JPH03143393A - Production of alpha-galactosidase having strong saccharide transition activity - Google Patents

Abstract

PURPOSE:To obtain the title composition usable for synthesis of alpha -galactosyl group-containing oligosaccharide and glycoside, having high transition activity, by culturing Pseudomonas fluorescence H-601 strain. CONSTITUTION:Pseudomonas fluorescence H-601 strain is aerobically cultured by using a liquid medium containing a carbon source and nitrogen source preferably at 20-40 deg.C for 2-14 days to give the objective compound.

Description

[Detailed description of the invention] [) Industrial application field] The present invention is directed to the use of Pseudomonas fluorescens (Pse.
udomonas fluorescens) H-6
This invention relates to a method for producing α-galactosidase with strong transglycosylation activity using the 01 strain.

[Prior Art] In recent years, various oligosaccharides have attracted attention as substances with new functionality such as bifidus factors. As methods for synthesizing such oligomers or glycosides, methods using the glycosyltransfer action of glycosyltransferases or hydrolases and methods using condensation reactions have been developed. Unlike the synthesis of oligosaccharides or glycosides by condensation reactions, methods that utilize transglycosylation can specifically synthesize only oligosaccharides or glycosides with a specific bonding mode. For example, fructooligosaccharides are produced using β-fructofuranosidase, and β-galactooligosaccharides are produced using β-galactosidase. On the other hand, α
- Galactosidase is an enzyme that is widely present in animal tissues, microorganisms, and higher plants, and catalyzes a reaction that hydrolyzes α-D-galactoside bonds to liberate D-galactose.

Since this enzyme was extracted from yeast as an enzyme (melibiase) that hydrolyzes melibiose at the end of the 19th century,
Those of various origins have been isolated and studied. In particular, it is used for the purpose of efficiently hydrolyzing raffinose, which prevents crystallization of sucrose, into sucrose and galactose in the separation and purification process of sucrose from beet sugar. However, the axis translocation effect of α-galactosidase has not been studied very much because the source of the inexpensive α-galactosyl group-containing compound as a substrate was not known. Although oligosaccharides containing α-galactosyl groups have attracted attention as substances with strong bifidus factor activity, an industrial method for producing oligosaccharides that utilizes the translocation action of α-galactosidase has not been established. Therefore, the present inventors obtained α-galactosyl group-containing oligosaccharide from soil with strong transglycosylation activity and for its effective use.
-An attempt was made to isolate a bacterium that produces galactosidase.

[Problems to be Solved by the Invention] Therefore, the purpose of the present invention is to obtain a strong α-
An object of the present invention is to provide a method for producing galactosidase at low cost and in large quantities.

The present inventors previously conducted a wide search in nature for microorganisms that produce α-galactosidase that has the ability to transfer α-galactosyl groups, and found that Pseudomonas bacteria have the ability to produce α-galactosidase that has transfer activity. We named it H-1 and presented our research [Collection of Abstracts of the 1988 Annual Conference of the Japanese Society of Agricultural Chemistry, page 296].

However, the α-galactosidase produced by Pseudomonas fluorescens H-1 has a problem in that when melibiose is used as a substrate, only about 60% of the galactose produced can be transferred when the substrate concentration is around 10. .

[Means for Solving the Problems] After that, the present inventors continued searching for Pseudomonas bacteria with stronger α-galactosyl group transfer activity, and found a new strain with extremely strong transfer activity.・Fluorescence (Pseudomon)
The present invention was completed by naming the strain H-601 (as fluorescens). That is, the present invention provides a method for producing a glycosyltransferase, which is characterized by culturing Pseudomonas fluorescens H-601 strain that produces α-galactosidase with strong glycosyltransfer activity, and collecting α-galactosidase with strong glycosyl transfer activity from the culture supernatant. This invention relates to a method for producing α-galactosidase with strong metastatic activity.

In addition, α-galactosidase with strong glycosyltransfer activity as used in the present invention means that when melibiose is used as a substrate, there is almost no substrate damage even when the substrate concentration is around 10, and more than 65% of the generated galactose is transferred. α-galactosidase that has the activity of

The content of the present invention will be explained in more detail below.

In the present invention, Pseudomonas fluorescens (Pseudomonas fluorescens) is used as an exemplary strain.
ore-scens) H-601 strain is effectively used. Pseudomonas fluorescens is isolated from soil, rivers, food, etc., and is well known to produce fluorescent pigments. However, no strain of Pseudomonas fluorescens having the ability to produce α-galactosidase has been reported other than the above two strains discovered by the present inventors.

Next, the scientific properties of the strain discovered by the present inventors are as follows.

