Acid Urease Research Articles

Isolation and Taxonomie Characterization of Acid Urease-producing Bacteria

About 16,000 isolates from animal feces and intestines were assayed for the production of acid urease, and the 700 strains selected as producers were examined as to their taxonomic properties. Of these 700 strains, 370 belonged to the genus Streptococcus, 312 to the genus Lactobacillus, 9 to the genus Escherichia, 6 to the genus Staphylococcus, 2 to the genus Morganella and 1 to the genus Bifidobacterium. The majority of the streptococci were identified as Streptococcus mitior, the remainder being Streptococcus salivarius, Streptococcus faecalis, Streptococcus faecium, Streptococcus avium and Streptococcus gallinarum. The majority of the lactobacilli were considered to be Lactobacillus reuteri or Lactobacillus fermentum and Lactobacillus animalis or Lactobacillus salivarius, the remainder being Lactobacillus ruminis, Lactobacillus viridescens, Lactobacillus vac- cinostercus and strains considered to be Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus or Lactobacillus gasseri.

Ethyl carbamate in alcoholic beverages.

Urethane, ethylcarbamate, which is known to be carcinogenic, teratogenic and mutagenic, was reported to be contaminated in various kinds of alcoholic beverages. Therefore, removal of urethane or prevention of its formation from alcoholic beverages is an urgent problem in all over the world. The present review describes, 1. toxicity of urethane, 2. methods of its measurement, 3. mechanism of its formation and 4. prevention of its formation. Urethane was chemically produced from carbamyl compounds and ethyl alcohol by heating in an acidic condition of fermentation. For example, urethane (20-60 ppb) in sake and wine was produced from urea as a major carbamyl source during sterilization process or long-term storage. Urethane contamination levels were dependent upon acidicity, temperature, periods of time and the concentrations of urea and of ethyl alcohol. Acid urease, whose optimal pH lies around 4, obtained from Lactobacillus reuterii lowered urea concentrations in sake and wine, whose pHs are acidic (pH 4.4 and 3.2, respectively). On the basis of these results, enzymatic removal of urea with acid urease during or after fermentation processes and before heating or storage prevented the urethane formation in sake and probably in other alcoholic beverages. Urethanase which hydrolyzes urethane in the acidic region in high ethyl alcohol concentrations is expected to be useful for the elimination of a potentially hazardous compound once formed in distilled alcoholic beverages.

Purification and characterization of acid urease from Lactobacillus reuteri.

Acid urease was purified from Lactobacillus reuteri and characterized. The enzyme preparation was electrophoretically homogeneous and the molecular weight of the enzyme was estimated to be 220, 000. The enzyme consisted of three kinds of polypeptides, designated as α, β and γ, with molecular weights of 68, 000, 16, 100 and 8, 800, respectively, in a (α1β2γ1)2 structure. The isoelectric point of the enzyme was 4.7. The nickel content was found to be 1.8 atoms of nickel per α1β2γ1 unit- The amino acid profile was different from those of known bacterial neutral ureases. The enzyme was most active at pH 2 and at around 65°C. It was stable between pH 3 and 8, and below 50°C. The Km for urea was 2.8 HIM at pH 2. The enzyme activity was inhibited by Ag+, Hg2+, Cu2+, p-chloromercuribenzoate and acetohydroxamate. The acid urease eliminated urea in alcoholic beverages that are acidic in general.

Causes for Inhibition of an Acid Urease fromLactobacillus fermentus

The acid urease from <i>Lactobacillus fermentum</i> was found to be inhibited to a minor degree Cu⁺⁺, Fe⁺⁺⁺, Ca⁺⁺, PO₄^---, phenolic compounds, SO₂, and ethanol. Significant inhibition was found from L-malic acid, L-lactic acid, D-lactic acid, pyruvic acid, α-ketoglutaric acid, and acetic acid. No inhibition was noted from tartaric acid or succinic acid. The efficiency varied greatly from wine to wine. The enzyme was shown to be extremely durable and capable of operating for extended lengths of time in wine without negative effect on the efficiency. A doubling in rate occurs with each 10°C rise in incubation temperature. It is very effective in pH 3.0 to 4.0 range. without inhibitors present.

Isolation and taxonomic characterization of acid urease-producing bacteria.

About 16, 000 isolates from animal feces and intestines were assayed for the production of acid urease, and the 700 strains selected as producers were examined as to their taxonomic properties. Of these 700 strains, 370 belonged to the genus Streptococcus, 312 to the genus Lactobacillus, 9 to the genus Escherichia, 6 to the genus Staphylococcus, 2 to the genus Morganella and 1 to the genus Bifidobacterium. The majority of the streptococci were identified as Streptococcus mitior, the remainder being Streptococcus salivarius, Streptococcus faecalis, Streptococcus faecium, Streptococcus avium and Streptococcus gallinarum. The majority of the lactobacilli were considered to be Lactobacillus reuteri or Lactobacillus fermentum and Lactobacillus animalis or Lactobacillus salivarius, the remainder being Lactobacillus ruminis, Lactobacillus viridescens, Lactobacillus vaccinostercus and strains considered to be Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus or Lactobacillus gasseri.

Removal of urea from alcoholic beverages with an acid urease.

Urethan, a cancer causing chemical, was reported to contaminate some alcoholic beverages. Since urethan is formed by heating urea with ethyl-alcohol, removal of urea is necessary to prevent urethan formation in alcoholic beverages. Acid urease, whose optimal pH lies around 4, lowered urea concentrations in Japanese sake. This finding indicates a protective effect of acid urease on urethan's potential hazards in alcohol.

Acid urease from Lactobacillus of rat intestine.

The effect of pH on the urease activity of the extract from ileum-cecum contents of rats revealed the presence of an enzyme having hign activiy in the acid region (pH 2-3). This type of enzyme was designated as acid urease to distinguish it from the enzyme having high activity in the neutral region. The bacterium producing acid urease was isolated from the cecum contents of rats and identified as Lactobacillus fermentum. Acid urease was partially purified from the extract of L. fermentum TK1214. The enzyme had the optimal pH at 2.4, the isoelectric point at 4.2, and a Km of 17 mM, and was inhibited by caprylohydroxamic acid, N-ethylmaleimide and p-chloromercuribenzoic acid, though these compounds were considerably less inhibitory to acid urease than to neutral urease.

Urea Removal from Wine by an Acid Urease

Urea, the main precursor for ethyl carbamate formation, is present naturally in certain wines. Treatment of these wines with a preparation of killed cells containing an acid urease will remove the major potential source of ethyl carbamate. The action of the enzyme is fairly rapid. The cells can be reused a number of times with little loss in activity. A number of different wines were tested. Variability of the response to the enzyme action was due to pH. In the range of wine pHs, it is most effective at higher pHs. In certain wines some inhibition was noted beyond the pH effect. Methods of use are suggested.