KR20000003189A - New oligosaccharide producing method using glucansucrases of leuconostoc mesenteroides mutant - Google Patents

Abstract

PURPOSE: A new method for producing oligosaccharide using glucansucrases of new mutant is provided to produce various oligosaccharides having a new structure by performing the receptor reaction of maltose, gentiobiose, raffinose and lactose. CONSTITUTION: A new method for producing oligosaccharide using glucansucrases of leuconostoc mesenteroides comprises the steps of: selecting the mutant of leuconostoc mesenteroides B-512FMCM, B-742CB and B-3155C; producing dextransucrase or alternansucrase by cultivating these bacteria; reacting enzyme with the receptor such as maltose, gentiobiose, raffinose, lactose and the like separated from each bacterium; and analyzing the structure of the oligosaccharide by methylating the oligosaccharide.

Description

Method for Producing Novel Oligosaccharides Using Glucan Sucrase of Leukonostock Mesenteroide Mutant Strains

The present invention relates to a method for producing novel oligosaccharides using glucansucrases. More specifically, the present invention relates to a method for producing oligosaccharides using dextran sucrases obtained from mutant strains of L. mesenteroides and maltose, genthiobio, raffinose or lactose as receptors.

In addition to sweeteners, oligosaccharides are useful in the food industry as growth agents for stabilizers, flocculants and small intestine strains. In order to produce this oligosaccharide on an industrial scale, extraction methods from plants and hydrolysis methods of acids or enzymes of vegetable or microbial carbohydrate polymers are used. In addition, there is a method of using dextran sucrose (dextransucrase) as an enzymatic synthesis method of oligosaccharides. Dextran sucrase obtained from Leuconostoc mesenteroides is an enzyme that industrially synthesizes dextran. The synthesized dextran is composed of glucose units in the main chain with α1-6, and according to the type of dextran sucrase, Proportions and lengths vary respectively. Since most of L. mesenteroides dextransucrase must be added to the enzyme production medium to induce the production of enzymes, dextran is produced in the supernatant, so the enzyme in the medium supernatant is bound to dextran. There is a problem in that dextranase must be processed. In order to solve the above problems, the present inventors have isolated a structurally mutant strain from L. mesenteroides B-742, B-1355, B-1299 or B-512FB, and B-512FMCM, which is mutated by vacuum ultraviolet irradiation. (Kim, D. et al. 1997. Development of constitutive dextransucrase hyper-producing mutants of Leuconostoc mesenteroides using the synchroton radiation in the 70-1000 eV region. Biotechnol. Techniq. 11: 319-321). Dextran biosynthesized from B-512FMCM dextran sucrase has 5% α1-3 structure to 95% α1-6 bond. In addition, B-742CB dextran sucrase synthesizes dextran having main bonds of α1-6 and branched structures of α1-2, α1-3 and α1-4, unlike dextransucrase of the parent strain. B-1355C Alternasucrase selectively produces only alternating alkanes, polysaccharides of alternating structures of α1-6 and α1-3.

Dextran sucrose, along with dextran biosynthesis, produces oligosaccharides by transferring glucose units of sugar to added carbohydrates when other carbohydrates are added to the enzyme reaction. At this time, the added carbohydrate is called a receptor and this reaction is called a receptor reaction. The most studied B-512F dextransucrase for receptor responses produces receptor products containing a series of oligosaccharides using maltose or isomaltose as receptors, but D-fructose, lactose and raffinose are used as receptors. In case it produces only one receptor product. Therefore, the present inventors have devised in view of the problems of the above enzyme purification and a single productivity of the receptor product, the object of the present invention is B-512FMCM dextran sucrase, B-742CB dextran, which is a mutant strain of an oligosaccharide producing strain It is used to produce various receptor products (oligosaccharides) of novel structure by using sucrase and B-1355C alternasu sucrose and maltose, genthiobio, raffinose and lactose as receptors.

The above object of the present invention is a B-512FMCM dextran sucrase, B-742CB dextran sucrase or B-1355C alternan sucrase, maltose, genthio obtained from a structural mutant strain of L. mesenteroides producing oligosaccharides. Using bios, raffinose and lactose as receptors, this was achieved by comparing and analyzing oligosaccharide productivity according to the ratio of sugar and receptor and the amount of enzyme.

Hereinafter, the configuration and operation of the present invention will be described in detail.

