JP5345766B2 - Novel maltose phosphorylase, method for producing the same, and method for using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new maltose phosphorylase having optimum temperature overlapping with that of a trehalose phosphorylase to be combined therewith, the range of the optimum temperature being in a temperature region expectable of sterilizing effect, and temperature stability suitable for producing the trehalose and deactivable at a moderate temperature and to provide a method for utilizing the new maltose phosphorylase. <P>SOLUTION: The new maltose phosphorylase has the optimum temperature and the temperature stability within the range of 50.0-58&deg;C and is deactivated by &ge;90% through a heat treatment at 65&deg;C for 15 min. The method for producing the trehalose using the enzyme is provided. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a novel maltose phosphorylase, a method for producing the maltose phosphorylase, and a method for producing β-glucose-1-phosphate or trehalose using the maltose phosphorylase.

  Trehalose is a substance that is expected to be widely used for pharmaceuticals, cosmetics, foods and the like because it is more stable than other disaccharides. Therefore, many attempts have been made to industrially produce trehalose. These technologies can be broadly classified into two types: fermentation methods and enzymatic methods.

  The fermentation method is a method of extracting and purifying the substance after culturing a microorganism having a property of accumulating trehalose inside and outside the cell body (see, for example, Patent Document 1). In this method, mass production of trehalose is difficult in terms of operation or equipment, and even if these are solved, production efficiency is poor, and further, removal of impurities is required, resulting in high production costs and economical disadvantages. Met. In particular, in order to use trehalose for food applications, it is necessary to produce it in a large amount at a low cost, and this disadvantage has been a serious problem to be solved.

  On the other hand, the enzyme method is a method that solves the above various problems of the fermentation method at once. As one of them, maltose phosphorylase (Maltose: orthophosphatase β-D-glucosyltransferase; Maltose phosphorylase, EC 2.4.1.8) and trehalose phosphorylase (α, α-Trepharose β-D-glucosylase, orthophosphatase β-D-glucosylase; There is a simultaneous reaction method using phosphorylase (EC 2.4. 1.64) (see, for example, Patent Document 2).

  This method uses a reaction in which two types of phosphorylase act on maltose and trehalose to reversibly phosphorolyze to produce glucose and β-glucose-1-phosphate, and is an inexpensive raw material. Trehalose is produced when both enzymes are allowed to act on maltose at the same time. According to this method, it has been reported that trehalose is produced from maltose with a high yield of 60 to 65%. In addition, since the raw material used is a purified high-purity saccharide, it is easy to purify trehalose obtained by an enzymatic reaction, which is considered to be an industrially advantageous method compared to other methods. It has been. However, maltose phosphorylase and trehalose phosphorylase used in combination in the method are greatly different in the optimum pH range of each enzyme, and are very low in temperature stability, and the reaction for producing trehalose is about 25 to 37 ° C. It was possible only at low temperatures. This is not only very difficult to control the pH when using two types of enzymes in combination, but also causes contamination of bacteria during the enzyme reaction performed using an open reaction tank due to the low reaction temperature. In order to prevent such a secondary reaction, there were problems such as the need for strict hygiene management. For this reason, this method cannot be said to be an economically efficient method.

  Based on the above, if a combination of maltose phosphorylase and trehalose phosphorylase suitable for high-efficiency production of trehalose can be found, trehalose can be easily and efficiently produced in high yield from maltose that can be obtained easily and in large quantities. Can do. Specifically, in the maltose phosphorylase and trehalose phosphorylase used for the production of trehalose, the optimum pH and the optimum temperature range overlap, and if the pH stability and the temperature stability suitable for production are present, High-efficiency production of trehalose is possible.

  On the other hand, the present inventors searched for new maltose phosphorylase and trehalose phosphorylase in order to find an enzyme having the above properties. As a result of intensive studies, the present inventors have found novel maltose phosphorylase and trehalose phosphorylase that overlap at an optimum pH of around 7.0 to 7.5 and an optimum temperature of around 45 to 55 ° C. (for example, Patent Document 3) See).

In addition, thermostable maltose phosphorylase has been developed so far (see, for example, Patent Documents 4 and 5; Non-Patent Document 1). The maltose phosphorylase has an optimum (optimum) pH and an optimum temperature of 6.0 to 7.0 and 55 to 70 ° C., respectively, and is stable in the range of 5.5 to 8.0 with respect to pH. The temperature is extremely stable up to 60 ° C.
Japanese Patent Laid-Open No. 5-91890 Japanese Patent No. 1513517 WO2005 / 003343 Japanese Patent No. 3691875 JP-A-10-262683 Yasushi, Inoue et al. (2001) Biosci Biotechnol Biochem 65: 2644-2649

  However, in the case of producing trehalose using the maltose phosphorylase and trehalose phosphorylase described in Patent Document 3 described above, since the extremely stable temperature of the maltose phosphorylase is up to 50 ° C, the reaction temperature is set to a temperature exceeding 50 ° C. It could not be set, and therefore a sufficient bactericidal effect could not be obtained. Therefore, in order to produce trehalose in combination with the trehalose phosphorylase, the optimum temperature overlaps with the trehalose phosphorylase in a wide range, and has high stability at a temperature of 50 ° C. or more, which is a temperature range where a bactericidal effect can be expected. It has been found that maltose phosphorylase is required.

  It is also considered that trehalose can be efficiently produced from maltose by combining the thermostable maltose phosphorylase described in Patent Documents 4 and 5 and Non-patent Document 1 with the trehalose phosphorylase described in Patent Document 3. However, since the maltose phosphorylases described in Patent Documents 4 and 5 and Non-Patent Document 1 have residual activity up to a high temperature range, the enzyme deactivation process in trehalose production must be set to a higher temperature. It has been found that a new problem arises.

  Therefore, the present inventors have searched for a novel maltose phosphorylase that has solved the various problems described above.

  From the above, the first object of the present invention is to provide a novel maltose phosphorylase that solves the various problems of the conventional techniques as described above and satisfies these various requirements. More specifically, the optimum temperature overlaps with the trehalose phosphorylase to be combined, and the optimum temperature is a temperature range of 50 ° C. or higher where the bactericidal effect can be expected, and has temperature stability suitable for the production of trehalose. It is to provide a novel maltose phosphorylase that can be inactivated at an appropriate temperature.

  The second object of the present invention is to provide a production method for obtaining the maltose phosphorylase easily and in high yield using a polynucleotide or nucleic acid molecule encoding the maltose phosphorylase.

  The third object of the present invention is to provide a method for producing β-glucose-1-phosphate or trehalose using the obtained maltose phosphorylase.

As a result of diligent research to solve the above problems, the present inventors have found a novel maltose phosphorylase having the following properties [A] to [C], and have completed the present invention.
[I] The optimum temperature for maltose phosphorolysis reaction activity is 50-60 ° C.
[B] Maltose phosphorolysis reaction activity is stable up to 58 ° C. under heating conditions of pH 6.0 and 30 minutes.
[C] Maltose phosphorolysis activity is deactivated by 90% or more by heat treatment at 65 ° C. for 15 minutes.

That is, according to the present invention, maltose phosphorylase having any one of the following amino acid sequences (a) to (c) is provided.
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 70% or more homology with the modified amino acid sequence and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes.

  Preferably, the modified amino acid sequence described above is a modified amino acid sequence in which asparagine at position 72 and threonine at position 679 are replaced with isoleucine and isoleucine, isoleucine and valine, valine and isoleucine, or a combination of valine and valine, respectively. .

Furthermore, according to the present invention, a polynucleotide encoding maltose phosphorylase comprising any one of the following amino acid sequences (a) to (c) is provided.
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 70% or more homology with the modified amino acid sequence and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes.

Furthermore, according to the present invention, a polynucleotide comprising any one of the following base sequences (a) to (c) is provided.
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 70% or more homology with the modified amino acid sequence and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes.

