Modified sericin protein and nanofibre composite production from this protein

Abstract

The invention relates to the modified sericin obtained as a result of modification of the sericin protein, one of the silk proteins, with amines, the nanofiber composite produced from said modified sericin, and the production method of this composite. In the invention, the functionality of the sericin is increased as a result of nucleophilic substitution with amines, and composites are produced from the modified sericin with increased functionality. The nanofiber composite subject to the invention can be used in many fields, especially chemistry, biomedicine and medicine, with its high oxygen permeability, similarity to extracellular matrix (ECM) structural components, high surface / volume ratio and adjustable pore size.

Description

[0001] MODIFIED SERICIN PROTEIN AND NANOFIBRE COMPOSITE PRODUCTION FROM THIS PROTEIN

[0002] Technical Field

[0003] The invention relates to the modified sericin obtained as a result of modification of the sericin protein, one of the silk proteins, with amines, the nanofibre composite produced from said modified sericin, and the production method of this composite. In the invention, the functionality of the sericin is increased as a result of nucleophilic substitution with amines, and nanofibre composites are produced from the modified sericin with increased functionality. The nanofibre composite subject to the invention can be used in many fields, especially chemistry, biomedicine and medicine, with its high oxygen permeability, similarity to extracellular matrix (ECM) structural components, high surface / volume ratio and adjustable pore size.

[0004] State of the Art

[0005] Silk protein fibres are a composite material consisting of a sericin protein and two fibroin proteins surrounded by sericin and are obtained from the silkworm of the Bombyx Mori species. Fibroin and sericin, both proteins, are found in fibres at the rate of 75% and 25%, respectively

[0001] . Silk fibroin fibres are approximately 10-25 pm in diameter and consist of two proteins, a light chain (-26 kDa) and a heavy chain (-390 kDa), linked in a 1 :1 ratio by a single disulphide bond. These proteins are covered with sericins (20-310 kDa), which are a family of hydrophilic proteins and have much more nucleophilic groups than fibroin [2], Sericin, with its 35% serine content, acts as a protein glue to fix fibroin fibres together in the cocoon. By means of the facts that the silk proteins, dipeptides and tripeptides can easily penetrate into the bloodstream from the dermis layer of the skin, also have antioxidant and antibacterial properties, are resistant to UV rays and easily absorb and release moisture, they have a wide range of applications in medicine, cosmetics, textile industry, microbial field and also in tissue engineering [3]. Additionally, controllable and stable biomaterials can be prepared by modification of the sericin, cross-linking and precipitation with other polymers, etc. However, during the processing of raw silk in all applications worldwide, sericin is separated from fibroin by the silk industry to improve the smoothness, luster, lightness and dyeability of the fibers [4], and for this reason, tons of sericin are turned into waste in silk processing facilities, especially by being thrown into sewers, causing environmental damage and not being reused. In the annual production of silk cocoons, which is reported as 600,000 tons, 150,000 tons of sericin are converted into waste from the processing process [5].

[0006] Chemical modification is frequently used in the state of the art as a powerful tool to add new properties to natural polymers. In the state of the art, various functional materials have been developed from polysaccharides such as cellulose, starch or chitin through chemical modification of hydroxyl groups [6]. Enzyme immobilisation support [7], biocompatible materials [8], anti-HIV reagents [9], anticoagulant materials

[0010] or inorganic-organic composites

[0011] are being developed by modifications of the fibroin protein, one of the silk proteins. Sericin, on the other hand, contains amino acids with much more nucleophilic groups than fibroin, reaching 50% of all amino acids, and this property distinguishes sericin from other proteins and makes sericin show high chemical reactivity [6] and is prone to modifications. In addition, by means of the polar side chains of the hydroxyl, carboxyl and amino groups in the structure of the sericin, it can be cross-linked, copolymerised or blended with other polymers and used in the production of new generation biodegradable materials with advanced properties.

[0007] In a study conducted by Teramoto et al. [6] in the state of the art, the synthesis of 4- cyanophenyl isocyanate-modified sericin derivatives using the lithium chloride / dimethyl sulfoxide (LiCI / DMSO) solvent system is described. In said document, sericin is modified with 4-cyanophenyl isocyanate in its solvent after being dissolved in LiCI / dimethyl sulphoxide (DMSO). In the study, it is said that chemical modification to the hydroxyl groups abundant in LiCI / DMSO is very useful to add new properties to the sericin. Furthermore, FTIR analysis shows that most 4-cyanophenyl groups are bonded with sericin molecules in the hydroxyl side chains via urethane linkages. It is stated that the modification makes the sericin less hygroscopic and more thermally unstable than before the modification. In said study, modification is made using the isocyanate molecule. The patent application numbered JP2012136795A in the state of the art relates to a method for producing sericin nanofibre and the nanofibre produced by this method. In said document, sericin is modified by treatment with an epoxy compound or a dibasic acid anhydride, and sericin nanofibre is produced using the electrospinning method. In said study, the experimental method and the resulting products are completely different. At the end of the reaction, fibres that can be used to remove water-insoluble cations are obtained.