1 is a photograph of B-512FMCM dextran sucrase receptor reaction results with maltose by TLC.

FIG. 2 is a photograph of B-512FMCM dextran sucrase receptor reaction with genthiobio.

Figure 3 is a TLC analysis of the results of the receptor reaction of lactose of B-512FMCM dextran sucrase.

Figure 4 is a photograph of the reaction of the receptor reaction with raffinose of B-1355C Alterna sucrase by TLC.

5 is a TLC analysis photograph of the structural analysis of maltose, genthiobiose, lactose and the receptor product.

The present invention comprises the steps of selecting the mutant strains of Leuconostoc mesenteroides B-512FMCM, B-742CB, B-1355C bacteria and culturing them to produce dextran sucrase or alternan sucrase; A reaction step of an enzyme and a receptor such as maltose, genthiobiose, raffinose and lactose isolated from the respective strains; And a step of methylation of the oligosaccharide which is a receptor product obtained through the reaction. In addition, the carbohydrate used in the following example used what was purchased from Sigma.

Hereinafter, the configuration and operation of the present invention will be described in detail by way of examples, but the scope of the present invention is not limited to these examples.

Example 1 Selection of Mutant Strains and Preparation of Enzyme Solution

According to Kim et al. (Kim, D., et al. 1997., Biotechnol. Techniq. 11: 319-321), Leuconostoc mesenteroides B-512FMC and its mutant strains contain 2% glucose or fructose as a carbon source. LM medium (4 g yeast extract per liter of distilled water, 2 g peptone, 0.2 g MgSO ₄ · 7H ₂ O, 0.015 g CaCl ₂ · 2H ₂ O, 0.01 g FeSO ₄ · 7H ₂ O, 0.01 g NaCl, 2 g K ₂ HPO ₄ Incubated in 0.01 g MnSO ₄ H ₂ O) and using an electron emission chamber (Photoemission Spectroscopy Beamline of the Pohang Light Source, Pohang, Korea), energy ranging from 70 eV to 1 keV (electrons placed between UV and X-rays). Magnetic (VUV) light) was used to induce mutations to obtain mutant strain B-512FMCM. In addition, Leucomostoc mesenteroides B-742 and B-1355 were distributed by the USDA Northern Regional Laboratory (NRRL; Peoria, IL), and were used as a carbon source. g MgSO ₄ · 7H ₂ O, 0.015g CaCl ₂ · 2H ₂ O, 0.01g FeSO ₄ · 7H ₂ O, 0.01g NaCl, 2g K ₂ HPO ₄ , 0.01g MnSO ₄ · H ₂ O) at 28 ° C. Each culture was incubated at a density of about 3 x 10 ⁷ cell / mL. Each cell was suspended and washed twice with 1.5 mL of sterile 100 mM Sodium phosphate buffer (pH 7.0), and 40 μl of ethyl methane sulfate (hereinafter referred to as EMS) was added to the cell suspension (15 mL) to induce mutation. . After incubating the cell suspension added with EMS for 1 hour at 28 ° C., the cell suspension (1 mL) was transferred to 2 mL of sterilized 10 parts by weight sodium thiosulfate to remove residual EMS. The cells thus treated were centrifuged (10.4 (x) g), washed twice with sodium phosphate buffer, and then plated onto LM agar medium containing 2% glucose. Colonies were selected during structural analysis of glucans sucrase obtained by inoculation in LM medium containing 2% glucose or fructose and incubated at 28 ° C. for 24 hours. The medium supernatant was obtained by glucan sucrose activity analysis by radioactive assay using ¹⁴ C-sucrose, to obtain structural mutants Leucomostoc mesenteroides B-742CB, B-1299CB or B-1355C. In addition, Leuconostoc mesenteroides B-512FMCM, B-742CB, B-1355C by Kim and Robyt's method (Kim, D. and JF Robyt. 1994., Enzyme Microbiol Technical. 16: 659-664) using the selected strain. The bacteria were cultured to produce and purified dextran sucrose or alternan sucrose. The B-512FMCM, B-742CB, and B-1355C mutants are all stored in the Department of Biological Science and Engineering, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju, and especially the B-512FMCM mutant is a glycerol stock. It is stored in the form.

