JPH0336512B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a novel method for producing glycosyltransferase A using Bacillus bacteria. (Prior Art) Enzymes that transfer sugars include those that use transferases (transferases) and those that use reverse reactions of hydrolases. Furthermore, transferases are divided into seven types depending on the type of residue transferred. Among these, enzymes involved in sugar transfer are enzymes that use oligosaccharides or polysaccharides as donors and transfer their sugar residues to appropriate acceptors such as other monosaccharides, oligosaccharides, and various alcohols. In addition, glucose residues, xylose residues, galactose residues, etc.
There are enzymes that transfer various sugars. They are also classified according to the type of bond in which the transition occurs. As an example of industrial application of glycosyltransferase,
Examples include the production of cyclodextrin using cyclodextrin glucanotransferase, the production of coupling juice using the same enzyme, and the production of glucosyl stevioside. Examples of methods that utilize the reverse reaction of hydrolytic enzymes include the production of branched cyclodextrin using pullulanase and the production of isomaltose using glucoamylase. The present inventors have recognized that certain glycosyltransferases have a remarkable effect on increasing the yield of glucose in starch decomposition by glucoamylase. Glucose is currently produced by liquefying starch with α-amylase and then saccharifying it with glugoamylase. Glucoamylase can hydrolyze both α-1,4-glucosidic bonds and α-1,6-glucosidic bonds in starch, so in the case of a dilute starch solution, it almost completely converts it into glucose. Can be disassembled. However, on an industrial scale, saccharification is carried out in the presence of starch at a high concentration of 30 to 35%, and the resulting glucose is polymerized due to the retrosynthetic action of glucoamylase itself. The yield of glucose is usually
It is about 93-95%. The remainder remains as maltose, isomaltose, panose, and other higher molecular weight oligosaccharides. Therefore, it has been a long-held desire in glucose production to increase the yield of glucose by decomposing these remaining oligosaccharides. (Problems to be solved by the invention) In starch saccharification using glucoamylase,
It is known that when starch is saccharified in the presence of pullulanase, which has the ability to decompose α-1,6-glucoside bonds, the saccharification reaction is accelerated and glucose yield can be increased by 0.5 to 2%.
−29570, 57−39, 57−174089, etc.). In addition, the yield of glucose can also be increased by using a certain kind of α-amylase (Japanese Patent Publication No. 19498, 1983). Pullulanase is an enzyme that decomposes α-1,6-glucoside bonds in amylopectin or its derivatives in addition to pullulan, and ultimately produces maltotriose from pullulan. However, pullulanase is also known to be retrosynthesized.
For example, it produces hexasaccharide (G6) from maltotriose. For this reason, when pullulanase is used in combination with starch saccharification using glucoamylase, an increase in glucose yield is observed, but on the other hand, there is a problem in that the retrosynthetic action of pullulanase also produces oligos that are difficult to be degraded by glucoamylase. (Means for Solving the Problem) The present inventor has been searching for a new enzyme that is effective in increasing the yield of glucose when saccharifying starch using glucoamylase. It was found that enzymes produced by bacteria identified as belonging to the genus had an extremely significant effect on increasing glucose yield. When this enzyme acts on soluble starch, it produces a saccharide whose main component is maltose, but when it acts on maltooligosaccharides larger than maltose, it produces maltooligosaccharides with smaller molecular weights and various maltooligosaccharides with larger molecular weights. produces sugar. In other words, it was confirmed that it is a novel glycosyltransferase that simultaneously performs decomposition and glycosyltransfer. For example, when this enzyme is applied to maltotriose (G3), maltose (G2), maltotetraose (G4), maltopentaose ( G5) and maltohexaose (G6). Figure 1 is a liquid chromatogram showing the composition of maltoheptaose (G7) among the sugars produced by the action of this enzyme on 1% maltotriose. Sugar was also produced. 4-α-D-glucanotransferase (EC2.4.1.25) is known as an enzyme that has a similar effect.
As detailed below, significant differences were observed between this enzyme and properties such as action, temperature resistance, and molecular weight. The present invention has been made based on this knowledge. (Structure) The present invention relates to a method for producing glycosyltransferase A, which comprises culturing a Bacillus bacterium that produces glycosyltransferase A, and collecting glycosyltransferase A from the culture. The method of the present invention will be explained in more detail below. Glycosyltransferase A of the present invention has the enzymatic properties described below. (1) Action When acting on soluble starch, amylose, amylopectin, etc., it mainly produces maltose, but also produces a series of malto-oligosaccharides such as maltotriose, maltotetraose, maltopentaose, and maltohexaose. . However, less glucose is