Recovery and Purification of Silk proteins from waste of silk industry.

OBJECTIVE:
To recover and purify silk protein sericin from waste silk from silk industry by various processes and equipment.

ABSTRACT

Sericin is the second main component in cocoons, which are removed in the silk reeling process of the raw silk industry and in the silk waste degumming of the spun silk industry. The main amino acid of sericin, serine, exhibits a skin moistening and anti-wrinkle action, which is interesting to use for film formation in this study. The extraction conditions of sericin from two silk wastes, pieced cocoon and inferior knubbs were studied to find the optimum extraction conditions. Boiling water extraction was considered based on the response surface methodology (RSM) in order to identify the important factors for the sericin extraction. The two factors considered were time and temperature. Both factors were needed to be independent parameters in the predicted equation in order to improve the model fit with R2 = 0.84. The components of extracted sericin were 18.24% serine, 9.83% aspartate, and 5.51% glycine with a molecular weight of 132 kDa. .

1 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry.

INTRODUCTION
Silk has been of great scientific interest for centuries and a new insight about these polymers have been surfacing with improved analytical methods and molecular biology tools. Silk is composed of broad range of primarily protein based high molecular weight polymers.Silk is produced by silkworm Bombyx mori which belongs to Bombycidae which is one of the families that produce silk.Certain spiders belonging to Saturnidae family also produce silk. Bombycidae produces a delicate twin thread of silk fibroin, which is coated by a protective cover of sericin. Silk protein is a similar to collagen, elastin, keratin, fibroin, sporgin etc. which is an essential constituent of cocoon filament. Sericulture in India is an essential cottage industry. The post rearing operations are fairly cost effective and silkworm rearing is still only considered as a side activity to main farm activity. India is the second largest producer of silk in the world and has the distinction of producing all the four varities of silk.Presently,India produces nearly 16,700mt silk and reeled silk prices are in the range of Rs 900-1300/Kg,the pierced cocoons and waste silk generated at the rearing are sold at Rs 80-100/kg,The waste contributes nearly 30 % of total cocoon production. Waste silk can be classified as waste from the cocoon, rearing waste and thread waste. Silk waste can be used as coarse yarn and spun silk, which can be incorporated in natural rubber to achieve the physiochemical properties. It is also possible to utilize the silk waste by extracting fibroin and sericin from silk polymer. This helps to make sericulture a viable agro industry. Structure of Silk Silk is a continuous strand of two-filaments cemented together forming the cocoon of silkworm, Bombyx mori.Silk filament is a double strand of fibroin, which is held together by gummy substance called silk sericine or silk gum.Silk fibroin is the protein that forms the filament of silkworm and gives its unique physical and chemical properties.Silk adapts various secondary structures,including -helix,-sheet,and crossed -sheet. Fibroin is a glycol protein composed of two equimolar protein subunits of 370 and 25 kDa covalently linked by disulphide bonds.Fibroin filament is made of both crystalline and amorphous domains. The use of recovered sericin from silk reeling and degumming processes in the silk industry will add more value to sericin. Moreover, the recovery of sericin will minimize the BOD and COD values in the waste water. Since sericin is composed of 80% amino acids that contain hydrophilic groups, such as serine, aspartate, and glycine, it can absorb moisture very well. Sericin is also a good antioxidant. Therefore, sericin is used in many applications, 2 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. for example, in the medical field as an anticoagulation agent and as an anticancer agent. In cosmetics, sericin is used in skin and hair products. Sericin that has a molecular weight of more than 20 kDa can be used as medical biomaterials, degradable biomaterials, compound polymers, functional bio membranes and hydrogels (Zhang, 2002; Lee et al., 2004; Kongdee et al., 2005). Sericin is interesting to determine its use as a biopolymer film. Biopolymer films are generally made from thin sheets of biomaterials such as proteins, carbohydrates, fats and biomass. These films can be used for food coatings to prevent water and oxygen permeation between food products and the atmosphere. In this research, the extraction conditions in boiling water of sericin from silk waste were studied in order to predict the equations for computing the amount of extracted sericin under a defined condition.

