Shigeya Kakimoto

Purification and characterization of acid urease from Lactobacillus fermentum

SummaryAcid urease was purified to an electrophoretically homogeneous state, and the molecular weight was estimated to be 220 000. The enzyme consisted of three kinds of subunits, designated α, β and λ, with molecular weights of 67 000, 16 800 and 8600, respectively, in a (α1 \2λ1)2 structure. The isoelectric point of the enzyme was 4.8. The nickel content was found to be 1.9 atoms of nickel per α1β2λ1 unit. The amino acid profile was different from those of known bacterial neutral ureases. The enzyme was most active at pH 2 and around 65° C. It was stable between pH 3 and 9, and below 50° C. The Km for urea was 2.7 mM at pH 2. The enzyme activity was inhibited by Ag+, Hg2+, Cu2+, p-chloromercuribenzoate and acetohydroxamate. The enzyme was separated into three subunits by reverse phase HPLC. The amino terminal amino acid sequences of the subunits α, β and λ were Ser-Phe-Asp-Met-, Met-Val-Pro-Gly- and Met-Arg-Leu-Thr-, respectively.

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, s 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 mM 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.

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.