JPH0545233B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to oligosaccharide dehydrogenase, which can be used as a conjugate enzyme when measuring serum lipase activity, amylase activity, etc., in clinical tests, for example. It is.

[Conventional technology]

Examples of dehydrogenases that act on sugar include ^glucose dehydrogenase (King. ^TE , Meth.
Enzymol., vol.9, p98 (1966)), NAD ⁺ and
Maltose dehydrogenase with NADP ⁺ as an electron acceptor (Y.Kobayahi and K.Horikoshi,
Agric, Biol.Chem., vol.44, p41 (1980)), glycose dehydrogenase with ferricyanide as an electron acceptor (M.Ameyama, E.Shinagawa,
K. Matushita and O. Odachi, Agric. Biol.
Chem., vol.45, p851 (1981)).

On the other hand, recently, microorganisms of the genus Staphylococcus have developed oligosaccharide dehydrogenase, a novel substance that acts on substrates such as maltose, D-glucose, maltotriose, and maltopentaose using phenazine methosulfate as an electron acceptor. It was also reported that it can be produced (Japanese Patent Application Laid-open No. 114193/1983).

[Problems to be Solved by the Invention] In clinical analysis using glucose oxidase or maltose oxidase, as with other analytical methods, it has been desired to further increase detection sensitivity.

Furthermore, since the degradation products of amylase contain various oligosaccharides in addition to glucose, it is desired to develop more means to measure these for analysis or analysis of various amylases. Ta.

[Means for solving problems]

As a result of intensive studies by the present inventor to develop excellent new glucose oxidase and maltose oxidase, it was discovered that a microorganism isolated from soil produces oligosaccharide dehydrogenase, which has a number of excellent properties.
We were able to complete the present invention by knowing that the above-mentioned problems could be solved by using this enzyme.

Next, the physicochemical properties of the enzyme of the present invention obtained in Examples will be shown.

(1) Action The enzyme of the present invention acts on various monosaccharides and oligosaccharides, including maltose and D-glucose, using phenazine methosulfate as an electron acceptor to perform a dehydrogenation reaction.

(2) Substrate specificity When the substrates shown in the table below were used in place of maltose and measurements were performed according to the activity measurement method described below, the following results were obtained. Substrate Relative activity (%) Maltose 100 D-glucose 100 D-galactose 95 D-xylose 94 D-mannoma 100 D-sorbitol 0 D-fructose 0 D-glycerol 0 Maltotriose 29 Maltopentaose 24 (3) Electron acceptor Specificity of the activity When the electron acceptors shown in the table below were used in place of phenazine methosulfate (PMS), measurements were performed according to the activity measurement method described below, and the following results were obtained. Electron acceptor Relative activity (%) PMS 100 NAD 0 NADP 0 NTB ^* 0 Ferricyanide 0 *Nitroblue tetrazolium (4) Optimal PH and stable PH range The optimal PH of this enzyme is as shown in Figure 1. PH6.5
It's nearby. In the same figure, PH4-5 is 0.1M
Acetate buffer (triangle), 0.1M sodium phosphate buffer (black circle) for pH 5.5-8, and pH 7-9
was measured using 0.1M Tris-HCl buffer (white circles).

As shown in FIG. 2, this enzyme is stable at pH 5 to 8, particularly at pH 6 to 7. The buffers used for the measurements were the same as those shown in Figure 1, and the residual activity was determined after treatment in each buffer at 37°C for 1 hour.

(5) Activity measurement method 0.08 μmol phenazine methosulfate,
Contains 0.3μmol nitroblue-tetrazolium, 0.2% Triton X-100, 0.1mol maltose
1ml of 20μmol sodium phosphate buffer (PH6.5)
Add 10 μl of oxygen solution to the solution and incubate at 37°C for 10 minutes. Add 1 ml of 0.1M hydrochloric acid to stop the reaction,
Determine the enzyme activity by measuring the absorbance at 530 nm. The amount of enzyme that reduces 1 μmol of nitroblue tetrazolium per minute is defined as 2 units.

(6) Molecular weight The molecular weight determined by gel filtration method was approximately 80,000.

(7) Isoelectric point The isoelectric point of this enzyme is at PH4.4.

Comparing the physical and chemical properties of this enzyme with the enzyme described in JP-A-60-114193, which is closest to this enzyme, we find that in terms of substrate specificity, this enzyme acts on fructose, whereas this enzyme does not. They differ in some respects. In addition, the optimum pH of this enzyme is 7.0~
8.0, stable PH range 7.5-8.5, and isoelectric point PH
9.5, this enzyme has an optimum pH of 6.5 and is stable.
They are also different in that their PH range is 5 to 8, and their isoelectric point is PH4.4. Therefore, this enzyme was recognized as a new enzyme.

This enzyme was isolated from soil, Serratia marcescens No.
It can be produced and obtained from C886-2 (FERM P-8370).

The Dutch properties of Serratia marsetscens No. C886-2 are shown below.

(a) Morphology Cell shape and size, Bacillus (0.8-1.0×
1.3 to 1.5 μm) Cell pleomorphism: None Motility: Yes Spores: None Bram staining: Negative (b) Growth on various media Broth agar plate: Colonies are plate-shaped Size: 2.0 to 0.2 mm in diameter, all edges are glossy, the surface is glossy, red pigment is produced, it grows well in a linear shape on a gravy agar slope, and even in condensed water with glossy edges.

