JPH02182187A - Immobilized enzyme - Google Patents

Abstract

PURPOSE:To provide an immobilized enzyme having excellent reactivity and good storage stability without causing any side reaction and useful reactors, etc., in a high immobilization degree by immobilizing an enzyme on an anion exchange group-containing carrier, the enzyme generating NAD(P) and H2O2 on an action on NAD(P)H. CONSTITUTION:An enzyme [NAD(P)H oxidase] generating oxidation type nicotinamide adenine dinucleotide(NAD) or oxidation type nicotinamide adenine dinucleotide phosphate(NADP) and hydrogen peroxide on an action on reduction type nicotinamide adenine dinucleotide(NADH) or reduction type nicotinamide dinucleotide phosphate(NADPH) is immobilized on an action exchange group- containing carrier (e.g. DEAE-cephalose CL-6B) to provide the objective immobilized enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immobilized enzyme used as a beam actor or reagent.

[Conventional technology]

Immobilized enzymes are commonly used to efficiently convert substances using enzymes. However, in order to produce an immobilized enzyme, it is generally necessary to carry out steps such as preparing the enzyme of interest to a nearly uniform consistency and then bonding it to a carrier that is encapsulated within the carrier.

In most species, microorganisms generally act on constitutively reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate to produce oxidized nicotine, as previously discovered by the present inventors. It produces an enzyme that generates amide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate and hydrogen peroxide [hereinafter abbreviated as NAD(P)H oxidase], but the NAD that accounts for the total protein produced by the bacterial cell Although the proportion of (P)H oxidase is small and it takes considerable effort to purify the main fermentation product, the purified M enzyme often contains extra enzymes such as catalase and other enzymes or proteins.

The catalytic efficiency of NAD(P,)H oxidase mixed with contaminants such as the above may decrease over time due to the influence of the coexisting quality.

In order to avoid the effects of coexisting substances, it is possible to perform high-level chromatography by using affinity chromatography when producing this enzyme, but there are problems in terms of cost to purify this enzyme on an industrial scale. Roru.

[Problem to be solved by the invention]

As mentioned above, it takes considerable effort to purify NAD(P)H oxidase to a high degree, and if the purification is insufficient, the catalytic function of the enzyme is impaired.

The purpose of the present invention is to provide an immobilized enzyme in which only the present enzyme is specifically and easily immobilized.
Enzyme with AD(P)H as coenzyme or NAD(P)H
Useful for constant voltage.

[Means to solve the problem]

To summarize the present invention, the present invention relates to immobilized NAD(P)H oxidase, in which reduced 7[l: type nicotinamide adenine dinucleotide or reduced nicotine It is characterized by immobilized NAD(P)H oxidase that acts on amide adenine dinucleotide phosphate to generate oxidized nicotinamide adenine dinucleotide or dioxidized nicotinamide adenine nucleotide phosphate and hydrogen atomide. do.

As a result of intensive research, the present inventors found that NAD(P)H oxidase binds firmly to a carrier having an anion exchange group, and that this binding is maintained even in a saline bath solution of about 20%. . Generally, in a saline bath solution of about 20%, substances ionically bound to anion exchange groups dissociate. By washing with a 20% saline solution, only NAD(P)H oxidase is specifically immobilized on the carrier having anion exchange groups.

Examples of the NAD(P)H oxidase in the present invention include those of the genus Brevibacterium (BrθVibac-ter
ium), Corynebacterium (Corynθti
aC-terium', l, Arthrobacterium (Ar
throbacter), Micrococcus spp. (MiCr
OCoccu8, Pseuaomo
Achromobacter spp.
er ), Agrobacterium spp.
rium), Flavobacterium (Flavobacterium
cterium), Stregutomyces (Strep
tomyces) reproduces <JP-A-65-44
No. 882, No. 63-157675), or rf1' derived from Lactobacillus (Agricultural &amp;
Agricutural
andBiol,ogica'l Chemistry
y) Vol. 25, p. 876, 1961], or A'cholep'1.asma
) derived enzyme, [European Journal of
Biochemistry ('Eurcipean Journal)
al of BiocMmi8tr7) Volume 120,
329, 1981], Bacillus spp.
Journal of Biochemistry (Journal of Biochemistry)
stry) Volume 98, Page 1433, 1985, JP-A No. 65-25+082], and these enzymes may be purified products or undiluted products.

Further, the enzyme preparation may be in the form of a solution or suspension.