  Furthermore, according to the present invention, a recombinant vector containing the above-described polynucleotide of the present invention is provided.

  Furthermore, according to the present invention, there is provided a transformant introduced with the above-described polynucleotide of the present invention or containing the above-described recombinant vector of the present invention.

Furthermore, according to this invention, the manufacturing method of the maltose phosphorylase of the above-mentioned this invention which has the process of following (a)-(c) is provided.
(A) a step of culturing the transformant of the present invention;
(B) production / accumulation step of the maltose phosphorylase; and (c) collection step of the maltose phosphorylase:

  Preferably, in the collection step, a transformant is separated from the culture, and (a) the separated transformant is used as a crude enzyme of maltose phosphorylase as it is, or (b) maltose is extracted from the separated transformant. Either a crude enzyme of phosphorylase or (c) the obtained culture supernatant is used as a crude enzyme of maltose phosphorylase.

  Furthermore, according to the present invention, β-glucose-1-phosphate is obtained by allowing the maltose phosphorylase of the present invention to act on maltose in the presence of phosphoric acid to obtain β-glucose-1-phosphate. A method for producing an acid is provided.

  Furthermore, according to the present invention, there is provided a method for producing trehalose, characterized in that trehalose is obtained by allowing the maltose phosphorylase and trehalose phosphorylase of the present invention to act on maltose in the presence of phosphoric acid.

Preferably, the trehalose phosphorylase is characterized by having the following properties (a) to (d).
(A) Catalytic action: reversibly phosphorolytically decomposes the α-1,1-glucopyranoside bond in trehalose to produce glucose and β-glucose-1-phosphate;
(B) Optimum pH and stable pH range: The optimum pH for the trehalose synthesis reaction is 5.8 to 7.8, and is stable within the range of pH 5.5 to 9.5 under heating conditions at 50 ° C. for 10 minutes. is there;
(C) Optimal temperature: The optimal temperature for the trehalose synthesis reaction is 45-60 ° C; and (d) Temperature stability: pH 7.0, stable to 60 ° C under heating conditions for 15 minutes, and 90 ° C at 70 ° C. % Inactivated.

The novel maltose phosphorylase according to the present invention maintains high activity in a wide pH range, has about 80% of untreated activity for 150 minutes at 55 ° C. or lower, and rapidly loses when it exceeds 65 ° C. Live. From this, the maltose phosphorylase of the present invention has a great advantage in industrial production of β-glucose-1-phosphate from maltose, or utilization of industrial production of trehalose in combination with the trehalose phosphorylase described above.
In addition, the maltose phosphorylase of the present invention can produce β-glucose-1-phosphate more rapidly than the corresponding wild-type maltose phosphorylase. Thereby, if the maltose phosphorylase of this invention is used, the enzyme reaction process which spends much time in trehalose manufacture can be shortened, and rapid production of trehalose can be achieved. One possible reason for this is that the maltose phosphorylase of the present invention has a higher affinity for the substrate phosphate than the corresponding wild-type maltose phosphorylase.

Hereinafter, embodiments of the present invention will be described in detail.
(A) Enzyme of the present invention The present inventors randomly selected a maltose phosphorylase gene derived from Paenibacillus sp. SH-55 (deposition number FERM BP-8420) described in WO2005 / 003343. Mutants were introduced, and a transformant library transformed with the obtained mutant maltose phosphorylase gene was constructed. From the transformant library, transformants expressing the mutant maltose phosphorylase were screened for the purpose of obtaining a mutant maltose phosphorylase that retains enzyme activity even under high temperature conditions of 50 ° C. The inventors of the present invention have succeeded in constructing the maltose phosphorylase of the present invention by specifying the site and type of amino acid residues contributing to heat resistance from the amino acid sequence of the mutant maltose phosphorylase obtained by screening.

That is, the maltose phosphorylase of the present invention is a maltose phosphorylase having any one of the following amino acid sequences (a) to (c).
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at pH 6.0, 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 70% or more homology with the modified amino acid sequence and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at pH 6.0, 65 ° C. for 15 minutes.

The maltose phosphorylase of the present invention is an enzyme having the following properties.
(A) Catalytic action: In the presence of phosphoric acid, α-1,4-glucopyranoside bond in maltose is reversibly phosphorylated to produce glucose and β-glucose-1-phosphate.
(B) Substrate specificity: It acts on maltose in the degradation reaction, and does not act on trehalose, sucrose, lactose, or cellobiose.
(C) Optimum pH and pH stability range: The optimum pH in the decomposition reaction is around 6.5 to 7.5, and is stable within the range of pH 5.0 to 8.5 under heating conditions for 15 minutes. .
(D) Optimal temperature: It has an optimal temperature for the decomposition reaction in the vicinity of 50 to 60 ° C.
(E) Temperature stability: stable to 58 ° C. under heating conditions of pH 6.0 and 30 minutes, and deactivated by 90% or more by heat treatment at 65 ° C. for 15 minutes.
(F) substrate affinity: K _m values by plotting the Lineweaver-Burk, for phosphoric acid with respect to about 5.0～6.0MM, maltose about 4.0～5.0MM.
(G) Inhibitory: Inhibited by copper, mercury, N-bromosuccinimide, p-chloromercuribenzoic acid (1 mM each), sodium dodecylbenzenesulfonate (1%).
(H) Isoelectric point: in the range of pH 4.8 to 5.0.
(I) Molecular weight: The molecular weight by SDS-polyacrylamide electrophoresis is about 89,000-90,000 daltons, the molecular weight by gel filtration is about 190,000 daltons, and is composed of homodimers.

  The term “catalytic action” as used in the present specification means an embodiment of a catalytic reaction possessed by an enzyme.

  Unless otherwise stated, “maltose phosphorolytic activity”, “activity of maltose phosphorylase” and “activity” in this specification are β-glucose-1-phosphate among the catalytic actions of maltose phosphorylase. It means an embodiment of a decomposition reaction for producing glucose (referred to as “maltose phosphorolysis reaction” or simply “decomposition reaction”). Further, the “enzyme” in the present specification means maltose phosphorylase unless otherwise stated.

The action of maltose phosphorylase is not particularly limited with respect to the measurement method, but for example, it can be measured by the following method.
5 units of maltose phosphorylase was added to 1 g of a substrate in 1% (w / v) maltose solution dissolved in 10 mM phosphate buffer (pH 7.0), reacted at 50 ° C. for 5 hours, and then boiled. Heat in water bath for 3 minutes. The sugar in the obtained saccharified solution is measured by high performance liquid chromatography to detect glucose and glucose-1-phosphate, respectively. Also, 1% (w / v) glucose and β-glucose-1-phosphate sodium salt or α-glucose-1-phosphate sodium salt dissolved in 10 mM Tris-HCl buffer (pH 7.0) As a substrate, 5 units of maltose phosphorylase is added to 1 g of the substrate and reacted at 50 ° C. for 5 hours. The synthesis reaction by maltose phosphorylase can be confirmed by measuring the sugar composition produced by the treatment as described above and detecting maltose from glucose and β-glucose-1-phosphate. However, the synthesis reaction of disaccharides from glucose and α-glucose-1-phosphate is not detected. The produced sugar can be analyzed by the following method. Unreacted maltose in the heat-inactivated saccharified solution is completely decomposed into glucose by glucoamylase or the like, then heated at 100 ° C. for 10 minutes to completely inactivate glucoamylase or the like and then generated The trehalose content is measured by high performance liquid chromatography. A differential refractometer is used for detection.

