Application And Mechanism Study Of Laccase-assisted Tyrosine Grafting/Cross-linking On Wool Modification

Wool,as a kind of natural keratin fiber,shows excellent elasticity and gentle luster.Wool fabric is favored by consumers since it is comfortable and warmth to wear.However,wool could be unavoidably damaged in the spinning and finishing process,affecting the service life of its fabrics.Enzymatic method to repair the wool damage has obviously advantages,such as mild conditions,eco-friendly,comparing to the traditional chemical and physical measurements.Transglutaminases is widely used in strength remediation of wool,which shows a low efficiency and leads to by-reactions.Therefore,it is necessary to explore an eco-friendly and high-efficient approach to remediate wool damage.Amines and thiols in wool are used as reactive groups and the cross-linking structure“wool-[tyrosine]_n-wool”is formed via laccase-assisted tyrosine grafting/cross-linking to enhance the stability of the protein structure and then improve the mechanical properties of wool.The strength loss of anti-felting wool is remediated with satisfactory shrinkage resistance.Furthermore,when disulfide bonds are broken and new bonds are rearranged between wool keratin and tyrosine,curls are induced in wool,helping to improve wool’s wearability without compromising the fibers’strength.The main contents and results are as follows:（1）Laccase-assisted tyrosine grafting/cross-linking reaction system is constructed and its influence on the remediation of anti-felting wool’s strength damage is discussed.The structure of self-assembly tyrosine is observed by LC-MS,FTIR and fluorescence spectra.The effects of tyrosine grafting/cross-linking on the structure,thermal properties and mechanical properties of wool are evaluated by ATR-FTIR,XRD,DSC and stretching curves.The results shows that,tyrosine monomers are linked via a C-C biphenyl bond to form dityrosine;grafting/crosslinking modification of wool is be accomplished on the premise of tyrosine radical or dityrosine radical formation;tyrosine monomer is grafted/cross-linked on wool,leading to strength and elongation loss of fibers;when dityrosine is grafted/cross-linked on wool,improving the strength and elongation of fibers,while the increase of yarn’s strength and Young’s modulus is accompanied by the decline of stretch;tyrosine grafting/cross-linking is applied to strength remediation of anti-felting wool yarns;using 30 U/m L of laccase to promote tyrosine grafting/cross-linking for 1 h at p H 5.0 and 40℃,anti-felting wool achieves the highest strength,increasing by 16.42%,with the shrinkage area of 2.50%.（2）Wool keratin and bovine serum albumin（BSA）are adopted as model proteins to reveal the mechanism of wool’s mechanical properties improving via grafting/cross-linking modification using tyrosine as a moiety.FTIR,CD,SDS-PAGE,MALDI-TOF,and SEC are used to investigate the effects of tyrosine grafting/cross-linking on the secondary structure and molecular weight distribution of proteins;the grafting/cross-linking residues are determined by amino acid composition analysis and trypsin hydrolysis combined with LC-MS test technology.The results indicate that tyrosine/dityrosine grafting promote the cross-linked wool keratin and cross-linked BSA;tyrosine grafted/cross-linked BSA present two absorption peaks at 32 min and 24 min in SEC spectra,corresponding to molecular weights of 67.73 k Da and 137.02 k Da in MALDI-TOF spectra and assisting with BSA-tyrosine conjugate（BSA-[Tyrosine]₅）and cross-linked BSA（[Dityrosine]₄-BSA-Dityrosine-BSA-[Dityrosine]₄）;the specific reactive residues are Lys-136,Lys-204,Lys-224,Lys-322,and Lys-537.In essence,the grafting/cross-linking of wool is attributed to wool keratin modification.The newly cross-linking bonds（-NH-dityrosne-NH-）allow forming of a better three-dimensional network within wool fibers,resulting in strength improvement because the external force required for the slippage of macromolecular chains in wool is increased.（3）Based on of clarifying the effect of tyrosine grafting/cross-linking on wool,the ability of L-cysteine pre-treatment combined with laccase-assisted tyrosine grafting/cross-linking is further investigated to improve curly twists and warmth retention of wool.The effects of solvent composition and p H value of L-cysteine solution,pre-treatment time consuming,laccase usage,grafting/cross-linking time,and tyrosine concentration on the curling formation of wool yarns were discussed.The shape modification is evaluated by the number of twists and length of wool yarns.When p H is 10.0,L-cysteine pre-treatment for 10 min can reduce plenty of disulfide bonds in wool,the reduced thiols would react with tyrosine via laccase-assisted.The L-cysteine pre-treatment by a solution composed by 20%ethanol,15%propylene glycol and 0.5%benzyl alcohol in phosphate buffer promote the higher entrapment of the L-cysteine into the wool fibers,resulting in deepening of curls amplitude in tyrosine grafted/cross-linked wool yarns.Adopting3.0 mmo L/L of tyrosine as cross-linking agent and 18 U/m L of laccase as catalyst,the 10 cm of wool yarns obtain 11 curls and hold 54.5%curls after washing.The disulfide bonds in wool are broken and then form the S-Tyrosine-S structures,which enhance the curly twists of wool yarns and partial recovery of the strength loss caused by L-cysteine pre-treatment.In wearing characteristics,curly wool has better fluffiness and warmth retention.（4）The mechanism of L-cysteine pre-treatment combined with laccase-assisted tyrosine grafting/cross-linking to induce curls in wool is explored.SDS-PAGE and MALDI-TOF are used to analyze tyrosine grafting/cross-linking degree of L-cysteine pre-treated BSA（BSA（cys））in qualitatively and quantitatively.The reduced disulfide bonds and residues of tyrosine grafting/cross-linking in BSA（cys）are found to reveal how cross-linked structure impact on wool performance.The results showed that three disulfide bonds in BSA are reduced,resulting in a decrease of the disulfide bond content by 29.89μmo L/L·mg;nine tyrosines are grafted/cross-linked to BSA（cys）,resulting in a molecule weight increase by 1546 Da;among then,three tyrosines are linked to thiol groups of Cys-101,Cys-315,and Cys-176 in BSA（cys）by Michael addition,leading to a decline of disulfide bond content by 10.72μmo L/L·mg.Dityrosine act as a“bridge”between amines and thiols in BSA（cys）.The pre-exist amines and reduced thiols in wool as the tyrosine grafting/cross-linking site,leading to a better three-dimensional network between macromolecular chains in wool and enhancing the mechanical properties of wool.L-cysteine pre-treatment attack disulfide bonds specifically,making the wool fiber stretching and twisting.Through tyrosine grafting/cross-linking,the cross-linking structure between thiols is changed via laccase-assisted tyrosine grafting/cross-linking,inducing curls to wool fibers and providing a novel approach to accomplish the multi-modification of wool with satisfactory strength.