KR100914861B1 - Cyclic oligosaccharide for catalyzing the lysis of benzoate - Google Patents

Abstract

The present invention relates to a carbohydrate catalyst for catalyzing a reaction for decomposing benzoate and a method for decomposing benzoate using the catalyst.

Description

Cyclic oligosaccharide for catalyzing the lysis of benzoate

The present invention relates to a carbohydrate catalyst for catalyzing a reaction for decomposing benzoate and a method for decomposing benzoate or salicylic acid using the catalyst. More specifically, the present invention relates to alpha from a microorganism of genus Xanthomonas catalyzing the reaction for decomposing benzoate. It relates to a cyclophosphohexadechaose catalyst.

In general, the alpha-cyclosophorohexadecaose (α-C16), an cyclic osmotic cytoplasmic glucan (OPG) produced by the genus Xanthomonas , has 15 beta- (1 → 2) -linkages and 1 alpha- ( Branched cyclooligosaccharides consisting of 1 → 6) -linkages function as osmotic regulators.

Single alpha- (1 → 6) -linkages enable ring closure and induce structural limitations of this cyclic OPG. Such constraints can greatly simplify the structure analysis of the cyclic OPG and impart a unique physical properties to them (Talaga, P., and so on (1996). Journal of Bacteriology , 178 , 2263-2271).

Methanol decomposition is a type of transesterification using methanol and esters as substrates. Transesterification is an important organic reaction in which an ester is converted to another ester through the interaction of an alkoxy moiety. The transesterification is an equilibrium reaction and the conversion takes place by mixing the reactants. However, the presence of the catalyst accelerates the adaptation of the equilibrium state by lowering the activation energy and stabilizing the transition state of the reaction. The so far used in the transesterification catalysts are acids (Freedman, B., etc. (1986). Journal of the american oil Chemists's Society, 63, 1375-1380), alkali metal hydroxide, alkoxide (Gryglewicz, S. (2000). Applied Catalysis A:. General 192, 23-28 ), carbonates (. Mouloungui, Z., such as Tetrahedron, 48, 1219-1232), non-ionic base (Flynn, KG, etc. (1963) Journal of Organic Chemistry, 28, 3527-3530) and mainly serine protease enzyme (Glazer, AN (1966). Journal of Biological Chemistry , 241 , 635-638; Gryglewicz, S., et al. (2000). Biotechnology Letters , 22 , 1379-1382; Zaks, A. , et al . (1985). Proceedings of the National Academy of Sciences USA , 82 , 3192-3196) and acyl intermediates can be formed by the catalyst during the catalytic reaction. In this regard, artificial enzymes have been reported to increase the rate ( k cat / k uncat) between 10 ^{1 and} 10 ⁵ (Rousseau, C., et al. (2004) Tetrahedron Letters , 45 , 8709-8711), but this rate acceleration is weak compared to natural enzyme equivalents.

In the present invention, rice pathogenic microorganisms catalyzing the decomposition of benzoate methanol, X. oryzae pv. It relates to alpha-C16, a catalytic carbohydrate isolated from oryzae .

The present invention solves the above problems, and the object of the present invention is to provide a catalyst for promoting a benzoate or salicylic acid decomposition reaction.

Another object of the present invention is to provide a method for promoting a benzoate or salicylic acid decomposition reaction.

The present invention to achieve the above object

It provides a catalyst for benzoate decomposition described in the following formula.

In a preferred embodiment of the invention, the compound of Formula may be obtained from the organisms existing in nature or synthesized, not limited One preferred isolated from microorganisms of the Xanthomonas genus that this, in the Xanthomonas oryzae pv microorganism X. . oryzae KACC 10331 is most preferred.

In one embodiment of the present invention, the benzoate is preferably 2-hydroxy benzoate.

In addition, in one embodiment of the present invention, the benzoate or 2-hydroxy benzoate (salicylic acid) is a benzoate or 2-hydroxybenzoate having a vinyl group, a phenyl group, a halogenated phenyl group, or methoxyphenyl (that is, vinyl Benzoate or 2-hydroxybenzoate; phenyl benzoate or 2-hydroxybenzoate; halogenated phenyl benzoate or 2-hydroxybenzoate; methoxyphenyl benzoate or 2-hydroxybenzoate) The methoxyphenyl benzoate or 2-hydroxybenzoate is preferably 2-, 3-, or 4-methoxyphenyl benzoate or 2-hydroxybenzoate , and the halogenated phenyl benzoate or 2-hydroxy. Phenyl benzoate Preferred is 2-chlorophenyl benzoate, 3-chlorophenyl benzoate or 4-chlorophenyl benzoate or 2-hydroxybenzoate, and 4-chlorophenyl benzoate or 4-bromophenyl benzoate or 2-hydroxy Benzoate is most preferred but not limited thereto.