Example 2 Preparation of Immobilized Yeast

40 mL of 25 parts by weight yeast suspension was mixed with 100 mL of 35 parts by weight sodium acetate solution. At this time, the solution was uniformed by rapid stirring and left at 4 ° C. for 1 hour to remove bubbles. The prepared yeast-alginate solution was added dropwise to the stirring 2 parts by weight calcium chloride solution, followed by further stirring for 2 hours to strengthen the strength of the beads and refrigerated.

Example 3. Receptor Response Test

Experimental Example 1. Receptor reaction test of B-512FMCM dextran sucrose

Receptor reaction solution was prepared by mixing maltose, genthiobio, raffinose and lactose as sugar and the receptor prepared by dissolving in 20mM sodium acetate buffer (pH 5.2). After fixing the total carbohydrate concentration to 100 mM, the receptor and sugar ratio were changed to 1/10, 1/5, 1/1, 4/1 and 10/1, and B-512FMCM dextran sucrase obtained in Example 1 was used. After the receptor reaction, the effect of the concentration ratio of the sugar and the receptor on the type and yield of the receptor product was investigated. When maltose was used as a receptor, as shown in Fig. 1 and Table 1, the number of receptor products decreased as maltose / sugar (M / S) ratio increased. As shown in FIG. 2 and Table 2, the results of the receptor reaction of B-512FMCM dextran sucrase with genthiobios showed that the genthiobios exhibited the same behavior as maltose. Products: 6 ² -α-D-glucopyranosylgentiobiose (P _G 3), 6 ² -α-D-isomaltosylgentiobiose (P _G 4), 6 ² -α-D-isomaltosylgentiobiose (P _G 5), 6 ² -α -D-isomaltosylgentiobiose (P _G 6), 6 ² -α-D-isomaltosylgentiobiose (P _G 7), etc. were generated, and at a ratio above 10/1, only two receptor products, 6 ² -α-D-glucopyranosylgentiobiose (P _G 6), were produced. 3) and 6 ² -α-D-glucopyranosylgentiobiose (P _G 4) were obtained only, and the result of the receptor reaction of lactose was shown to be a single receptor product 2'-α-D-glucopyranosyllactose (P ₁ 3). ) Was created. In addition, the effect of the amount of enzyme on the receptor reaction was fixed to a fixed total carbohydrate concentration (100mM) and the sugar ratio to 1/1 and the amount of enzyme prepared in Example 2 was 0.45, 4.5, 45, 90 and 450 ( U / digest). In this case, one unit of enzyme is expressed as the number of micromoles of fructose liberated from sugar per mL of enzyme per minute. After reacting at 28 ° C. for 20 hours, 1 μl of the reaction solution was added to the Merck K6F TLC plate, and then developed in a developing solution in which nitromethane / 1-propanol / water was mixed at a volume ratio of 2/5 / 2.5, respectively. It was. The components of the separated carbohydrates were identified by using a coloring reagent containing 0.5 parts by weight of α-naphthol and 5 parts by weight of sulfuric acid. As can be seen from Table 4, the amount of dextran production decreased as the amount of enzyme product increased as the amount of enzyme increased. As can be seen in Table 5, the number and amount of receptor products increased with the increase of the amount of enzyme as in the case of maltose, and the amount of dextran production decreased, but the structure of the produced receptor product was It was an isomaltoligosaccharide with bios and lactose, as shown in Table 6, when the amount of enzyme increased to 45 U / digest, there was no significant change in the amount of the product of the receptor, but when the amount of enzyme increased above 45 U / digest, The amount of tran was sharply reduced to 34.5%.

Relationship between dextran and receptor product (maltose / sucrose) formed by B-512FMCM dextran sucrase M / S ^b Dextran P3 P4 P5 P6 P7 P8 10/1 10.6 66.8 22.6 4/1 8.6 56.3 25.0 10.1 1/1 11.6 34.7 25.1 16.1 8.4 4.1 1/5 19.6 22.8 19.6 17.5 12.9 7.6 1/10 28.3 16.5 15.0 14.2 12.1 8.1 5.8

Note; M / S ^b is the concentration ratio of maltose to sucrose.

