JP2006246827A - Separation and purification of enzymatic product and hydrolyzed composition obtained by using the enzyme obtained by separation and purification - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating enzymatic product based on the α-glucuronidase activity, the enzyme obtained thereby and the hydrolytic composition of polysaccharide originating from enzymatically hydrolyzed plant cell wall and/or tree sap by using the resultant enzyme. <P>SOLUTION: An enzymatic product having hemicellulase activity and α-glucuronidase activity is subjected to the ultra-filtration treatment to separate the treated produce into the enzyme having the α-glucuronidase activity and the enzyme having no α-glucuronidase activity. Plant cell wall and/or tree sap polysaccharide are hydrolyzed with the enzyme having the α-glucuronidase activity or with the enzyme having no α-glucuronidase to obtain hydrolytic compositions. One example of the composition hydrolyzed with the enzyme having no α-glucuronidase activity comprises as a minimum unit, a tetramer in which one of glucuronic acid or 4-O-methyl glucuronate bonds to xylotriose with an average polymerization degree is 4 to 10. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for separating an enzyme product based on α-glucuronidase activity, an enzyme obtained thereby, and a composition for decomposing a plant cell wall and / or sap-derived polysaccharide by the enzyme.

  The demand for functional health foods for preventing lifestyle-related diseases and beauty is increasing year by year. Many of the materials are extracted and purified from the natural world, or have been processed by simple processes such as degradation and enzymatic synthesis.

  For example, indigestible oligosaccharides that have the function of escaping the digestive fluid in the body, reaching the large intestine, and being utilized by the intestinal bacterial flora in the large intestine to condition the stomach, Extracts of components that are difficult to be decomposed (see Patent Document 1), cell wall polysaccharides and sap-derived polysaccharides that are difficult to be digested by digestive juice (see Patent Documents 2 and 3), or those that are digested and absorbed, such as starch, lactose, and sugar Is changed to a structure that is difficult to digest using enzymatic synthesis (see Patent Document 4, Patent Document 5, and Patent Document 6).

  Soybean oligosaccharides and lactulose for extracts, cellooligosaccharides, xylooligosaccharides, arabinoxylooligosaccharides, arabinogalactooligosaccharides, arabinooligosaccharides, galactomannooligosaccharides, glucomannooligosaccharides, mannooligosaccharides, chitin / chitosan oligosaccharide, etc. Enzymatic products include isomalt-oligosaccharides, fructooligosaccharides, galactooligosaccharides, and dairy oligosaccharides.

  Acid treatment and enzyme treatment are used for the decomposition treatment of the indigestible product. Cellulase, hemicellulase, pectinase, mannanase, chitinase, etc. are used as the enzyme to be decomposed depending on the raw material to be decomposed. Many of the commercially available enzymes are complex enzymes. For example, even those sold as cellulases usually have some hemicellulase activity. Further, arabinosidase is often included in pectinase used for the purpose of preventing turbidity such as fruit juice.

  Plant cell wall polysaccharides and sap-derived polysaccharides such as corn fiber, beet fiber and gum arabic have weakly acidic components such as glucuronic acid and phenolic acid in their constituent components. Therefore, when oligosaccharides are produced by degrading such polysaccharides with acids or enzymes, the fractions that contain weakly acidic components (acidic oligosaccharides) and the fractions that do not contain (neutral oligosaccharides). Will exist in a mixture. Neutral oligosaccharides include cellooligosaccharides, xylo-oligosaccharides, arabinoxylo-oligosaccharides, arabinogalacto-oligosaccharides, arabino-oligosaccharides, galactomanno-oligosaccharides, glucomanno-oligosaccharides, manno-oligosaccharides, etc. , Α-glucuronogalactooligosaccharide, α-glucuronoarabinooligosaccharide, ferric arabinooligosaccharide and the like. When an enzyme that releases an acidic component such as glucuronidase or esterase is allowed to act on plant cell wall polysaccharides or sap-derived polysaccharides, only the acidic components glucuronic acid and ferulic acid are released alone, and the oligosaccharide is neutral. It will become. Therefore, when it is desired to produce a large amount of neutral oligosaccharides, it is desirable to allow an enzyme having a large activity to liberate acidic components to act, and when it is desired to produce a large amount of acidic oligosaccharides, it contains as much activity as possible to liberate acidic components. It is desirable to have no enzyme working.