The present invention also provides a process for the decomposition or 2-hydroxy benzoate (salicylic acid) comprising the addition of a catalyst of the formula in the presence of a solvent.

In one embodiment of the present invention, the solvent is preferably alcohol, more preferably methanol, ethanol or propanol, methanol is most preferred, but not limited thereto.

In the method of the present invention, the compound of formula (I) may be obtained from the organisms existing in nature or synthesized, not limited one preferably isolated from the microorganisms of this genus Xanthomonas, the genus Xanthomonas oryzae pv microorganism X. . oryzae KACC 10331 is most preferred.

The present invention will be briefly described below.

By "benzoate" is meant herein a benzoate compound comprising a vinyl, phenyl, halogenated phenyl, or methoxy phenyl group.

By "2-hydroxybenzoate (salicylic acid)" is meant herein a benzoate compound comprising a vinyl, phenyl, halogenated phenyl, or methoxy phenyl group.

"Decomposition" in the present invention means that the benzoate or 2-hydroxy benzoates are decomposed in the presence of alcohol (preferably methanol, ethanol, propanol, more preferably methanol).

The benzoate methanolysis of the present invention is alpha-cyclosophorohexadecaose, a neutral cyclic hexadecaglucoside containing 15 beta- (1 → 2) -linkages and 1 alpha- (1 → 6) -linkages. (Hereinafter referred to as "α-C16"). This α-C16 is Xanthomonas Cytolytic cyclooligosaccharides isolated from oryzae , for methanolysis of several benzoates (e.g., vinyl benzoate, phenyl benzoate, and 4-chlorophenyl benzoate), respectively, from about 14 to about 69 times as compared to the control. To promote.

Possible intermediates formed during the reaction were detected by MALDI-TOF spectra.

Through the present invention, the inventors found that alpha-C16 has the effect of acting as a carbohydrate catalyst for catalyzing the methanolysis of various ester compounds.

1 shows X. oryzae pv . oryzae is a structural identification of alpha-C16 isolated from KACC 10331. (a) ¹ H NMR spectrum of Alpha-C16. The peak at 5.18 ppm is indicative of the anomeric protons (H-1) of alpha-glucose residues, and the peaks from 4.67 to 5.06 ppm represent H-1 protons of glucose residues involved in beta- (1 → 2) -linkage. It is an indicator. Other proton signals of glucose are found between 3.3 and 4.0 ppm. (b) positive-ion MALDI-TOF MS of alpha-C16. Neutral alpha-C16 has m / z 2616 and 2632 calculated masses representing [M + Na + H] ⁺ and [M + K + H] ⁺ , respectively. (Esterisk (*) means cyclic glucan ( m / z 2778) of DP 17 isolated from X. oryzae pv . Oryzae KACC 10331) (c) X. oryzae pv . Chemical structure of alpha-C16 isolated by oryzae KACC 10331.

2 is a diagram of methanolysis of phenyl benzoate (1), 4-chlorophenyl benzoate (2) and vinyl benzoate (3) by alpha-C16.

FIG. 3 shows partial ¹ H NMR spectra of vinyl benzoate (3) when (a) alpha-C16 was treated with catalyst for 0, 0.25, 0.5, 1, 2, 4, and 8 hours. FIG. The reaction was carried out in methanol at 40 ° C. in the presence or absence of 0.2 equivalents of alpha-C16. (b) Time course plot of methanolysis of vinyl benzoate (3) in the presence or absence of 0.2 equivalents of alpha-C16. Substrate concentration used was fixed at 20 mM and the data was fitted to one phase exponential association.

4 is a mass spectrum of (a) original alpha-C16, (b) alpha-C16 used for methanolysis of phenyl benzoate (1), and (c) alpha-C16 used for methanolysis of phenyl salicylate. MALDI-TOF MS in positive ion mode with 2,5-DHB as matrix. (b) m / z 2721 [acylated alpha-C16 + Na + 2H] ⁺ and m / z 2737 [acylated alpha-C16 + K + 2H] ⁺ Mass spectrum. (Esterisk (*) means cyclic glucan of DP 17 corresponding to sodium and potassium adducts isolated from X. oryzae pv . Oryzae KACC 10331, respectively ( m / z 2779 and 2795)) (c) m / z 2737 [Acylated alpha-C16 + Na + 2H] ⁺ and 2753 [acylated alpha-C16 + K + 2H] ⁺ .