1.1

Sericin extraction and properties

1.11 Sericin extraction in boiling water

Two types of silk waste, pierced cocoons (the result of breeding moths that have emerged from their cocoons to produce eggs required for the next crops) and inferior knubbs (outer portion of the cocoon layer obtained in the first process of reeling cocoons), from Bombyx mori (Thai Golden Silk, Multivoltine strain) were used in this study.These raw materials were analyzed moisture and sericin contents following the standard method (ISA, 1993). The sericin contents were determined based on the gravimetric method. Sericin extraction in boiling water was studied with two important factors, which were temperature in the range of 82-120C (above 105C using autoclave) and time in the range of 10-60 min. The experimental design chosen for this study was the central composite design (CCD) and then was performed by response surface methodology (RSM). This design was a full factorial design with all combinations of the factors at two levels (high, +1, and low, -1). The centre point (coded level 0), which was the midpoint between the high and low levels, was repeated with five points. The range and levels of the independent variables with actual andcoded levels were given in Table 1. The total number of test runs needed for this design was 13. The sericin extraction was carried out by subjection silk waste of 20 grams in boiling water at a liquid ratio of

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Recovery and Purification of Silk proteins from waste of silk industry. 1:30 with various temperatures and times. After extraction, sericin solution was separated by hydraulic pressing at 2.5 MPa for 1 min. The silk waste was washed with hot water (100 ml) at 80C and then pressed again for 1 min to separate the extracted solution. The collected solution was measured for volume and the % dry solid was determined by hot air oven method at 105C in order to calculate the weight of total extracted soluble solid. Protein concentration in the sericin solution was determined using the Lowry method (Lowry et al., 1951). The percentage of sericin extraction was calculated based on sericin content in the raw material at different experimental designs. RSM was applied to analyze the effect of independent variables on the response parameter (% sericin extraction) by matching the response studied (Y) with the code factors using the polynomial model associated with the experimental design as defined in Equation 1. (1) where Y is the dependent variable, A0 is the constant coefficient, Ai is the linear coefficient, Aii is the quadratic coefficient, and Aij is the two factors interaction coefficient. 2.1.2 Statistical analyses

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Recovery and Purification of Silk proteins from waste of silk industry. Statgraphic 3.0 for Windows (Statistical Graphics Corp.) was used for regression and ANOVA analysis. Response surface graphs were obtained from the regression equation in actual levels of variables, keeping the response function on the Z axis with X and Y axes representing the independent variables. a yield of 77% for both. For pierced cocoons, Y = -948.66+17.27X1 +1.726X2 -0.0763X1 2 +0.0078X1X2 -0.0352X2 2 (4) R 2 = 0.84, p-value 0.0097 Maximum condition at 115C, 37 min, %Sericin extraction = 77.14% For inferior knubbs, Z = -1344.24+25.73X1

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Recovery and Purification of Silk proteins from waste of silk industry. +1.56X2 -0.1181X1 2 -0.0060X1X2 -0.0106X2 2 (5) R 2 = 0.83, p-value = 0.0120 Maximum condition at 108C, 43 min; %Sericin extraction = 77.10% As Y = %sericin extraction, X1 = Temperature (C), X2 = Time (min) 3.1.2 Properties of extracted sericin SDS gel electrophoresis was used to determine the molecular weight of sericin from pierced cocoons. It was found that the extracted sericin had a molecular weight distribution above 132 kDa compared with a standard marker as shown in Figure 2. This result was different from sericin extracted from Bombyx mori cocoons with saturated aqueous salt solution, which had molecular weights of 150, 250, and 6 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. 400 kDa (Takasu et al., 2002). It was implied that the extracted sericin was partly degraded by boiling water at high temperatures. The amino acid compositions of sericin were given for 18 kinds as shown in Table 2. The main amino acids in the sericin extracted from the pierced cocoons by boiling water were serine (32.74%), aspartic acid (17.64%), and glycine (9.89%). This composition was similar to those sericin extracted from the Bivoltine strain of different sources; 1) raw material with serine 31.99%, aspartic acid 15.74%, and glycine 14.20% (Vaithamsat and Kitpreechavanich, 2008), 2) degumming solution with serine 27.30%, aspartic acid 18.80%, and glycine 10.70% (Wu et al., 2007), and 3) hot water-soluble sericin with serine 28.00%, aspartic acid 17.97%, and glycine 16.29% (Zhang et al., 2004). The extracted sericin was used to make biopolymer since it had a high molecular weight (more than 20 kDa) and can absorb moisture well. These properties can be used for film formation

MATERIALS AND METHODS

Equipments
Principle of operation: The rotary evaporator is a device for gently and efficiently evaporating solvents from a mixture. It consists of a heated rotating vessel (usually a large flask) which is maintained under a vacuum through a tube connecting it to a condenser. The rotating flask is heated by partial emersion in a hot water bath. The flask's rotation provides improved heat transfer to the contained liquid; the rotation also strongly reduces the occurance of 'bumps' caused by superheating of the liquid. The solvent vapours leave the 7 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. flask by the connecting tube and are condensed in the condenser section. The condenser section is arranged so that the condensed vapours drain into another flask where they are collected. It is a very efficient way of rapidly removing large quantities of solvent.

