Study On The Effect Of Fluorinated Surfactant And Chitosan Phenol Derivatives On The Activity Of α-chymotrypsin

The α-chymotrypsin is widely used in industries,such as medicine,chemical industry,environmental protection,food,feed,textile,and so on.Consequently,it’s of great practical value and theoretical significance to study the activity of α-chymotrypsin.Because α-chymotrypsin is a protein,its activity is easily affected by physical and chemical factors.In order to improve the activity of α-chymotrypsin,a lot of work has been done.It is a simple and convenient way to add addictives to protect α-chymotrypsin,but most of the previous studies have focused on homogeneous systems.Enzyme immobilization is a promising way with good enzyme stability and high recycling rate,but enzyme load rate regulation is not convenient,enzyme is inactivated easily,enzyme immobilization operation time is relatively long.There are few reports on the refolding of α-chymotrypsin by artificial chaperones.In this paper,the interaction between three types of functional materials and α-chymotrypsin are studied.The three types of materials are:fluorinated Pluronic surfactant,phenol-modified chitosans and catechol-modified chitosans.The main works of this thesis are as follows:1.The protein and protein structure,the basic structure and applications ofα-chymotrypsin,the methods for improving enzyme stability,the research progress of addictives and enzyme refolding are reviewed.2.The study on the interaction between fluorinated Pluronic surfactant andα-chymotrypsin.Enzyme activity was measured by enzymatic reaction kinetic method.The changes of molecular structure of enzyme were characterized by fluorescence spectroscopy.The thermal stability of enzyme was also measured.The enzyme activity data showed that fluorinated Pluronic surfactant had little effect on the activity ofα-chymotrypsin.The fluorescence spectra of α-chymotrypsin showed that fluorinated Pluronic surfactant could induce minor changes in the structure of α-chymotrypsin molecule.The variation of activity of α-chymotrypsin with time at different temperatures showed that fluorinated Pluronic surfactant couldn’t change the thermal stability ofα-chymotrypsin.3.The study on the refolding of α-chymotrypsin by the fluorinated Pluronic surfactant.It was determined that the fluorinated Pluronic surfactant couldn’t refold completely denatured α-chymotrypsin with the orthogonal experiment.The fluorinated Pluronic surfactant couldn’t also refold the partially denatured α-chymotrypsin.4.Study on the interaction between chitosan phenol derivatives and α-chymotrypsin.The phenol-modified chitosans and the catechol-modified chitosans were synthesized by reductive amination,1H NMR spectrum proved that the target products were obtained,and the degree of substitution on chitosan was also determined.The α-chymotrypsin was immobilized by in situ co-precipitation.The results showed that the rate of enzyme loading and encapsulation of phenol-modified chitosan decreased firstly and then increased with the increase of the degree of substitution.The rate of loading and encapsulation of α-chymotrypsin of catechol-modified chitosan increased with the increase of the degree of substitution.The enzyme activity was determined by enzymatic reaction kinetics method.The activity of α-chymotrypsin in phenol-modified chitosan was increased at the low degree of substitution,but decreased at high degree of substitution.It was found that the enzyme activity of α-chymotrypsin increased with the increase of the degree of substitution in catechol-modified chitosan,the activity ofα-chymotrypsin in the chitosan with the substitution of 67%was 20 times higher than that in the acetic acid buffer.The results show that a further study need to be done on α-chymotrypsin protection and renaturation by using fluorinated Pluronic surfactant.Phenol-modified chitosans have a little effect on the protection of α-chymotrypsin,and catechol-modified chitosans have the ability to protect the α-chymotrypsin.