The activity of maltose phosphorylase can be measured by the following method or a method equivalent to the following method.
0.1 ml of maltose phosphorylase solution having a concentration of 5 μg / ml was added to 0.4 ml of 20 mM maltose dissolved in 200 mM phosphate buffer (pH 7.0), reacted at 50 ° C. for 15 minutes, and then boiled Stop the enzyme reaction by heating in a water bath for 3 minutes. After cooling in ice water, 0.5 ml of the resulting enzyme reaction stop solution is collected, 2 ml of glucose measuring reagent (Wako Pure Chemical Industries, Ltd., glucose C-II test Wako) is added, and the mixture is incubated at 37 ° C. for 10 minutes. After the reaction, the absorbance at 505 nm is measured. Here, one unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of glucose per minute under the same conditions.
If the maltose phosphorylase solution contains α-glucosidase (maltase), glucoamylase, trehalase, etc. that hydrolyzes maltose or trehalose, the above method is carried out by inactivating these enzymes in advance. This method is used.

The substrate affinity of maltose phosphorylase is not particularly limited with respect to the measurement method, but can be measured, for example, by the following method.
In the enzyme activity measurement method described above, the substrate to be used is measured by examining the decomposition activity of these substrates using trehalose, isomaltose, neotrehalose, sucrose, lactose, or cellobiose as a substrate instead of maltose.

The optimum pH and pH stability range of maltose phosphorylase can be measured by the following method or a method equivalent thereto.
The optimum pH was obtained after treatment with 50 mM phosphate-citrate buffer (pH 4.0 to 8.0) and 50 mM phosphate-borate buffer (pH 8.0 to 9.0) at 50 ° C. for 15 minutes. The residual activity of the enzyme is measured. The pH having a value of about 80% or more with the value of the highest enzyme activity under the above conditions (hereinafter abbreviated as “maximum activity”) as the standard (100%) is defined as the optimum pH.
On the other hand, pH stability was obtained after diluting the purified enzyme 10 times in 50 mM phosphate-citrate buffer (pH 4.0-8.0) and 50 mM phosphate-borate buffer (pH 8.0-9.0). The sample is treated at 50 ° C. for 15 minutes, and the remaining enzyme activity is determined and measured. A pH exhibiting an activity of about 80% or more on the basis of the value of the maximum activity under the above conditions (100%) is referred to as a pH exhibiting pH stability or simply a stable pH.

The range of the working temperature, the optimum temperature and the temperature stability of maltose phosphorylase can be measured by the following method or a method equivalent thereto.
The optimum temperature is determined by measuring the enzyme activity under the condition of adding 0.1 ml of purified enzyme to 0.4 ml of 50 mM phosphate buffer (pH 7.0) and reacting at each temperature for 15 minutes. The temperature at which the activity is about 80% or more of the maximum activity under the above conditions is taken as the optimum temperature.
On the other hand, temperature stability is measured by dissolving the purified enzyme in 20 mM phosphate buffer (pH 6.0), treating at various temperatures for 15 minutes and 30 minutes, and then determining the remaining enzyme activity. The temperature at which the enzyme activity value is about 80% or higher with the untreated enzyme activity value as the reference (100%) is referred to as temperature at which temperature stability is exhibited or simply stable temperature.

  The term “inactivation” as used herein refers to the state of an enzyme that exhibits an activity of 50% or less with reference to the untreated enzyme activity (100%) when the enzyme activity before and after treatment is measured. However, in the deactivation range, 30% or less is preferable, 10% or less is more preferable, 5% or less is particularly preferable, and 3% or less is most preferable. In particular, “90% or more inactivated” means an enzyme state exhibiting an activity of 10% or less based on the untreated enzyme activity (100%) when the enzyme activity before and after treatment is measured.

The K _m value of maltose phosphorylase is not particularly limited with respect to the measurement method, but can be measured by, for example, a line Weber-Burk plot.

  The inhibitor of maltose phosphorylase is not particularly limited with respect to the measurement method, and for example, it can be measured by determining enzyme activity in the presence of various compounds.

  The isoelectric point of maltose phosphorylase is not particularly limited with respect to the measurement method, but can be measured, for example, by isoelectrofocusing method isogel (manufactured by FMC BioProduct).

  The molecular weight of maltose phosphorylase is not particularly limited with respect to the measurement method, and can be measured, for example, by gel filtration using Sephacryl S-200.

  As used herein, “modified amino acid sequence” means an amino acid sequence in which asparagine at position 72 and threonine at position 679 are each replaced with another amino acid in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. The method for obtaining the modified amino acid sequence is not limited, and may be synthesized physicochemically, or may be prepared from a polynucleotide encoding the biologically modified amino acid sequence, and is generally known. You may acquire by various screening using a means.

The range of “1 to several” in the “amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids” as used herein is not particularly limited. It means 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably about 1 to 3. In addition, “amino acid deletion” means deletion or disappearance of an amino acid residue in the sequence, and “amino acid substitution” means that an amino acid residue in the sequence is replaced with another amino acid residue, “ “Inverted amino acid” means that the positions of two or more adjacent amino acid residues are reversed, “Addition of amino acid” means that an amino acid residue is added, and “Insertion of amino acid” means sequence. It means that another amino acid residue is inserted between the amino acid residues inside.
In the amino acid sequence, asparagine at position 72 and threonine at position 679 in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing are each substituted with another amino acid, and in addition, one to several amino acids An amino acid sequence having a deletion or the like is meant.

  The homology in the “amino acid sequence including substitution of amino acids at positions 72 and 679 having 70% or more homology with the modified amino acid sequence” as used herein is not particularly limited as long as it is 70% or more. Is 80% or more, more preferably 85% or more, still more preferably 90% or more, and particularly preferably 95% or more. However, it is necessary that the asparagine at position 72 and the threonine at position 679 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing are respectively substituted with amino acids other than asparagine and threonine.

  Specific examples of the modified amino acid sequence described above include asparagine at position 72 and threonine at position 679, isoleucine at position 72, isoleucine at position 679, isoleucine at position 72, valine at position 679, valine at position 72 and position 679, respectively. Isoleucine, or a modified amino acid substituted with valine at position 72 and valine at position 679.

The method for obtaining maltose phosphorylase of the present invention may be a protein synthesized by chemical synthesis in addition to a recombinant protein produced by gene recombination technology.
When producing a recombinant protein, first, a polynucleotide encoding maltose phosphorylase is obtained. By introducing this polynucleotide into an appropriate expression system, the maltose phosphorylase of the present invention can be produced. The expression of the protein in the expression system will be described later in this specification.

(B) Polynucleotide of the Present Invention The polynucleotide of the present invention is a polynucleotide encoding maltose phosphorylase having the amino acid sequence of any of the following (a) to (c).
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at pH 6.0, 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 70% or more homology with the modified amino acid sequence and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at pH 6.0, 65 ° C. for 15 minutes.

A specific example of the polynucleotide of the present invention is a polynucleotide comprising any one of the following base sequences (a) to (c).
(A) the base sequence according to any one of SEQ ID NOs: 2 to 5 in the sequence listing;
(B) deletion, substitution, inversion, addition of one to several bases at positions other than 214 to 216 bases and 2035 to 2037 bases in the base sequence described in any of SEQ ID NOs: 2 to 5 in the sequence listing And / or an amino acid sequence having an insertion and having a maltose phosphorolysis activity having the following properties [a] to [c]:
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at pH 6.0, 65 ° C. for 15 minutes; or
(C) hybridizes with the base sequence described in any one of SEQ ID NOs: 2 to 5 under a stringent condition, and 214 to 216 bases in the base sequence described in any one of SEQ ID NOs: 2 to 5; An amino acid sequence having a base complementary to 2035 to 2037 and having a maltose phosphorolysis activity having the following properties [a] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at pH 6.0, 65 ° C. for 15 minutes.