Procedure
1. The solvent collection flask of the unit should always be emptied prior use to prevent 8 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. accidentally mixing of incompatible chemicals. SAFETY FIRST! 2. The flask with the solution is placed on the rotary evaporator. The use of a bump trap prevents the solution from accidentally splashing into the condenser (and being contaminated). It is highly advisable to start with a clean bump bulb in case something bumps over after all! This would allow the experimenter to recover the solution or solid.

3. A metal or Keck clip is used to secure the flask and the bump trap. The green one shown below fits 24/40 ground glass joints. Similar blue clips fit 19/22 joints and the yellow ones fit 14/20 joints, which will most likely used in the lab. If you break the bump trap, you will have to pay for it! 4. The dial on the motor is used for speed control of the flask rotation. A typical rotavap uses a variable speed sparkless induction motor that spins at 0-220 rpm and provides high constant torque. A good setting here is 7-8. 5. The aspirator vacuum is turned on. On most models, the vacuum on/off control is managed by turning a stopcock at the top of the condenser (left side of the above diagram). This stopcock is later also used to vent the setup after the solvent is removed. 6. The flask is lowered into the water bath (or the water bath is raised to immerse the flask in the warm water. (On most models, a convenient handle (with height locking mechanism) moves the entire condenser/motor/flask assembly up and down. Often the tilt of the condenser assembly can also be adjusted. The water bath temperature should not exceed the boiling point of the solvent!! For small amounts of common solvents the bath heater is not needed. 7. The solvent should start collecting on the condenser and drip into the receiving flask. Some solvents (such as diethyl ether or dichloromethane) are so volatile that they will also evaporate from the receiving flask and be discharged down the drain. To prevent this, a cooling bath on the receiver or (on some models) use a dry-ice condenser can be used. In addition, an additional trap (with dry-ice or liquid nitrogen) can be placed between the vacuum source and the condenser unit. This is particularly important of a membrane pump is used as vacuum source. 9 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry.

8. Once all the solvent evaporated (or whatever is desired at this point), the vacuum is released,. The flask is raised out of the water bath and the spinning is discontinued. 9. The bump trap has to be cleaned and the receiving flask is emptied upon completion of the evaporation

Vacuum Drier
Principle Of Operation:The term drying generally refers to the removal of moisture from substance. Vacuum tray drying is a batch drying processing many cases it may be desirable to dry materials on trays more rapidly than can be done by passing a stream of air over them and yet to maintain the temperature lower than would correspond to the evaporation of water at atmospheric pressure. Vacuum tray drier are used for materials that cannot be subjected to high temperature as much be reached in a compartment dryer such as pharmaceuticals. They are also suitable for materials which are sensitive when contacted to air or any oxidizing gases. If the liquid to be vaporized is a valuable solvent that can be easily collected in a condenser. The Vacuum tray drier is suited for drying of materials where the material is to be obtained in a powder form Procedure: 1. The sample taken in petri plate Is placed in the tray. 2.The petri plate contains sample that has to be dried and brought to powder form. 3.The tray is placed inside the vacuum drying chamber and the door is closed and tightened. 4.The heater is switched on and the vapor line is closed by immersing the coil in ice,so that the vapors coming out is condensed. 5.The vacuum pump is switched on, required pressure should be set . 6.The temperature is maintained at or below 50. 7.When the sample is dried,the tray is taken out and the dried sample is scraped out into powder form. Agarose gel electrophoresis Electrophoresis is a technique used to separate and sometimes purify macromolecules especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.

When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have 10 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value

The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks: Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders). Acrylamide is a potent neurotoxin and should be handled with care!Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide. Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins. Protein estimation by Lowrys method Objective: To determine the concentration of proteins by Lowrys method.

