Purification, characterization, and application of an acid urease from Arthrobacter mobilis

Katsuro Miyagawa, Motoo Sumida, Masahiro Nakao, Masami Harada, Hiroshi Yamamoto, Takaaki Kusumi, Kiyoshi Yoshizawa, Teruo Amachi, Toru Nakayama

Research output: Contribution to journalArticlepeer-review

53 Citations (Scopus)

Abstract

It has been shown that urea in fermented beverages and foods can serve as a precursor of ethylcarbamate, a potential carcinogen, and acid urease is an effective agent for removing urea in such products. We describe herein the purification and characterization of a novel acid urease from Arthrobacter mobilis SAM 0752 and show its unique application for the removal of urea from fermented beverages using the Japanese rice wine, sake, as an example. The purified acid urease showed an optimum pH for activity at pH 4.2. The enzyme exhibited an apparent K(m) for urea of 3.0 mM and a V(max) of 2370 μmol of urea per mg and min at 37°C and pH 4.2. Gel permeation chromatographic and sodium dodecyl sulfate gel electrophoretic analyses showed that the enzyme has an apparent native molecular weight (M(r)) of 290 000 and consisted of three types of subunit proteins (M(r), 67 000, 16 600, 14 100) denoted by α, β, and γ. The most probable stoichiometry of the subunits was estimated to be α:β:γ=1:1:1, suggesting the enzyme subunit structure of (αβγ)3. The enzyme also existed as an aggregated form with an M(r) of 580,000. The purified enzyme contained 2 g-atom of nickel per αβγ unit of the enzyme. Enzyme activity was inhibited by acetohydroxamic acid, HgCl2, and CuCl2. The isoelectric point of the native enzyme was estimated by gel electrofocusing to be 6.8. Urea (50 ppm), which was exogenously added to sake (pH 4.4, 17±1% (v/v) ethanol), was completely decomposed by incubation with the enzyme (0.09 U ml-1) at 15°C for 13 days. The enzyme was unstable at temperatures higher than 65°C and pHs lower than 4, and was completely inactivated under the conditions of a pasteurization step involved in the traditional sake-making processes. These results indicate that the enzyme is applicable to the elimination of urea in fermented beverages with minimal modification to the conventional process. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)227-236
Number of pages10
JournalJournal of Biotechnology
Volume68
Issue number2-3
DOIs
Publication statusPublished - 1999 Feb 19
Externally publishedYes

Keywords

  • Acid urease
  • Arthrobacter mobilis
  • Ethyl carbamate
  • Fermented foods and beverages
  • Japanese rice wine (sake)
  • Urea

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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