(a) Morphological properties ■Cell shape Bacilli ■Durham staining Negative ■Presence of spores None ■Motility Yes (b) Growth on medium Agar flat medium Round, regular, smooth surroundings Surface smooth, semi-oblate Transparent, pale yellow (C) Growth temperature 37°C Grows 41°C No growth (d) Biochemical properties Catalase + - Oxidase (Kovacs) + 0-F test 0 (e) Ravid test (API) [ 30℃ 48 hours culture] Nitrate reduction indole raw! Production of acid from glucose Arginine dehydrase Urease Hydrolysis of esculin Hydrolysis of gelatin Beta-galactosidase Assimilation of sugar Glucose Results Arabinose Mannose Mannitol N-acetylglucosamine Maltose Gluconate Caprate Deca Adipate Malate Ten Citrate 1- Acetate phenyl ester cytochrome oxidase + [Culture at 30°C for 7 days] Hydrolysis of gelatin Production of ten-bioverdein Production of ten levan Reduction of nitrate Production of nitrite Various properties of residual nitrogen and above are described in Bergei's Manual of Determinative Bacteriology 8th edition (1974)
By comparing the description with the description, it was confirmed that this bacterium was Pseudomonas
Confirm that it is fluorescent and Pseudomonas
Fluorescence (Pseudomonas fluo)
It was named H-601.

In addition, this strain has been approved by FIKEN Bacteria No. 11027 (FERM
P-11027) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Pseudomonas fluorescens H-601 of the present invention
In order to produce α-galactosidase with extremely strong transglycosylation activity depending on the strain, melibiose, raffinose, stachyose, galactomannan, or their decomposition products alone, or succulents, powdered candy, lactose, or starch are used as an inducer. Additions of melibiose, raffinose, stachyose, galactomannan, or their decomposition products are used as carbon sources, and organic or inorganic nitrogen sources such as whole or defatted soybeans, cornstarch liquor polypeptone, soyton, or yeast extract are used. What is necessary is just to culture|cultivate aerobically for about 2 to 14 days at 20-40 degreeC using the liquid 1st medium containing. Since α-galactosidase, which has a strong transglycosylation activity, is an enzyme produced outside the bacterial cell, in the case of a liquid medium, the culture supernatant obtained by filtration or centrifugation after culturing can be used as is as a complementary solution. The crude enzyme solution is
Although it can be used as it is, a crude enzyme concentrate can also be obtained by a known method such as ammonium sulfate precipitation, solvent precipitation, or ultrafiltration. The thus obtained α-galactosidase preparation of the present invention with strong transglycosylation activity has the following properties.

(1) Effect α-Galactocida Lse is an enzyme that catalyzes the reaction of hydrolyzing the α-D-galac I cytobond to liberate D-galactose. This enzyme was used to convert paranitrophenyl-α- into a compound with α-galactosyl group at a final concentration of 44 mM
Galactoside〉orthoni], lophenylu-α-galactoside〉melibiose〉raffinose〉stachyose worked well in this order.

(2) Optimal action pH range The optimal working pH range of this enzyme was found to be approximately 6-7.

(3) Stable pH range McHvaine buffer, Tris buffer, Glycj
The stability of this enzyme when left at 40° C. for 2 hours under ne-NaOH buffer was pH 6 to 9.

(4) Action temperature range and optimum action temperature 0 This enzyme works well at temperatures up to about 60°C, but the optimum action temperature was about 45°C.

(5) Thermostability The enzyme was heat-treated at each temperature for 15 minutes under 0.02M phosphate buffer (pH 6,5).
More than 0% activity was maintained.

(6) As a donor for the glycosyltransfer activity α-galactosyl group, any oligo Ijf or all derivatives containing an α-galactosyl group, which is used as a substrate for this enzyme, can be used. Here, the glycosyltransfer activity of this enzyme was measured using melibiose as a substrate. This enzyme is applied to melibiose at various concentrations, and the free glucose and lactose produced by the reaction are removed by Boehringer
Quantification was performed using Mannheim's F kit (trade name). As shown in Figure 1, this enzyme begins to utilize the galactose generated by cleavage of the substrate for the transfer reaction when the substrate concentration is around 50mM, and even at around 1M, more than 65% of the generated galactose is unharmed by the substrate. It had strong metastatic activity. In FIG. 1, the horizontal axis represents the substrate concentration, the vertical axis represents the amount of product, the number represents the amount of glucose produced by the decomposition of melibiose, and the number represents the amount of galactose. In other words, this difference indicates the amount of galactose used for transglycosylation.

(7) This enzyme acts on 1% paranitrophenyl-α-galactoside as a receptor-specific receptor, 4% galac I-su as a receptor, glucose, mannose, fructose, sucrose, and glycerin. The reaction product was confirmed by thin layer chromatography. As a result, transfer products were confirmed in all cases of this receptor, confirming that this enzyme has broad receptor specificity.

(8) Activity measurement method for α-galac I sidase 10mM varanitrophenyl-α-galactoside 02ml and 40m
Add 0.05 ml of α-galactosidase solution to 0.2 ml of M phosphate buffer (pH 6.5) and mix with 40'C1
, O minutes. After the reaction, 0.5 ml of 0.2M sodium carbonate was added to stop the reaction, and the amount of paranitrophenol liberated was measured by measuring the absorbance at 400 nm with a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that produces 1 μmole of palani I lophenol per minute under these conditions.

(9) Purification method The culture supernatant obtained by centrifugation was subjected to hydrophobic chromatography (Butyl Toyovar 650M manufactured by Tosoh Corporation), ion exchange chromatography (DEAE-Toyovar 650M manufactured by Tosoh Corporation) in the presence of saturated ammonium sulfate, and,
Gel chromatography (Toyobar H manufactured by Tosoh (a)
W-55F) to purify this enzyme.