5 shows TLC analysis of the reaction mixture of various carbohydrates including alpha-C16 and substrates (1, 2 and 3) after 10 hours at 40 ° C. Reaction conditions include 0.2 equivalents of 20 mM substrate (1, 2 and 3) in 0.3 mL methanol with 0.2 equivalents of alpha-C16 (lane E), no carbohydrates (lane D), the same mass of glucose (lane C), Treatment with alpha-CD (lane B) and amylose (lane A). Lane E represents the reaction product in addition to the reactants. Arrows indicate the product. (TLC solvent condition, hexanes: methylene chloride = 1: 1)

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Phenyl benzoate ( 1 ), 4-chlorophenyl benzoate ( 2 ), vinyl benzoate ( 3 ) and glucose used in the present invention are Aldrich Chemical Co. (Milwaukee, WI, USA). Alpha-CD and amylose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol and N, N-dimethylformylamide are described in Sigma-Aldrich. (St. Louis, MO, USA) and the solvents were obtained in anhydrous form.

Example  1: Bacteria Culture and Neutral Alpha- C16 Manufacture

X. oryzae pv. oryzae KACC 10331 was obtained from Korean Agricultural Culture Collection (KACC) and grown in TGY medium at 28 ° C. with stirring at 150 rpm (Sunish Kumar R, et al. (2001). Applied Microbiology and Biotechnology , 55 , 782-786). Incubate the microorganisms for 2 days Collected by centrifugation at 4 ℃ 10 minutes at 8000 rpm. The cell pellet was extracted with 5% trichloroacetic acid and centrifuged, then the supernatant was neutralized with NH ₄ OH and chromatographed at a rate of 1 ml per minute on a Sephadex G-25 (2.5 x 34 cm) column. Fractions containing the putative cyclic glucan were collected and concentrated and applied to a DEAE-Sephadex column (2 x 35 cm) to separate alpha C16 in anionic form. Neutral forms were collected and desalted on a Bio-Gel P-4 column (Bio-Rad). The column (2.4 x 54 cm) was run at room temperature with distilled water at a flow rate of 18 mL / h and the desalted material was finally lyophilized.

Example  2: Thin - layer chromatography  ( TLC )

TLC analysis was performed to monitor benzoate methanolysis. Analyte was spotted on silica gel G-60 (E. Merck, 400-240 mesh) plates and developed under solvent system (1: 1 hexane-methylene chloride). The reactants and products were detected by ultraviolet radiation (254 nm).

Example  3: MALDI - TOF  Mass spectrometer analysis

MALDI-TOF mass spectrometer (Voyager-DE ^TM ) in cationic mode using 2,5-dihydroxybenzoic acid (DHB) as a matrix Mass spectra of oligosaccharides were obtained by STR BioSpectrometry, PerSeptive Biosystems, Framingham, Mass., USA.

Example  4: Acylated  Alpha- C16 Pity

To measure acyl intermediates, Phenyl benzoate (100 mM) was dissolved in 0.5 mL of MeOH and alpha-C16 (5.2 mg) was added. The reaction mixture was magnetically stirred at 40 ° C., after 3 hours the liquid was removed from the precipitate by centrifugation and the residual liquid was evaporated to dryness with N ₂ gas completely. Residual pellets containing the presumed acyl alpha-C16 were dissolved in ultrapure water. The intermediate dissolved in the aqueous layer was detected by MALDI-TOF MS analysis in positive mode using 2,5-dihydroxybenzoic acid (DHB) as a matrix.

For another acyl intermediate, phenyl salicylate (100 mM) was dissolved in 0.5 mL N, N-dimethylformamide and then alpha-C16 (5.2 mg) was added. The reaction mixture was stirred at 40 ° C. After 2 hours, the solution was detected by MALDI-TOF MS analysis in positive mode using 2,5-dihydroxybenzoic acid (DHB) as the matrix.

Example  5: NMR  Spectroscopic analysis

For NMR spectroscopy, we used a Bruker Avance 500 spectrometer. vinyl benzoate conversion By 500-MHz ¹ H NMR spectroscopy 7.99 ppm ortho proton of [reaction (vinyl benzoate) in the ortho protons (a)] decreases and 7.92 ppm [product (methyl benzoate) the integral area (integral area) of the resonance ( a ')] was determined by measuring the increase in the integral area of resonance. The conversion of phenyl benzoate ( 1 ) and 4-chlorophenyl benzoate ) was determined in the same way. The reaction fractions were then quantified. For these three reactions, aliquots were taken at appropriate time intervals and immediately frozen immediately after centrifugation to remove the catalyst to stop the reaction. All NMR spectroscopic analyzes were performed in CDCl ₃ . The time course of the three reactions was fitted to a one phase exponential association to obtain k _cat or k _uncat .