produced. Moreover, even if it is made to act on malto-oligosaccharides greater than maltotriose, a series of maltooligosaccharides greater than maltose are produced, but the production of glucose is small. Figure 1 shows the sugar composition up to maltoheptaose among the sugars produced when this enzyme acts on 1% maltotriose, but oligosaccharides with even larger molecular weights are also produced. . Furthermore, when it acts on maltooligosaccharides of maltotetraose or higher, decomposition products with a similar composition are produced. In other words, this enzyme is a type of glycosyltransferase that redistributes substrate molecules by producing maltooligosaccharides with smaller molecular weights and maltooligosaccharides with larger molecular weights from substrate molecules by simultaneous decomposition and transfer action. . 4-α-D-glucanotransferase (EC2.4.1.25) is known as an enzyme with a similar action, but in the case of this enzyme, when maltotriose is used as a substrate, glucose Generates {Used as a method for measuring activity (edited by Shiro Akahori et al., Enzyme Handbook, page 272, Asakura Shoten)}. However, in the case of the enzyme of the present invention, as is clear from FIG. 1, less glucose is produced, and maltose and maltotetraose are mainly produced at the early stage of the reaction. This indicates that the transfer by this enzyme occurs mostly in glucosyl units. This enzyme also acts on maltose, albeit weakly, and produces various maltooligosaccharides that have a higher molecular weight than maltose. Furthermore, when this enzyme was allowed to act on maltopentaose (G5),
From a relatively early stage of the reaction, maltotetraose (G4), then maltotriose (G3), maltose (G2), maltohexaose (G6), then maltoheptaose (G7), maltose Since octaose (G8) etc. are produced, it is thought that in addition to glucosyl groups, maltosyl and maltotriosyl groups are also transferred. Water is also considered to be a receptor because it acts on polymeric soluble starch to mainly produce maltose, and acts on pullulan and the like to increase its reducing power. In contrast, 4-α-D-glucanotransferase produced by Bacillus subtilis is reported to transfer from maltosyl to maltopentaosyl units {Journal of
Biological Chemistry, 243, 4732 (1968)}. (2) Action PH and optimal action PH It works in a wide range of pH from about 3 to about 9, and has an optimal action.
The pH was found to be around 5 (Figure 3, 1% soluble starch, 0.1M acetate buffer or phosphate buffer, 1% soluble starch, 0.1M acetate buffer or phosphate buffer,
Reaction for 1 hour at 50°C under ×10 ^-2 M calcium chloride). (3) Working temperature range and optimum working temperature It works up to a temperature of about 80℃, and the optimum working temperature is about 80℃.
60℃ {Figure 4, 1% soluble starch,
React for 30 minutes under 2.5×10 ^-2 M acetate buffer (PH5)}. (4) Thermostability The enzyme aqueous solution was heat-treated at 50, 55, and 60°C for 10 minutes each, and then the residual activity was measured. As a result, approximately 0%, approximately 30%, and approximately 80% inactivation occurred, respectively. At 50°C, almost no inactivation was observed even after heating for 30 minutes. On the other hand, Bacillus subtilis 4-α-D-glucanotransferase is unstable to heat, and when heated at 50°C for 15 minutes,
It has been reported that % inactivation {Journal of
Biological Chemistry, 243, 4732, (1968)}. (5) PH stability Stable at pH about 4.5 to about 7 (under 5x10 ^-2 M acetate buffer or Tris buffer at room temperature)
After standing for a period of time, measure the remaining activity). (6) Stabilizers and activators The presence of calcium and iron ions stabilizes the enzyme. Calcium ions also increase enzyme activity. Such properties have not been observed in the enzyme of Bacillus subtilis {Journal of Biological Chemistry, 243,
4732 (1968)}. (7) Inhibitor Strongly inhibited by 1x10 ^-3 M copper, mercury, and silver ions. (8) Molecular weight The molecular weight measured by gel filtration using Cephadex G-200 was approximately 500,000. The number of enzymes of Bacillus subtilis is reported to be 70,000 to 80,000. {Journal of Biology
Chemistry, 243, 4732 (1968)}. (9) Purification method This enzyme is prepared by adding ammonium sulfate to the culture supernatant for 70 min.
% saturation, the resulting precipitate fraction is collected by centrifugation, dialyzed, concentrated, and then subjected to Sephadex G-200 column chromatography and rechromatography using the same column to produce a homogeneous product. (10) Titer measurement method Add an appropriate amount of enzyme to 0.5 ml of a 2% soluble starch (or pullulan) solution dissolved in 0.1 M acetate buffer (PH5.0) containing 1x10 ^-2 M calcium chloride, and add it with distilled water. Make the total volume 1 ml and react at 60°C. Under these conditions, the amount of enzyme that generated a reducing power equivalent to 1 μM glucose per minute was defined as 1 A unit (1 P unit when pullulan was used as a substrate).
In the case of purified enzyme, the P unit/A unit is approximately 1/4. (Inventive effect) Table 1 shows that dextrin at a concentration of 30% is used at pH 4.5,
When saccharified for 72 hours using commercially available Aspergillus niger glucoamylase at a temperature of 60℃,
Figure 2 shows the effect of the enzyme of the invention and commercially available Bacillus pullulanase on glucose yield.