However, commercially available enzymes often have both an activity for oligosaccharide-forming a neutral component and an activity for releasing an acidic component, and it has been extremely difficult to properly use them. Especially regarding α-glucuronidase, there are few reports (see Patent Document 7), and there is no commercially available α-glucuronidase.
JP-A-3-287594 Japanese Patent Laid-Open No. 2-100694 JP-A-4-309501 Japanese Patent Laid-Open No. 61-219345 JP-A-3-290197 JP 62-14792 A Special table 2002-510485 gazette

  The present invention was devised in view of the current state of the prior art, and its purpose is to provide an enzyme having α-glucuronidase and an enzyme having no α-glucuronidase by purifying and separating a commercially available enzyme product by a simple method. And the degradation composition of the plant cell wall and sap origin polysaccharide obtained by each such enzyme is provided.

  As a result of intensive studies to achieve the above object, the present inventor has an activity (α-glucuronidase activity) for releasing acidic components in commercially available hemicellulase degrading enzymes, and the molecular weights of these enzymes are greatly different. I found. Furthermore, the enzyme is subjected to an ultrafiltration treatment to separate an enzyme having an activity that releases an acidic component (α-glucuronidase activity) and an enzyme that does not have an activity that releases an acidic component (α-glucuronidase activity) at all. The present inventors have found that this can be easily performed, and have completed the present invention.

  That is, the present invention is characterized in that an enzyme product having hemicellulase activity and α-glucuronidase activity is separated into an enzyme having α-glucuronidase activity and an enzyme not having α-glucuronidase activity by ultrafiltration treatment. Is the method.

  In addition, the present invention is an enzyme having α-glucuronidase activity and an enzyme not having α-glucuronidase activity, which are obtained by the above method.

  Furthermore, the present invention is a degradation composition obtained by degrading a plant cell wall and / or sap polysaccharide with the enzyme having the α-glucuronidase activity or the enzyme not having the α-glucuronidase activity. One example of the enzyme-degrading composition of the present invention having no α-glucuronidase activity is a tetramer in which one glucuronic acid or 4-O-methylglucuronic acid is bound to xylotriose, and the average degree of polymerization. Is 4-10.

  According to the present invention, by using a commercially available enzyme product having a complex activity as a raw material, an enzyme having a high α-glucuronidase activity and two enzymes having no α-glucuronidase activity are produced. Effective use of the enzyme enables efficient production of either acidic oligosaccharides or neutral oligosaccharides.

  The enzyme product used in the present invention is not particularly limited in the type and product name of the basic enzyme, but has at least two activities of hemicellulase activity and α-glucuronidase activity. Examples of such commercially available enzymes include cellulase A-Amano, cellulase T-Amano (above, Amano Enzyme), cellulase XP-415, cellulase XP-425 (above, Nagase Seikagaku), Viscozyme L, Pectinex Ultra SP-L, Ultra Furo L, Cell Crust (above, Novo Nordisk Industry), Cellulosin HC-100, Cellulosin PE60, Cellulosin PEL (above, Hankyu Bioindustry), Sumiteam X, Sumiteam C, Sumiteam AC40, Sumiteam ARS, Sumiteam X, Sumiteam PX ( As described above, there are New Nippon Chemical Co., Ltd.), Doricerase KSM (Kyowa Enzyme), GODO-TCL, GODO TCD-H (above, joint sake), Cellulase Y-NC, Cellulase Onozuka (Yakult) and the like. Among these, Sumiteam X (Nippon Nippon Kagaku), which is a hemicellulase derived from Tricoderderma sp, can be preferably used.

  In the method of the present invention, an enzyme product is subjected to ultrafiltration with a membrane having a molecular weight cut off of 1 to 100,000, preferably 50,000, so that a fraction (enzyme) having α-glucuronidase activity is used as a concentrate, and α-glucuronidase is used. A fraction (enzyme) having no activity can be collected as a permeate. The material and type of the ultrafiltration membrane to be used are not particularly limited. Examples of the material include membrane and ceramic, and examples of the material include a flat membrane type and a hollow paper type.

  The substrate on which the fraction (concentrated solution) having α-glucuronidase activity acts is not limited to its origin or state, but it retains α-glucuronic acid in the side chain. Desirably, it is a polysaccharide derived from a plant cell wall or a polysaccharide derived from sap. Examples of its origin include woody hemicellulose, wheat bran, rice bran, corn fiber, beet fiber, gum arabic and the like, and examples of polysaccharides include xylan, arabinoxylan, arabinoglucan, arabinan, arabinogalactan and the like. The substrate may be used as it is before the enzymatic decomposition, or it may be pretreated by acid, alkali, explosion, steaming, supercritical treatment or the like. Moreover, when it has a cellulose in a substrate raw material, it is not necessary to remove a cellulose beforehand. Examples of the removal method include alkali extraction treatment and limited acid decomposition treatment.