Meat broth...Produces a uniformly cloudy sediment that forms a film.

Meat juice gelatin puncture culture...Grows well in the upper and middle layers. liquefy.

Litmus milk... coagulates and turns into peptone.

(c) Physiological properties Nitrate reduction...Positive MR test...Negative VP test...Positive Indole production...Negative Hydrogen sulfide production...Negative Citric acid utilization...Positive Pigment production...Red pigment production Urease …Negative Oxidase…Negative Catalase…Positive Growth at 42°C…Growth Attitude towards oxygen…Aerobic OF test…Fermentative Production of gas (glucose)…Negative Production of acid from sugar Glucose, Acid is produced from galactose, kinloose, mannose, arabinose, and fructose.

Does not produce acids from glycerose, maltose, trepas, sucrose, inositol, mannitol, raftose, sorbitol, fructose.

Serratia marsetuscens No. C886-2 may be cultured by the conventional method of culturing Serratia microorganisms, including carbon sources such as glucose, maltose, sucrose, and glycerol, nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate, and phosphorus. It may be cultured in a medium containing inorganic salts such as potassium acid, magnesium sulfate, and sodium chloride, and to which organic nutrients such as yeast extract, meat extract, peptone, and soybean protein hydrolyzate are appropriately added. The culture temperature may be about 20 to 35°C, and the pH may be about 5 to 8. By culturing with aeration and stirring for about 20 to 48 hours, the enzyme is mainly produced and accumulated within the bacterial cells.

The method for isolating and purifying the present enzyme from the culture may be carried out in accordance with the general method for separating and purifying enzymes in bacterial cells. For example, bacterial cells are collected from the culture by centrifugation or filtration and destroyed. As a method for destroying bacterial cells, physical methods such as grinding with a dyno mill, methods using ultrasound, oxygen digestion methods, autolysis methods, and other chemical methods are appropriately used. After destroying the bacterial cells, remove bacterial cell residue by centrifugation to obtain an enzyme extract, and perform ion exchange chromatography, affinity chromatography, ammonium sulfate salting out, centrifugal density gradient method, gel filtration, dialysis, etc. as necessary. What is necessary is just to refine|purify by combining suitably.

〔Example〕

1% glucose, 0.2% sodium nitrate, 0.2%
Yeast extract, 0.1% dipotassium phosphate, 0.05% sodium chloride, 0.05% magnesium sulfate, 0.4%
Medium consisting of calcium carbonate (PH7.0) 30 to 90
The mixture was placed in a jar and sterilized at 120°C for 20 minutes. After cooling Serratia marcescens No.C886−
2 (FERMP-8370) was inoculated, and cultured with aeration and stirring at 28°C for 20 hours.

The amount of ventilation during this time was 0.5 vvm, and the rotation speed of the stirring blade was 300 rpm. After the culture was completed, the culture solution was centrifuged to obtain about 500 g of wet bacterial cells. This bacterial body
Suspend in 2g of 10mM sodium phosphate buffer (PH7.0) and use Dyno Mill (KDL type).
Bacterial cells were disrupted at a flow rate of 3000 rpm and 30 ml/mm. The enzyme extract 2 was obtained by centrifugation to remove bacterial cell residue. Add Triton X-100 to this enzyme extract to a concentration of 1%.
was added and stirred at 6°C for 2 hours, followed by equilibration with 10mM sodium phosphate buffer. Pass through a diethylaminoethyl cellulose column to adsorb impure proteins, and calculate the yield from the fraction that passes through the enzyme.
Obtained with 80%.

〔Effect of the invention〕

The enzyme of the present invention can approximately double the color concentration in reaction with maltose, glucose, etc. in terms of molar extinction coefficient, and can improve the detection sensitivity of these sugars. Therefore, it goes without saying that the detection sensitivity of methods for measuring the activities of various compounds, such as lipase and amylase, which utilize reactions that produce these sugars, can also be improved. In addition, NADH and
Unlike when NADPH is used, the coloring range is in the visible region, so an inexpensive spectrophotometer can be used, and measurements can be easily performed at various locations. Since the optimum pH of enzymes is 6 to 7, clinical test samples can be stably measured in the most preferred pH range. In addition, new uses are possible, such as the ability to more accurately evaluate amylase activity since it acts on a wide variety of monosaccharides and oligosaccharides.

[Brief explanation of the drawing]

FIG. 1 shows a curve showing the optimum pH of the enzyme of the present invention, and FIG. 2 shows a curve showing the PH stability.

Claims (1)

[Scope of Claims] 1 Mono-, oligosaccharide dehydrogenase substrate specificity having the following physicochemical properties: at least maltose, D-glucose,
Acts on D-galactose, D-xylose, D-mannose, maltotriose and maltopentaose. Specificity for electron acceptors: phenazine methosulfate can be used as an electron acceptor when maltose is used as a substrate, but nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, nitro blue tetrazolium and Anide is not used. Isoelectric point: Around PH4.4 Optimal PH: PH6.5 Stable PH range: PH5-8 Molecular weight: Approximately 80,000 (gel filtration method)