The range of “1 to several” in the “base sequence having deletion, substitution, inversion, addition and / or insertion of one to several bases” as used herein is not particularly limited. To 40, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably about 1 to 3. Also, “base deletion” means the loss or disappearance of a base in the sequence, “base replacement” means that the base in the sequence is replaced with another base, “base inversion” Indicates that the positions of two or more adjacent bases are reversed, “adding a base” means adding a base, and “inserting a base” means that another base is inserted between the bases in the sequence. It means that it is inserted.
In the “base sequence having deletion, substitution, inversion, addition and / or insertion of 1 to several bases”, 214 to 216 bases and 2035 in the base sequences described in SEQ ID NOs: 2 to 5 in the Sequence Listing. Has deletions, substitutions, inversions, additions and / or insertions for bases other than ~ 2037 bases.

As used herein, “hybridizes under stringent conditions” means DNA obtained by using a DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. After performing hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, for example, Examples include DNA that can be identified by washing a filter at 65 ° C. using a 0.1 to 2 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Hybridization, Molecular Cloning:.. A laboratory Mannual, 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 ( hereinafter, abbreviated as Molecular Cloning 2nd Edition), or, Current Protocols in Molecular Biology, Supplement 1 ~ 38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as "Current Protocols in Molecular Biology") and the like.

Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 70% or more, preferably 80% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.
However, the base sequence to hybridize necessarily includes 214 to 216 bases and 2035 to 2037 bases in the base sequences described in SEQ ID NOs: 2 to 5 in the Sequence Listing.

  The method for obtaining the polynucleotide of the present invention is not particularly limited. For example, appropriate probes and primers are prepared based on the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing in this specification and the base sequence information shown in SEQ ID NOs: 2 to 5, and using them, Paenibacillus sp. The gene of the present invention can be isolated by screening a chromosomal DNA library derived from SH-55. The chromosomal DNA library can be prepared by introducing a mutation into a maltose phosphorylase gene derived from Paenibacillus sp. SH-55 expressing the wild type of the maltose phosphorylase of the present invention by a generally known method.

  If the above chromosomal DNA library can be obtained, the polynucleotide of the present invention can also be obtained by PCR. Using the chromosomal DNA library as a template, PCR is carried out using a pair of primers designed so that the base sequences described in SEQ ID NOs: 2 to 5 can be amplified. PCR reaction conditions can be appropriately set, and include, for example, 94 ° C. for 15 seconds to 30 seconds (denaturation), 63 ° C. for 30 seconds to 1 minute (annealing), and 68 ° C. for 2-3 minutes (extension). One example of the reaction step is 50 cycles, followed by a reaction at 72 ° C. for 10 minutes. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

  Operations such as the preparation of the probe or primer, construction of a chromosomal DNA library, screening of a cDNA library, and cloning of a target gene are known to those skilled in the art. For example, Molecular Cloning 2nd Edition, or Current Protocols -It can be performed according to the method described in In-Molecular Biology etc.

  In addition, the maltose phosphorylase and polynucleotide of the present invention are derived from the maltose phosphorylase gene derived from Paenibacillus sp. SH-55 expressing the wild type of the maltose phosphorylase of the present invention. Can be prepared by any method commonly known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, or mutagenesis.

  For example, a method of contacting a maltose phosphorylase gene derived from Paenibacillus sp. SH-55 expressing a wild type of maltose phosphorylase with a mutagen agent, a method of irradiating ultraviolet rays, a genetic engineering method, etc. Can be done. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce mutations at target positions. Saturation mutagenesis, Molecular Cloning 2nd edition and Current Protocols It can be performed according to the method described in in-molecular biology and the like.

  Even if the polynucleotide of the present invention consists of any nucleotide sequence, chemical synthesis, genetic engineering technique or mutagenesis is performed based on the modified amino acid sequence and the nucleotide sequence information shown in SEQ ID NOs: 2 to 5 in the sequence listing. It can be prepared by any method commonly known to those skilled in the art.

  For example, using a method in which a polynucleotide having any one of the sequence numbers 2 to 5 in the sequence listing is brought into contact with a mutagen agent, a method of irradiating ultraviolet rays, a genetic engineering method, etc. It can be carried out. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce mutations at target positions. Molecular cloning 2nd edition, Current Protocols in Molecular Bio It can be carried out according to the method described in LOGI.

(C) Recombinant vector of the present invention The gene of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited, and may be, for example, a vector that can replicate autonomously (for example, a plasmid), or may be integrated into the genome of a host cell when introduced into the host cell. And may be replicated with the integrated chromosome. Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.

  Specific examples of vectors that can replicate autonomously include plasmid vectors such as pRSETA, pCR8, pBR322, pBluescriptIISK (+), pUC18, pCR2.1, pLEX, pJL3, pSW1, pSE280, pSE420, and pHY300PLK. And phage vectors such as λgt11, λZAP, etc., but pRSETA, pCR8, pBR322, pBluescriptII SK (+), pUC18, pKK223-3, and pCR2.1 are preferred for expression in E. coli. For expression, pHY300PLK is preferred.

As a promoter which can operate in bacterial cells, Bacillus stearothermophilus maltogenic amylase gene (Bacillus stearothermophilus maltogenic amylase gene), Bacillus licheniformis α-amylase gene (Bacillus licheniformis alpha-amylase gene) , Bacillus Amirorikefachi Enns · BAN amylase gene (Bacillus amyloliquefaciens BAN amylase gene), Bacillus subtilis alkaline protease gene (Bacillus subtilis alkaline protease gene) or Bacillus pumilus-xylosidase gene (Baci lus pumilus xylosidase gene) promoter or phage lambda P _R or P _L promoters,, lac of E.coli, such as trp or tac promoter and the like. Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene. There are promoters. Examples of promoters operable in yeast host cells include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, TPI1 promoters, ADH2-4c promoters, and the like. Examples of promoters that can operate in filamentous fungal cells include the ADH3 promoter or the tpi A promoter.

  The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, genes whose complement is lacking in host cells such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin. Methods of ligating the gene of the present invention, promoter, and, if desired, terminator, enhancer and / or secretory signal sequence and inserting them into an appropriate vector are generally known to those skilled in the art.

(D) Transformant of the present invention A transformant can be prepared by introducing the polynucleotide or recombinant vector of the present invention into a suitable host.

  That is, the transformant of the present invention is a transformant obtained by introducing the polynucleotide of the present invention or containing the recombinant vector of the present invention.

  As used herein, “introducing a polynucleotide” in “introducing a polynucleotide” refers to introducing the polynucleotide of the present invention into a host cell by bacteriophage or the like, preferably the polynucleotide of the present invention. Is integrated into the chromosomal DNA of the host cell.

  The host cell into which the polynucleotide or recombinant vector of the present invention is introduced may be any host cell that does not produce maltase under the culture conditions of the transformant in the production of maltose phosphorylase of the present invention. Bacteria, yeast, fungi and higher eukaryotes Examples thereof include cells.

Examples of bacterial cells include gram positive bacteria such as Bacillus subtilis or actinomycetes or gram negative bacteria such as E. coli. Transformation of these bacteria may be performed by a protoplast method, an electroporation method, a method using calcium ions, or the like.
Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells. A method of transforming a mammalian cell and expressing a DNA sequence introduced into the cell is also known. For example, an electroporation method, a calcium phosphate method, a lipofection method, or the like can be used.

Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevisiae or Saccharomyces kluyveri . Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

  Examples of other fungal cells are cells belonging to filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be carried out according to a generally known method, for example, by homologous recombination or heterologous recombination.

  When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. The protein can be expressed.

As the baculovirus, for example, Autographa californica nucleopolyhedrosis virus, which is a virus that infects Coleoptera insects, can be used. As the insect cells, can be used is an ovarian cell of Trichoplusia ni HiFive (Invitrogen) and the like. Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include a calcium phosphate method and a lipofection method.

(E) Production of recombinant protein using the transformant of the present invention The transformant of the present invention is inoculated and cultured in an appropriate medium according to a conventional method, and the maltose phosphorylase of the present invention is collected from the culture. And a method for producing recombinant maltose phosphorylase.