[0008] It has become necessary to make a development in the relevant field due to reasons such as the limitations and inadequacies of the solutions in the state of the art, sericin protein becoming waste and therefore causing economic losses, environmental damage and limited usage areas.

[0009] Brief Description and Aims of the Invention

[0010] In the invention, a modified sericin obtained as a result of modification of the sericin protein, one of the silk proteins, with amines, the nanofibre composite produced from said modified sericin, and the production method of this composite are disclosed. In the invention, nanofiber composites with high oxygen permeability, structural similarity to extracellular matrix (ECM), high surface / volume ratio and adjustable pore size are produced from the sericin the functionality of which was increased as a result of nucleophilic substitution with naphthylamine, methylamine, ethylamine, ammonia, butylamine, aniline, benzylamine or furfurylamine. Each amine-modified sericin is reinforced separately into the nanofibre membrane. However, sericin produced by combining different amounts of amines can also be used in nanofibre production.

[0011] Formula X The aim of the invention is to reuse and recycle sericin waste. The nanofibre composite that is the subject of the invention comprises modified sericin as raw material. In this way, high value-added products are produced by utilising sericin waste and eliminating its negative impact on the environment.

[0012] Another aim of the invention is to produce a nanofibre composite for use in the fields of chemistry, biomedical, cosmetics and medicine. By means of the presence of amine groups in the modified sericin that is the subject of the invention, the basic feature of the structure and its affinity towards acidic structures (such as metal surfaces) have increased. For this reason, it can also be used in cancer studies with special metal complexes or in targeted drug delivery systems. In addition, said composite can be used in membrane production due to its smooth surface, non-agglomeration and morphological properties with a tight network structure at low pore diameters.

[0013] Another aim of the invention is to produce a nanofibre composite with high thermal and mechanical characteristics. In the invention, the mechanical and thermal properties of the composite that is the subject of the invention are increased by means of the contribution of modified sericins. Because it has been determined that modified sericin- doped nanofibre mats have a significant increase in strength compared to sericin- doped nanofibre mats and have almost 20 times more strength than sericin-doped nanofibre mats alone.

[0014] Description of Drawings

[0015] Figure 1. Sericin modified with naphthylamine

[0016] Figure 2. Sericin modified with methylamine

[0017] Figure 3. Sericin modified with ethylamine

[0018] Figure 4. Sericin modified with ammonia

[0019] Figure 5. Sericin modified with butylamine

[0020] Figure 6. Sericin modified with aniline

[0021] Figure 7. Sericin modified with benzylamine Figure 8. Sericin modified with furfurylamine

[0022] Figure 9. FTIR analysis of ammonia modified sericin (SAMT)

[0023] Figure 10. FTIR analysis of methylamine modified sericin (SMAT)

[0024] Figure 11. FTIR analysis of ethylamine modified sericin (SEAT)

[0025] Figure 12. FTIR analysis of butylamine modified sericin (SBUAT)

[0026] Figure 13. FTIR analysis of aniline-modified sericin (SANT)

[0027] Figure 14. FTIR analysis of benzylamine modified sericin (SBAT)

[0028] Figure 15. FTIR analysis of furfurylamine modified sericin (SFUAT)

[0029] Figure 16. FTIR analysis of naphthylamine modified sericin (SNAT)

[0030] Figure 17. Sericin FTIR Analysis

[0031] Figure 18. H-NMR analysis of the sericin

[0032] Figure 19. H-NMR analysis of ammonia-modified sericin

[0033] Figure 20. H-NMR analysis of the methylamine-modified sericin

[0034] Figure 21. H-NMR analysis of ethylamine-modified sericin

[0035] Figure 22. H-NMR analysis of butylamine modified sericin

[0036] Figure 23. H-NMR analysis of aniline-modified sericin

[0037] Figure 24. H-NMR analysis of benzylamine modified sericin

[0038] Figure 25. H-NMR analysis of furfurylamine modified sericin

[0039] Figure 26. H-NMR analysis of naphthylamine modified sericin

[0040] Figure 27. Sericin thermal degradation curve (Thermogram)

[0041] Figure 28. Thermal decomposition curve (%TG%) of the sericin modified with ammonia

[0042] Figure 29. Thermal decomposition curve of the methylamine-modified sericin Figure 30. Thermal decomposition curve of the ethylamine-modified sericin

[0043] Figure 31. Thermal decomposition curve of butylamine modified sericin

[0044] Figure 32. Thermal decomposition curve of aniline-modified sericin

[0045] Figure 33. Thermal decomposition curve of benzylamine modified sericin

[0046] Figure 34. Thermal decomposition curve of furfurylamine modified sericin

[0047] Figure 35. Thermal decomposition curve of the sericin modified with naphthylamine

[0048] Detailed Description of the Invention

[0049] The invention relates to the modified sericin obtained as a result of modification of the sericin protein, one of the silk proteins, with amines, the nanofiber composite produced from said modified sericin, and the production method of this composite. In the invention, nanofiber composites with high oxygen permeability, structural similarity to extracellular matrix (ECM), high surface / volume ratio and adjustable pore size are produced from the sericin the functionality of which was increased as a result of nucleophilic substitution with naphthylamine, methylamine, ethylamine, ammonia, butylamine, aniline, benzylamine or furfurylamine.