Reagents required: 1. BSA stock solution (0.2mg/ml), 11 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. 2. Analytical reagents:

(a) 50 ml of 2% sodium carbonate mixed with 50 ml of 0.1 N NaOH solution (0.4 gm in 100 ml distilled water.) (b) 10 ml of 1.56% copper sulphate solution mixed with 10 ml of 2.37% sodium potassium tartarate solution. (c)Prepare analytical reagents by mixing 1 ml of (b) with 50 ml of (a)

3. Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N) with an equal volume of water on the day of use (2 ml of commercial reagent + 2 ml distilled water) Principle:

The phenolic group of tyrosine and tryptophan residues ( amino acid) in a protein will produce a blue purple color complex , with maximum absorption in the region of 660 nm wavelength, with Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and phosphate.

Thus the intensity of color depends on the amount of these aromatic amino acids present and will thus vary for dierent proteins.

Most proteins estimation techniques use Bovine Serum Albumin (BSA) universally as a standard protein, because of its low cost, high purity and ready availability.

The method is sensitive down to about 10 g/ml and is probably the most widely used protein assay despite its being only a relative method , subject to interference from Tris buer, EDTA, non ionic and cationic detergents, carbohydrate, lipids and some salts. The incubation time is very critical for a reproducible assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5 is essential. Procedure 12 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. Dierent aliquotes of BSA solutions were prepared by mixing stock BSA solution (1 mg/ ml) 0, 0.2, 0.4,0.6, 0.8 and 1 ml

The nal volume in each of the test tubes was made upto 1ml by adding distilled water.

5ml of reagent C was added. Solutions were mixed well.

This solution was incubated at room temperature for 10 minutes.

0.5ml of reagent Folin Ciocalteau solution was added to each tube and incubated in dark for 30 minutes.

Absorbance was taken at 660 nm.

The absorbance against protein concentration graph was plotted to get a standard calibration curve.

From the absorbance values of unknown samples , concentration was determined from the standard calibration curve. What is SDS-PAGE? SDS (Sodium dodecyl sulphate) is a detergent used to denature proteins and give them a negative charge PAGE: Polyacrylamide Gel Electrophoresis

It is a technique to separate proteins by their molecular weight

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Recovery and Purification of Silk proteins from waste of silk industry.

Polyacrylamide gel

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Recovery and Purification of Silk proteins from waste of silk industry.

How can we interpret our SDS-PAGE results Percent commonality is a measure of the similarity between the protein composition of 2 samples

How could this help us solve a question of identity in the case of different types of seafood? Introduction of Spectrometric Analyses

15 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. The study how the chemical compound interacts with different wavelengths in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis. The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum

REVIVAL OF LITERATURE

Silk thread produced by the domesticated silkworm, Bombyx mori; is composed of two kinds of protein: a fibrous protein (fibroin) which forms the thread core, and a gum-like protein (sericin) that surrounds the fibroin fibers to join them together. To obtain shiny aspect, soft handle, and elegant drape associated with silk fibers serisin is removed by boiling aqueous solutions containing soap, alkali, synthetic detergents, or organic acids, or recently by proteolytic enzymes (1) and discarded in the silk processing wastewater. Lately, there is a growing interest in the recovery of sericin from silk industry waste water not only to reduce the environmental impact of silk manufacturing (2) but also to utilize it as a valuable resource for many industries including cosmetics, pharmaceutical, biomedical, and food (3-6). For decades, much biochemical research has focused on the use of fibroin in biotechnological materials and biomedical applications (7, 8). Sericin, which has excellent moisture absorption and release properties, is antimicrobial (9), UV resistant and has pharmacological functions such as anticoagulation (10), anti-cancer and antioxidant activities (11, 12) with inhibitory action of tyrosinase (13), can find applications in functional biomaterials and textiles as well. It can impart useful and unusual properties to polymer gels, membranes, foams, fibers, and other composite materials (2). It can be used to produce cryopreservatives, anticoagulants, and biocompatible materials. With its unique properties, sericin can be used in the surface modifications of fibers and fabrics. In fact, it was used as a coating material for cellulose fibers and the treated textiles exhibited a decrease in free formaldehyde content, resistance to electricity, skin irritation and allergic reactions with increased water retention and only a negligible decrease in the textile tensile strength (14). Sericin was also impregnated into polyester fabric to overcome polyester hydrophobicity and to improve UV absorption properties of the sericin treated fabrics (15). Sericin, proposed as a candidate material for antimicrobial finishing of the textile by incorporating its powder form into synthetic fibers during process, may offer some additional advantages (16). It is a water-soluble natural protein constituting 25%-30% of a versatile 16 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. material like silk. The protein can be cross-linked, copolymerized, and blended with other macromolecular materials to produce materials with improved properties (2, 17). Considerable scientific research has demonstrated that improved functional property is achievable for carrier-based textile finishing applications by manipulation of the carrier particle size and morphology (18). Manufacturing of carrier-based dry powder formulations can be achieved by various methods including milling, freeze-drying and spray drying (19). The advantages of the spray drying process include the negligible possibility of degradation of heat-sensitive molecules and rapid production of dry powders from the solution in a onestep process with controlled particle characteristics (20-24); the disadvantages were reported to be the difficulty of obtaining a narrow size distribution and the tendency to agglomerate (19). Optimization and control of the powder dispersion and deposition properties could be an important phenomenon in the development of dry powder sericin microspheres, which may be attached to fabrics to impart unique properties. The aim of this study was to examine the physical characteristics--including particle size distribution, shape and moisture content as well as agglomeration degree of the sericin powders produced by a lab-scale spray dryer under various conditions. Since these characteristics are governed by formulation and process variables, the significance of these variables, as well as interactions between them, were examined using a factorial experimental design and linear regression analysis. The sericin feed concentration was chosen as formulation variable, and the solution feed rate and drying temperature chosen as process variables. This information will be useful for the optimization of the spray drying manufacturing of sericin powders with different dispersion and deposition properties. Degumming process - Silk degumming with dried papayas latex Raw silk was degummed by using degumming liquor with various concentrations of dried latex of papaya fruit. Degumming liquor was comprised papayas dried latex ranges 0%, 1%, 2%, 3%, and 4 % owf respectively. The liquor ratio of raw silk to degumming liquor was 1 : 25 (g : ml). Specimens were degummed at the temperature ranges 55, 65, 75 and 85 and time 10, 20, 30 and 40 minutes, then rinsed with cool and hot water and dried at 60 OC - Silk degumming with soap and Sodium carbonate Specimens were subjected to degum in a boiled alkaline solution containing 12 % owf soap and 6 %owf sodium carbonate for 60 minutes at a liquor ratio of 1 : 30 (g : ml). Degummed silks were rinsed with cool and hot water and dried at 60 OC Staining test with direct dyes 17 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. Hirus Supra Red 3BL 140% (C.I. Direct Red 80) was used to evaluate the remaining of sericin after degumming. The degummed silk samples were dyed in an dye liquor comprised Hirus Supra Red 3BL 140% 1 g/L with a liquor ratio of 1 : 200 (degummed silk : dye liquor) at 100 OC for 2 minutes. The color strength of dyed samples (K/S values) was measured by a spectrophotometer (Color Quest XE) under illuminant D65 with a 10O standard observer. Color strength was calculated from the reflectance values using Kubelka-Munk equation as follows R2)R1(S/K2= (1) Where R is the decimal fraction of the reflection of the dye fabric, K is the absorption coefficient and S is scattering coefficient. 2.5 Fiber surface morphology analysis Scanning electron microscope (SEM) was used to study surface morphology of degummed silk. The samples were coated with gold by sputtering at room temperature. Scanning electron micrographs of fibers were taken by scanning electron microscope (JSM-5410LV). The instrument was operated at 15 kv.

SUMMARY
Sericin is the second main component in cocoons, which are removed in the silk reeling process of the raw silk industry and in the silk waste degumming of the spun silk industry. The main amino acid of sericin, serine, exhibits a skin moistening and anti-wrinkle action, which is interesting to use for film formation in this study. The extraction conditions of sericin from two silk wastes, pieced cocoon and inferior knubbs were studied to find the optimum extraction conditions. Boiling water extraction was considered based on the response surface methodology (RSM) in order to identify the important factors for the sericin extraction. The two factors considered were time and temperature. Both factors were needed to be independent parameters in the predicted equation in order to improve the model fit with R2 = 0.84. The components of extracted sericin were 18.24% serine, 9.83% aspartate, and 5.51% glycine with a molecular weight of 132 kDa. Silk sericin microparticles were prepared by precipitation in organic solvent (methanol, ethanol and iso-propanol). Effect of organic solvent on the molecular characteristics of silk sericin microparticle were investigated by ATR FT-IR microspectroscopy. The results suggested that the conformation of the microparticle were changing from random coil to sheet and -turn after the treatment. After being treated with methanol, ethanol and iso-

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Recovery and Purification of Silk proteins from waste of silk industry. propanol, the -sheet and -turn proportion of microparticles were increased. The appearance of -sheet and -turn conformation may be associated with the packing of molecular chains.