That is, the method for producing maltose phosphorylase of the present invention is a method for producing maltose phosphorylase having the following steps (a) to (c).
(A) a step of culturing the transformant of the present invention;
(B) Step of producing and accumulating maltose phosphorylase of the present invention; and (c) Step of collecting the maltose phosphorylase:

  As a nutrient medium used for culturing the transformant of the present invention, a natural medium or a synthetic medium may be used as long as the medium contains a carbon source, a nitrogen source, an inorganic substance, and if necessary, micronutrients required by the strain used. Either is acceptable.

  The carbon source of the nutrient medium used for culturing the transformant of the present invention may be any material that can be assimilated by the transformant, such as glucose, maltose, fructose, mannose, trehalose, sucrose, mannitol, sorbitol, Sugars such as starch, dextrin, molasses, or organic acids such as citric acid and succinic acid, or fatty acids such as glycerin can also be used.

  As a nitrogen source of the nutrient medium used for culturing the transformant of the present invention, various organic and inorganic nitrogen compounds, and the medium may contain various inorganic salts. For example, compounds such as organic nitrogen sources such as corn steep liquor, soybean meal or various peptones, and inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, and ammonium phosphate can be used. Needless to say, amino acids such as glutamic acid and organic nitrogen sources such as urea also serve as carbon sources. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, bactopeptone, meat extract, yeast extract, corn steep liquor, soy flour, soybean meal, dried yeast, casamino acid, soluble vegetable protein and the like can also be used as a nitrogen source.

  As an inorganic substance of the nutrient medium used for culturing the transformant of the present invention, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, A cobalt salt or the like is appropriately used. Specifically, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium chloride, potassium chloride, calcium chloride and the like are used. Further, amino acids and micronutrient vitamins such as biotin and thiamine are also used as necessary.

  As a culture method, a liquid culture method (shaking culture method or aeration stirring culture method) is preferable, and the aeration stirring culture method is preferable industrially. The culture temperature and pH may be selected under conditions most suitable for the growth of the transformant to be used. For example, culture when the transformant is a microorganism is usually aerobically performed under conditions selected from a temperature of 20 to 45 ° C., preferably 25 to 42 ° C., pH 5 to 9, preferably 6 to 8. The culture time may be a time longer than the time when the microorganism starts to grow, preferably 8 to 120 hours, and more preferably the time until the maltose phosphorylase of the present invention is produced to the maximum. The method for confirming the growth of microorganisms is not particularly limited. For example, the culture may be collected and observed with a microscope, or may be observed with absorbance. Moreover, although there is no restriction | limiting in particular in the dissolved oxygen concentration of a culture solution, Usually, 0.5-20 ppm is preferable. For that purpose, it is only necessary to adjust the aeration amount, stir, or add oxygen to the aeration. The culture method may be any of batch culture, fed-batch culture, continuous culture or perfusion culture.

  In the culture of the transformant of the present invention, when a selection marker is contained in the recombinant vector, an antibiotic corresponding to the selection marker is added together with a nutrient medium. For example, when an ampicillin resistance gene and a chloramphenicol resistance gene are contained as selection markers, an ampicillin solution and a chloramphenicol solution prepared at appropriate concentrations are added, respectively. If necessary, an expression inducer that induces the expression of the gene having the polynucleotide of the present invention is added at the start of the culture or after confirming the growth of the transformant from the start of the culture. For example, isopropyl-β-D-thiogalactopyranoside (IPTG) is used as an expression inducer.

  The maltose phosphorylase of the present invention is collected from the culture thus obtained. Depending on the type of transformant of the present invention, the maltose phosphorylase is accumulated inside and outside the cell. Therefore, maltose phosphorylase produced and accumulated inside or outside the cell is collected.

  The method for collecting maltose phosphorylase of the present invention can be carried out according to a general means for collecting enzymes. When the maltose phosphorylase is produced and accumulated extracellularly, the culture supernatant can be used as a crude enzyme after removing the cells by a generally known means. When it is produced and accumulated in cells, it is not particularly limited to the following methods. For example, a method of lysing cells with an enzyme such as an organic solvent or lysozyme, an ultrasonic crushing method, a French press method, a glass A cell disruption product and / or culture obtained by a cell disruption method such as a bead disruption method or a dynomill disruption method is separated into cells and a culture supernatant by an operation such as a centrifugal separation method or a filtration method. The culture supernatant thus obtained can be used as a crude enzyme. In addition, the separated cells can be used as a crude enzyme of maltose phosphorylase as it is.

  The crude enzyme can be used as it is, but if necessary, it can be concentrated for industrial use by combining a known method such as a salting-out method, a precipitation method, a dialysis method, and an ultrafiltration method alone or in combination. Enzymes can be prepared.

  Further, the concentrated enzyme is generally known, for example, ion exchange chromatography, isoelectric point chromatography, hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, reverse phase chromatography, resin column method and the like. A purified enzyme can be obtained by subjecting it to a combination of isolation and purification methods.

(F) Process for producing β-glucose-1-phosphate or trehalose of the present invention In the presence of the maltose phosphorylase of the present invention, the transformant of the present invention (for example, a microorganism) or a culture thereof, a substrate (for example, maltose) ) To react to synthesize β-glucose-1-phosphate, or another product (eg trehalose) from the substrate by combining with other enzymes (eg trehalose phosphorylase). it can. The above synthesis method is also included in the scope of the present invention.

  That is, the method for producing β-glucose-1-phosphate of the present invention is characterized in that β-glucose-1-phosphate is obtained by allowing the maltose phosphorylase of the present invention to act on maltose in the presence of phosphoric acid. This is a method for producing β-glucose-1-phosphate.

The method for producing trehalose of the present invention is a method for producing trehalose characterized in that trehalose is obtained by allowing the maltose phosphorylase and trehalose phosphorylase of the present invention to act on maltose in the presence of phosphoric acid. Preferably, the trehalose phosphorylase is characterized by having the following properties (a) to (d), and most preferably trehalose phosphorylase having the following properties (a) to (d): .
(A) Catalytic action: reversibly phosphorolytically decomposes the α-1,1-glucopyranoside bond in trehalose to produce glucose and β-glucose-1-phosphate;
(B) Optimum pH and stable pH range: The optimum pH for the trehalose synthesis reaction is 5.8 to 7.8, and is stable within the range of pH 5.5 to 9.5 under heating conditions at 50 ° C. for 10 minutes. is there;
(C) Optimal temperature: The optimal temperature for the trehalose synthesis reaction is 45-60 ° C; and (d) Temperature stability: pH 7.0, stable to 60 ° C under heating conditions for 15 minutes, % Inactivated.
In the production of the trehalose of the present invention, specific examples of the trehalose phosphorylase used together with the maltose phosphorylase of the present invention include trehalose phosphorylase having the properties (a) to (d) described in Patent Document 3. .

  As used herein, “trehalose phosphorylase” is an enzyme having an activity of reversibly catalyzing in a reaction for producing trehalose and phosphate from glucose and β-glucose-1-phosphate. The catalytic action, optimum pH and stable pH range, optimum temperature and temperature stability of the trehalose phosphorylase are measured by a generally known method as described in Patent Document 3, for example.

  For example, in the presence of the maltose phosphorylase of the present invention and trehalose phosphorylase having the properties (a) to (d) described above, maltose and phosphoric acid or phosphate are reacted in an aqueous medium to give trehalose and / or Alternatively, β-glucose-1-phosphate is produced.

  The maltose phosphorylase of the present invention can be used as a solid or liquid crude enzyme and / or purified enzyme. However, the crude enzyme needs to be a crude enzyme that does not contain an enzyme that adversely affects the production of trehalose such as maltase. Furthermore, the production of trehalose and / or β-glucose-1-phosphate using a cell having the activity of the maltose phosphorylase and an immobilized cell obtained by encapsulating, adsorbing or chemically binding the cell with an appropriate carrier Can be used for Further, the maltose phosphorylase can also be used as an immobilized enzyme that is immobilized by a generally known method.