[Table] As is clear from the table, only 95.2% of glucose was obtained using glucoamylase alone, but when the enzyme of the present invention was present, glucose was obtained with a yield of 97.1%. This yield was almost the same as the yield (97.2%) when pullulanase was used. However, in the case of the enzyme of the present invention, the disaccharide (G2) is slightly more abundant than in the case of pullulanase;
A characteristic feature was observed in which there were fewer oligosaccharides of trisaccharide (G3) or higher than in the case of pullulanase. When high molecular weight oligosaccharides are present in the saccharification solution,
It is desirable that the amount of high-molecular oligosaccharides be as small as possible, since this will impair the filterability of the sugar solution and cause it to become cloudy. Therefore, the enzyme of the present invention not only has the effect of increasing the yield of glucose, but also has the advantage that a sugar solution containing less oligosaccharides with large molecular weights can be obtained. Figure 2 shows the time course of liquefied starch saccharification when glucoamylase was used alone and when the enzyme of the present invention was used in combination, and as is clear from the figure, the presence of this enzyme also promoted the effect of saccharification. It was done. This means that glucoamylase can be reduced. The role played by the enzyme of the present invention in starch saccharification by glucoamylase can be understood as follows. That is, it is known that the affinity (reactivity) of glucoamylase for a substrate is greatly influenced by the degree of polymerization of glucose, and has a greater affinity for a substrate with a large molecular weight than a substrate with a small molecular weight. In the latter stage of starch saccharification by glucoamylase, oligosaccharides with various degrees of polymerization that are difficult to decompose by glucoamylase are produced, but the enzyme of the present invention can convert substrates that are easily decomposed by glucoamylase through decomposition and transfer actions. It is thought that by supplying it, the decomposition rate can be improved and the yield of glucose can be increased. Therefore, an enzyme having such a transfer effect can achieve an increased yield of glucose in the same manner as the enzymes exemplified in the present invention. but,
Bacillus, for example, which does not have such a metastatic effect
Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae
Even when using α-amylase such as, the effect of increasing glucose yield was not observed. On the other hand, the promotion of saccharification reaction by pullulanase is
Glucoamylase weak branched bonds (α-1, 6-
It is thought that the purpose of this enzyme is to compensate for the ability to cleave glucosidic bonds), so the mechanism for increasing glucose yield is different from that of the enzyme of the present invention. Therefore, the sugar compositions of the two starch saccharides are naturally considered to be different. As an exemplary bacterium that produces the enzyme of the present invention, Bacillus megaterium isolated from soil is used.
megaterium). This strain has the following enzymatic properties. (1) Morphology: Bacillus, motile (2) Spores: Sporangium; non-swelling, spores; oval, position: neutral to sub-erect (3) Gram staining: + (4) Catalase: + (5) V-P reaction: - PH5.0 (6) Growth under anaerobic conditions: - (7) Starch decomposition: + (8) Gelatin liquefaction: + (9) Egg yolk reaction: - (10) Acid production from glucose: + ( 11) Gas production from glucose: - (12) Growth at PH5.7: + (13) Growth at 50℃: - (14) Utilization of citrate: + (15) Growth in the presence of 5% NaCl :+ (16) Nitrate reduction:- Regarding the above mycological properties, Bergey's
Referring to the 8th edition of the Manual of Determinative Bacteriology (The William & Wilkins Co., 1974), this bacterium was identified as Bacillus megaterium.
megaterium) and fixed. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM BP-1672. To produce the enzyme of the present invention using this strain, peptone, meat extract, yeast extract, corn extract,
Organic nitrogen sources such as staple liquor, urea,
Inorganic nitrogen compounds such as ammonium chloride, ammonium sulfate, ammonium phosphate, sodium nitrate, potassium nitrate, and carbon sources such as soluble starch, dextrin, sugar, maltose, and glucose are used. In addition, a medium containing a small amount of phosphate, magnesium salt, calcium salt, etc. is adjusted to a pH of 5 to 8, and the pH is usually 1 to 1 at 30°C.
Incubate aerobically for 4 days. Most of the enzymes are produced outside the bacterial cells, so after culturing, bacteria are removed by filtration or centrifugation, and the supernatant is concentrated, or salted out with sodium sulfate, ammonium sulfate, etc., or methanol, An organic solvent such as ethanol, propanol, isopropanol, or acetone is added to precipitate and recover the enzyme. This strain produces maltogenic amylase in addition to the enzyme of the present invention. This enzyme does not have the effect of increasing glucose yield and interferes with the activity measurement of the enzyme of the present invention. Therefore, in the case of a culture solution or crude enzyme, using pullulan as a substrate makes it possible to accurately measure the enzyme of the present invention. be able to. Using the enzyme of the present invention in combination with glucoamylase,
The reaction to saccharify starch usually involves a liquefied starch concentration of 30~
35 (W/V)%, PH4~5, temperature 55~60℃, 2
It will be held for ~4 days. Next, the details of the present invention will be explained with reference to examples. Example 1 2% polypeptone, 2% soluble starch,
Pour 300 ml of a medium (PH5) consisting of 0.3% K ₂ HPO ₄ and 0.1% MgSO ₄ 7H ₂ O into a 2-liter Erlenmeyer flask, sterilize it using a conventional method, and then prepare Bacillus megaterium FERM BP-1672. The cells were inoculated and cultured with shaking at 30°C for 4 days. After culturing, the amount of enzyme produced in the centrifuged supernatant was measured, and the result was 1.5 P units per ml of medium. Ammonium sulfate was added to the culture supernatant to reach 70% saturation, and the resulting precipitate was collected by centrifugation, dissolved in distilled water, dialyzed against distilled water, and used in the following saccharification experiment. Example 2 Commercially available dextrin (manufactured by Nippon Shiryo Co., Ltd.,
NSD DE approx. 13) and glucoamylase derived from Aspergillus niger, commercially available from Nihon Huynh Schüger Co., Ltd., were used. In addition, pullulanase is manufactured by Novo Japan Co., Ltd.
We used a product derived from Bacillus acidopulliticus sold by Bacillus acidopulliticus. The composition of the reaction solution is shown in Table 2.

