JPS62155085A - Production of l-fucose dehydrogenase - Google Patents

Abstract

PURPOSE:To produce L-fucose dehydrogenase useful for the research of the structure and function of composite sugar chain, on an industrial scale at a low cost, by culturing an L-fucose dehydrogenase-producing strain belonging to Corynebacterium genus in a medium. CONSTITUTION:A bacterial strain such as Corynebacterium sp FS-0077 having the following bacteriological properties is cultured in a medium containing about 0.1wt% L-fucose, etc., peptone as a nitrogen source and phosphate, etc., as an inorganic substance at 20-35 deg.C and 5-7pH for 10-48hr under agitation and aeration. The size of cell, (0.3-0.5)X(3-5)mum; shape of cell, polymorphic such as linear, rod-like, club-like, etc.; locomotivity, none; positive to reduction of nitrate; utilizing L-fucose, etc.; etc. The obtained culture cell is extracted to obtain L-fucose dehydrogenase capable of oxidizing L-fucose to L-fucono-sigma- lactone and having optimum pH of 7.5-8, a molecular weight of about 26,000, an isoelectric point of 5.8pH, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing L-7 course dehydrogenase. More specifically, the present invention relates to a method for culturing bacteria and producing L-7 course dehydrogenase from the culture.

[Conventional technology]

L-fucose dehydrogenase is an enzyme that oxidizes L-7 course to produce L-fucono δ-lactone, and its presence in microorganisms and animal tissues has been reported. α-L-fucosyl groups are frequently found at the non-reducing ends or branched parts of complex sugar chains derived from higher animals, and in order to elucidate the structure and function of these sugar chains, it is necessary to After releasing the L-7 course residue from the sugar chain, it is very important to quantify the L-fucose content. As a method for quantifying this free L-fucose (methods in which both free L-fucose in a sample are purified by gel filtration or ion exchange method and quantified by gas chromatography (Methods in Engineering Vol. 28, Vol. 738, 1972), and a method for quantifying acetaldehyde produced by periodate oxidation ζ (Journal of Biological Chemistry, Vol. 245, p. 1659, 1970), but the procedures are very complicated. There is also a problem with accuracy.

Compared to these chemical methods, the enzymatic method using L-fucose dehydrogenase does not require purification of free ML-fucose in the sample and can be quantified quickly and accurately.

[Problem that the invention seeks to solve]

This L-fucose dehydrogenase has already been reported to be produced by the microorganism Pluralia pullulan (IR ('J Mad, Sci, Libr).
, Compend.

Volume 1, page 73, 1973). However, since the optimum reaction pH of this enzyme is 10.2, which is quite alkaline, there is a desire to search for microorganisms that produce L-fucose dehydrogenase having an optimum pH near neutrality. In addition, among animal tissues, pig liver (Journal of Biological Chemistry Vol. 244, p. 4785, 1
Its existence has been reported in rabbit liver (Journal of Biochemistry, Vol. 86, p. 1559, 1979), but this enzyme also has an optimum pH around pH 10, and is Furthermore, when animal-derived enzymes are industrially produced, they are disadvantageous compared to microbial enzymes in terms of stable supply of raw materials.

In view of the above-mentioned current situation, the purpose of the present invention is to
An object of the present invention is to provide a method for industrially producing L-fucose dehydrogenase containing H at low cost.

[Means for solving problems]

To summarize the present invention, the present invention relates to a method for producing L-fucose dehydrogenase, in which microorganisms belonging to the Corynebacterium group and having the ability to produce L-fucose dehydrogenase are cultivated, and a culture medium f5' It consists of collecting L-fucose dehydrogenase from 4/g.

The bacteria belonging to the Corynebacterium genus used in the present invention can produce L-fucose dehydrogenase in large quantities in culture, and as described later, it is very easy to purify and has excellent properties. are doing.

First, the bacterial strain used in the present invention may be any strain as long as it belongs to the genus Corynebacterium and has the ability to produce L-fucose dehydrogenase, or it may be a variant of these strains. Specific examples of strains belonging to the genus Corynebacterium and having the ability to produce L-fucose dehydrogenase include, for example, Coryn@baaterium sp, F
S-0077 is mentioned. This bacterium is a strain newly found by the present inventors in soil in Shiga Prefecture, and its mycological properties are as follows.

a. Morphological properties Microscopic observation (cultured on broth agar medium at 30°C) (1) Cell shape and size: Normal cell size is 0.3-0.5 x 3-5 fin
, straight or slightly curved rods; some have a club-shaped curve at one or both ends, and are polymorphous.

(2) Presence of motile #: None 3) Presence of periflagella: None 4 Gram staining: Positive (5) Presence of spores: None 6) Acid-fastness: Negative Growth status in each medium (1 ) Broth agar plate culture: When cultured at 30°C, convex circular colonies with a diameter of 2 to 4 wR are formed. The surface and edges are smooth. Colony color is white-yellow, opaque, and shiny.

