Direct Effect Of Ethylene And Nano-TiO₂ On The Conformation,Kinetics And Thermodynamics Of Enzyme In Vitro

?-Amylase?EC.3.2.1.1?,named 1,4-?-D-glucan-glucanhydrolase,is a very important enzyme that could hydrolyze ?-1,4-glucoside bond of starch.?-Amylase is widely used in many industry technologies,such as glucose production,beer brewing,ethanol industry,fermentation industry,textile industry and so on.Pectinase is a kind of light yellow powder and is applied in industrial production,which is extracted by fermentation using various moulds,such as Aspergillus Niger and Rhizopus.Pectinase is mainly used to catalyze the decomposition of pectin from fruit,vegetable juice and wine.Ethylene,as the simplest unsaturated hydrocarbon,is a plant hormone which plays an important role in plant development and growth,such as promoting the seed germination,leaf growth and fruit mature.It is well known that ethylene can regulate enzyme activity by signal pathways of four receptors ETR2,EIN4,ERS1 and ERS2 in plant growth process.Up to now,ethephon has been widely used in most fruits and vegetables in order to ripen them in the world and the mechanism is very clear in plant.Nanometer titanium dioxide?nano-TiO₂?is a kind of white powder.At present,it has been extensively used in foods industry,medicine production and cosmetics manufacture.But there were few researches on the toxicity of ethephon releasing ethylene and the effect of nano-TiO₂ on enzyme activity and conformation in vitro,which maybe affect the health of humans and animals.This article mainly investigated the effect of ethylene and nano-TiO₂ on ?-amylase and pectinase in vitro,respectively,from aspects of activity,conformation,kinetics and thermodynamics.The conformation of enzymes have been studied using viscosity,fluorescence spectra,ultraviolet absorption spectrum and circular dichroism spectrum.This article was divided into the following three sections: 1.The effects of ethylene on the activity of ?-amylase and nano-TiO₂ on the activity of pectinase in vitro were investigated by ultraviolet absorption spectrum.2.The effect of ethylene and nano-TiO₂ on secondary structure of ?-amylase and pectinase,respectively,were researched utilizing fluorescence spectra and circular dichroism spectrum.3.The effect of ethylene and nano-TiO₂ on the kinetics and thermodynamics of ?-amylase and pectinase were investigated,respectively.This paper was investigated from 2 systems:System 1: Direct effect of ethylene on the activity,conformation,kinetics and thermodynamics of ?-amylase in vitro.System 2: Direct effect of nano-TiO₂ on the activity,conformation,kinetics and thermodynamics of pectinase in vitro.This report mainly includes the following contents: Chapter 1:?1?This article briefly introduced the sources and application of enzyme,?-amylase and pectinase;catalysis mechanism of enzyme;the recent development of enzyme conformation and the influencing factors of enzymes catalysis.?2?This article also briefly introduced the sources of the ethylene and its effect on the animal and plant as well as its application in foods.?3?The applications and mechanism of nano-TiO₂ on photocatalytic degradation,sterilization,foods and cosmetics were also included.Chapter 2: The research background,research objectives,research content and technical route of the paper were simply introduced.Chapter 3 The materials and methods relating to the experiment of ethylene on ?-amylase were introduced in this chapter.Chapter 4 The materials and methods relating to the experiment of nano-TiO₂ on pectinase were introduced in this chapter.Chapter 5?1?The effects of ethylene on ?-amylase activity,optimum temperature,optimum pH and reaction time were mainly investigated in this chapter.?2?The effects of ethylene on ?-amylase viscosity,fluorescence spectra,ultraviolet absorption spectrum and circular dichroism spectrum were investigated.?3?The effects of ethylene on ?-amylase kinetics and thermodynamics were investigated.Chapter 6?1?The effects of nano-TiO₂ on pectinase activity,optimum temperature,optimum pH and reaction time were mainly investigated.?2?The effects of nano-TiO₂ on pectinase viscosity,fluorescence spectra,ultraviolet absorption spectrum and circular dichroism spectrum were investigated.?3?The effects of nano-TiO₂ on pectinase kinetics and thermodynamics were investigated.Chapter 7 Conclusion:?1?The results showed that ethylene can affect ?-amylase's optimum temperature which was increased by 5 oC,however,optimum pH?6.0?was barely influenced by ethylene.Compared with the control group,low concentration ethylene can promote the catalyzation activity of ?-amylase,while high concentration ethylene inhibits the activity of ?-amylase.And the ultraviolet absorption of ?-amylase at 278 nm was blue-shifted 1nm.The fluorescence emission of treated ?-amylase was obviously enhanced with increases of ethylene concentrations.The viscosity of ?-amylase solution was declined obviously than that of the control group with the increases of the ethylene concentrations.Circular dichroism spectra indicates that the conformations of ?-amylase treated with 29.32 and 262.11 ?mol/L ethylene were obviously changed.In especial,?-helix content of ?-amylase were decreased by 20 and 31%,respectively,and ?-sheet,?-turn and random coil contents were increased by contrast.The kinetics and thermodynamics results indicate that hydrophobic interactions may play a main role in the binding process of ethylene and ?-amylase.The possible reason is that ethylene molecules were easily embedded into the interior of ?-amylase,thus changing the hydrophobic force in ?-amylase,leading to the conformation and thermodynamics changes of ?-amylase.?2?Nano-TiO₂ can obviously affect pectinase activity,compared with the control group,promoting the pectinase activity at low concentration and inhibiting the pectinase activity at high concentration.However,nano-TiO₂ has no effect on optimum pH?5.0?and the optimum temperature of pectinase.The ultraviolet absorption of pectinase at 197 nm red-shifted 1nm;the fluorescence emission and viscosities of treated pectinase were obviously enhanced with increases of nano-TiO₂ concentrations.Circular dichroism spectra indicates that the conformations of ?-amylase treated with 30mg/L nano-TiO₂ were obviously changed.To be specific,its ?-helix and ?-sheet content were increased by 2.6 and 37.8% respectively,while its ?-turn and random coil contents were decreased by 5.6% and 35.5% respectively.In addition,the group treated with 100mg/L nano-TiO₂ were also changed in which ?-helix,?-sheet and ?-turn content were decreased by 0.2,0.6 and 0.1% respectively and random coil contents was increased by 0.1% by contrast.The data of kinetics and thermodynamics indicate that hydrogen bonding and van der Waals force of pectinase were altered by nano-Ti O₂.The results indicate that nano-TiO₂ is conducive to the stability of pectinase conformation at low concentration,but nano-TiO₂ slightly decreased the stability of pectinase conformation at high concentration.