[0050] The structure of the sericin modified here is specified below with Formula Y.

[0051] Formula Y

[0052] With the modification applied in the invention, the functional groups designated as R in Formula X and -OH groups in Formula Y are changed. That is, the R groups in Formula X are the -OH groups in Formula Y before modification. The general structure of the modified sericins that are the subject of the invention obtained after said nucleophilic substitution is shown by Formula X.

[0053] Formula X

[0054] The general structure of the modified sericins that are the subject of the invention is shown by Formula X. In Formula X R'= can be any of methane sulphonate, para toluene sulphonate or mesityl sulphonate. Preferably, in the invention R' = methane sulphonate. In said Formula X, If R is R= NH2-, R'= methane sulphonate, the resulting compound is shown with Formula 1 . Formula 1 is called ammoni 1 derivative of sericin protein (SAMT).

[0055] Formula 1 In said Formula X, If R is R=NH" and R'= methane sulphonate, the compound obtained is shown in Formula 2. Formula 2 is called methylamine derivative of sericin protein (SMAT).

[0056]

[0057] Formula 2

[0058] In said Formula X, If R is R= and R'= methane sulphonate, the compound obtained is shown in Formula 3. Formula 3 is called methylamine derivative of sericin protein (SEAT).

[0059] Formula 3

[0060] In said Formula X, If R is R= and R'= methane sulphonate, the compound obtained is shown in Formula 4. Formula 4 is called methylamine derivative of sericin protein (SBUT).

[0061]

[0062] Formula 4

[0063] In said Formula X, If R is R= and R'= methane sulphonate, the compound obtained is shown in Formula 5. Formula 5 is called methylamine derivative of sericin protein (SANT).

[0064] Formula 5 In said Formula methane sulphonate, the compound obtained is shown in Formula 6. Formula 6 is called methylamine derivative of sericin protein (SBAT).

[0065] Formula 6 ln said Formula X, If R is R= and R'= methane sulphonate, the compound obtained is shown in Formula 7. Formula 7 is called methylamine derivative of sericin protein (SFUAT).

[0066] Formula 7 In said Formula methane sulphonate, the compound obtained is shown in Formula 8. Formula 8 is called methylamine derivative of sericin protein (SNAT).

[0067] Formula 8

[0068] Production method of the nanofiber composite subject to the invention comprises the process steps of: i. boiling the waste liquids of cocoons obtained from silk production, ii. filtering the solution concentrated by boiling with filter paper to remove the cocoons and / or solid particles remaining in the solution, iii. removing the water of the filtered aqueous solution by evaporator, iv. removing the water remaining in the solution by freezing with a lyophiliser and obtaining powder sericin, v. mixing the powder sericin in LiCI / DMSO (lithium chloride / dimethyl sulphoxide), vi. mixing aliphatic or aromatic sulphonyl chloride in Li / DMSO, vii. adding the solution prepared in the process step (v) to the solution prepared in the process step (vi) and stirring overnight, viii. adding TEA (triethylamine) drop by drop to the mixture prepared in (vii) and mixing, ix. synthesising modified sericins by adding and mixing primary and / or secondary amine and TEA dropwise simultaneously to the mixture prepared in (viii), x. pouring the mixture containing modified sericins into DCM (dichloromethane) and keeping it in the refrigerator to precipitate, xi. separation of the waited mixture by centrifugation, xii. lyophilising the modified sericin separated by centrifugation and simultaneously doping it with both hydrophilic and hydrophobic polymers to obtain nanofibres, and xiii. producing nanofibre composites using the electrospinning technique with the obtained nanofibres.

[0069] In another embodiment of the invention, the production method of the nanofiber composite that is the subject of the invention comprises the process steps of: i. boiling the waste liquids of cocoons obtained from silk production for 3-24 hours, ii. filtering the solution concentrated by boiling with filter paper to remove the cocoons and / or solid particles remaining in the solution, iii. removing the water of the filtered aqueous solution by evaporator, iv. removing the remaining water in the solution by freezing it with a lyophiliser at minimum -120°C and maximum -20°C and obtaining powder sericin, v. mixing 0,1 -1 gr powder sericin in 5-50 ml LiCI / DMSO (lithium chloride / dimethyl sulphoxide) for 2-6 hours, vi. mixing 0.001 -0.1 mol of aliphatic or aromatic sulphonyl chloride in 5-50 ml Li / DMSO for 1 -5 hours, vii. adding the solution prepared in the process step (v) to the solution prepared in the process step (vi) and stirring overnight, viii. Adding 0.001-0.1 mol of TEA (triethylamine) dropwise to 0.1 -1 mL of the mixture prepared in (vii), ix. synthesising modified sericins by adding primary and / or secondary amine and 0.1 -1 mL TEA dropwise simultaneously to the mixture prepared in (viii) at 40-80 °C and stirring for 1 night, x. pouring the mixture containing modified sericin into 20-100 mL DCM (dichloromethane) and keeping it in the refrigerator for 1 -5 hours to precipitate, xi. separating the mixture waited by centrifugation at 1000-10000 rpm for 5-50 minutes, xii. lyophilising the remaining products at -100-(-20)°C and by simultaneously doping them with both hydrophilic and hydrophobic polymers, obtaining nanofibres, and xiii. producing nanofibre composites using the electrospinning technique with the obtained nanofibres.

