Research On Function Routes And Mechanisms Of Ultrasound In The Enzymatic Degradation Of Pectin

Pectin is an acidic heteropolysaccharide in the plants cell walls with extensive sources and easy availability.As more and more researchers are focused on the structure-function relationships of pectin,pectin is degraded using chemical,enzymatic,or physical methods in order to design bioactive products,which are called "modified pectin".However,the chemical method is polluting and hard to control;the physical method often shows a low efficiency,and the related studies still stay in an experimental stage.Enzymatic hydrolysis is an environmentally-friendly method and it has high efficiency and specificity.However,applications of enzymatic preparation are limited by the expensive price of pectinase enzymes and the resulted high production cost.The application of ultrasound in enzymatic reactions has recently become a research focus in polymer degradation,because of its strong selectivity,high efficiency and less pollution.However,applications of ultrasound in enzymatic degradation of pectin are only reported by a few studies.This study focuses on the application of ultrasound in the enzymatic degradation of pectin.With the aid of chemical analysis methods,kinetics and thermodynamics models and various structural analysis methods for protein and polysaccharide,ultrasound modifications of the enzyme and substrate,and the effect of ultrasound on the enzymatic reactions are investigated systematically.The main contents and results are summarized as follows:(1)Modifications and mechanisms of free pectinase under ultrasound irradiationLow-intensity and short-duration ultrasonic field was proved to increase the enzyme activity of pectinase,whereas high ultrasound intensity or prolonged treatment duration would deactivate the enzyme.The maximum activity of pectinase was observed at 4.5 W mL-1 intensity and ultrasound duration of 15 min.Results of degradation kinetics and thermodynamics of hydrolysis reactions catalysed by the modified pectin certified that ultrasound treatment could make pectinase exhibit higher reaction ability,which was evidenced from the increased rate constants and reduced activation energy(Ea),enthalpy changes(?H),entropy changes(?S)and free energy changes(?G).Meanwhile,after ultrasound treatment,the substrate was converted into the product at a higher rate and efficiency,and the enzyme displayed better affinity to the substrate.Ultrasound improved the reaction stability of pectinase at the temperature range of 20-60 ? without affecting its optimum temperature.In addition,a biphasic first order model was chosen to fit the inactivation process for pectinase at the temperature range of 40-60 ?,and parameters proved that ultrasound could prolong pectianse lifetime.Fluorescence spectra and circular dichroism spectra revealed that ultrasound treatment decreased the amount of tryptophan on the pectinase surface but increased the P-sheet and random coil in its secondary conformation,possibly by forming more orderly structures and affecting its active centres;modifications in the pectinase structures were irreversible within 24 h.Free radical effects and mechanical effects during ultrasound modification of pectinase were studied.When ultrasound was introduced to a liquid system,the number of free radicals was positively correlated with ultrasound intensity and treatment duration,but negatively correlated with temperature.Thiourea with a concentration of 4 mM was selected as a free radical scavenger to effectively eliminated the free radical effects of ultrasound.Concentrations of pectinase solutions did not affect the optimum ultrasound conditions for enzyme modification;but the lower the concentration was,the more significant the modification effect would be.During modification,both free radical effects and mechanical effects played a role to some extent.In general,mechanical effects dominated in pectinase activation process,while free radical effects dominated in the inactivation process.(2)Ultrasound-assisted pectinase immobilization and characteristics of the immobilized pectinaseWhen pectinase was immobilized using entrapment method,the highest yield of immobilized pectinase was achieved with concentrations of 2%and 0.15 M for sodium alginate and calcium chloride,respectively.When introduced during the immobilization process,ultrasound at an intensity of 9 W mL-1 for 20 min increased the immobilization yield to the greatest extent.When introduced to the already immobilized pectinase,ultrasound at an intensity of 4.5 W mL-1 for 10 min increased the pectinase activity to the greatest extent.Ultrasound increased surface area and loosened structures of immobilized enzymes,allowing more access to the enzyme's active sites.Changes in the enzymatic kinetics parameters indicated that ultrasound increased catalytic efficiency and enhanced affinity of immobilized pectinase.Furthermore,the optimum temperature and pH for pectinase remained unchanged under immobilization and ultrasound treatments.Immobilization allowed the enzyme to be more tolerable to high temperature and pH environments;furthermore,the two ultrasound-treated enzyme samples showed higher activity than the untreated immobilized pectinase at a specific temperature or pH.Immobilized enzymes can be re-used and have overall long-term thermostability and reaction stability when compared to free enzymes.However,ultrasound slightly decreased the stability of the immobilized pectinase.(3)Effect of ultrasound pretreatment of pectin substrate on the enzymatic reactions and structures and productsGiven the variation patterns of pectin molecular weight and its polydispersity as well as the hydrolysis rate from the enzymatic reactions under different parameters,ultrasound at an intensity of 18.0 W mL-1 and a time of 30 min were selected as the pretreatment conditions for pectin substrates.Ultrasound pretreatment of pectin could increase catalytic efficiency of the enzymatic reactions and enhance the affinity of pectinase.On the other hand,the pretreated pectin showed significantly lower molecular weight and polydispersity and degree of methoxylation(DM),which was more favored by pectinase enzymes.When the ultrasound-pretreated pectin was introduced to enzymatic reactions,main chains of the enzymolysis products were degraded more completely resulting in a higher content of galactan,which was beneficial to the increase in the bioactivity of the modified pectin.(4)Synergistic effects and mechanisms of combining ultrasound and pectinase on pectin degradationThe hydrolysis rate of pectin achieved maximum value with ultrasound treatment at 4.5 W mL-1 intensity and ultrasound time of 10 min.Under ultrasound irradiation,pectin was hydrolyzed at an elevated rate and the pectinase exhibited stronger affinity to the substrate.Furthermore,ultrasound favorably modified the tertiary and secondary structures of pectinase.Degradation(including the sonolysis,enzymolysis and sonoenzymolysis reactions)of pectin molecular weight followed the second-order kinetics model,suggesting the randomness of degradation processes.With the concept of the "synergistic coefficient",the combination effects between ultrasound and pectinase were studied both from perspectives of productions in reducing sugar and variations in pectin molecular weight;synergistic effects were observed more effective at lower temperatures.Furthermore,the degree of methoxylation of sonoenzymolysis pectin significantly decreased whereas the degree of acetylation remained unchanged compared to the original and enzymolysis pectin.Simultaneous functions of ultrasound and pectinase caused severe decomposition in pectin homogalacturonan(HG)regions without altering the rhamnogalacturonan ?(RG-?)regions.The complex polymeric and multi-branches structures of pectin transformed into smaller units with simpler branches and shorter chains after sonoenzymolysis reactions.This research work suggested an innovative idea for modified pectin production and will provide basic theories for applications of ultrasound in every part of enzymatic reactions,which is of significant importance in expanding the application range of ultrasound in the food industry.