Study On The Role Of Ultrasound In The Enzymatic Degradation Of Chitin

Chitin is the second most abundant natural biopolysaccharides on earth.However,due to its strong intermolecular and intramolecular hydrogen bond network structure,a highly ordered crystal structure is formed,which results in poor solubility,thereby limiting its application.Nowadays,many studies have found that the crystalline structure of chitin is destroyed after degradation,and low molecular weight chitin with excellent adsorption capacity could be obtained.Nowadays,there have been many studies proving the efficacy of ultrasound when used in enzymatic reactions,which can accelerate the enzymatic reaction rate and achieve effective degradation of macromolecules.Therefore,in order to obtain an effective way to degrade chitin to broaden its application,this study applies ultrasound to the enzymatic degradation process of chitin,and explores the effect of ultrasound on the enzymatic reaction process,activity and properties of chitinase,as well as the structure and functional properties of the chitin product.By analyzing degradation products capacity adsorption to Congo red and their adsorption characteristics,it could provide a certain application basis for the ultrasonic-assisted enzymatic degradation of chitin.The main research contents and results of this study are as follows:(1)The effect of ultrasound on the enzymatic degradation efficiency of chitinBy exploring the influence of different ultrasonic conditions on the enzymatic degradation rate of chitin,a sonoenzymatic degradation system of chitin was established.Ultrasound was helpful to accelerate the enzymatic reactions under suitable conditions.At the ultrasonic intensity of 25 W/m L at 50°C,the ultrasonic treatment within 35 min was beneficial to the enzymatic reaction process.And the enzymatic hydrolysis efficiency reached the maximum 38.72%in 20 min,which was 27.93%higher than that without ultrasound.When the ultrasound intensities and durations were further increased,the promotion effect of ultrasound gradually weakened,and the reaction efficiency with ultrasound even became lower than the enzymatic reaction.The reaction rate constant gradually increased with the increase in temperature(from 20 to50°C),while with the temperature continuing to rise,the reaction rate constant began to decrease,and the rate constant of sonoenzymolysis was even lower than that of enzymolysis,indicating that ultrasound was beneficial to the enzymatic reaction without changing the optimal reaction temperature of chitinase.Under the same condition,the activation energy E_a,enthalpy change?H and entropy change?S of the sonozymolysis were all lower than those of the enzymolysis reaction,showing that ultrasound was benefical to accelerate the collision between the enzyme and the substrate and improved the conversion efficiency of the product.Comparing with the enzymolysis,molecular weight(M_v)of the chitin product was reduced from 247.78 k Da to 92.98 k Da during the sonoenzymolysis reaction,while no obvious change was observed when ultrasound was used alone,indicating that the improved degradation extent of chitin with sonoenzymolysis was attributed to the improved enzymatic efficiency with ultrasound,instead of the degradation of chitin in the ultrasonic field.(2)The effect of ultrasound on the properties and structure of chitinaseDifferent ultrasonic conditions were used to treat chitinase in order to analyze the effect of ultrasound on chitinase activity.Ultrasound could activate chitinase under certain conditions.The chitinase activity was increased by 19.17%treated after ultrasound treatment at 25 W/m L intensity for 20 min(increased from 1.22 U to 1.45U).Moreover,the maximum reaction rate V_max of chitinase was increased by 10.91%,and the Michaelis constant K_m was reduced by 11.84%,indicating that ultrasound increased the affinity of chitinase and substrate.However,no change was found on the tolerance of chitinase to most metal ions(except Mn²⁺and Mg²⁺).Ultrasonic treatment could change the content of chitinase secondary structures,including the decrease of?-helix and?-turn and the increase of?-sheets and random coils.Furthermore,ultrasound could reduce the content of tryptophan(Trp)on the surface of chitinase.Atomic force microscopy(AFM)force spectroscopy technology was applied to estimate the binding ability of chitinase and chitin.Through analyzing the typical force-distance curves,it was found that at a loading rate of 0.3?m/s,ultrasound increased the binding force between chitinase and chitin from 71±12 p N(untreated)to 105±23 p N(sonication for 20 min).With the increase of ultrasonic durations,the binding force between chitinase and chitin gradually increased,exhibiting a similar trend with that for chitinase activity.(3)The effect of ultrasound on the structure and functional properties of the sonoenzymolysis product of chitinDifferent methods were used to measure the effects of enzymolysis and sonoenzymolysis on the structural,physical,chemical and rheological properties of chitin degradation products.After enzymolysis,the chitin particles became smaller,and Sonoenzymolysis even accelerated the destruction of the crystal structure of chitin,making it easier for chitinase to degrade it.Therefore,the crystallinity,microscopic crystal size and the d-spacing were reduced without changing its basic structure of chitin after enzymolysis and sonoenzymolysis.It was also observed that chitinase or ultrasound would not cause severe deacetylation of chitin,but enzymolysis and sonoenzymolysis could significantly reduce the particle size(D_4,3 of 301.80?m from untreated reduced to 175.40?m and 128.60?m after enzymolysis and sonoenzymolysis,respectively).According to the analysis of thermal stability,it was found that ultrasound and chitinase reduced the heat stability of chitin due to the decrease of molecular weight.With the increase of shear rate at low shear rate,enzymolysis and sonoenzymolysis did not change the shear thinning phenomenon,as well as the fluid type and elastic gel characteristics of chitin.(4)The effect of sonoenzymolysis of chitin on the adsorption of Congo RedThe chitin samples before and after enzymolysis and sonoenzymolysis were used as Congo Red adsorbents to analyze the influence on the adsorption capacity of chitin.As the concentration of Congo Red increased from 100 mg/L to 300 mg/L(constant chitin dosage),or as the dosage of chitin increased from 1 mg to 2 mg(constant Congo Red concentration),the removal rate of Congo Red gradually increased,and the products of enzymolysis and sonoenzymolysis showed higher adsorption capacities and removal efficiencies.Langmuir isotherm adsorption model and pseudo-second-order kinetic model were the most suitable model to describe the adsorption of chitin to Congo Red,indicating a single layer of heat physical absorption.Due to enzymolysis and sonoenzymolysis,chitin showed a larger specific surface area and higher porosity,which was conducive to the diffusion and filling of Congo Red,thereby improving the adsorption capacity without changing its adsorption characteristics.