REFERENCES
This work was supported by the Kasetsart UniversityResearch Development Institute (KURDI). The authors wish to thank Shinano Kenshi (Thailand) Co., Ltd., for supportingthe raw materials, the Faculty of Ago-Industry, Faculty ofScience, and KAPI, Kasetsart University, for the degummingprocess and instrument analyses. References Chollakup, R., Sinoimeri, A. and Dran, J-Y. 2004. Characteristics of Thai Hybrid Silk Fibres from different portions of the cocoon layer wastes: Feasibility in blending with cotton fibre. Journal of Insect Biotechnology Sericology 73, 39-45.Cuq, B., Gontard, N., Cuq, J. L. and Guilbert, S. 1997. Selected functional properties of fish myofibrillar protein-based films as affected by hydrophilic plasticizers. Journal of Agricultural and Food Chemistry. 45, 622-626.International Silk Association (ISA). 1993. Spun silk: Qualitylimits, testing procedures and recommendation sregarding quality, Group of Section IV, ZellwegerUster AG, Switzerland, pp. 5-8. Kongdee, A., Bechtold, T. and Teufel, L. 2005. Modification of cellulose fiber with silk sericin. Journal of Applied Polymer Science. 96, 1421-1428.Lee S.R, Miyazaki K., Hisada K. and Hori T. 2004. Applicationof silk sericin to finishing of synthetic fabrics.Sen-I Gakkaishi. 60, 9-15.Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.1951. Protein measurement with the Folin phenolreagent. Journal of Biological Chemistry. 93, 265-275.McHugh, T.H., Avena-Bustillas, R. and Krochta, J.M. 1993.Hydrophilic edible films: modified procedure forwater vapor permeability and explanation of thickness effects. Journal of Food Science. 58, 899-903.Schultz, T.H., Miers, J.C., Owens, H.S. and Maclay, W.D.1949. Permeability of pectinate films to water vapor. Journal of Physical Colloid Chemistry. 53, 1320-1330.Sothornvit, R. and Krochta, J.M. 2000. Water vapor permeability and solubility of films from hydrolyzed whey protein. Journal of Food Science. 65, 700-703.Takasu, Y., Yamada, H. and Tsubouchi, K. 2002.Isolation of three main sericin components from the cocoon ofthe silkworm, Bombyx mori. Bioscience Biotechnology Biochemistry. 66, 2715-2718.Vaithamsat, P. and Kitpreechavanich, V. 2008. Sericin separation from silk degumming wastewater. Separationand Purification Technology. 59, 129-133.Wu, J-H., Wang, Z. and Xu, S-Y. 19 Department of Biotechnology, RVCE, Bangalore.

Recovery and Purification of Silk proteins from waste of silk industry. 2007. Preparation and characterization of sericin powder extracted from silkindustry wastewater. Food Chemistry 103, 1255-1262.Zhang, Y-Q 2002. Applications of natural silk protein sericinin biomaterials. Biotechnology Advance. 20, 91-100.Zhang, Y.-Q., Tao, M.-L., Shen, W.-D., Zhou, Y.-Z., Ding, Y.,Ma, Y. and Zhou, W.-L. 2004. Immobilization of Lasparaginase on the microparticles of the natural silksericin protein and its characters. Biomaterials. 25,3751-3759. Y.Q. Zhang, Biotech Adv., 2002, 20, 91-100. 2. J.H. Wu, Z. Wang and S.Y. Xu, Food Chem., 2007, 103, 1255-1262. 3. X. Hu, D.Kaplan and P. Cebe, Macromolecules.,2006, 39, 6161-6170. 4. K.Y. Cho, J.Y. Moon, Y.W. Lee, K.G. Lee, J.H. Yeo, H.Y. Kweon, K.H. Kim and C.S. Cho, Biol Macromol., 2003, 32, 36-42. 5. K. Lee, H.Y. Kweon, J.H. Yeo, S.O. Woo, Y.W. Lee, C.S. Cho, K.H. Kim and Y.H. Park, Biol Macromol., 2003, 33, 75-80. 6. A. Kurioka, F. Kurioka and M. Yamazaki, Biosci Biotech Biochem., 2004, 68, 774-780.

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