The result of the above embodiment is as follows.

Neutral alpha C16 Ok

The structure of alpha -C16 (FIG. 1) is reported before (Talaga, P., and so on (1996). Journal of Bacteriology , 178 , 2263-2271) and identified by NMR spectroscopy and MALDI-TOF MS analysis.

Alpha- C16 By Catalyzed phenyl benzoate  (1), 4- chlorophenyl benzoate  (2) vinyl benzoate  (3) methanolysis of Methanolysis )

The reaction was carried out in methanol (3 mL) at 40 ° C. in the presence or absence of 0.2 equivalents of alpha-C16. Each substrate concentration was fixed at 20 mM. Alpha-C16 catalyzes methanolysis of three benzoates with different reaction rates (Table 1) (FIG. 2). The substrates of different R groups (Fig. 2.) were k _cat And k _uncat had a unique effect. In the presence of alpha-C16, vinyl benzoate ( 3 ) methanolysis was completed at least 95% (≧ 95%) after 4 hours but conversion to product was less than 5% in the absence of alpha-C16 (FIG. 3 (b)). Therefore, the reaction rate was increased 69 times ( k _cat / k _uncat ). 3 (a) shows partial ¹ H nuclear magnetic resonance (NMR) spectra that monitor the progress of the catalytic reaction over several time periods.

After 24 hours, methanolysis of 4-chlorophenyl benzoate ( 2 ) by alpha- _C16 is 14 times more complete ( k _cat / k _uncat ) than the reaction without alpha C-16 (Table 1). ). After 34 hours, phenyl benzoate ( 1 ) methanolysis was more than 95% complete and 24 times faster ( k _cat / k _uncat ) compared to the reaction without alpha C-16 (Table 1). Comparing phenyl ( 1 R) and 4-chlorophenyl groups ( 2 R) with vinyl groups (3 R), the catalytic methanolysis of benzoates comprising vinyl groups is faster than the other groups. The inventors speculate that larger R groups inhibit alpha-C16 from accessing the substrate to form intermediates, so that smaller groups can give favorable space for the catalysis of alpha C-16. In a 4-chlorophenyl group (R 2) other phenyl group (R 1) When comparing, the k _cat / k _uncat is the first to the k _uncat value of 2 larger than 1 will of k _cat / k _uncat 1 a Higher than two's. This would be due to the electrical and resonance effects of the 2 R para-chloro substituent.

In conclusion, alpha-C16 promotes methanolysis of three different benzoates as compared to controls (about 24 times for 1, about 14 times for 2, and about 69 times for 3).

We therefore showed that alpha-C16 acts as a carbohydrate that catalyzes the methanolysis of benzoate.

Possible Intermediates

To investigate how alpha-C16 is involved in the reaction, MALDI-TOF MS analysis was performed. MS analysis is often performed in order to demonstrate a proof of the existence of a covalent acyl polymer (Ashton, DS, etc. (1991). Federation of European Biochemical Societies , 292, 201-204. Possible intermediate peaks were detected in the present invention on the MALDI-TOF spectrum. 4 (a) shows the original alpha-C16 mass data, with no peak observed between the cyclic glucans of alpha-C16 and DP 17.

Figure 4 (b) shows the mass spectrum of alpha C16 used for phenyl benzoate ( 1 ) methanolysis, and the values measured at m / z 2721 and 2737 are [acylated a-C16 + Na + 2H] ⁺ and It is represented by [acylated a-C16 + K + 2H] ⁺ . To confirm the presence of the intermediate again, phenyl salicylate methanolysis by alpha C16 was performed by monitoring the reaction by TLC analysis (data not shown), and alpha-C16 also catalyzed the reaction. Phenyl salicylate has an OH group at the ortho position of benzoate and thus the mass difference between the intermediate of 1,2, or 3 methanolysis and the acyl intermediate of phenyl salicylate is 16. 4 (c) shows m / z 2737 [acylated alpha-C16 + Na + 2H] ⁺ and 2753 [acylated alpha-C16 + K + 2H] ⁺ show this mass difference.

Based on Figures 4 (b) and (c), the MALDI-TOF spectrum shows a possible intermediate that clearly shows the presence of covalent compounds between alpha C16 and its substrate.

