4 AU/g was procured from Novonordisk , Denmark .

2.2 Methods

2.2.1 Preparation of Samples

Samples from each silk were cut weighing 3 g each. They were conditioned at 30% RH and 25°C for 48 h and weighed again.

2.2.2 Fabric Treatment

The recipes used for treatment of the fabric with lipase, protease and soap are given below:

70 INDIAN 1. FIBRE TEXT. RES., MARCH 2000

Dewaxing (with lipase) Buffer solution Lipase Temperature pH Time MLR

Degumming (with protease) Alcalase Sandopan DTC NaHCOJ

Temperature pH Time MLR

O.IM NaHC03

10% owf 40%C 8-8.5 2h 1:50

10% owf 1.5 gpl 7.5% owf 60°C 8.5 2h 1:50

Degumming (with Marseilles ' soap) Marseilles' soap 25% owf Temperature 93°C pH 10 Time MLR

1.5 h 1:50

2.2.3 Optimization of Lipase Concentration The samples were treated with 0%, 5% and 10%

lipase enzyme. The effectiveness of lipase was esti­mated by measuring the weight loss .

2.2.4 Optimization of Conditions for Alcalase Treatment Degummi ng of dewaxed and raw silk samples was

carried out with two concentrations of Alcalase, viz. 10% and 15 % (owf), for 45 min and 3 h.

2.2.5 Combined Treatment of Lipase and Alcalase To see the effect of combined treatment of lipase

and protease in the same bath the samples were treated under the following conditions: Alcalase 10% owf Lipase 10% owf Sandopan DTC 1.5 gpl Buffer solution 0.1 M NaHC03

Temperature 60°C pH 8.5 Time 2h MLR 1:50

2.2.6 Determination of Weight Loss Samples were weighed accurately before and after

the enzyme treatment using Sartorius moisture ana­lyzer and the weight loss was calculated as follows:

Weight loss (%) = (WI - W2 ) x 100 WI

where WI and W2 are the weights of fabric before and after the enzyme treatment.

2.2.7 Determination of Wettability

Five samples (I in.x3 in .) were cut from both warp and weft directions. On each sample, two lines were drawn I inch apart with an ink marker. With the help of a clamp stand the samples were immersed in dis­tilled water till the lower line. The spreading of col­our showed the rise of water. The time taken by water to travel from lower mark to upper mark was re­corded.

2.2.8 Determination of Increase in Dye Uptake To see the extent of wax removal from silk, the

ease by which the sample will get dyed was meas­ured. Samples were dyed with reacti ve dye (Remazol Red BS ill, 3% owf) for 5, IS and 45 min at 80°C with 60 gpl Glauber's salt, maintaining the material­to-liquor ratio at I :50. Dye uptake was measured by taking the K/S values of the samples on the spectro­photometer.

2.2.9 Determination of Yellowing

Samples were cut in 2 in.x4 in . size and their ini­tial whiteness was determined by taking Hunter' s whiteness index using spectrophotometer. The sam­ples were than exposed to light equ ivalent to sunlight for 24 h. Their whiteness was again measured.

2.2.10 SEM Studies The extent of degumming can be qualitatively as­

sessed by viewing the degummed fibres under Cam­bridge scanning electron microscope. Residual sericin appears as lumps on the surface of the fibre . The ex­tent of fibre damaged can also be assessed by this method . The splitting of fibres into minute fibrillae is frequently observed in si lk. This has been attributed to the mechanical injury of the fibre caused by chaffing and abrasion during degumming l.

2.2.11 Determination of Handle and Lustre

The samples of equal size (2 in.x2 in.) were mounted on black sheet and ten people were asked to evaluate these samples on the basis of handle and lustre. Mean ranking was taken.

3 Results and Discussion

3.1 Optimization of Lipase Concentration Treatment of Silk A with 5% owf enzyme concen­

tration gave a weight loss of 1.37% and the control

GULRAJANI et af.: DEGUMMING OF SILK WITH LIPASE AND PROTEASE 71

sample gave a weight loss of 1.33%. Whereas the treatment with 10% enzyme concentration gave a weight loss of 4.62%. As the weight loss with 5% enzyme concentration was very close to that of the control sample, 10% owf enzyme concentration was chosen. Further, the results were not reproducible. Therefore, lipase treatmf'nt was tried as pretreatment for degumming process.

3.2 Optimization of AJcalase Concentration Due to the erratic weight loss, the conditions for

dewaxing treatment could [lOt be standardized. There: fore, the conditions for the degumming treatment which could be applied to the lipase pretreated sam­ples were optimized. Table I shows the weight loss obtained after the treatment of silk with AIcalase. As the weight loss obtained after 45 min and 180 min was comparable, time period of 45 min was taken. Weight loss obtained after dewaxing was compared with the weight loss obtained without dewaxing treatment. Table 2 shows that the lipase pretreated samples show only marginal increase in weight loss after degumming. Therefore, lipase was not used as pretreatment.

3.3 Effect of Combined Dewaxing and Degumming

3.3.1 Weight Loss Table 3 shows that the maximum weight loss is

obtained by Marseilles' soap treatment and the weight loss on combined treatment of lipase and protease is comparable with that of soap treatment. Though the Marseilles' soap treatment results in complete re­moval of serecin, the quantity of soap required is high (25% owf) which is not economical. Al so, the high temperature(at boil) and alkaline pH (10.5) used might cause degradation of fibroin . The enzymatic treatment, on the other hand, is milder (temp. 600 e and pH 9) and gives fairly good weight loss .