(2) Meat juice agar needle culture: Grows well when cultured at 30°C.

(3) Meat juice liquid culture: No film is formed in static culture at 30°C, but a precipitate is formed.

(4) Meat juice gelatin puncture culture: Static culture at 20°C to grow on the surface and along the puncture line,
Slowly liquefy the gelatin.

(9 Lidmus milk culture: Alkalinization of milk can be seen during static culture at 30°C.

C1 physiological properties (1) Reduction of nitrate: Positive (2) Denitrification reaction: Negative (33M R test: Negative (4)’/P test: Negative (5) Indole production: Negative (6) Generation of hydrogen sulfide: Positive (7) Hydrolysis of starch: Very small amount or does not decompose.

(8) Utilization of citric acid: Positive (9) Utilization of inorganic nitrogen sources: Positive (Ammonium salts and nitrates) (10) Production of pigment: Negative αl) Urease: Negative (12) Catalase: Positive (13) Oxidase: Negative (14) a Suitable growth conditions = 22°C to 37°C, pH 7 to 8
．． 5 (19) Attitude towards oxygen: Aerobic (16) OF test: Negative (17) Vitamin requirements: Negative (18) Utilization of carbon sources: L-fucose, D-arabinose, L-galactose,
L-arabinose, D-fructose, D-glucose, D-galactose, maltose, lactose, glycerol, sucrose, dextrin (19) GC content of DNA: 58% (by Tm method) As mentioned above, this bacterium From the properties, Bergsy'e Manual of'
Dst@rminatfve Bqotsrlog
y) When compared with the 8th edition (1974), it is recognized that it belongs to the genus Corynebacterium. Therefore, this strain was designated as Corynebacterium sp, FS-0077. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the FERMP-854
It has been deposited as 5).

In the present invention, the nutrient source added to the culture medium may be one that can be used by the strain used to produce L-fucose dehydrogenase, and carbon sources include, for example, L-fucose, glycerol, glucose, sucrose, maltose, Lactose, etc. can be used, and nitrogen sources include peptone, yeast extract, cornstarch liquor, meat extract,
Defatted soybeans, ammonium sulfate, ammonium chloride, etc. are suitable.

In addition, inorganic and metal salts such as phosphates, potassium salts, and magnesium salts may be added. In addition, since the L-fucose-dehydrogenase of the present invention is an N-conductor,
Addition of L-7 course to the medium significantly increases enzyme production. For example, when 0.1% of L-fucose is added, about 100 times as much L-fucose dehydrogenase is produced as compared to when no addition is made.

When culturing L-fucose Q dehydrogenase-producing bacteria, the production amount varies greatly depending on the culture conditions, but generally the culture temperature is 20-35°C and the pH of the medium is 5-7.
The production of L-fucose dehydrogenase according to the present invention reaches i&high after 10 to 48 hours of aerated agitation culture. It is natural that the culture conditions should be set so as to maximize the production amount of L-fucose dehydrogenase depending on the bacterial strain used, medium composition, etc.

Since the L-fucose dehydrogenase produced by the bacterium of the present invention exists within the bacterium, the culture is solid-liquid separated and the resulting wet bacterium is subjected to various commonly used methods such as ultrasonication, French press, and Dynamyl. An f@cell extract can be obtained by destroying the bacterial cells using the following destruction means or by dissolving the bacterial cell walls using a cell wall lytic enzyme such as lysozyme. Next, a purified enzyme preparation can be obtained from this extract by a commonly used purification method. For example, salting out,
Nucleic acid removal, ion exchange column chromatography, hydrophobic binding column chromatography, adsorbent column chromatography, gel filtration, frozen P
Purification is carried out by drying, etc., and a single fucose dehydrogenase can be obtained by polyacrylamide gel disk electrophoresis.

The enzyme-chemical and physical-chemical properties of L-fucose dehydrogenase obtained according to the present invention are as follows.

(1) Action: This enzyme oxidizes L-fucose to produce L-fucono δ-lactone as shown in the reaction formula below.

L-fucose + NAD+ → L-fucono δ-lactone + NADH-1-H+ (2) Substrate specificity: This enzyme has the highest specificity for L-fucose, but also acts on L-galactose, D-arabinose, etc. (Table 1).

Substrate (10mM) Relative activity (lol)
Fucose 100L- is lactose
74D-arabinose
37 In addition, this enzyme requires NAD as a coenzyme and produces NAD
It has no effect on P.

(3) Optimal pH and pH stability: The optimal pH of this enzyme is around 7.5 to 8.0 (Figure 1). In addition, this enzyme was tested at 30°C and at each pH.
When treated for 0 minutes, the pH was stable from 6.0 to 10.5 (Figure 3).

