Study On The Interaction Between Cyclodextrins And α-Amylase

Cyclodextrins(CDs)are a type of cyclic oligosaccharides,produced by certain species of bacillus,in which CDs glucose transferase ACTS on starch and is composed of D-glucagon.The glucose unit is in chair conformation,which makes the cyclodextrin have the special ring structure of hydrophobic inside and hydrophilic outside,and can form the host and object inclusion compound with a variety of object substances through the intermolecular force,thus playing the role of shielding and protective activity to the object substances,and has been widely used in many fields.α-Amylase is an endonuclease that breaks down starch by cutting α-1,4-glycosidic bonds.As one of the most widely used enzyme preparations,it is widely used in food,washing and textile.The application of α-amylase to fermented flour products can make the stomatal structure of the fermented dough more uniform,improve the elasticity and taste,and increase the volume.However,when the activity of α-amylase is too high,the starch will be excessively hydrolyzed,which will adversely affect the quality of the dough.At present,α-amylase inhibitors are widely used to inhibit the activity of α-amylase.The main sources of α-amylase inhibitors are wheat flour and white kidney bean.If the α-amylase inhibitor is extracted from it for a long time,the utilization rate of its crops can’t be maximized.At the same time,studies have shown that CDs can inhibit the activity of some enzyme under certain conditions,but the inhibition of α-amylase activity has been rarely reported.The effects of α-,β-and γ-CD on α-amylase were studied.The effect of CDs concentration,time of action,temperature and pH on the activity of α-amylase was investigated by using relative residual enzyme activity as the evaluation index.Fluorescence spectrum and circular dichroism were used to characterize the change of α-amylase under different action conditions.The hydrolysis of CDs by α-amylase was studied by high performance liquid-mass spectrometry.Finally,dough and steamed bread were used as carriers to explore the macroscopic performance of the two and their interactions.The main research results are as follows:(1)By setting the single factor experiment to investigate the α-,β-and γ-CD influence on α-amylase activity,according to the experimental results,the overall β-CD evident effects of α-amylase activity,so choose β-CD orthogonal experiment was carried out,it is concluded that the biggest restrain conditions: concentration of 1 mM,120 min,temperature 45 ℃,pH 5.9,relative residual enzyme activity is close to 41%.(2)Fluorescence spectra were used to investigate the effect of CDs on the endogenous fluorescence intensity of α-amylase under different conditions.Under certain conditions,the addition of CDs can reduce the endogenous fluorescence intensity of the enzyme molecule,that is,change the microenvironment of tryptophan.The effect of different CDs is different,partly because the different structures of the three CDs lead to different interaction strength,and different conditions also affect the structure of the enzyme molecule itself.(3)The results of circular dichromatogram showed that the addition of CDs caused the transformation of secondary structure of α-amylase.On the whole,the action rules of α-CD and γ-CD were similar,which might be caused by the different cavity size and solubility of CDs.In addition,NMR was used to investigate the hydrogen proton displacement of CDs before and after the two effects.The results showed that in this process,some amino acids of the enzyme molecules entered the hydrophobic cavity,while some amino acids reacted with the hydrophilic surface of CDs to form non-inclusion complexes.(4)Hydrolysis of β-CD by α-amylase was determined by high performance liquid chromatography-mass spectrometry(HPLC-ESI-MS).The results showed that the hydrolysate produced in the experiment was glucose.By measuring the glucose content under the condition of different roles,the conditions of the hydrolysis rate respectively are: the lowest β-CD concentration of 2 mg/mL,temperature 45 ℃,and the degree of hydrolysis increased with the extension of time,after 3 h is relatively stable,and pH is not main factor.(5)Set the α-amylase,β-CD,α-amylase and β-CD physical mixture,α-amylase interact with β-CD certain conditions forming compounds are added to the dough as experimental group,compared with the blank group,change the properties of the dough,including formation characteristics,tensile property and the nature of the fermentation,the experimental group compared with blank group on dough properties have certain to improve,through the quality of steamed bread test also said that the existence of the interaction.