[0070] In another embodiment of the invention, the production method of the nanofiber composite that is the subject of the invention comprises the process steps of: i. boiling the waste liquids of cocoons obtained from silk production for 6 hours, ii. filtering the solution concentrated by boiling with filter paper to remove the cocoons and / or solid particles remaining in the solution, iii. removing the water of the filtered aqueous solution by evaporator, iv. removing the water remaining in the solution by freezing with a lyophiliser at -80°C and obtaining powder sericin, v. mixing 0.5 gr powder sericin in 10 ml LiCI / DMSO (lithium chloride / dimethyl sulphoxide) for 3 hours, vi. mixing 0.005 mol of aliphatic or aromatic sulphonyl chloride in 10 ml Li / DMSO for 2 hours, vii. adding the solution prepared in the process step (v) to the solution prepared in the process step (vi) and stirring overnight, viii. adding 0.005 mol of TEA (triethylamine) dropwise to 0.069 ml of the mixture prepared in (vii) and mixing for 2 hours, ix. synthesising modified sericins by adding primary and / or secondary amine and 0.1 -1 mL TEA dropwise simultaneously to the mixture prepared in (viii) at 40-80 °C and stirring for 1 night, x. pouring the mixture containing modified sericin into 50 mL DCM (dichloromethane) and keeping it in the refrigerator for 1 hour to precipitate, xi. separating the mixture waited by centrifugation at 3000 rpm for 10 minutes, xii. lyophilising the remaining products at -80°C and by simultaneously doping them with both hydrophilic and hydrophobic polymers, obtaining nanofibres, and xiii. producing nanofibre composites using the electrospinning technique with the obtained nanofibres.

[0071] In the method of the invention, the primary and / or secondary amines mentioned in the process step (ix) are naphthylamine, methylamine, ethylamine, ammonia, butylamine, aniline, benzylamine or furfurylamine, but various primary and secondary amines can also be used in this derivatisation. Moreover, in the process step (xii) of said method, the hydrophilic polymer mentioned is PVA and the hydrophobic polymer is PCL or PLA.

[0072] The solutions given in Table 1 were produced and optimised with the electrospinning system containing a coaxial nozzle according to the nanofibre composite production parameters in Table 1 . Double syringe pump and coaxial nozzle were preferred in the electrospinning system. Using the coaxial nozzle, hydrophobic polymer solutions such as PCL and PLA were fed from the outer nozzle, while solutions containing PVA (polyvinyl alcohol) hydrophilic polymer, sericin and modified sericin substances were fed from the inner nozzle to obtain nanofibre composites. In addition, in the invention, nanofibre production was achieved by feeding hydrophobic polymer solutions such as PCL (polycaprolactone), PLA (polylactic acid) through the inner nozzle, while solutions with PVA hydrophilic polymer, sericin and modified sericin substances added were fed from the outer nozzle. The produced nanofibre composites were dried in a vacuum oven at 40-70°C for 12-36 hours before characterisation studies, and solvents were removed from the nanofibre composite membranes.

[0073] Table 1. Parameters of nanofibre composite solution preparation by electrospinning technique with coaxial nozzle Table 2. Nanofibre composite production parameters by electrospinning technique with coaxial nozzle.

[0074] The solutions prepared according to Table 1 were produced with the electrospinning system containing a coaxial nozzle, according to the nanofibre composite production parameters in Table 2. Double syringe pump and coaxial nozzle were preferred in the electrospinning system. Using the coaxial nozzle, hydrophobic polymer solutions such as PCL and PLA were fed from the outer nozzle, while solutions containing PVA hydrophilic polymer, sericin and modified sericin substances were fed from the inner nozzle to obtain nanofibre composites. In addition, in the invention, nanofibre production was achieved by feeding hydrophobic polymer solutions such as PCL, PLA through the inner nozzle, while solutions with PVA hydrophilic polymer, sericin and modified sericin substances added were fed from the outer nozzle. The produced nanofibre composites were dried in a vacuum oven at 50°C for 24 hours before characterisation studies, and solvents were removed from the nanofibre composite membranes.

[0075] Table 3. Mechanical tensile test results of nanofibres obtained in the invention.