[Table] Each sample was made into a total volume of 10 ml and saccharified for 65 and ⁷² hours at pH _4.5 and 60°C. It was determined by high performance liquid chromatography.The results are shown in Table 3.

[Table] As is clear from the table, when this enzyme is used, the yield of glucose is approximately 2
% increase. This increase was almost the same as when pullulanase was used. When this enzyme was used, the amount of disaccharides was slightly higher than when pullulanase was used, but the amount of trisaccharides and higher was lower. Example 3 3 g of liquefied starch (DE approximately 13) obtained by liquefying corn starch with bacterial α-amylase as a solid content,
0.1% glucoamylase per solid content, and 0.75 units of the enzyme prepared in Example 1,
CaCl ₂ 1x10 ^-2 M added, each total volume 10
ml and reacted at PH4.5 and 60°C. A fixed amount was taken over time, and the produced glucose was quantified by liquid chromatography. The results are shown in Figure 2. As is clear from the figure, when this enzyme was present, the saccharification reaction was promoted compared to when it was not present. Glucose was also obtained in high yield.

[Brief explanation of drawings]

FIG. 1 shows the sugar composition of the product obtained when transferase A is allowed to act on 1% maltotriose. Figure 2 shows the influence of transferase A on liquefied starch saccharification by glucoamylase.
FIG. 3 shows the optimal action PH of transferase A. FIG. 4 shows the optimum operating temperature of transferase A.

Claims (1)

[Scope of Claims] 1. A glycosyltransferase A characterized by culturing a microorganism belonging to the genus Bacillus that produces glycosyltransferase A having the following enzymatic properties, and collecting the glycosyltransferase A from the culture. manufacturing method. (1) When acting on maltooligosaccharides greater than maltose, a series of maltooligosaccharides with a higher degree of polymerization than the added substrate are produced, and the amount of glucose produced is small. (2) When it acts on a mixture of maltooligosaccharides containing maltose or higher, it redistributes among the substrate molecules and produces a series of maltooligosaccharides with a high degree of polymerization. (3) The optimal action pH is around 5. (4) The optimum working temperature is approximately 60°C. (5) Stable after heat treatment at 50℃ for 30 minutes. (6) Stabilized by the presence of calcium and iron ions. (7) Inhibited by copper, mercury, and silver ions. (8) The molecular weight is approximately 500,000.