Action of other carbohydrates

Through TLC analysis, we investigated methanolysis to identify the action of different carbohydrates (each using amylose, alpha CD, glucose, and alpha-C16). Figure 5 shows TLC analysis of methanolysis for three different benzoates after 10 hours at 40 ° C. The top spots represent substrates 1, 2 and 3 from the left, respectively. The lower spots in substrates E and 2 represent phenol and 4-chlorophenol, respectively, and the intermediate spots in lanes E in substrates 1, 2 and 3 represent methyl benzoate. Alpha-C16 is a product of methyl benzoate in common with all three reaction mixtures. Many control carbohydrates such as glucose, amylose and alpha-CD did not show any catalytic effect on these reactions.

The above results may be caused by structural differences in the carbohydrates used in the reaction. Glucose and amylose lack adequate space for the substrate to bind. We used alpha-CD as another control cyclic carbohydrate, but it was also ineffective in the reaction. In the complexation of alpha-CD with fixed space and substrate, no suitable localization for interaction between nucleophilic —OH and carbonyl carbon is formed. As a microbial cyclic glucan, alpha-C16 has a backbone of cyclic beta- (1 → 2) glucan and a single alpha- (1 → 6) linkage, which gives more flexibility than alpha-CD. The unique scaffolding induced by the alpha-C16 beta- (1 → 2) glucan and the single alpha- (1 → 6) linkage provides a suitable space for substrate binding, resulting in a catalyst that occurs through the alpha-C16-acyl intermediates. Reactions are possible.

As can be seen from the above configuration, it can be seen that alpha-C16 of the present invention promotes methanolysis of three different benzoates as compared to the control group (about 24 times for 1 and 2 times). Is about 14 times, 3 is about 69 times). It is understood that alpha-C16 can be used as a catalyst for the methanolysis of benzoate and thus can be applied to a variety of applications.

Claims (15)

Benzoate decomposition catalyst as described in the following formula. The benzoate decomposition catalyst according to claim 1, wherein the compound of formula is isolated from microorganisms of the genus Xanthomonas . According to claim 2, wherein the genus Xanthomonas microorganism is X. oryzae pv. oryzae KACC 10331, characterized in that the catalyst for benzoate decomposition. The method of claim 1, wherein the benzoate is selected from vinyl benzoate; Phenyl benzoate; Halogenated phenyl benzoate; And benzoate selected from the group consisting of methoxyphenyl benzoate. The method of claim 4, wherein the halogenated phenyl benzoate is 2-chlorophenyl benzoate, 2-bromophenyl benzoate, 3-chlorophenyl benzoate, 3-bromophenyl benzoate, 4-chlorophenyl benzoate, And benzoate selected from the group consisting of 4-bromophenyl benzoate. delete The benzoate decomposition catalyst according to claim 4, wherein the methoxyphenyl benzoate is 2-methoxyphenyl benzoate, 3-methoxyphenyl benzoate, or 4-methoxyphenyl benzoate. The benzoate decomposition catalyst according to claim 1, wherein the benzoate is 2-hydroxybenzoate. The compound of claim 8, wherein the 2-hydroxybenzoate is selected from vinyl 2-hydroxybenzoate; Phenyl 2-hydroxybenzoate; Halogenated phenyl 2-hydroxybenzoate; And 2-hydroxybenzoate selected from the group consisting of methoxyphenyl 2-hydroxybenzoate. 10. The method of claim 9, wherein the halogenated phenyl 2-hydroxybenzoate is 2-chlorophenyl 2-hydroxybenzoate, 2-bromophenyl 2-hydroxybenzoate, 3-chlorophenyl 2-hydroxybenzoate Benzoate, wherein the benzoate is selected from the group consisting of 3-bromophenyl 2-hydroxybenzoate, 4-chlorophenyl 2-hydroxybenzoate, and 4-bromophenyl 2-hydroxybenzoate. Decomposition catalyst. 10. The method of claim 9, wherein the methoxyphenyl 2-hydroxybenzoate is 2-methoxyphenyl 2-hydroxybenzoate, 3-methoxyphenyl 2-hydroxybenzoate, or 4-methoxyphenyl 2-hydroxy. It is oxybenzoate, The catalyst for benzoate decomposition characterized by the above-mentioned. A benzoate decomposition process comprising the step of adding a catalyst of the formula in the presence of a solvent. 13. The process of claim 12 wherein the solvent is an alcohol. The method of claim 13 wherein the alcohol is methanol, ethanol or propanol. 13. The method of claim 12, wherein said benzoate is 2-hydroxybenzoate.