In addition, as the carrier having an anion exchange group in the present invention, for example, DEAE-Sepharose (Sepharos'
e) CL-6B, DEAE-Sephadex (5
ephadex')'A-25, DEAE-Sephadex A-50, DEAE-Sephadex A/ (5eph
ace1. ), QAE-Sephadex A-25, Q
AE-Sephadex A-50 (manufactured by Pharmacia), DEAE-TOYOPEARL
) 650M, DEAE-Toyobar 65O8 (
(manufactured by Toyo Soda Co., Ltd.), DEAF-cellulose (Ce11)
ulose), Amberlite
) IRA-400 [Dowex]
Any carrier having an ion exchange group may be used. Further, the shape of the carrier only needs to be the same phase, and may be resin-like or film-like.

For immobilization, for example, the present enzyme is prepared in a 10 mM potassium phosphate buffer, and the DE equilibrated with the present buffer is
Gently mix AE-Sephadex A-25 and add 1
Just leave it for about 0 minutes. The buffer may be a Tris-hydrochloric acid buffer, a glycine-sodium hydroxide buffer, or the like. Next, the immobilized enzyme is washed. As a cleaning agent, for example, a 5-20% saline solution, preferably a 10%-20% saline solution may be used to thoroughly wash the enzyme-immobilized carrier. Further, as long as the solution has a similar ionic strength, for example, a 5-20% potassium chloride aqueous solution or a 1M potassium phosphate mild trlJ solution may be used.

The immobilized NAD (P) H oxidase is free of contaminant enzymes such as catalase and grotease, and is
The hydrogen peroxide produced by the action of oxidase on NAD(P)H is not degraded by the action of contaminants in catalase, and the fixed violet property of this immobilized enzyme is good (21NA).
D(P)H oxidase is not degraded by the action of contaminants such as grotease, and the immobilized enzyme has good stability.

In fact, when we conducted an experiment to oxidize NADH using this immobilized enzyme, we found that the amount of decrease in NADH was determined by measuring the absorbance at the specific absorption wavelength of 340 nm of NADH, and the increase in hydrogen peroxide was determined using a hydrogen peroxide electrode. The lids matched. Furthermore, when the present enzyme was stored at 4°C after preparation, 100% activity was maintained even after 60 days.

〔Example〕

The present invention will be explained below with reference to examples, but the present invention is not limited to the scope of the following examples.

Example 1 Brevibacterium ammoniagenes (Brevib
acterium ammoniagenes) I
AM1645 strain was cultured and collected according to the culture method described in JP-A No. 63-44882, and 2f of this bacterial body was grown in a 10 m
M suspended in potassium phosphate buffer fi pH 7.0,
20 pieces of crude enzyme solution were obtained by ultrasonication. Add 10mM potassium phosphate buffer (pH 7) to the above crude enzyme solution in advance.
Equilibrated at 0 fC, DEAE-Sepharose CL-6B
(10 g) was added, and after stirring well, the resin was filtered with a glass filter, and the resin was washed with 20% saline bath solution 50-50%, and then thoroughly washed with 50 mM potassium phosphate buffer pH 7.0. An immobilized enzyme was obtained.

Example 2 Bacillus sphaericus (Bacillus 5pha)
e-ricus) IFo 3341 strain was published in 1986
The cells were cultured and collected according to the culture method described in Publication No. 251082, and the cells 101 were suspended in 60mM potassium phosphate buffer pH 7.2 and subjected to ultrasonication to produce 40ml of crude enzyme solution.
I got it. To this crude enzyme solution, add DEAE-
Sepharose CL-6B (10g) was added, stirred thoroughly, and filtered through a glass filter. The resin was washed with a 10% sodium chloride solution, 50%, and a 60% potassium phosphate mild WJ solution, pH 7.
2 to obtain an immobilized enzyme.

Example 3 NAD produced by various microorganisms in the same manner as in Example 1
(P)H oxidase was immobilized.

As shown in Table 1, the identified N
AD(P)H oxidase was obtained.

Dragon Example 4 (a) The immobilized enzyme obtained in Example 1 and Example 2 was added to an NADH solution with a concentration of 0.2 mM, the reaction was carried out at 37°C, and the absorbance at 540 nm of the decrease in NADH was measured. When the amount of hydrogen peroxide produced was measured using a hydrogen peroxide electrode, the number of moles of NADH decreased and the number of moles of hydrogen peroxide produced matched.

(The immobilized enzyme obtained in Example 1 and Example 2 was
When stored at ℃, even after 30 days the activity immediately after identification is 100
% retained.

〔Effect of the invention〕

As explained in detail above, according to the present invention, NAD(P)
Provided is an immobilized enzyme that easily and efficiently stores H oxidase, has good reactivity, does not cause side reactions, and has good storage stability.

Claims (1)

[Claims] 1. Oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide is added to a carrier having an anion exchange group by acting on reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate. An immobilized enzyme characterized by immobilizing an enzyme that generates nucleotide phosphate and hydrogen peroxide.