  Further, in the production of trehalose and / or β-glucose-1-phosphate of the present invention, the maltose phosphorylase of the present invention is preferably used in combination with trehalose phosphorylase having the properties (a) to (d) described above. However, the nature of trehalose phosphorylase is, for example, the optimum pH in the trehalose synthesis reaction is between 6.5 and 7.5, stable within the range of pH 5.0 to 8.5, and optimum at around 50 to 60 ° C. If it has a temperature and is extremely stable up to 58 ° C., it can be used in combination with the maltose phosphorylase of the present invention.

  The trehalose phosphorylase used for the production of trehalose and / or β-glucose-1-phosphate of the present invention may be a purified enzyme or a crude enzyme in the same manner as the maltose phosphorylase of the present invention. The enzyme may be immobilized and used.

  As maltose, maltose or a substance containing maltose (for example, a maltose-rich sugar solution) can be used. As the phosphate, water-soluble phosphates such as tripotassium phosphate (or sodium), dipotassium hydrogen phosphate (or sodium), and potassium dihydrogen phosphate (or sodium) can be used. Examples of the aqueous medium include water and a buffer solution. As the buffer solution, an acetate buffer solution, a phosphate buffer solution, a citrate buffer solution, a succinate buffer solution, a Tris-HCl buffer solution, or the like can be used.

  Although there is no restriction | limiting in particular about the usage-amount of an enzyme, It is suitable to use 0.1-50 units for each enzyme with respect to 1 g of maltose, Preferably it is 1-20 units. The ratio of use of maltose phosphorylase and trehalose phosphorylase of the present invention is not particularly limited, but the ratio of the former is preferably 1: 5 to 5: 1, preferably 1: 2 to 2: 1. is there.

  Phosphoric acid and / or phosphate is not particularly limited with respect to maltose, but it is appropriate to use 0.001 to 1 mol, preferably 0.005 to 0.5 mol. When the buffer solution contains phosphoric acid (salt), the total amount of phosphoric acid and phosphate in the system may be in the above range.

  The reaction is preferably performed at 50 to 60 ° C., more preferably 55 to 60 ° C., and still more preferably 55 to 58 ° C. in order to further avoid the temperature and contamination with bacteria and increase the yield. The pH is generally 6.0 to 8.0, preferably 6.5 to 7.5. The reaction is completed when sufficient trehalose production is observed under the above conditions, but the reaction is usually completed in 1 to 144 hours.

  After completion of the reaction, the reaction solution is heated to 60 to 135 ° C., preferably 65 to 100 ° C. to inactivate the enzyme, or the enzyme is inactivated by appropriate means such as pH reduction (addition of acid such as hydrochloric acid). To stop the reaction. Thereafter, trehalose can be obtained by appropriately combining the reaction stopping solution with isolation / purification means such as activated carbon treatment and ion exchange resin treatment.

  In the present invention, β-glucose-1-phosphate can also be produced by reacting maltose with phosphoric acid or phosphate in the presence of the maltose phosphorylase of the present invention in an aqueous medium.

  The maltose phosphorylase, maltose and aqueous medium of the present invention used for the reaction can be carried out in the same manner as in the production of trehalose. The enzyme is 0.1 to 50 units, preferably 1 to 20 units, based on 1 g of maltose.

The reaction temperature, reaction pH, and reaction time can be carried out in the same manner as in the production of trehalose. After completion of the reaction, β-glucose-1-phosphate can be obtained by appropriately combining isolation / purification means such as ion exchange resin treatment.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[Example 1] Acquisition of thermostable maltose phosphorylase (1) Construction of mutant gene library Recombination containing a maltose phosphorylase gene derived from Paenibacillus sp. SH-55 of deposit number FERM BP-8420 ( Paenibacillus sp. SH-55) The body plasmid pRSMP1 (described in International Publication No. WO2005 / 00343) was extracted from E. coli DH5α and purified by using GFX Plasmid prep Kit (GE Healthcare Biosciences). Using the obtained recombinant plasmid as a template, using the primer shown in SEQ ID NO: 6 and SEQ ID NO: 7 in the sequence listing, using Diversity PCR Random Mutagenesis Kit (Clontech) according to the protocol of the kit, maltose phosphorylase A random mutation was introduced into the gene. The resulting 2.3 k base pair DNA fragment containing the mutated maltose phosphorylase was purified and then cleaved with restriction enzymes Xho I and Kpn I. This and plasmid vector pRSETA (Invitrogen) cleaved with restriction enzymes Xho I and Kpn I were ligated for 5 minutes at room temperature using Quick Ligation Kit (NEB), and then E. coli BL21 (DE3) pLysS (F ^{-, ompT hsdSB (rB - mB} -) by performing transformation into gal dcm (DE3) pLysS (Cam R)) strain, were constructed recombinant E. coli library with the introduced recombinant plasmid random mutations.

(2) Screening for thermostable maltose phosphorylase The transformant obtained by the mutation operation of (1) above was subjected to LB-Amp-Cm agar medium (1% bactopeptone, 0.5% yeast extract, 1% sodium chloride, Bactagar 1.5%, ampicillin 50 μg / ml, chloramphenicol 35 μg / ml) were cultured overnight at 37 ° C. to form colonies. Among each colony, 1 ml of LB-Amp-Cm medium (1% each of LB-Amp-Cm medium (bactopeptone 1%, yeast extract 0.5%, sodium chloride 1%, ampicillin 50 μg / ml, chloramphenicol 35 μg / ml)) The 96-well deep well plate was inoculated and cultured with shaking at 25 ° C. for 8 hours. Thereafter, 10 μl of 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to each well, and further cultured with shaking at 25 ° C. for 16 hours. After completion of the culture, the culture solution was centrifuged at 3000 rpm for 10 minutes to obtain bacterial cells. 100 μl of BugBuster (Novagen) was added to each well of the obtained cells, stirred at room temperature for 20 minutes, and then centrifuged at 3000 rpm for 10 minutes to obtain a crude enzyme extract. The obtained crude enzyme extract was subjected to a heat treatment at 55 ° C. for 15 minutes. As a control, the same operation was performed for a transformant (RSMP1) having a plasmid (pRSMP1) into which no random mutation was introduced. About the obtained enzyme liquid, the activity measurement of the maltose phosphorylase before and behind heat processing was performed.
The activity was measured by adding 0.1 ml of enzyme solution to 0.4 ml of 20 mM maltose dissolved in 200 mM phosphate buffer (pH 7.0), reacting at 50 ° C. for 15 minutes, and then in a boiling water bath for 3 minutes. The enzyme reaction was stopped by heating. After cooling in ice water, the produced glucose was measured by the glucose oxidase method (Wako Pure Chemical Industries, Ltd., Glucose C-II Test Wako). Here, 1 unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of glucose per minute under the same conditions.
Mutant enzymes with higher residual activity than the control were selected. As a result, 6 strains (RSMP2-7) had a high residual activity.

(3) Analysis of mutation site The following analysis was performed to confirm which part of the gene was replaced by the mutation operation for the maltose phosphorylase gene contained in the excellent mutant obtained as described above. went. Plasmid pRSMP2-7 contained in recombinant RSMP2-7 obtained in (2) above was purified using GFX Plasmid prep Kit. The base sequence of this plasmid was determined using MegaBACE 1000 (GE Healthcare Bioscience). The results are shown in Table 1.

(4) Measurement of residual activity at 55 ° C. The crude enzyme solution of RSMP1-7 obtained in (2) above was allowed to stand in a constant temperature bath at 55 ° C. for 30 minutes, and then left on ice for enzyme activity (residual activity). ) The measured results are shown in Table 2.

