Effects Of Protease Degumming On The Structure And Properties Of Silk Fibroin Materials

Neutral soap or sodium carbonate solution is widely used in silk degumming,but its disadvantage is that it will cause obvious damage to silk fibroin.Neutral proteases can catalyze the hydrolysis of sericin in neutral or near neutral environment and at relatively low temperatures.Some of neutral proteases have the potential to be widely used in silk degumming due to the certain specificity for the hydrolysis of sericin.Choosing appropriate neutral protease and degumning with appropriate process conditionsto achieve the removal of sericin and minimize damage to silk fibroin,is an urgent issue in the field of silk fabric refining and medical silk fibroin preparation.In this paper,different concentrations of trypsin,subtilisin,bromelain and papain were used to degum raw silk,and compared with sodium carbonate degumming,and the effects of protease types and concentrations on the degumming degree,the physical and chemical properties,and the molecular weight of silk fibroin were investigated.The silk fibroin obtained after degumming,dissolving and dialysis was separated according to the molecular weight,and then the porous silk fibroin material was prepared by freeze-drying method.The effects of molecular weight on the pore structure and mechanical properties of porous materials were investigated.Firstly,we studied the degmming effect of different concentrations of proteases(0.1g/L,1g/L,2g/L and 4g/L)on silk and the effects on the surface morphology,mechanical properties,structure and composition of silk fibroin fibers at the optimal temperature and pH that can keep the highest activity of trypsin,bacillus subtilis protease,bromelain and papain.The results of degumming rate test showed that with the increase of protease concentration in degumming,the degumming rate of silkwas increased.The order of degumming rate was:papain>trypsin>subtilisin>bromelain,under the same enzyme concentration;The result of scanning electron microscope showed that the surface of the silk was smooth without sericin residue after the degumming of 2g/L and 4g/L trypsin,4g/L of subtilisin and bromelain,and 2g/L of papain;The results of infrared absorption spectrum and amino acid composition analysis of silk fibroin content in degummed silk fibroin fiber showed that,2g/L and 4g/L of trypsin,4g/L of bacillus subtilis protease and bromelain,and 2g/L of papain can completely remove the sericin around the silk;the mechanical properties test shows that the tensile strength of the degumming silk is decreased compared with raw silk,but the strength decreased slightly after degumming with bromelain.Secondly,silk degumming was conducted with trypsin,bacillus subtilis protease,bromelain at the concentration of 4g/L,papain at the concentration of 2g/L and Na2CO3 at the concentration of 0.5g/L.SDS-polyacrylamide electrophoresis,Ubbeloh viscosity Analytical,gel chromatography and rheological shear viscosity were used to analyze and compare the effects of five degumming schemes on silk fibroin molecular weight and aqueous solution viscosity.The results showed that the average molecular weights of the silk fibroin obtained by degumming with subtilisin,trypsin,bromelain and papain were 183.5kDa,183 kDa,184.5kDa and 170kDa,respectively,which were higher than the molecular weight of silk fibroin obtained by degumming sodium carbonate 142.5kDa.The results of shear viscosity test showed that the viscosity of silk fibroin obtained by bromelaindegumming was the highest,and the viscosity of silk fibroin obtained by protease degumming was higher than that of degummed by sodium carbonate,indicating that the bromelain degumming has obvious protective effect on silk fibroin.Subtilisin,papain and trypsin also inhibited the hydrolysis of silk fibroin.The degumming of sodium carbonate has obvious hydrolysis to silk fibroin.Finally,the silk fibroin fiber obtained by degumming with 4g/L trypsin was dissolved in 9.3M LiBr,and then the disulfide bond between the heavy chain and the light chain was broken by dithiothreitol.The low molecular weight component of silk fibroin was separated and removed by double-layer dialysis to obtain high molecular weight silk fibroin,and the isolated high molecular weight silk fibroin component was identified by SDS-Polyacrylamide gel electrophoresis.The porous material was prepared by using the high molecular weight silk fibroin component,and the unseoerated silk fibroin was used as the control group to investigate the pore structure and mechanical properties of the high molecular weight silk fibroin porous material.The results of SDS-polyacrylamide electrophoresis showed that the disulfide bond in the silk fibroin was blocked by dithiothreitol and the double-layer dialysis method was used to separate the low molecular weight component of the silk fibroin obtained by trypsin degumming to obtain the high molecular weight componrnt of silk fibroin.Compared with the silk fibroin which has not removed the low molecular weight component,the fibrous material and the network structure are substantially reduced inside the porous material prepared by the high molecular weight silk fibroin component,and the compressive strength and compressive modulus of the porous material are significantly increased.