Fructosylamine deglycase and a method of producing it

• US 5,387,109 A
• Filed: 05/24/1993
• Issued: 02/07/1995
• Est. Priority Date: 06/05/1992
• Status: Expired due to Fees

• Current Assignee: Nakano Vinegar Co., Ltd.
• Original Assignee: Nakano Vinegar Co., Ltd.
• Inventors: Okumura, Hajime, Ishikawa, Atsushi, Yamada, Mikio, Ohshima, Yoshifumi, Kawamura, Yoshiya
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): LANKFORD JR, LEON B
• Application Number:
  US08/066,499
• Time in Patent Office: 624 Days
• Field of Search: 435/189, 435/190, 435/254.22, 435/26, 435/191, 435/192, 435/200, 435/71.1, 435/71.2
• US Class Current: 435/191
• CPC Class Codes: C12N 9/0012 acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase

Abstract

Disclosed are a novel fructosylamine deglycase characterized by the specificity to amadori compounds of catalyzing oxidation of the compounds to produce an α-ketoaldehyde, an amine derivative and hydrogen peroxide; a method of producing the novel enzyme by cultivating microorganisms belonging to the genus Candida and having an ability of producing the novel enzyme; and a method of quantitative determination of amadori compounds by applying the novel enzyme to a sample containing amadori compounds to measure the amount of the hydrogen peroxide to be formed by the oxidation reaction or measure the amount of the oxygen to be consumed by the reaction to thereby obtain the amount of the amadori compounds from the measured value. The invention provides a novel enzyme characterized by the high specificity to the reaction with amadori compounds, especially that having therein a saccharide moiety as bonded to the ε-amino group, and also provides a method of producing the enzyme and a method of quantitative determination of amadori compounds with the enzyme. Using the enzyme, quantitative determination of amadori compounds, which is difficult by conventional enzymatic methods, is possible with ease. In particular, in measurement of the amount of a saccharified protein in a sample from a living body, which is an important index substance in diagnosis of diabetes, the enzymatic method of the invention of measuring fructosamine in the sample is hardly influenced by other interfering substances or impurities in the sample, the influence by them having been inevitable in conventional chemical methods. Accurate determination of the amount is possible by the method, and the method is free from the inconvenience of staining the kits and instruments used. For measuring glycohemoglobin, the method displays another characteristic feature that its operation is more simple and needs less labor and time than conventional methods.

Claims

1. A fructosylamine deglycase having the following physico-chemical properties:

(a) effect and substrate specificity;

the fructosylamine deglycase acts on amadori compounds, especially effectively on an ε

-amino acid amadori compound to catalyze oxidation of the amadori compounds to produce an α

-ketoaldehyde, an amine compound and hydrogen peroxide;

(b) optimum pH and pH stability;

the fructosylamine deglycase is stable at a pH of 5.0 to 8.0 with respect to a substrate of di-fructosyl-lysine, and having an optimum pH with respect to the substrate of 7.0 to 8.0 in a phosphate buffer;

(c) range of reaction temperature;

the range of the reaction temperature of the fructosylamine deglycase is 15°

to 45°

C.;

(d) thermostability;

the fructosylamine deglycase is stable to heat treatment of 40°

C. for 10 minutes, and when the fructosylamine deglycase is heated at 50°

C. for 10 minutes, 80% or more of the fructosylamine deglycase is inactivated;

(e) molecular weight;

the fructosylamine deglycase has a molecular weight of about 43000, as measured by gel filtration on a column of a porous, semirigid, spherical gel.

2. The fructosylamine deglycase of claim 1, having an isoelectric point of 4.8, as measured by disc isoelectric point electrography.

3. A method of producing a fructosylamine deglycase of claim 1, comprising

(a) cultivating microorganism Canada guilliermondii FERM BP-3878, said microorganism having a capacity of producing the fructosylamine deglycase, in a medium and

(b) collecting the produced fructosylamine deglycase from the culture.

4. The method of claim 3, wherein the cultivating is carried out at a temperature of 25°

C. to 35°

C. at a pH of 4.0 to 7.0 for 12 hours to 36 hours.

5. The method of claim 4, wherein the temperature is 30°

C. and the pH is 5.0.