[Example 2] Preparation and evaluation of point mutants based on RSMP3 Three types of point mutants were prepared in which the three mutant amino acid residues of RSMP3 (N72I, V127I, D308E) were made one by one. PRSMP8 (N72I) in which the asparagine at the 72nd amino acid residue was replaced with isoleucine, pRSMP9 (V127I) in which the valine at the 127th amino acid residue was replaced with isoleucine, and the aspartic acid at the 308th amino acid residue were replaced with glutamic acid pRSMP10 (D308E) was performed using a site-directed mutagenesis method, which is a commonly known technique.
More specifically, pRSMP1 is used as a template plasmid for introducing a mutation, and three kinds of sense primers designed to cause a predetermined mutation: FWN72I (SEQ ID NO: 8), FWV127I (SEQ ID NO: 9), FWD308E (SEQ ID NO: 9) 10) and three types of antisense primers RVN72I (SEQ ID NO: 11), RVV127I (SEQ ID NO: 12), and RVD308E (SEQ ID NO: 13), and FWN72I and RVN72I, FWV127I and RVV127I, and FWD308E and RV308E were used as one set, respectively. .

PCR with Pfu polymerase was performed according to QuickChange Site-Directed Mutagenesis Kit (Stratagene) using the above-mentioned primer pair and pRSMP1, and then treated with Dpn I at 37 ° C. for 1 hour. This Dpn I-treated PCR product was transformed into Escherichia coli XL1-Blue and cultured overnight at 37 ° C. on an LB-Amp agar medium. The obtained colony was cultured overnight at 37 ° C. in LB-Amp medium, and extracted and purified by using GFX Plasmid prep Kit. Finally, the base sequence of the obtained plasmid was determined, and it was confirmed that the target mutation was introduced. In this way, a point mutant pRSMP8-10 was obtained.
Recombinant E. coli RSMP8-10 was obtained by transforming pRSMP8-10 into E. coli BL21 (DE3) pLysS strain. The obtained recombinant was examined for heat resistance in the same manner as in Example 1 (4). As a comparative control, the same operation was performed for a transformant (RSMP1) having a plasmid (pRSMP1) into which no mutation was introduced and a transformant (RSMP3) having a triple mutant (pRSMP3). The results are shown in Table 3.

[Example 3] Preparation and evaluation of pRSMP11 mutant pRSMP11 obtained by multiplying pRSMP5 of Example 1 and pRSMP8 prepared in Example 2 was prepared. Specifically, mutants were prepared by site-directed mutagenesis as in Example 2 using pRSMP5 as a template and FWN72I and RVN72I as primers. The obtained mutant pRSMP11 was transformed into E. coli BL21 (DE3) pLysS strain to obtain recombinant E. coli RSMP11. The obtained recombinant was examined for heat resistance in the same manner as in Example 1 (4). As a result, the residual activity was 94.6%.

[Example 4] Saturation mutation at amino acids 72 and 679 Saturation mutation was performed on amino acids 72 and 679. Two types of sense primers designed to cause a predetermined mutation: FWN72X (SEQ ID NO: 14), FWT679X (SEQ ID NO: 15) and two types of antisense primers; RVN72X (SEQ ID NO: 16) and RVT679X (SEQ ID NO: 17) Using FWN72X and RVN72X, FWT679X and RVT679X as a set, and pRSMP1 as a template, the operation was performed according to the usual method of Quickchange Site-Directed Mutagenesis Kit. The plasmid of the obtained transformant was extracted using GFX Plasmid prep Kit, and transformed into E. coli BL21 (DE3) pLysS strain. For each of the 390 clones, the same operation as in Example 1 (2) (3) was performed. As a result, 11 mutants with higher residual activity than the control were obtained at position 72. The amino acid sequence was confirmed to be 8 isoleucine (ATT) and 3 valine (GTT). It was. Similarly, at position 679, 27 were obtained, and when the amino acid sequence was confirmed, the breakdown was 3 isoleucine (ATT) and 24 valines (GTG, GTT). Therefore, using a site-specific mutation, a mutant in which position 72 is valine and 679 is replaced with isoleucine (RSMP12), position 72 is isoleucine, position 679 is replaced with valine (RSMP13), and position 72 is valine. , A mutant (RSMP14) in which position 679 was substituted with valine was prepared.

[Example 5] Expression of mutant enzyme and preparation of enzyme solution The maltose phosphorylase mutant RSMP11-14 obtained in Examples 3 and 4 was cultured, and a recombinant enzyme was prepared using Escherichia coli. The culture is sterilized by adding 0.5 L of LB medium (bactopeptone 1%, yeast extract 0.5%, sodium chloride 1%) to a 2 L Erlenmeyer flask, and then the ampicillin solution at a final concentration of 50 μg / ml, chloram. The phenicol solution was added to a final concentration of 35 μg / ml, the seed culture was inoculated to 1%, and cultured at a temperature of 37 ° C. and 180 rpm until OD600 was 0.5. Thereafter, IPTG was added to a final concentration of 1 mM, and further cultured for 3 hours. After completion of the culture, the culture solution was centrifuged at 5000 g for 10 minutes to obtain bacterial cells. 20 ml of BugBuster was added to the resulting Escherichia coli, and the mixture was stirred at room temperature for 20 minutes, followed by centrifugation at 12000 g for 15 minutes. The obtained crude extract was subjected to chromatography (washing, 5 mM imidazole; elution, 150 mM imidazole) using an affinity column (TALON Resin, manufactured by Clontech). Fractions showing maltose phosphorylase activity are collected and applied to a HiPrep 26/10 Desalting column (GE Healthcare Biosciences) equilibrated with 50 mM phosphate buffer and 0.15 M NaCl (pH 7.0) to remove imidazole. And subjected to gel filtration chromatography HiLoad 16/10 Superdex 200 column (GE Healthcare Bioscience Co., Ltd.) equilibrated with the same buffer, and maltose phosphorylase was purified to homogeneity to obtain an enzyme solution. .
As a control, RSMP1 was cultured and purified as described above.

[Example 6] Measurement of half-life of enzyme The enzyme solution of the present invention obtained by the method of Example 5 was allowed to stand in a constant temperature bath at 55 ° C for a certain period of time and then left on ice to determine the enzyme activity (residual activity). It was measured. The half-life of the enzyme is shown in Table 4, and the time course of residual activity is shown in FIG.

[Example 7] Properties of thermostable maltose phosphorylase Using the enzyme solution obtained in Example 5, various properties of thermostable maltose phosphorylase RSMP13 were examined.
(1) Optimum pH and pH stability The optimal pH is 50 mM phosphate-citrate buffer (pH 4.0-8.0), 50 mM phosphate-borate buffer (pH 8.0-9.0). The activity of this enzyme at each pH was measured. The results are as shown in FIG. 2, and the optimum pH of this enzyme was around 6.5 to 7.5.
The pH stability was determined after treatment with 50 mM phosphate-citrate buffer (pH 4.0-8.0) and 50 mM phosphate-borate buffer (pH 8.0-9.0) at 50 ° C. for 15 minutes, respectively. This was determined by measuring the residual activity of the enzyme. The results are as shown in FIG. 3, and the enzyme was stable in the range of pH 5.0 to 8.5.
(2) Optimum temperature As shown in FIG. 4, the optimum temperature was around 50 to 60 ° C. under the reaction conditions of 50 mM phosphate buffer (pH 7.0) for 15 minutes.
(3) Temperature stability The enzyme was measured by treating the enzyme with 20 mM phosphate buffer (pH 6.0) at various temperatures for 15 to 30 minutes, and then determining the residual activity by a conventional method. The remaining activities at 58 ° C., 60 ° C., and 65 ° C. after 15 minutes were 85.3%, 62.5%, and 0%, respectively. The results after 30 minutes of treatment are as shown in FIG. It had about 80% residual activity.
(4) Deactivation 90% or more was deactivated by treatment at 70 ° C. for 10 minutes.
(5) K _m value by the plot of K _m value Lineweaver-Burk, relative to the phosphoric acid 5.9 mM for maltose was 4.4 mM. On the other hand, the K _m value of maltose phosphorylase RSMP1 (corresponding wild-type maltose phosphorylase) derived from a wild strain measured as a control was 16.7 mM for phosphate and 5.7 mM for maltose. In this regard, FIG. 6 is a plot of the time course of maltose-degrading activity in RSMP13 and wild-type maltose phosphorylase RSMP1.