(4) Optimal temperature and thermostability: This enzyme has an optimal temperature at 43°C (Figure 2). Furthermore, when this enzyme was treated at pH 8 and 0 for 10 minutes at each temperature, it was stable up to 40°C, but was completely inactivated at 60°C (Figure 4).

(5) Molecular weight: The molecular weight of this enzyme was approximately 26,000 when measured by gel filtration using Sephacryl S-200 (manufactured by Pharmacia).

(6) Isoelectric point: Pharmalite (pH 5.0-8.0 manufactured by Pharmacia)
The isoelectric point of this enzyme was determined to be 5.8 by isoelectric focusing using .

(7) Effect of metal ions: As shown in Table 2, this enzyme was strongly inhibited by Ag+ and Cu''+.

Table 2 No additives 110 0A+5 Cu"+ 23Hg"
0(8)K m value: Linesaver-Park
The Km value of this enzyme for L-fucose was determined by Burk) plot and was found to be 1.3×10 −'M.

(9) Measuring method for lactic acid activity: L-fucose dehydrogenase activity was determined as follows.

That is, 30mML-7 course 1.0me, 2QQmM glycine-sodium hydroxide mild &0 (pH 8,0) 1.8
〃+e, 1.5 +nM NAD Oll me
and appropriate diluted enzyme solution 0.1 me, , Fi
After reacting at 37°C for 10 minutes, measure the absorbance at 340nlll (0. D, S, PULL).In addition, as a control, distilled water was used instead of the enzyme solution. Add 0.0 and measure the absorbance using the same procedure. D, plastic, χΔ0・D,
The 0L-fucose dehydrogenase activity determined by Ha(0.D-Numpho.D.Blank) is determined by the following calculation formula.

6.22: Millimolar molecular extinction coefficient of NADH at 340 Lm d: Optical path length (am) df: Dilution rate [Example] Below, Example 5 of the method for producing L-fucose dehydrogenase according to the present invention is shown. The invention is not limited to the scope of the following examples. Note that % is v/v% unless otherwise specified.

Example I L-fucose 0.1%, peptone 0.5%, KH, PO
.

0.3% Mg5Oi” 7 HzO0.1%,
Dispense 100me of medium with pH 7.0 and sterilize (1
Corynebacterium sp, FS-0077 (Feikoken Bacteria No. 8545) was inoculated into a 500πE Erlenmeyer flask (20°C for 20 minutes) and cultured at 30°C for 48 hours. Shifufus dehydrogenase activity in this culture was 1.
02 units/me.

Example 2 Corynebacterium sp, FS-0077 (Feikoken Bacteria No. 8545) was mixed with yeast extract 0.5% and peptone 1.0
%, KH! PO4Q, 3% and MgSO4Fist 7H,
00.1%, pH 7.0 medium was dispensed and sterilized (120°C, 20 minutes).
The cells were inoculated into an Erlenmeyer flask and cultured at 30°C for 24 hours to obtain a seed culture. L-fucose O, 1%, peptone 0
5%, KH, PO, Q, 3%, MgSO4.7 H, 0
0.1% and antifoaming agent (CB-442 manufactured by NOF Corporation) 0
．． 01″/%, medium consisting of pH 7,0 15/30
The mixture was placed in a 1 volume jar fermentor and sterilized at 120°C for 20 minutes. After cooling, 100 mg of the above seed culture was inoculated and incubated at 30°C for 36 hours with an aeration rate of 151/min and 3/min.
Culture was carried out at a stirring speed of 0.00 rpm. After culturing, centrifuge the culture solution to collect the bacterial cells, suspend them in 500 ml of 5Q+mM phosphate buffer (pH 8,0), and then centrifuge the bacterial cell suspension obtained by crushing the bacterial cells by ultrasonication. Separate and supernatant liquid 5
Got 40me. The L-fucose dehydrogenase activity of this supernatant was 37.8 units/me. This supernatant was adsorbed onto a DEAE-Sepharose CL-6B (manufactured by Pharmacia) column (diameter 5.0α x length 1001) buffered in advance with 50mMIJ acid buffer, and the adsorbed matter was added to 100mMIJ acid buffer. After washing with
Active fractions were collected by elution with +nM phosphate buffer. Next, sodium chloride was added to this active fraction to make it 4M6 degrees. Add this in advance to 100+ containing 4M sodium chloride.
Phenyl Sepharose CL buffered with M phosphate buffer
-4B (manufactured by Pharmacia) column (diameter 2, 5
cra
Elution was performed with 2QmMIJ acid buffer containing M sodium chloride, and active fractions were collected. This active fraction was again adsorbed onto a Phenyl Sepharose CL-5E column (diameter 2.5 CIIX, length 10α), and 5mM sodium chloride containing 2M sodium chloride was added.
After elution with J acid buffer, it was concentrated using a collodion membrane.

