**3. Results and discussion**

#### **3.1. Characteristics of the enzymes used for degumming**

The commercial enzyme preparations used for degumming of the silk fabric are listed in Table 6. Novozymes launched Lipolase® in 1988, the first commercial lipase developed for the detergent Industry. Lipolase® was the first lipase produced by recombinant DNA tech‐ nology. This lipase, originating from *Thermomyces lanuginosus*, formerly *Humicola lanuginosa*, was expressed in *Aspergillus oryzae*. This enzyme is widely used in detergent formulations to remove fat-containing stains and it also has a broad range of substrate specificity. Further‐ more is stable in proteolytic wash solutions. Novozymes launched two variants of Lipolase® issued from rational protein design: Lipolase® Ultra and LipoPrime™. These variants were also expressed in *A. oryzae* [64]. The enzyme preparation used in the present study (Lipo‐ lase® Ultra 50T) exhibited optimal pH and temperature of 9.0 and 50o C respectively. Further‐ more, it was stable at temperatures 30-50o C and pH values of 7.0-10.0 (the remaining activity was measured after incubation for 24 h at the above mentioned temperatures and the pH values).

Esperase® 8.0L is also a product of Novozymes. It is a bacterial serine type alkaline protease produced by *Bacillus* sp. Esperase® is characterized by excellent perfomance at elevated tem‐ perature and pH. It exhibited optimal pH and temperature of 10.0 and 70o C respectively. The enzyme preparation was stable (>90% of its original activity) at pH values of 7.0 and 8.0 and temperatures of 30-50o C (the remaining activity was measured after incubation for 24 h at the above mentioned temperatures and the pH values).

Papain is a cysteine protease, isolated from Papaya (*Carica papaya*) Latex. Papain consists of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for activity of the enzyme. The pH optimum of papain was found 8.0 while temperature opti‐ mum at 50o C. Papain was stable at pH values of 7.0 and 8.0 and temperatures of 30-50o C (the remaining activity was measured after incubation for 24 h at the above mentioned tempera‐ tures and the pH values).


**Table 6.** Enzymes used for silk degumming

#### **3.2. Protease treatment of silk fabrics: effect of enzyme dosage**

Raw silk fabrics were degummed with Papain and Esperase® 8.0L using different enzyme loadings (expressed as Units of protease per g of silk fabric, U. g-1 fabric) and the physicochemi‐ cal (weight loss, crystallinity index, whiteness, whiteness after bleaching) and low-stress me‐ chanical properties (Kawabata evaluation system) of the preterated fabrics were assessed. Untreated and conventionally degummed (using Marseille soap) silk fabrics were used as controls.

#### *3.2.1. Weight loss*

002 002 ( ) 100 *am I I CrI I*

phous phases.

fraction method [63].

244 Eco-Friendly Textile Dyeing and Finishing

**2.11. The Kawabata evaluation system**

bending rigidity per unit length (*B*, gf.

**3. Results and discussion**

measurements were performed in triplicate.

more, it was stable at temperatures 30-50o

and temperatures of 30-50o

values).

**3.1. Characteristics of the enzymes used for degumming**

lase® Ultra 50T) exhibited optimal pH and temperature of 9.0 and 50o

at the above mentioned temperatures and the pH values).

perature and pH. It exhibited optimal pH and temperature of 10.0 and 70o

where I002 is the peak intensity from the lattice plane, and Iam is the peak intensity of amor‐

Triplicate sets of data were used to establish the relative error associated with the X-ray dif‐

Basic mechanical properties namely tensile, bending and shearing were measured by the

The commercial enzyme preparations used for degumming of the silk fabric are listed in Table 6. Novozymes launched Lipolase® in 1988, the first commercial lipase developed for the detergent Industry. Lipolase® was the first lipase produced by recombinant DNA tech‐ nology. This lipase, originating from *Thermomyces lanuginosus*, formerly *Humicola lanuginosa*, was expressed in *Aspergillus oryzae*. This enzyme is widely used in detergent formulations to remove fat-containing stains and it also has a broad range of substrate specificity. Further‐ more is stable in proteolytic wash solutions. Novozymes launched two variants of Lipolase® issued from rational protein design: Lipolase® Ultra and LipoPrime™. These variants were also expressed in *A. oryzae* [64]. The enzyme preparation used in the present study (Lipo‐

was measured after incubation for 24 h at the above mentioned temperatures and the pH

Esperase® 8.0L is also a product of Novozymes. It is a bacterial serine type alkaline protease produced by *Bacillus* sp. Esperase® is characterized by excellent perfomance at elevated tem‐

The enzyme preparation was stable (>90% of its original activity) at pH values of 7.0 and 8.0

Papain is a cysteine protease, isolated from Papaya (*Carica papaya*) Latex. Papain consists of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for

KES-FB system under high sensitivity conditions. The temperature was 20 ± 0.5o

tive humidity is 65% ± 5. The properties measured were shear rigidity (*G*, gf.


cm2.cm-1) and extensibility (*EMT*, %) at 500 gf.

C, and rela‐

degree-1),

cm-1. All

cm.

C respectively. Further‐

C respectively.

