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Fundamental Research On Hydrolysis Of Corn Starch By Microwave Inducement Catalysis

Posted on:2013-01-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:X Y ZhangFull Text:PDF
GTID:1111330374987372Subject:Biology
Abstract/Summary:PDF Full Text Request
Microwave inducement catalysis can strengthen the corn starch hydrolysis catalyzed by a-amylase and glucoamylase. Hydrolysis of corn starch by double enzyme catalyzed under microwave inducement was studied detailedly in this paper, comparing with the conventional water bath heating. The main contents and conclusions are as follows:(1) The optimum conditions of corn starch hydrolysis were studied by the microwave irradiation and double-enzyme. First, the study of the pH value, temperature, substrate concentration, enzyme's dosage, reaction time and microwave power on the starch hydrolysis rate, compared with the conventional water bath heating, it was found that the hydrolysis rate of corn starch by microwave irradiation was more than20%higher than the water bath; Secondly, the corn starch hydrolysis process under microwave irradiation condition was optimized by orthogonal test. Range and variance analysis were used to finalize the optimal conditions of corn starch hydrolysis under microwave irradiation, the reaction temperature65℃, reaction time30min, the dosage of a-amylase90U/g dry starch, the dosage of glucoamylase110U/g dry starch.(2) The kinetics of corn starch hydrolysis by microwave irradiation and double-enzyme was studied. The influence of different factors on the initial reaction rate of corn starch hydrolysis under microwave irradiation and water bath conditions with double-enzyme, the conclusion was found that the initial reaction rate under microwave irradiation increased over20%of all cases. In addition, the two-enzyme's Michaelis constants Km and the maximum initial reaction velocity Vm were obtained by experiments, the Km of a-amylase under microwave irradiation is12times higher than water bath, the Vm is14times higher than water bath, the Km of glucoamylase under microwave irradiation is10times higher than water bath, the Vm is about8times higher than water bath, at the same time, the influence of glucose as a inhibitor on glucoamylase decreased under microwave irradiation.(3) SDS-polyacrylamide gel electrophoresis experimental results showed that, microwave irradiation did not change the double-enzyme's molecular size compared with water bath. That is, the peptide bonds of doubleenzymes did not break, and their primary structure did not change. That is, microwave irradiation did not have the impact on the primary structure of the double enzymes, so microwave irradiation will help the reaction of double-enzyme's corn starch hydrolysis.(4) α-Amylase's circular dichroism (CD) and secondary structure changes under the condition of microwave irradiation and water bath was studied by circular dichroism spectra. The results showed that, the peak height (at λ,=193nm) of the CD spectra of the samples treated by microwave irradiation and water bath both reduced, the reduced rate by microwave irradiation ranged from140%to220%, while the reduced rate by water bath ranged from60%to140%; the peak of the sample treated by microwave irradiation for60min disappeared at λ=193nm, while showed a wake peak by water bath; the peak position by microwave irradiation emerged a blue shift in the range of between5-8nm at λ=204nm and λ=220nm, while emerged in the range of between3-5nm by water bath. With time going, microwave irradiation and water bath have prompted the secondary structure of α-helix, β-sheet, β-turn and random coil's mutual transformations, but the trends were different.Glucoamylase's circular dichroism and secondary structure changes under the condition of microwave irradiation and water bath were studied too. Results have shown that, the peak height of the CD spectra of samples treated by microwave irradiation and water bath reduced by36.4%~68.2%at λ=193nm, and the peak height of the CD spectra of samples treated increased by10.8%~31.4%at λ=206nm and λ=220nm, and the peak position emerged a blue shift at λ=193nm in the range of between0.2-3nm nm, but the peak position chang a little by water bath. With the time going, microwave irradiation and water bath have prompted the secondary structure of a-helix, β-sheet, β-turn and random coil's mutual transformations, but the trends were different. Conclusions indicated that microwave radiation affected the distribution of hydrogen bonds in peptide chains, resulting in relaxation, fracturing and recombinant of hydrogen bonds. The orientation of hydrogen bonds maintaining peptide secondary structure stability changed, so that the structural stability of peptide reduced, and flexibility larger, which provided an important mechanism of the theory for the macroscopic biological effects of microwave irradiation.(5) According to the difference between the UV absorption spectrums of double enzyme under microwave irradiation and water bath, it was concluded that microwave irradiation induced the changes in double enzymes'three-dimensional structure, so that microwave irradiation enables the exposure of the double enzymes'active site. This is very conducive to the combination of the active site and substrate molecules and to enhance the activity of double enzymes. It was concluded that microwave irradiation changed the micro-environment of double enzymes'active site conformation to enhance the starch hydrolysis reaction.(6) After being processed by microwave radiation and water bath respectively, the enzyme activity of both a-amylase and glucoamylase changed. While processed by water bath, the enzyme activity of a-amylase first increased and then decreased as the temperature of the water bath increased. It had the most enzyme activity around60℃. The enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the temperature of the water bath kept increasing. It had the most enzyme activity around40℃. When the temperature of the water bath was kept constant, the enzyme activity of a-amylase first increased and then decreased as the time of heating increased. It had the most enzyme activity around20min and then It's enzyme activity decreased rapidly after25min; the enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the time of heating increased. It had the most enzyme activity around15min and then It's enzyme activity decreased rapidly after20min. While processed by microwave radiation, the enzyme activity of a-amylase first increased and then decreased as the temperature of the radiation increased. It had the most enzyme activity around60℃. The enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the temperature of the water bath kept increasing. It had the most enzyme activity around40℃. The enzyme activity of a-amylase first increased and then decreased as the time of radiation increased. It had the most enzyme activity around20min; the enzyme activity of glucoamylase had the same pattern as that of the a-amylase as the time of heating increased. It had the most enzyme activity around15min. The appropriate radiation temperature for a-amylase is between55~65℃and for glucoamylase is between35~45℃; the appropriate radiation time for a-amylase is between15~25min and for glucoamylase is around15min.(7)The effect of radiation time,temperature,substrate concentration and microwave power on the crystal structure,chemical structure and morphological configuration of corn starch were studied in different hydrolysis systems (with no enzyme,with a-amylase and with double enzyme) respectively. The crystallinity of corn starch had changed after its solution had been dealt with microwave radiation by different lengths of time. After the corn starch had been dealt with microwave radiation, it had been observed under400times lens of optical microscope that even it started to paste and its surface became rough, its morphology configuration was still a complete granule. Pit was also observed on the surface of the corn starch that had been dealt with enzyme. This effect was more obvious for the corn starch that had been dealt with double enzyme than that had been dealt with one, and it was also reflected in the infrared absorption spectrum:the former had low crystallinity and its infrared absorption peak was broader.Conclusions indicated that microwave irradiation can induce the structure changes in a-amylase and glucoamylase and strengthen the double-enzyme synergy effect. Compared with the traditional methods of starch hydrolysis, such as acid hydrolysis, enzymatic hydrolysis and acid-enzyme combined hydrolysis, microwave inducement catalysis can enhance the starch hydrolysis reaction and shorten the reaction time.It is a new technique superior to the tradition hydrolysis method on corn starch hydrolysis.
Keywords/Search Tags:microwave induce catalysis, corn starch, α-amylase, glucoamylase, hydrolysis kinetics, UV absorptionspectra, SDS-polyacrylamide gel electrophoresis, circular dichroism spectra, enzyme activity, starchstructure
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