[0076] Max

[0077] F rnorrccpe - Tr-ensile r Eilast-ic Mod -1u 1le Extension

[0078] Sample name Strength E ' °

[0079] Waxed paper (The Essential Strength of the Paper is the strength of the remaining material when the active ingredient is in its sprayed form and falls 6,116 out of the waxed paper tare) The nanofiber structures of the composites produced by the method of the invention were evaluated morphologically with scanning electron microscope (SEM) images, and while only the nanofibres in the sericin-doped composite vary in the range of 60-500 nm, the ammonia and methylamine modified nanofibres vary in the range of 100-800 nm, and the others vary in the range of 100-400 nm. Additionally, in the invention, the biological activities of nanofibres were examined. Cytotoxicity, ISO 10993-5 Cell Viability (MTT)) and cell viability tests were performed. After the characterisations, the biological activities of sericin and modified sericin-doped nanofibre mats were examined. While none of the mats examined showed a cytotoxic effect, cell retention was evaluated to be quite high in most of them. While the least amount of cell retention occurred on furfurylamine and benzylamine derivative fibres, significantly more cell retention occurred on the methylamine derivative sericin, even compared to the normal sericin. It has been proven that all products can be used in biological applications as they are both biocompatible and have cell retention.

[0080] Table 4. FTIR peaks of modified sericin derivatives

[0081] Sericin Amide I Amide II Amide I Amide II Other (1400-400)

[0082] Derivati ve(N-H) (N-H) (C-N) C-N peak (cm4) peak (cm4) peak (cm4) peak (cm’ peak (cm’

[0083] ') ')

[0084] SAMT 3380 3261 1644 1535 1406, 1318, 1265, 1051

[0085] SMAT 3335 3239 1638 1529 1406, 1315, 1267, 1015

[0086] SEAT 3360 3260 1648 1531 1404, 1316, 1223, 1012

[0087] SBUAT 3343 3267 1647 1505 1407, 1318, 1225, 1014

[0088] SANT 3335 3245-3045 1649 1529 1403, 1315, 1224, 1013

[0089] SBAT 3343 3218-3049 1648 1524 1405, 1314, 1231, 1015

[0090] SFUAT 3355 3255-3063 1644 1538 1404, 1316, 1231, 1012

[0091] SNAT 3386 3262 1645 1534 1405, 1318, 1235, 1012

[0092] FTIR and NMR analyses were performed to examine the structural properties of silk sericin (SS) at the molecular level. According to the absorption bands in the spectrum seen in Figure 17, the characteristic peaks of the sericin are observed as 3201 cm-1, 3056 cm-1, 2929 cm-1, 1638 cm-1, 1513 cm-1, and 1251 cm-1

[0012] , When the groups in the molecular structure of these bands are examined in detail, the most intense absorption bands in the SS structure belong to amide groups. The peaks with wavelengths 3201 , 3056 cm-1are N-H stretching vibrations belonging to amide groups. The O-H stretching band is located at 3600-3200 cm-1, which is in the region overlapping with the peak of the N-H stretching vibration band in the range of 3500- 3000 cm-1

[0013] . The peaks at 1638, 1514 cm-1are the vibrations of amide I and amide II. The wavelength of the amide I group is in the range of 1600-1700 cm-1, and they are the stretching vibrations of the C=O and C-N groups. The peak at 1638 cm-1confirms the presence of the amide I group. Amide II is located in the 1510-1580 cm-1region. The peak at 1514 cm-1indicates the presence of amide II [14-15]. Other peaks located in the range of 1500-400 cm-1may be related to complex amide III and / or IV bands. The band at 1452 cm-1is C-H and O-H bending vibrations, and the band at 1055 cm-1is C-OH stretching vibration absorption bands and may be attributed to the relatively high content of carboxylic acid and alkyl hydroxyl-containing amino acid side chains in this sericin of bands

[0016] . It was determined that the peaks seen in the FTIR spectrum of the sericin powder prepared in this study were compatible with the results obtained by other researchers.

[0093] Looking at the characteristic peaks that occur according to the FTIR spectrum of modified sericins (M-SS), aromatic and aliphatic C-H peaks are in the range of 3100- 2800 cm-1, N-H peaks of amines are in the range of 3400-3200 cm-1, N-H peaks of amide groups are in the range of 3300-3400 cm-1, the peaks belonging to the C=N amide group are in the range of 1650-1500, and the stretching vibration bands of C-N bonds are in the range of 1200-1300 cm-1

[0012] , When the FTIR spectra of modified sericin, to which aromatic amines (benzylamine, naphthylamine, furfurylamine, aniline) are bound, are examined, 3100-300 cm-1aromatic C-H stretching bands are seen in all 4 spectra [17-18].