Through this procedure, it was possible to obtain a single sample of α-galactosidase using disk gel electrophoresis.

(10) Molecular weight About this enzyme Tosoh (User rack Toyobar HW-55F
When the molecular weight was measured by a gel filtration method using (trade name), the molecular weight was equivalent to 3.9×10'.

(11) About isoelectric focusing enzyme Pharmacia PhastGel IEF
The isoelectric point 3 was determined to be 63 by isofocus electrophoresis using 3-9 (trade name).

[Effects of the Invention] As described in Section 2 below, the α-galactosidase of the present invention with strong transfer activity is not harmed by the substrate even when the substrate concentration is around 1M.
It had extremely strong translocation activity, transferring more than 65% of the galactose generated by cleavage of the substrate. In addition, the receptor specificity was very wide for galactose, glucose, mannose, fructose, sucrose, and glycerin, and its enzymatic properties were superior to those of previously known enzymes. The characteristics of this enzyme are as follows:
This work has brought about significant technological progress in the synthesis of oligosaccharides or glycosides containing α-galactosyl groups using the transglycosylation action of α-galactosidase. Furthermore, since this enzyme is a hydrolase, it can naturally be used in the synthesis of oligosaccharides or glycosides containing an α-galactosyl group using a condensation reaction.

[Example] Next, the present invention will be explained with reference to Examples. is not limited to these.

Example 1 1% melibiose, 1% polypeptone, 0.05% 2
HP040.01%MgS Oa・7)1.0.0
．． Liquid medium containing 01% KCI (pH 7,0 HOOmL)
was placed in a 500 m1 slope flask and sterilized by autoclaving in a conventional manner, followed by Pseudomonas fluorescens.
H-601 strain was inoculated and cultured with shaking at 30°C for 4 days.

After culturing, bacteria are removed by centrifugation, and the resulting culture soil l+'
α-galactosidase activity was measured for j.

As a result, the enzyme activity was 075 units per ml of culture solution.

Example 2 In Example 1, instead of melibiose, Pseudomonas fluorescens was grown in a medium prepared by adding 0.1% melibiose to 1% powdered candy as an induction source.
As fluorescens) Toro 01 strain was inoculated and cultured with shaking at 30°C for 4 days. α-galactosidase activity was measured for the culture supernatant obtained by centrifugation after culturing. As a result, the enzyme activity was 0.3 per ml of culture solution.
It was 8 units.

Example 3 2000 ml of the crude enzyme preparation containing 760 units of α-galactosidase obtained by 4"J in Example 2 was subjected to hydrophobic chromatography (butyltoyobal 650M manufactured by Tosoh Corporation) and ion exchange chromatography (Tosoh Corporation) in the presence of saturated ammonium sulfate. 300 units of enzyme preparation 10 were prepared using gel chromatography (DEAE-)Yopal 650M (manufactured by Tosoh Corporation) and gel chromatography (Toyobal HW-55F, manufactured by Tosoh Corporation).
m1 was obtained.

This enzyme preparation was disk electrophoretically uniform.

The yield of α-galactosidase by such purification is approximately 4
It was 0%.

Example 4 pH containing 1g melibiose and 1g sucrose
5 units of the purified enzyme preparation obtained in Example 3 were added to 5 ml of the phosphate buffer solution of Example 6 and 5, and the mixture was reacted at 40°C for 50 hours. After heating the reaction solution by boiling for 10 minutes to inactivate the enzyme, 1 μl of the reaction solution was spotted on a filter paper and n-7''l/-
4 in a solvent system with a composition of 7 water and 6:43
After double development, when ketose coloring was performed using the phloroglucin method, subo-soto was detected at the position corresponding to raffinose. This reaction solution was subjected to activated carbon column chromatography to adsorb oligo-rice grains I, and then eluted with an ethyl alcohol concentration gradient.

This trimer fraction was collected, concentrated, and freeze-dried sample 0.8g
I got it. This specimen (a) produces equimolar amounts of galactose and sucrose when hydrolyzed with α-galactosidase; (b) produces equimolar amounts of melibiose and fructose when hydrolyzed with β-fructosidase; (c) that when hydrolyzed by α-galac 1-sidase and β-fructosidase, 1 mole each of galactose, glucose, and fructose are produced; (d) that the mobility in paper chromatography matches that of raffinose; Therefore, it was identified as raffinose.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of investigating the effect of substrate concentration on the transglycosylation action of this enzyme using melibiose as a substrate.

Claims (2)

[Claims] (1) Pseudomonas fluorescens (￥Pseu
-domonas￥￥fluorescens￥)H-
α- with strong transglycosylation activity was obtained by culturing strain 601.
Method for producing galactosidase (2) α-galactosidase is derived from Pseudomonas fluorescens (\Pseudomonas\\fluorescens).
The method for producing α-galactosidase with strong transglycosylation activity according to claim (1), which is a culture supernatant of S. escens￥) H-601 strain.