3.3.2 Wettability It is observed from Table 4 that for both Silk A

and Silk B, the minimum wicking time is in case of

Table I-Weight loss obtained after treatment with A1calase

Alcalase Time Weight loss, % (owf), % min Plain silk Crepe si lk

10 45 17.98 15.45 10 180 17.68 16.84 15 45 19.59 16.49 15 180 18.50 16.30

combined treatment with aIcalase and lipase. This shows that there is improved degumming by the com­bined treatment and hence improvement in absorb­ency. Both the silk samples show better wicking property in warp direction due to formation of capil­laries .

3.3.3 Dye Uptake Table 5 shows that the KlS values of the combined

enzyme treated samples are comparable to that of the samples treated with Marseilles' soap.

3.3.4 Microscopic Structure The scanning electron micrographs of control sam­

ple, aIcalase-treated sample, lipase-treated sample, combined enzyme treated sample and soap-treated sample of Silk A and Silk B are shown in Figs 1 and 2 respectively. It is observed that aIcalase-treated sam­ples are equally clean as the samples obtained on combined enzyme treatment. But in case of only al­calase enzyme treatment, certain small remnants of sericin are noticed. Marseilles ' soap treated samples show fibrillation of fibre. There is not much differ­ence in Silk A and Silk B samples. In case of plain

Table 2-Weight loss obtained after sequential treatment of li pase and alcalase

Lipase conc. A1calase conc. Weight loss (owf) % (owf) % %

10 17.98 15 19.54

10 10 18.21 10 15 20.33

Table 3-Effect of enzyme treatment on weight loss

Treatment

Only buffer Only lipase Only A1calase Lipase and A1calase Marseilles' soap

Weight loss, % Plain silk Crepe silk

2.44 5.73 19. 11 20.57 23 .55

2.30 4.09

20.40 21.50 21 .83

Table 4-Effect of enzyme treatment on wicking time

Treatment Wicking time, min Plain sil k Crepe silk

Warp- Weft- Warp- Weft-wise wise wise wise

Only buffer 30.0+ 30.0+ 30.0+ 30.0+ Only lipase 30.0+ 30.0+ 30.0+ 30.0+ Only A1calase 1.38 1.23 1.10 3.80 Lipase and Alcalase 1.39 1.98 0.90 3.10 Marseilles' soap 6.15 5.93 4.20 15.0+

72 INDIAN 1. FIBRE TEXT. RES., MARCH 2000

Table 5-Effect of enzyme treatment on K/S value

Treatment K/S value Plain silk Crepe silk

5 min 15 min 45 mi n 5 min 15 min 45 min

Only Alcalase 1.6827 3. 162 1 6.8706 1.1 294 2.5440 7.1161 Lipase and Alcalase 1.6803 3.2209 6.7543 1.175 1 2.6 195 7.1554 Marseilles' soap 4.07 10 6. 168 1 8.5500 1.51 92 3.5905 8.919 1

Fig. I-Scanning e lectron micrographs of Silk A samples : (a) untreated , (b) treated with buffer or control , (c) treated with lipase, Cd) treated with Alcalase, (e) treated with Alcalase and lipase, and (f) treated with Marseilles ' soap

GULRAJANI e/ al.: DEGUMMING OF SILK WITH LIPASE AND PROTEASE 73

Fig. 2-Seallning c1eetrOl , mi crogrnphs of Silk B samples: (n) untreated, (b) treated with buffer or control , (c) treated with li p3~e,

(d) treated with Alcalase, (e) tn:ated with Alcalase 3nd lipase, and (f) ueated with Marsei ll es' soap

Table 6-Effect of enzyme treatment on handle and lu stre

Treatment Pl ain silk Crepe silk Handle Lu stre Handie Lustre

Only buffer 5 5 5 5 Only lipase 4 4 4 4 Only Alcalase 3 I 2-3 Lipase and Alealase 2 2 2-3 2

Marseilles' soap 3 3

woven silk samples (Silk A), more crimp is observed than in case of crepe silk or satin weave samples (Silk B) .

3.3.5 Haudle and Lustre The data in Table 6 shows that Marseilles ' soap treated

sample has been ranked first in case of handle and third in case of lustre. Good handle could be due to uniform dis­solution of sericin by the action of alkali, resulting in a

74 INDIAN J. FIBRE TEXT. RES., MARCH 2000

smooth surface. Excess of alkali of the hydrolyzed soap diminishes the lustre2

. Best results in terms of lustre is ob­served for the samples treated with only alcaIase. The combined treatment gives better results in terms of lustre but the samples are ranked lower in terms of handle as compared to the soap-treated samples.

4 Conclusion Lipase enzyme can be used along with protease to

improve the degumming of silk. The combined en­zyme treatment gives the same weight loss, better wettability and cleaner longitudinal surface as com­pared to Marseilles' soap treatment. This combined

enzyme treatment can be a useful replacement to the traditional method of treatment with Marseilles' soap.

Acknowledgement

The authors acknowledge the financial support re­ceived from the Department of Biotechnology, Min­istry of Science and technology, Govt. of India, for conducting this study.

References I Gulrajani M L, Degumming of silk in Chemical processing

of silk, edited by M L Gulrajani (Indian Institute of Tech-nology, New Delhi), 1993,63.

2 Tsunkaye V D, J Soc Dyers Colour, 48 (1932) 280.