[0094] Table 5. TGA peaks of modified sericin derivatives

[0095] Sericin Temperature ranges (°C) where segmental

[0096] Derivative losses are observed

[0097] SAMT 50-105 105-270 270-500 500-850

[0098] SMAT 50-105 105-280 280-520 520-850

[0099] SEAT 45-105 105-320 320-520 520-850

[0100] SBUAT 50-150 150-315 315-540 540-850 SANT 60-122 122-210 210-521 521-621

[0101] SBAT 65-110 110-280 280-535 535-850

[0102] SFUAT 55-125 125-280 280-520 520-850

[0103] SNAT 50-150 150-360 360-450 450-850

[0104] Figure 27 shows the TGA chart of the raw sericin. Looking at the TGA thermogram, 4 mass loss regions were determined and the temperatures at which these transitions started were determined as T1 60, T2 153, T3 243, T4 366 °C. The results are consistent with those published by Balcao et al., (2020) on the thermogravimetric analysis of peptide moieties of crude sericin extract

[0019] . TGA graphs of modified sericins (M-SS) are given in Figure 28-35. When we look at the TGA thermograms, 4 mass loss regions are determined and the temperature ranges where these transitions occur are given in Table 5.

[0105] When the1H Spectrum of the sericin in Figure 18 was examined, it was observed that there were 3 main peaks for the sample prepared in D-DMSO solution. Peaks arising from amide Hs in the sericin protein are observed around 8-9 ppm. Since the structure is polymeric and consists of many isomers, a broad broad peak was observed instead of a single sharp peak. The hydrogen of the carbon to which the nitrogen atom is bonded gives another characteristic peak. This peak was observed at approximately 4.6 ppm. In the other characteristic peak, the peaks of hydrogens attached to the -CH2 group to which -OH groups are attached are observed at approximately 3.8 ppm. Normally, if sericin consisted only of serine amino acids, no other peak should be observed, but there are various peaks between 1 -3. This is due to the peaks given by the aliphatic hydrogens of different amino acids in the sericin group. The methyl group of amino acids such as valine, isoleucine, threonine and valine gives peaks in the range of approximately 0.5-1 ppm. It is also found in other amino acids like this [20-21],

[0106] Detailed information1H NMR analysis was carried out for the purpose of molecular structure determination based on the structural arrangement of M-SS and the hydrogen contents in the molecular structure. DMSO was used as the solvent in this analysis. According to the1H Spectra in Figure 19-26, the peaks belonging to the - CONH proton in the amide group formed instead of the carboxylic acid in the structure are in the range of 8-9 ppm. The proton peaks of the S-NH group, which is in the sulphonamide group formed as a result of the Hinsberg reaction of the free amine group in the protein structure, are seen in the range of 7-8 ppm, and sulphonamide methyls (-O2S-CH3) are seen in the spectra as a single sharp peak at 2.9-3.0 ppm. The -NH group in the sulphonamide structure is in the range of 3-3.5. -NH belonging to the naphthylamine and aniline group is in the range of 7-7.5 ppm, and NH belonging to other amines is in the range of 3-4.5 ppm [22-23].

[0107] When the DSC (thermal analysis) results of the fibres produced in the invention were evaluated (Table 6), the differences between the melting and crystallisation temperatures of sericin-containing fibres and modified sericin-containing fibres were determined. According to the sericin-doped nanofibre structure, the methyl amine- modified nanofibre with the highest crystallisation temperature was determined.

[0108] Table 6. Thermal analysis (DSC) results of the fibres that are the subject of the invention REFERENCES

[0109] [1] Kerim DURAN, D. 0. (1970, January 1 ). ipek liflerindeki Serisinin Enzimatik Olarak uzakla§tinlmasi. Tekstil ve Konfeksiyon.

[0110] [2] Zhou CZ, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, Jacquet M,

[0111] Janin J, Duguet M, Perasso R, Li ZG. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 2000;28(12):2413-2419.

[0112] [3] Baby Joseph & S. Justin Raj (2012) Therapeutic applications and properties of silk proteins from Bombyx mori, Frontiers in Life Science, 6:3-4, 55-60, DOI:

[0113] 10.1080 / 21553769.2012.760491

[0114] [4] Lamboni, L.; Gauthier, M.; Yang, G.; Wang, Q. Silk sericin: A versatile material for tissue engineering and drug delivery. Biotechnol. Adv. 2015, 33, 1855-1867.

[0115] [5] Oh, H., Lee, J. Y., Kim, M. K., Um, I. C., Lee, K. H. 2011. “Refining hot-water extracted silk sericin by ethanol-induced precipitation”, International Journal of Biological Macromolecules, 48(1), 32-37.

[0116] [6] C;, T. H. K. (n.d.). Chemical modification of silk sericin in lithium chloride / dimethyl sulfoxide solvent with 4-cyanophenyl isocyanate. Biomacromolecules.

[0117] [7] A;, D. M. T. T. (n.d.). Characterization of low-temperature-plasma treated silk fibroin fabrics by Esca

[0118] [8] N;, G. Y. M. (n.d.). Effect of the chemical modification of the arginyl residue in

[0119] Bombyx Mori silk fibroin on the attachment and growth of fibroblast cells. Journal of biomedical materials research.

[0120] [9] Gotoh KJzumi H;Kanamoto T;Tamada Y;Nakashima H; (n.d.). Sulfated fibroin, a novel sulfated peptide derived from silk, inhibits human immunodeficiency virus replication in vitro. Bioscience, biotechnology, and biochemistry.