C and pH values of 7.0-10.0 (the remaining activity

C (the remaining activity was measured after incubation for 24 h

The weight loss (or degumming loss) represents a quantitative evaluation of the degum‐ ming efficiency after standard or enzymatic degumming. The effect of enzyme dosage on the extent of sericin removal was studied by treating silk fabric samples for 60 min with dif‐ ferent amounts of the two proteases. The results are depicted in Figure 2 both as weight loss (Fig. 2a) and degumming efficiency (Fig. 2b).

Degumming loss increased linearly as the amount of papain increased, attaining a value of 10.2 % (w/w) (degumming efficiency 50%). Esperase® 8.0L was more efficient in sericin re‐ moval as judged by the weight loss (Fig. 2a). Maximum value (20.1%, w/w, degumming effi‐ ciency nearly 99%) was obtained at enzyme loading of 75 U. g-1 fabric. Degumming loss of silk fabrics treated with Marseille soap attained a value of 20.4% (w/w).

Without enzymes, the degumming loss was negligible, owing to the low treatment tempera‐ ture. In fact, it is well known that sericin can be removed by using water alone, but high temperature is needed to attain complete degumming (110-120o C, under pressure).

The microstructure of cotton fabrics was investigated by X-ray diffraction. The results are presented on Table 7. The diffraction curves of all silk fibers exhibit the typical pattern of a

Physichochemical and Low Stress Mechanical Properties of Silk Fabrics Degummed by Enzymes

http://dx.doi.org/10.5772/53730

247

Silk fabrics treated with Esperase® 8.0L exhibited higher Crystallinity Index values com‐ pared to papain treated ones. Furthermore, at higher Esperase® 8.0L the crystallinity of the

Natural coloring matters present in silk are associated mainly with sericin and hence are eliminated during degumming. The natural colouring matter of silk can be roughly divided into yellow, green and brown pigments. However the residual pigments are adsorbed by fi‐ broin and hence silk fabrics made from yellow raw silk after degumming are not white but have a cream colour [65]. Lustre is one of the most important properties of silk, hence the

A slight increase in whiteness was observed after treatment with the two different proteases compared to the fabric treated in the absence of enzyme. Esperase® 8.0L exhibited better re‐ sults compared to papain. However, whiteness of silk fabrics treated with Marseille soap

The results are in contrast to those reported by Chopra et al., [49] who demonstrated that

Bleaching of silk is for white and pastel shades only. Degummed silk fabrics present a slight‐ ly off-white in colour, because of some sericin, which is stubbornly stuck to the fibrin [65]. After enzymatic and conventional degumming the silk fabrics were bleached with H2O2. The results indicated that the whiteness after bleaching of all Esperase® 8.0L treated fabrics were

8.0L at different loadings.

**Whiteness Index after bleaching** 

**(Berger degree)**

25 50 75 marseille

**Esperase 8.0L and Papain Activities (Units/ gfabric)**

soap

Papain Esperase 8.0L Marseille soap No enzyme **(b)**

> No enzyme

enzyme treated samples rate marginally better than the soap-treated samples.

fabric was superior to that treated conventionally (Marseille soap).

was superior to that of enzymatically treated fabrics (Figure 3a).

*3.2.3. Whiteness and whiteness after bleaching with H2O2*

silk II crystal with high crystallinity [19].

method of degumming is significant.

the highest (Figure 3b).

**Whiteness Index (Berger degree)**

25 50 75 marseille

seille soap (conventional treatment) or papain and Esperase®

Papain Esperase 8.0L Marseille soap No enzyme

**Esperase 8.0L and Papain Activities (Units/ gfabric)**

soap

No enzyme

**Figure 3.** a) Whiteness Index and (b) Whiteness Index after bleaching with Η2Ο2 of degummed silk fabrics using Mar‐

**(a)**

**Figure 2.** a) weight loss of silk fabrics treated with different types of proteases at enzyme dosages ranged from 25 to 74 U. g-1fabric and (b) degumming efficiency of the enzymatic process

The amount of sericin in raw silk measured in terms of weight loss varies between 17and 38%. Some of the good Chinese and Japanese varieties show about 17 to 17.5%, while yellow Italian silk has about 23% sericin and some Thai varieties have as high as 38% [27].

#### *3.2.2. Wettability and crystallinity*

Wettability was a function of both enzyme dosage and type of protease. Silk fabrics treated with Esperase® 8.0L at 75 U. g-1 fabric showed adequate water absorbency (<1 sec). On the other hand silk fabrics treated with papain showed higher wetting times namely more hydropho‐ bic fabrics. Although the implementation of higher papain activities formed silk fabrics with lower wetting times (Table 7) these values were higher than 1 sec, which is the maximum wetting time required for efficient dyeing and finishing. The lowest wetting time achieved using papain was 3.65 sec at the highest papain activity (75 U. g-1 fabric).


**Table 7.** Wetting time and Crystallinity Index of silk fabrics treated conventionally (Marseille soap) or enzymatically.

The microstructure of cotton fabrics was investigated by X-ray diffraction. The results are presented on Table 7. The diffraction curves of all silk fibers exhibit the typical pattern of a silk II crystal with high crystallinity [19].

Silk fabrics treated with Esperase® 8.0L exhibited higher Crystallinity Index values com‐ pared to papain treated ones. Furthermore, at higher Esperase® 8.0L the crystallinity of the fabric was superior to that treated conventionally (Marseille soap).
