| As a by-product of liquor brewing,the content of higher alcohol has an important effect on liquor flavor and taste.When its content is too high,the liquor will be bitter,spicy,and harmful to human health.Therefore,it is of great significance to control the content of higher alcohol in alcohol for the development of brewing industry.In this study,based on the principle of the formation of aldehydes and ketones from propanol by enzyme catalyzing in non-aqueous phase,the strain producing propanol dehydrogenase was isolated and purified,and the physicochemical properties of the enzyme were studied too.Moreover the immobilized enzyme and coenzyme regeneration system of the enzyme were constructed,which laid a foundation for the development and regulation of propanol content in alcohol.The results were as follows:1.The strains isolated from daqu,xiaoqu and fermented grains in the early stage were screened by the enzyme-labeling method and the headspace phase method.The strain 10-F being of both ethanol tolerance and propanol dehydrogenase production was obtained and identified to be Enterobacter by molecular biological method.The optimal culture conditions of 10-F were confirmed,the inoculum size of 2%,the rotating speed,130 r/min,pH 7.0,35? for 12h.The ethanol tolerance concentration of strain was 12%2.The effects of different ethanol concentrations,reaction temperatures,pH of different systems,different metal ions,different media,different substrates and different cofactors on the enzyme activity of propanol dehydrogenase produced by this strain were investigated.The optimum temperature of the propohydrin dehydrogenase was 40? and the optimum pH was 7,the enzyme activity was relatively stable between pH5 and 7,indicating that the enzyme can maintain high activity in the neutral or weak acid environment,and the environmental pH has a great influence on the enzyme activity when it leaves this range.The influence of different ethanol concentrations on the enzyme activity of propanol dehydrogenase was also very different.The maximum ethanol tolerance concentration of this enzyme was 10%.When the ethanol concentration was in the range of 0-10%,the catalytic capacity of propanol to propanol was gradually weakened with the increase of ethanol concentration.When the ethanol concentration was more than 10%,the ability to catalyze propanol was basically lost,and the specificity of the enzyme to the substrate may change as the ethanol concentration increases.In the study,it was found that different metal ions had different effects on enzyme activity.According to the promotion of enzyme activity,Zn2+>Mg2+>Mn2+>Fe2+>Cu2+,especially Zn2+had a significant promoting effect on enzyme activity,while Cu2+had a significant inhibiting effect on enzyme activity.In addition,the strain cultured in different media had no significant effect on overall enzyme activity.Compared with the catalytic capacity of two coenzymes NADP and NAD+combined with propanol dehydrogenase to catalyze the substrate n-propanol,the latter was more advantageous.3.Three absorption peaks appeared in the elution curve of the crude enzyme solution after Sephadex g-100 chromatography.The enzyme activity corresponding to the peak value of the eluent was measured and the highest enzyme activity was 1.76 U/ml,The eluent was concentrated and analyzed by SDS-PAGE electrophoresis.The results showed that the two bands of the target protein represented molecular weights of about 35 kD and 22 kD respectively,belonged to the short chain ADH isoenzyme,and the latter was more active.4.Kinetic studies showed that the enzyme had different affinity for different substrates.When ethanol was used as the substrate,Vmax was 1.01mmol/L/min and Km value was 55.56 mmol/L.When n propanol was used as substrate,Vmax was 1.03 mmol/L/min and Km value was 125 mmol/L.By comparing the Km values of the two substrates,it could be found that the affinity of the enzyme to ethanol was higher than that of propanol.The specificity of the enzyme to the substrate was also different under different ethanol concentrations.When isopropanol,n-butanol and isobutanol were used as substrates,the utilization of the substrate was similar,and the substrate content gradually decreased with the increase of ethanol concentration,which may be related to the solubility of the substrate in water.When n-pentanol and iso-pentanol were used as substrates,the substrate content increased first and then decreased with the increase of ethanol concentration.The reason for the difference may be related to the molecular weight of substrates and the arrangement of key groups.5.During enzyme immobilization,coenzyme NAD and propanol dehydrogenase were immobilized respectively,and then the two enzymes were combined by double enzyme embedding method,and their related enzymatic characteristics were investigated.The results showed that the fixation rate of immobilized NAD was the highest at pH6.0,while the enzyme activity of immobilized propanol dehydrogenase was the highest at pH6.5 at 117 U/ml.In addition,the immobilized enzyme was most stable at 35?,and the maximum enzyme activity was 125 U/ml.Propanol was catalyzed by enzyme to produce propanal when propanol was used as a single substrate.After recovery of the immobilized enzyme,acetaldehyde was used as a single substrate.The results showed that when the acetaldehyde concentration was 2%,acetaldehyde was catalyzed by the enzyme to produce ethanol,and the content reached a maximum of 0.45mg/L.Then after recycling of enzyme reaction with propyl alcohol,propyl alcohol can be catalytic propanal,but its content was lower,because the enzyme activity will decrease with the increase of application number,integrated the result after immobilized enzyme can realize recycling. |
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