[0121]

[0010] Y;, T. (n.d.). Sulfation of silk fibroin by chlorosulfonic acid and the anticoagulant activity. Biomaterials. https: / / pubmed.ncbi.nlm.nih.gOv / 14585685 /

[0011] J;, F. T. A. (n.d.). Nano-scaled hydroxyapatite / polymer composite I. Coating of sintered hydroxyapatite particles on poly(gamma-methacryloxypropyl trimethoxysilane)grafted silk fibroin fibers through chemical bonding. Journal of materials science. Materials in medicine. httDs: / / Dubmed.ncbi.nlm.nih.qov / 15338587 /

[0122]

[0012] Al-Tabakha, M. M„ Khan, S. A., Ashames, A., Ullah, H„ Ullah, K„ Murtaza, G„ Hassan, N. 2021. Synthesis, Characterization and Safety Evaluation of Sericin- Based Hydrogels for Controlled Delivery of Acyclovir. Pharmaceuticals, 14(3), 234.

[0123]

[0013] Saha, J., Mondal, M. I., Sheikh, M. K., & Habib, M. A. (2019). Extraction, structural and functional properties of silk sericin biopolymer from Bombyx mori silk cocoon waste. J. Text. Sci. Eng, 9(2).

[0124]

[0014] Zhang, X., & Wyeth, P. (2010). Using FTIR spectroscopy to detect sericin on historic silk. Science China Chemistry, 53, 626-631 .

[0125]

[0015] Teramoto, H., Nakajima, K. I., Takabayashi, C. 2004. Chemical modification of silk sericin in lithium chloride / dimethyl sulfoxide solvent with 4-cyanophenyl isocyanate. Biomacromolecules, 5(4), 1392-1398.

[0126]

[0016] Khan, M. M. R., Tsukada, M., Zhang, X., & Morikawa, H. (2013). Preparation and characterization of electrospun nanofibers based on silk sericin powders. Journal of Materials Science, 48, 3731 -3736.

[0127]

[0017] Li, X., Sun, C., & Wei, Z. (2005). Electrochemical properties of poly-1- naphthylamine synthesized in the presence of ferrocenesulfonic acid. Synthetic metals. 155(1 ), 45-50.

[0128]

[0018] Gao, G„ Wang, X., Chen, Q„ Xie, C„ Zheng, J., Fan, H„ ... & Hu, X. (2021 ). Synergy of surface fluorine and oxygen vacancy of TiO 2 nanosheets for O 2 activation in selective photocatalytic organic transformations. Journal of Materials Chemistry C, 9(5), 1593-1603.

[0019] Balcao, V. M., Harada, L. K., Jorge, L. R., Oliveira Jr, J. M., Tubino, M., & Vila, M.

[0129] M. (2020). Structural and functional stabilization of sericin from Bombyx mori cocoons in a biopolysaccharide film: Bioorigami for skin regeneration. Journal of the Brazilian Chemical Society, 31 , 833-848.

[0130]

[0020] Kovalenko, E. A., & Mainichev, D. A. (2015). Supramolecular system of aminoacids and cucurbit [7] uril: NMR studies in solution. Applied Magnetic Resonance, 46, 281 -293

[0131]

[0021] Zuge, L. C. B., Silva, V. R., Hamerski, F., Ribani, M., Gimenes, M. L., & Scheer, A. P. (2017). Emulsifying properties of sericin obtained from hot water degumming process. Journal of Food Process Engineering, 40(1 ), e12267.

[0132]

[0022] Das, D. (2020). Aromatic and Aliphatic Amines.

[0133]

[0023] Birchall, T., & Gillespie, R. J. (1963). Nuclear Magnetic Resonance Studies of The

[0134] Protonation Of Weak Bases In Fluorosulphuric Acid: II. Amides, Thioamides, And Sulphonamides. Canadian Journal of Chemistry, 41 (10), 2642-2650

Claims

CLAIMS1. A nanofibre composite comprising a modified sericin molecule having the chemical structure represented by Formula X or any combination of modified sericin molecules having the chemical structure represented by Formula XFormula X wherein R is selected from the formulas below:and R’= is selected from methane sulphonate, para toluene sulphonate or mesityl sulphonate.