Add this concentrated solution to 100mM phosphorus in advance! A column of Sepharose CL-5B buffered with V buffer (diameter 2.5
Gel filtration was performed using a column of Sephacryl S-200 (manufactured by Pharmacia) (diameter 2.5 cm x length 100 cm) after reconcentrating the obtained active fraction.
After repeating the gel filtration in step m), E[
TA was added to the final concentration of 1 mM and lyophilized to obtain 820 m'1 of purified enzyme powder. The specific activity of this powder was 8.41 units/t. This enzyme powder was electrophoretically active on polyacrylamide gel discs.

The above purification steps are shown in Table 3.

〔Effect of the invention〕

According to the present invention, L is useful for elucidating the structure and function of complex sugar chains.
- A new manufacturing method suitable for industrial production of fucose dehydrogenase has been provided.

[Brief Description of the Drawings] Figure 1 is a graph showing the relationship between pH and activity of L-fucose dehydrogenase obtained from the ζkoyosho of the present invention.
Figure 2 is a graph showing the relationship between temperature and activity;
The figure is a graph showing the relationship between pH and activity after L-fucose dehydrogenase was treated at 30°C for 60 minutes at each pH. Figure 4 is a graph showing the relationship between pH and activity after treatment at each temperature for 10 minutes at p) 18.0 It is a graph showing the relationship between temperature and activity after the reaction. Figure 1 pH fJ2r! J 1 degree (0C) Figure 3 pH Figure 4 Temperature (°C) Procedural amendment (spontaneous) Indication of the case of February 7, 1985 Patent application No. 295431 of 1985, title of invention L-fucose dehydrogenase Relation to the case of the person who amended the manufacturing method of the patent applicant 4. Drawings subject to the amendment by the agent 6. Drawings containing the content of the amendment (all drawings) are corrected as shown in the attached sheet. Above Figure 1 Compensation Figure 2 1 degree (C) Figure 3 H Figure 4 Yudo (@C) Procedural amendment (voluntary) February 10, 1985 Uga, Commissioner of the Japan Patent Office Mr. Dobe ;◇ 1. Indication of the incident 1985 Patent Application No. 295481 2
, Title of the invention: Process for producing L-fucose dehydrogenase 3, Person making the amendment 4 Relationship with the project Patent applicant: Kyoden Hesou 1, Attorney 5: Detailed description of the invention in the specification to be amended. , -, :, lt・'Otsupa°- 6. Contents of the amendment (1) "Bacillus" on page 5, line 9 of Specification 4 is corrected to "Bacillus J." (2) "Bacillus" on page 6, line 3 of the same specification Correct "needle surface culture" to "slope culture." (3) Insert rHP, J before rl"J in line 15 of page 12. (4) Insert r1.5mMJ in line 2 from the bottom of page 13 l"1
Correct it to 5mMJ. (5) Same page 16, line 4 r(pH8,0)J [(pH
8,0; All operations below were performed at pH 8). (6) Page 17, line 8 “cL-6BJ is “cL-4B”
Correct it with J. Notification of change in accession number December 19, 1985 1. Indication of the case Patent No. 295431 of 1985 2. Name of the invention Method for producing L-fucose dehydrogenase 3. Person who filed the procedure $ Relationship with the case Patent applicant representative Amami 1) Minoru 4, agent name (596 patent attorney Mitsuo Adachi
J5, old depository name Institute of Microbiology, Agency of Industrial Science and Technology 6, former accession number FERM P-8545 (FERM P-8545) 7, new depository name JM Agency of Industrial Science and Technology, Ministry of Commerce and Industry Industrial Technology Research Institute 8, new accession number FERM BP-1234 (FERM BP-1234) 9 Condolences I-1-Kichi 3:1 catalog (1) yq accession number, U1: Clear-1 ( )surface
1 copy Procedural amendment (voluntary) December 23, 1985 1 Indication of case 1985 Patent Application No. 295431 2
, Name of the invention L-7 Process for producing dehydrogenase of course 3, Relationship with the person making the amendment Contents (1) Page 8 of the specification, lines 10 to 11, “FyrRMP-8545 J” was replaced with “FyrRMP-8545 J” by “FyrRMP-8545 (Reference No. 8545)". (2) Page 15, lines 4 to 5 and lines 9 to 10
Correct the line ``(FEI Research Institute No. 8545)'' to ``(FEI Research Institute No. 1234).''that's all

Claims (1)

[Claims] 1. A method for producing L-fucose dehydrogenase, which comprises culturing L-fucose dehydrogenase-producing bacteria belonging to the genus Corynebacterium and collecting L-fucose dehydrogenase from the culture.