[Example 8] Production of β-glucose-1-phosphate using thermostable maltose phosphorylase 1 L of crystalline maltose dissolved in 50 mM phosphate buffer (pH 7.2) to a concentration of 10% (w / w) After maintaining the maltose solution at 60 ° C. for 30 minutes, the thermostable maltose phosphorylase (111 units) obtained in Example 5 was added and reacted for 40 hours. The reaction was stopped by maintaining at 100 ° C. for 15 minutes after adjusting to pH 4.5 with 1N hydrochloric acid. The supernatant liquid from which the insoluble fraction was removed by centrifugation was passed through 400 ml of a weakly basic anion exchange resin (Amberlite IRA-68) equilibrated with 20 mM acetate buffer (pH 4.5), and then the resin. Was washed with 10 times the amount of the same buffer as above to elute neutral sugars. Next, the adsorbed fraction is eluted with a 0.2 M sodium chloride aqueous solution, and the fraction is desalted by electrodialysis, further concentrated and freeze-dried, and finally 1.6 g of β-glucose-1 -Phosphoric acid was obtained.

[Example 9] Production of trehalose using thermostable maltose phosphorylase Maltose obtained in Example 5 in a 25% (w / w) maltose solution dissolved in 10 ml of 10 mM phosphate buffer (pH 6.5) 5 units of phosphorylase per gram of substrate weight and 10 units of trehalose phosphorylase described in International Publication No. WO2005 / 00343 per 10 g of substrate weight were added and reacted at 55 ° C. for 48 hours. After completion of the reaction, the trehalose content in the saccharified solution obtained by inactivating the enzyme by heating at 100 ° C. for 5 minutes was measured. As a result, 65% trehalose was generated with respect to the weight of the saccharified solution solid substrate.
Trehalose was quantified by the following method.
After adding water to the heat-inactivated saccharified solution to make it about 5% (w / w), 0.5 ml of the saccharified solution was added glucoamylase (Seikagaku Corporation) 70 units / ml 0.5 ml, The mixture was reacted at 55 ° C. and pH 5.0 for 30 minutes to completely decompose unreacted maltose into glucose. Next, after heating at 100 ° C. for 10 minutes to completely deactivate glucoamylase, the content of trehalose produced was measured by high performance liquid chromatography using a High-performance Carbohydrate column (manufactured by Waters). For measurement, acetonitrile / water (75/25) was used as an eluent, column temperature was 40 ° C., and a differential refractometer was used for detection.

Residual activity of maltose phosphorylase of the present invention Optimal pH of the maltose phosphorylase of the present invention PH stability of the maltose phosphorylase of the present invention Optimal temperature of the maltose phosphorylase of the present invention Temperature stability of maltose phosphorylase of the present invention Reaction time of maltose phosphorylase of the present invention

Claims (14)

The maltose phosphorylase which has an amino acid sequence in any one of the following (a)-(c).
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 90% or more homology with the modified amino acid sequence, and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes. The maltose phosphorylase according to claim 1, wherein the modified amino acid sequence has an amino acid sequence in which asparagine at position 72 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with isoleucine and threonine at position 679 is substituted with isoleucine. 2. The maltose phosphorylase according to claim 1, wherein the modified amino acid sequence has an amino acid sequence in which asparagine at position 72 in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing is substituted with isoleucine and threonine at position 679 is substituted with valine. The maltose phosphorylase according to claim 1, wherein the modified amino acid sequence has an amino acid sequence in which asparagine at position 72 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with valine and threonine at position 679 is substituted with isoleucine. The maltose according to claim 1, wherein the modified amino acid sequence has an amino acid sequence in which asparagine at position 72 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with valine and threonine at position 679 is substituted with valine, respectively. Phosphorylase. A polynucleotide encoding maltose phosphorylase comprising any one of the following amino acid sequences (a) to (c):
(A) a modified amino acid sequence in which asparagine at position 72 is replaced with isoleucine or valine and threonine at position 679 is replaced with isoleucine or valine in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) in the modified amino acid sequence, comprising the amino acid sequence having deletion, substitution, inversion, addition and / or insertion of one to several amino acids in addition to the substitution of the amino acids at positions 72 and 679, An amino acid sequence having maltose phosphorolysis activity having the properties [i] to [c],
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes; or
(C) a maltose phosphorolysis reaction comprising the amino acid sequence including substitution of the amino acids at positions 72 and 679 having 90% or more homology with the modified amino acid sequence, and having the following properties [a] to [c] An amino acid sequence having activity,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes. A polynucleotide comprising any one of the following base sequences (a) to (c):
(A) the base sequence according to any one of SEQ ID NOs: 2 to 5 in the sequence listing;
(B) deletion, substitution, inversion, addition of one to several bases at positions other than 214 to 216 bases and 2035 to 2037 bases in the base sequence described in any of SEQ ID NOs: 2 to 5 in the sequence listing And / or a base sequence having an insertion, the base sequence encoding maltose phosphorylase having the following properties [a] to [c]:
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes; or
(C) deletion, substitution, inversion, addition and / or insertion of a base other than 214 to 216 bases and 2035 to 2037 bases in the base sequence of any one of SEQ ID NOs: 2 to 5 a nucleotide sequence having a having a nucleotide sequence homology of 90% or more of any one of SEQ ID NO: 2-5 of the sequence Listing, the maltose phosphorylase properties under SL [i] - [c] Base sequence to encode,
[A] The optimum temperature is 50 to 60 ° C.
[B] Stable up to 58 ° C under heating conditions of pH 6.0 and 30 minutes,
[C] Deactivate 90% or more by heat treatment at 65 ° C. for 15 minutes. A recombinant vector containing the polynucleotide according to claim 6 or 7. A transformant obtained by introducing the polynucleotide according to claim 6 or 7, or containing the recombinant vector according to claim 8. The manufacturing method of the maltose phosphorylase of any one of Claims 1-5 which has the process of following (a)-(c).
(A) a step of culturing the transformant according to claim 9;
(B) production / accumulation step of the maltose phosphorylase; and (c) collection step of the maltose phosphorylase: The method for producing maltose phosphorylase according to claim 10, wherein in the collecting step, a transformant is separated from the culture, and extracted from the separated transformant to obtain a crude enzyme of maltose phosphorylase. A β-glucose-1-phosphate obtained by allowing the maltose phosphorylase according to any one of claims 1 to 5 to act on maltose in the presence of phosphoric acid to obtain β-glucose-1-phosphate Acid production method. A method for producing trehalose, wherein trehalose is obtained by allowing the maltose phosphorylase and trehalose phosphorylase according to any one of claims 1 to 5 to act on maltose in the presence of phosphoric acid. The method for producing trehalose according to claim 13 , wherein the trehalose phosphorylase is a trehalose phosphorylase having the following properties (a) to (d):
(A) Catalytic action: reversibly phosphorolytically decomposes the α-1,1-glucopyranoside bond in trehalose to produce glucose and β-glucose-1-phosphate;
(B) Optimum pH and stable pH range: The optimum pH for the trehalose synthesis reaction is 5.8 to 7.8, and is stable within the range of pH 5.5 to 9.5 under heating conditions at 50 ° C. for 10 minutes. is there;
(C) Optimum temperature: The optimum temperature for the trehalose synthesis reaction is 45-60 ° C; and (d) Temperature stability: pH 7.0, stable to 60 ° C under heating conditions for 15 minutes, 90 ° C at 70 ° C. % Inactivated.

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