2. A composite according to claim 1 , wherein said molecule is molecule shown byNH2- Formula 1 in the case that R= , R’= methane sulphonate.Formula 13. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 2 in the case that R=NH“, R’= methane sulphonate.Formula 24. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 3 in the case that, R’= methane sulphonate.Formula 35. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 4 in the case that, R’= methane sulphonate.Formula 46. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 5 in the case thatR’= methane sulphonate.Formula 57. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 6 in the case thatmethane sulphonate.Formula 68. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 7 in the case thatR’= methane sulphonate.Formula 79. A composite according to claim 1 , wherein said molecule is molecule shown byFormula 8 in the case thatmethane sulphonate.Formula 8 roduction method of nanofibre composite, comprising the process steps of: i. boiling the waste liquids of cocoons obtained from silk production, ii. filtering the solution concentrated by boiling with filter paper to remove the cocoons and / or solid particles remaining in the solution, iii. removing the water of the filtered aqueous solution by evaporator, iv. removing the water remaining in the solution by freezing with a lyophiliser and obtaining powder sericin, v. mixing the powder sericin in LiCI / DMSO (lithium chloride / dimethyl sulphoxide), vi. mixing aliphatic or aromatic sulphonyl chloride in Li / DMSO, vii. adding the solution prepared in the process step (v) to the solution prepared in the process step (vi) and stirring overnight, viii. adding TEA (triethylamine) drop by drop to the mixture prepared in (vii) and mixing, ix. synthesising modified sericins by adding and mixing primary and / or secondary amine and TEA dropwise simultaneously to the mixture prepared in (viii),x. pouring the mixture containing modified sericins into DCM (dichloromethane) and keeping it in the refrigerator to precipitate, xi. separation of the waited mixture by centrifugation, xii. lyophilising the modified sericin separated by centrifugation and simultaneously doping it with both hydrophilic and hydrophobic polymers to obtain nanofibres, and xiii. producing nanofibre composites using the electrospinning technique with the obtained nanofibres.

11. A method according to claim 10, comprising the steps of: i. boiling the waste liquids of cocoons obtained from silk production for 3-24 hours, ii. filtering the solution concentrated by boiling with filter paper to remove the cocoons and / or solid particles remaining in the solution, iii. removing the water of the filtered aqueous solution by evaporator, iv. removing the remaining water in the solution by freezing it with a lyophiliser at minimum -120°C and maximum -20°C and obtaining powder sericin, v. mixing 0,1 -1 gr powder sericin in 5-50 ml LiCI / DMSO (lithium chloride / dimethyl sulphoxide) for 2-6 hours, vi. mixing 0.001 -0.1 mol of aliphatic or aromatic sulphonyl chloride in 5- 50 ml Li / DMSO for 1 -5 hours, vii. adding the solution prepared in the process step (v) to the solution prepared in the process step (vi) and stirring overnight, viii. adding 0.001 -0.1 mol of TEA (triethylamine) dropwise to 0.1 -1 mL of the mixture prepared in (vii), ix. synthesising modified sericins by adding primary and / or secondary amine and 0.1 -1 mL TEA dropwise simultaneously to the mixture prepared in (viii) at 40-80 °C and stirring for 1 night,x. pouring the mixture containing modified sericin into 20-100 mL DCM (dichloromethane) and keeping it in the refrigerator for 1 -5 hours to precipitate, xi. separating the mixture waited by centrifugation at 1000-10000 rpm for 5-50 minutes, xii. lyophilising the remaining products at -100-(-20)°C and by simultaneously doping them with both hydrophilic and hydrophobic polymers, obtaining nanofibres, and xiii. producing nanofibre composites using the electrospinning technique with the obtained nanofibres.

12. A method according to claim 11 , comprising the process steps of: i. boiling the waste liquids of cocoons obtained from silk production for 6 hours, ii. filtering the solution concentrated by boiling with filter paper to remove the cocoons and / or solid particles remaining in the solution, iii. removing the water of the filtered aqueous solution by evaporator, iv. removing the water remaining in the solution by freezing with a lyophiliser at -80°C and obtaining powder sericin, v. mixing 0.5 gr powder sericin in 10 ml LiCI / DMSO (lithium chloride / dimethyl sulphoxide) for 3 hours, vi. mixing 0.005 mol of aliphatic or aromatic sulphonyl chloride in 10 ml Li / DMSO for 2 hours, vii. adding the solution prepared in the process step (v) to the solution prepared in the process step (vi) and stirring overnight, viii. adding 0.005 mol of TEA (triethylamine) dropwise to 0.069 ml of the mixture prepared in (vii) and mixing for 2 hours, ix. synthesising modified sericins by adding primary and / or secondary amine and 0.1 -1 mL TEA dropwise simultaneously to the mixture prepared in (viii) at 40-80 °C and stirring for 1 night,x. pouring the mixture containing modified sericin into 50 mL DCM (dichloromethane) and keeping it in the refrigerator for 1 hour to precipitate, xi. separating the mixture waited by centrifugation at 3000 rpm for 10 minutes, xii. lyophilising the remaining products at -80°C and by simultaneously doping them with both hydrophilic and hydrophobic polymers, obtaining nanofibres, and xiii. producing nanofibre composites using the electrospinning technique with the obtained nanofibres.

13. A method according to any of claims 10-12, wherein the primary and / or secondary amine mentioned in the process step (ix) is any primary and / or secondary amine.

14. A method according to any of claims 10-12, wherein the hydrophilic polymer mentioned in the process step (xii) is PVA and the hydrophobic polymer is PCL or PLA.

15. A method according to claim 13, wherein said primary and / or secondary amine in the process step (ix) is any of naphthylamine, methylamine, ethylamine, ammonia, butylamine, aniline, benzylamine or furfurylamine, or any combination thereof.

16. Nanofibre composite produced by a method according to claim 15.

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