The Isolation Of Acid Urease Producing Strain And Its Application In Chinese Rice Wine

Ethyl carbamate(EC),as a potential carcinogen,is widely found in fermented foods and alcoholic beverages.Among various fermented wines,the EC problem that Chinese rice wine faces is more prominent.Large number of methods and strategies to the problem of excessive content of EC in alcoholic beverages has been proposed and conducted by many scholars at home and abroad.As for all the methods,the removal of EC precursor material—urea by acid urease is considered to be the most effective method.The contents of this paper include screening and identification of high-yielding acid urease strains,optimization of enzyme production by fermentation of strians,purification and characterization of acid urease,study of enzymatic properties and application of enzyme in Chinese rice wine.The main findings are as follows:(1)Using mouse excrements,soil in a soybean products and dairy products factory(Hefei,China)as the source of the strains,a strain with higher acid urease production was screened by primary screening and secondary screening and the enzyme activity was 1.12 U/m L.The strain was identified as Staphylococcus cohnii based on the analysis of its 16 S r DNA gene sequence,morphological characteristics and physiological and biochemical properties,named as Staphylococcus cohnii HFUTY-08.(2)The optimum culture conditions were determined through single factor tests and orthogonal tests.The optimal medium was identified as follows: glucose 3%,peptone 1%,yeast extract 0.5%,beef extract 0.5%,KH2PO4 0.2%,Na Cl 0.5%,Na Ac 0.2%,urea 0.5%,Mn SO4·4H2O 0.005%,Ni SO4·6H2O 0.005%,initial p H 5.The optimal medium components were as follows: inoculation amount 5%,culture time 26 h,culture temperature 34 ? and shaking speed was 200 r/min.The optimum conditions for enzyme extraction as follows: ultrasonic power 400 W,ultrasonic time 20 min(open 2 S,stop 4 S).Under the optimal condition,the acid urese activity was increased from 1.12 U/m L-1 to 1.98 U/m L-1(3)The crude enzyme was purified to a certain purify after ethanol precipitation,DEAE 52 chromatograthy and superdex 200 chromatograthy.The purified enzyme with estimated molecular weight of 295 k Da,SDA-PAGE analysis revealed that the enzyme consisted of three subunits: ?(65 k Da),?(18 k Da),?(14.5 k Da),the structural features of the enzyme was defined as(???)3.The results of enzymatic properties shouwed that the Result of study on enzymatic properties was showed as follows: Optimum temperature of this enzyme was 35 ?,and its optimum p H was 5.Thermal stability of acid urease was higher between 25 ?~45 ?,and p H stability was higher between 4~6.The enzyme still retains more than 80% enzyme activity when stored at 4 ? for 60 days The Km value of the enzyme was 13.6 mmol/L and the Vmax was 1.68 ?mol/L/min.Besides,the enzyme also has good ethanol tolerance and can maintain more than 80% enzyme activity in the range of 5%~20% ethanol.(4)Finally,the preliminary study on the removal of urea by acid urease in Chinese rice wine showed that the enzyme could remove about 75% urea within 72 h at 37 ?;In addition,by the treatment of acid urease,the EC content of rice wine during frying process can be reduced by 75.7%,which has potential application value.