  When a fraction (concentrate) having α-glucuronidase activity is allowed to act on the aforementioned substrate, glucuronic acid is liberated and neutral oligosaccharides free from glucuron are efficiently produced.

  The substrate on which the fraction (permeate) that does not have α-glucuronidase activity is not limited to its origin or state, but has, for example, a structure in which the main chain has a β (1,4) bond to xylose. It is. In this case, α-glucuronic acid may or may not be held in the side chain. Desirably, it is a polysaccharide derived from a plant cell wall, and examples thereof include woody hemicellulose, wheat bran, rice bran, corn fiber, and polysaccharides such as xylan and arabinoxylan. The substrate may be used as it is before the enzymatic decomposition, or it may be pretreated by acid, alkali, explosion, steaming, supercritical treatment or the like. Moreover, when it has a cellulose in a substrate raw material, it is not necessary to remove a cellulose beforehand. Examples of the removal method include alkali extraction treatment and limited acid decomposition treatment.

  When a fraction that does not have α-glucuronidase activity (permeate) is allowed to act on a substrate that does not have α-glucuronic acid in the side chain, a product with the same yield with a smaller amount of enzyme than before purification. Can be obtained.

  When a fraction having no α-glucuronidase activity (permeate) is allowed to act on a substrate having α-glucuronic acid in the side chain, the acidic oligosaccharide has at least one glucuronic acid and xylose Have 3 or more. The smallest unit is a tetramer having a structure in which glucuronic acid is α (1,2) bonded to xylotriose (see FIG. 1). This enzyme produces the same composition even when used before separation and purification (having α-glucuronidase activity), but the product yield is low because side chain glucuronic acid is liberated. Become.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to this.

(Example of enzyme separation and purification)
A commercially available hemicellulase, Sumiteam X (Shin Nippon Chemical Co., Ltd.) is subjected to ultrafiltration using a UF-30UF membrane (fractionated molecular weight 30,000) (Tosoh Corporation) of an ultrafiltration device UF-PS. It was. Electrophoretic analysis of the enzyme in the stock solution and each fraction (concentrated solution and permeate) after ultrafiltration treatment revealed that a plurality of bands that were seen in the stock solution were subjected to ultrafiltration treatment. It changed into a wide band over the whole. Further, the permeate was purified to a component showing an almost single band of less than 30,000 (see FIG. 2).

  Table 1 shows changes in protein recovery rate and enzyme activity (hemicellulase activity and α-glucuronidase activity) of each fraction after ultrafiltration treatment (permeate after concentration). As is clear from Table 1, the protein recovery rate of the concentrated solution by ultrafiltration was 63%, and the α-glucuronidase activity was 1.5 times that of the stock solution. The protein recovery rate of the permeate by ultrafiltration was 37%, the hemicellulase activity was 2.0 times that of the stock solution, and no α-glucuronidase activity was observed.

(Example 1 of enzyme action)
Corn seed coat was acid-decomposed according to the method exemplified in JP-A-11-313700. Specifically, oxalic acid was added to the seed coat of corn to adjust the pH to around 2 and subjected to pressure decomposition at 130 ° C. for 30 minutes to 3 hours to obtain a decomposed sugar solution. The obtained decomposed sugar solution was treated with activated carbon, passed through an ion exchange resin, and further monosaccharide components were removed by continuous chromatographic separation using a strong acid ion exchange resin to obtain an oligosaccharide composition (see FIG. 3). ). The sugar composition analysis of this oligosaccharide composition was performed by Englyst, H. et al. et al, Analyst, 107; 307 (1982), and uronic acid species were examined by a reduction reaction with carbodiimide and confirmed to be glucuronic acid. Glucuronic acid was quantified by the m-hydroxy-diphenyl method (see Table 2). In order to examine the binding mode of glucuronic acid, commercially available β-glucuronidase was allowed to act, but glucuronic acid was not released (see FIG. 4).