Relationship between Dextran and Receptor Product (Genthiobios / Sucrose) Formed by B-512FMCM Dextran Sucrase G / S ^b Dextran P _G 3 P _G 4 P _G 5 P _G 6 P _G 7 10/1 15.7 69.0 15.3 4/1 20.4 58.8 16.3 4.5 1/1 25.4 44.2 18.5 8.7 3.2 1/5 34.7 33.2 16.1 9.4 4.4 2.2 1/10 44.1 28.3 13.6 8.1 4.4 1.5

Note; G / S ^b is the ratio of genthiobios to sucrose.

Relationship between dextran and receptor product (lactose / sucrose) formed by B-512FMCM dextran sucrase L / S ^b Dextran P _L 3 10/1 42.1 57.9 4/1 47.1 52.9 1/1 57.1 42.9 1/5 73.4 26.6 1/10 77.8 22.22

Note; L / S ^b is the ratio of lactose to sucrose.

Relationship between dextran and receptor product formed by B-512FMCM dextran sucrase when maltose and sucrose are equal Enzyme (U / digest) Dextran P3 P3a ^b P4 P5 P6 P7 0.45 12.9 38.6 30.2 13.9 4.4 4.5 11.7 38.1 26.3 16 7.9 45 8.1 35.7 24.3 16.5 9.7 5.7 90 7.8 35.3 25.4 15.8 10.3 5.4 450 5.0 41.4 8.6 25.2 13.4 6.4

Relationship ratio between dextran and the receptor product formed by B-512FMCM dextran sucrase when the ratio of genthiobios and sucrose is equal Enzyme (U / digest) Dextran P _G 3 P _G 4 P _G 5 P _G 6 0.45 32.6 50.1 12.9 4.4 4.5 28.0 48.5 15.5 6.2 1.8 45 19.3 46.9 19.6 10.6 3.6 90 13.6 43.7 23.3 13.1 6.3 450 7.9 53.2 22.4 11.4 5.1

Relationship between dextran and receptor product formed by B-512FMCM dextran sucrase when lactose and sucrose are equal Enzyme (U / digest) Dextran P _L 3 P _L 3a P _L 4 0.45 59.6 40.4 4.5 56.5 43.5 45 57.3 42.7 90 52.9 32.0 5.9 450 34.5 27.6 5.9

Experimental Example 2. Receptor reaction test of B-742CB dextransukrase

The receptor reaction solution was prepared by mixing maltose, genthiobio, raffinose and lactose with sugar prepared by dissolving in 20 mM sodium acetate buffer (pH 5.2). After fixing the total carbohydrate concentration at 100 mM, the receptor and sugar ratio were changed to 1/10, 1/5, 1/1, 4/1, and 10/1, and B-742CB dextran sucrase obtained in Example 1 was used. After the receptor reaction, the effect of concentration ratio of sugar and receptor on the type and yield of receptor product was investigated. As a result, B-512FMCM dextran sucrase was not significantly different from the case, but as shown in Table 7, B-742CB dextran sucrase synthesized various receptor products using lactose. When the ratio of receptor to sugar was 5/1, only one receptor product was synthesized and the amount of dextran was 29.4%. As the amount of sugar increased, the amount of dextran increased, showing the most synthesis of dextran at the ratio of 1/1. After that, when the amount of sugar increased further, the amount of dextran produced decreased and disaccharide, trisaccharide, etc. The production of small sized oligosaccharides was increased and the types were increased. In addition, as shown in Table 8, only one receptor product was produced when the ratio of receptor to sugar was 5/1, and the amount of dextran decreased when the amount of sugar increased by more than 1/5. The production of oligosaccharides of small size, etc., increased. That is, oligosaccharides containing raffinose were produced using B-742CB dextran sucrose.

Relationship between dextran and receptor product (lactose / sucrose) formed by B-742CB dextran sucrase L / S ^b Dextran P _L c P _L 3 P _L 4 P _L 5 P _L 6 P _L 7 P _L 8 5/1 29.4 70.6 1/1 50.6 49.4 1/5 44.7 15.7 24.3 8.0 5.0 2.3 1/10 36.3 15.9 15.4 15.4 7.3 8.3 6.1 10.7

Note; L / S ^b is the ratio of lactose to sucrose.

Relationship between Dextran and Receptor Product (Raffinose / Sucrose) Formed by B-742CB Dextran Sucrase R / S ^b Dextran PR4 PR5 PR6 PR7 PR8 PR9 PR10 5/1 49.1 50.9 1/1 60.7 39.3 1/5 44.8 39.8 8.9 6.5 1/10 34.2 24.1 6.6 5.7 7.8 3.4 10.8 7.3

Note; R / S ^b is the ratio of raffinose to sucrose.

