| As the main substance of alcohol sweet and flavorant in alcoholic beverages,higher alcohols have a fragrant smell when its content and proportion are coordinated.But when the content of higher alcohols is too high,it will not only lead to spicy bitterness,but also cause health risks such as headache after drinking.At present,the research and practice on the regulation and control of higher alcohol content in alcoholic beverages are mainly focused on the production process,but there are few reports on the post-treatment process.In this study,the common butanol in higher alcohols was isolated and purified to obtain a microorganism producing butanol dehydrogenase.The growth characteristics of the microorganism,the nature of the alcohol dehydrogenase produced and its preliminary application were studied,and the results are as follows:(1)A total of 200 culturable microorganisms were isolated from liquor koji and fermented grains.An ethanol-butanol dehydrogenase resistant microorganism DQ-54 was obtained by primary screening and secondary screening by enzyme-labeled instrument and headspace gas chromatography respectively.DQ-54 was identified as Enterobacter cancero genus by molecular biology.It can be found after further study on the growth law of DQ-54 that DQ-54 had the best growth when the amount of inoculation was 2%,culture time was 12 h,rotational speed was 130 r/min,temperature was 25? and pH was 7,and its logarithmic phase was in 2-8 h of culture.Ethanol tolerance test showed that the bacterial strain could tolerate 6%ethanol.(2)After further study on the enzymatic properties of Butanol-dehydrogenase(Ec-54ADH)produced by DQ-54,it was found that Ec-54ADH could convert n-butyl alcohol in the solution containing up to 5%ethanol,and with the increase of ethanol concentration(0-25%),the specificity of Ec-54ADH to n-butyl alcohol changed,and the catalytic ability decreased significantly.However,there was no significant difference in the catalytic ability of ethanol when the concentration of ethanol was 5?15%.Under the condition of 3%ethanol,Ec-54ADH has the highest catalytic activity for butanol when the pH value was 7 and the temperature was 40?.The addition of metal ions Fe2+,Mg2+,Mn2+,Zn2+ and Cu2+ could inhibit the catalytic activity of Ec-54ADH for n-butyl alcohol.According to the inhibitory effect on enzyme activity from high to low,the order is Mn2+>Cu2+>Fe2+>Mg2+>Zn2+.Ec-54ADH can catalyze isobutanol,n-propanol,isopropanol,n-amyl alcohol and isoamyl alcohol in the presence of ethanol(3%).NAD+is the best coenzyme for Ec-54ADH.Under the optimum conditions,the Vmax of the enzyme is 1.44/L/min,and the Km of the enzyme is 111.11mmol/L.In addition,it was found by SDS electrophoresis that DQ-54 could produce two kinds of alcohol dehydrogenase with molecular weights of 40KD and 22KD respectively,among which 40KD was one of the most common medium-chain alcohol dehydrogenase and 22KD may be a short-chain alcohol dehydrogenase.(3)Taking the commercially available beer as the research object,the crude enzyme was extracted and purified after being cultured for 12 hours under the conditions of inoculum size being 2%,pH being 7.0,temperature being 25? and rotational speed being 130r/min.The ratio of coenzyme NAD+added to Ec-54ADH was 1:1,and the ratio of enzyme to beer was 1:3.Compared with the control group with a water-to-beer ratio of 1:3,the addition of Ec-54ADH could significantly reduce the butanol content iri beer by 17.9%of the control.In this study,through isolation and screening,a microorganism capable of producing Ethanol-Butanol-dehydrogenase was obtained.On this basis,the enzymology properties of alcohol dehydrogenase produced by the screened bacterial strain and its preliminary application in beer were studied,which laid a foundation for the further development and utilization of the enzyme and for the establishment of a method for regulating the content of n-butyl alcohol in the production of finished alcohol. |
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