  Release of glucuronic acid was observed when Sumiteam X (Shin Nippon Chemical Co., Ltd.) was allowed to act on this oligosaccharide composition. Next, when each enzyme fraction (concentrate and permeate) obtained in the above-mentioned enzyme separation and purification example is allowed to act, glucuronic acid is released only when the concentrate is allowed to act, and when the permeate is allowed to act. Glucuronic acid was not released.

(Enzyme action example 2)
Corn seed coat was acid-decomposed according to the method exemplified in JP-A-11-313700. Specifically, oxalic acid was added to the seed coat of corn to adjust the pH to around 2 and subjected to pressure decomposition at 130 ° C. for 30 minutes to 3 hours to obtain a decomposed sugar solution. The obtained decomposed sugar solution was treated with activated carbon, passed through an ion exchange resin, and monosaccharide components were separated and removed by chromatographic separation using a Ca-type cation exchange resin to obtain a crude purified solution. This crudely purified solution was adsorbed on a weak base type ion exchange resin and desorbed with 4% NaCl to obtain a high-purity glucuronoxylo-oligosaccharide (purity 99% or more). Some (about 0.3%) phenolic acid was also observed in the obtained glucuronoxylo-oligosaccharide.

  The average degree of polymerization of the xylooligosaccharide serving as the main chain was 4.1, and it had a structure in which 1.4 glucuronic acids were bonded on average. Using this as a substrate, the permeate (the fraction in which α-glucuronidase was not found) separated in the above-described example of separation and purification of the enzyme was excessively added to perform complete decomposition (see FIG. 5). After the reaction, it was confirmed that the decomposition did not proceed any more even when the permeate was added again.

  In the decomposition sugar solution thus obtained, an acidic component to which glucuronic acid is bound and a neutral component released from a fragment to which glucuronic acid is bound by enzymatic degradation are mixed. The neutral component was separated by passing through a Na-type cation exchange resin to obtain a highly pure acidic component. The acidic component obtained here is a hemicellulase limit dextrin by commercially available hemicellulase (Sumiteam X (Shin Nihon Kagaku)), and the limit is due to the binding of glucuronic acid.

  When this limiting dextrin was analyzed by anion exchange HPLC (sugar analysis DX500 system, separation column Carbopak PA-1, Dionex), it was recognized that several kinds of components were mixed (see FIG. 6). Among these, acidic components (components to which glucuronic acid is bound) show elution that is slower than glucuronic acid, which is a monosaccharide. Therefore, acidic oligosaccharides are eluted later than glucuronic acid. Among the components in FIG. 6, F1 and F3 increased by enzymatic degradation, but F2 did not increase. Therefore, when F1 and F3 were collected and analyzed in detail, it was found that the ratio of xylose to glucuronic acid was 3: 1. Moreover, the average degree of polymerization of the xylose of component F1 was 3. That is, the minimum unit of acidic oligosaccharide obtained by degradation with Sumiteam X was a trimer in which one glucuronic acid was bound to three xyloses. The component F1 was determined to have the structure of FIG. 1 from the nature of the analysis technique.

The basic structure of the acidic oligosaccharide obtained by commercial hemicellulase is shown. The result of electrophoresis of a purified enzyme is shown. The purification result of acidic oligosaccharide is shown. It shows the release of glucuronic acid by β-glucuronidase degradation of acidic oligosaccharides. The result of the limit decomposition | disassembly by the refinement | purification hemicellulase (permeate) of acidic oligosaccharide is shown. The limit degradation product by the refinement | purification hemicellulase (permeate) of acidic oligosaccharide is shown.

Claims (7)

  A method comprising separating an enzyme product having hemicellulase activity and α-glucuronidase activity into an enzyme having α-glucuronidase activity and an enzyme not having α-glucuronidase activity by ultrafiltration treatment.   An enzyme having α-glucuronidase activity, which is obtained by the method according to claim 1.   An enzyme having no α-glucuronidase activity, which is obtained by the method according to claim 1.   A degradation composition obtained by degrading a plant cell wall and / or sap-derived polysaccharide with the enzyme having α-glucuronidase activity according to claim 2.   A degradation composition obtained by degrading a plant cell wall and / or sap-derived polysaccharide with the enzyme having no α-glucuronidase activity according to claim 3.   6. The decomposition composition according to claim 5, wherein a tetramer in which one glucuronic acid or 4-O-methylglucuronic acid is bound to xylotriose is used as a minimum unit. The decomposition composition according to claim 6, wherein the average degree of polymerization is 4 to 10.