Experimental Example 3 Receptor Reaction of B-1355C Alternasucrase

In the same manner as in Experimental Example 2, the receptor reaction using the B-1355C alternace sukrase obtained in Example 1 was examined. As a result, as shown in Figure 4 and Table 9, when the concentration of sugar in the raffinose receptor reaction significantly increased than the receptor oligosaccharide containing raffinose was synthesized. In addition to α1 Alter random sucrase generates 3 ² -α-D-glucopyranosyl raffinose with ² 2 -α-D-glucopyranosyl raffinose as the first acceptor product, and the ratio of the reducing end glucose-oligosaccharides bound glucose to 6 structure Produced.

B-1355C Relationship between dextran and receptor product (raffinose / sucrose) formed by alternan sucrase R / S ^b Dextran P _R 4a P _R 4b P _R 5a P _R 5b P _R 6a P _R 6b P _R 7a 5/1 58.9 27.8 13.3 1/1 59.3 23.7 17.0 1/5 46.2 23.7 22.5 7.6 1/10 39.2 21.4 17.3 3.8 4.4 5.3 4.6 4.0

Note; R / S ^b is the ratio of raffinose to sucrose.

Example 4. Isolation of Oligosaccharides

D-fructose, which is an unnecessary component in the reaction solutions in Experimental Examples 1 to 3 of Example 3, was removed by reacting the immobilized yeast prepared in Example 2 with the reaction solution at room temperature, and dextran synthesized during the reaction was The precipitate was precipitated using twice the volume of ethanol and then centrifuged (6000xg, 30 minutes) to remove. The supernatant was concentrated by rotary evaporator and freeze-dried. 2 mL of a dry sample of 5% concentration was injected into the activated carbon column to separate pure oligosaccharides by size through the concentration gradient of ethanol (2 to 16 parts by weight).

Example 5. Structural Analysis of Oligosaccharides

The oligosaccharides isolated in Example 4 were structurally analyzed using methylation analysis. That is, 10 mg of the dried oligosaccharide was dissolved by stirring with 0.8 mL Me ₂ SO. The solution was transferred to a tube, added with Hakomori reagent, blocked with septum, dissolved overnight, and then reacted for 2 hours by adding 0.2 mL of iodomethane. 4 mL of water was added thereto, and the mixed solution was extracted three times with 2 mL CHCl ₃ , followed by adding 2-3 pieces of Na ₂ S ₂ O ₃ crystals to remove the dull color, and then extracted twice with 10 mL of water. After drying the CHCl ₃ layer with air, the solution to which 1 mL of 4M CF ₃ CO ₂ H was added was transferred to a cap tube, heated at 121 ° C. for 30 minutes, dried at 60 ° C., and analyzed by TLC. And the receptor product including the maltose was methylated and acid hydrolyzed and analyzed by TLC. As shown in FIG. 5, the product of maltose methylation resulted in 6 ² -α-D-glucopyranosyl maltose in which D-glucose of sugar was bonded to α (1-6) at the non-reducing end of the receptor. The product of methylation of genthiobio was 6 ² -α-D-glucopyranosylgentiobiose, in which D-glucose of sugar was α (1-6) bound to the non-reducing end of the receptor. In the case of lactose, it was 2 ¹ -α-D-glucopyranosyl lactose in which D-glucose of sugar binds α (1-2) to glucose, the reducing end of the receptor.

As can be seen from the above examples, B-512FMCM dextran sucrase, B-742CB dextran sucrase, or B-1355C alternace sukrase obtained from structural mutant strains of oligosaccharide producing strains By carrying out the receptor reaction of maltose, genthiobio, raffinose and lactose using the effect of eliminating the separate enzyme purification process, the oligosaccharide production method of the present invention is also a novel structure that can be synthesized only enzymatically Because of the effect of synthesizing various oligosaccharides is a very useful invention in the food industry.

Claims (1)

Maltose using B-742CB dextransucrase, B-1355C alternansucrase or B-512FMCM dextransucrase obtained from the structural mutants B-742CB, B-1355C or B-512FMCM of the oligosaccharide producing strain. A method for producing an oligosaccharide, characterized in that it performs a receptor reaction with genthiobioses, raffinose and lactose.