JP2014100090A - Isomaltase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new enzyme that can decompose isomaltose to glucose even under conditions of high glucose concentration, and to provide a new method for industrial production of glucose from starch.SOLUTION: Provided is a mutant isomaltase derived from an isomaltase of a specified amino acid sequence, or an isomaltase having deletion, substitution or insertion of one or several amino acid residues in the specified sequence, where one or more amino acids in the amino acid sequence from #200 to #300 of the mutant isomaltose are mutated to be isoleucine, tryptophan or glutamic acid.

Description

  The present invention relates to isomaltase useful for the production of glucose from starch.

  In industrial glucose production from starch, various enzymes are used for liquefaction with α-amylase, saccharification with glucoamylase, debranching with isoamylase, pullulanase, and the like. In this series of saccharification processes for glucose production, isomaltose is produced as a by-product due to side reactions such as sugar transfer and condensation reaction of glucoamylase, which is a saccharifying enzyme, and all starch cannot be converted into glucose. There is a problem in that glucose productivity is reduced.

Isomaltose is an α-1,6 glucoside-bonded glucose, and it is considered that an increase in glucose yield can be expected if it can be decomposed into glucose. In order to decompose isomaltose into glucose, the function of isomaltase (isomaltosidase, oligo-1,6-glucosidase), an enzyme that degrades α-1,6-glucoside bonds, is expected.
For example, since oligo-1,6-glucosidase derived from Geobacillus thermoglucosidasius is excellent in heat resistance and has isomaltase activity, it is expected as isomaltase for producing glucose from starch (Non-patent Documents 1 and 2). .

Biochimica et Biophysica Acta, 445 (1976), 386-397 Journal of Bacteriology, Feb.1989, P.1219-1222

However, isomaltase is an enzyme that breaks down one molecule of isomaltose into two molecules of glucose. When the glucose concentration is about 2% in this reaction, the enzyme reaction that breaks down isomaltose does not proceed. is there. According to the study by the present inventor, it was also found that the thermostable isomaltase does not proceed at a glucose concentration of 2.2%.
Accordingly, an object of the present invention is to provide a new enzyme for decomposing isomaltose into glucose even under conditions of high glucose concentration, and a method for industrial production of glucose from starch.

  Therefore, the present inventor has searched for characteristics of various enzymes and modified the amino acids thereof, and improved the glucose tolerance of the isomaltase by 10 to 23% by mutating amino acids at specific sites of the thermostable isomaltase. The present invention has been completed.

That is, the present invention provides the following [1] to [8].
[1] In isomaltase consisting of the amino acid sequence represented by SEQ ID NO: 1 or isomaltase in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1, An isomaltase in which one or more amino acids of amino acid numbers 200 to 300 are mutated to an amino acid selected from isoleucine, tryptophan, and glutamic acid.
[2] One or more amino acid mutations selected from methionine, glutamine, glycine and arginine in amino acids 200 to 300 are mutated to amino acids selected from isoleucine, tryptophan and glutamic acid [1] ] Isomaltase as described.
[3] The isomaltase according to [1] or [2], wherein the amino acid mutation is 1 to 4 amino acids selected from M203W, Q216E, G259E, and R298I.
[4] A gene encoding the isomaltase according to any one of [1] to [3].
[5] A recombinant vector having the gene according to [4].
[6] A transformant transformed with the recombinant vector according to [5].
[7] A method for producing isomaltase, comprising culturing the transformant according to [6] and collecting isomaltase from the culture.
[8] Production of glucose characterized by allowing starch to react with an enzyme selected from α-amylase, glucoamylase, isoamylase and pullulanase, and the isomaltase according to any one of [1] to [3]. Law.

  If the isomaltase of the present invention is used, the production efficiency of glucose from starch is dramatically improved. Isomaltose produced by the degradation of starch by α-amylase or the like is about 3% of the total. When isomaltose produced as a by-product is separated and considered for reuse in the saccharification step, glucose can be obtained from isomaltose by isomaltase. At this time, it is required that the reaction is possible even at a higher glucose concentration. However, since wild-type isomaltase stops the reaction at a glucose concentration of 2.2%, isomaltose that can be decomposed is about 70% of the total reaction system. Is the limit. On the other hand, if the isomaltase of the present invention is used, the reaction can be performed up to a glucose concentration of 2.7%, and isomaltose corresponding to 90% of the whole reaction system can be decomposed, and the production of glucose from starch is extremely large. Considering certain things, the improvement in productivity is remarkable.

It is a figure which shows the heat resistance of variation | mutation isomaltase. It is a figure which shows the optimal temperature of a mutated isomaltase. It is a figure which shows the substrate specificity of a mutated isomaltase. It is a figure which shows the glucose tolerance of a mutant isomaltase. It is a figure which shows the example of application to the saccharification process of variation | mutation isomaltase.

  The isomaltase of the present invention has an isomaltase consisting of the amino acid sequence represented by SEQ ID NO: 1 or one to several amino acid residues deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1. Isomaltase is an isomaltase in which one or more of amino acids of amino acid numbers 200 to 300 are mutated to amino acids selected from isoleucine, tryptophan and glutamic acid.

  Here, the isomaltase consisting of the amino acid sequence represented by SEQ ID NO: 1 is an isomaltase produced by Geobacillus thermoglucosidasius described in Non-Patent Documents 1 and 2. This isomaltase includes isomaltase not derived from Geobacillus thermoglucosidasius as long as it has the same amino acid sequence. Moreover, as long as it has the same amino acid sequence, not only polypeptide but glycopeptide is contained.

  The number of amino acid residue deletions, substitutions or insertions in the isomaltase in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1 is SEQ ID NO: 1. Although it will not be limited if it shows the enzyme activity equivalent to isomaltase which consists of an amino acid sequence represented, 1-20 are preferable, 1-10 are more preferable, and 1-8 are more preferable. Further, the identity between the deleted, substituted or inserted isomaltase and the amino acid sequence of SEQ ID NO: 1 is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and 95% or more. Further preferred. Such sequence identity percentage can be calculated using publicly available or commercially available software with an algorithm that compares the reference sequence as a query sequence. For example, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) can be used.

In the isomaltase of the present invention, one or more of the amino acids of amino acid numbers 200 to 300 of the isomaltase are mutated to amino acids selected from isoleucine, tryptophan and glutamic acid. The mutation site is more preferably between M of amino acid number 203 to R of 298, and more preferably a position selected from 203 M, 216 Q, 259 G and 298 R. That is, it is more preferable that one to four amino acids selected from M203, Q216, G259 and R298 are mutated to amino acids selected from isoleucine, tryptophan and glutamic acid.
Further preferred amino acid mutations are 1 to 4 selected from M203W, Q216E, G259E and R298I. Furthermore, Q216E, G259E, R298I, G259E / R298I (double mutation), M203W / G259E / R298I (triple mutation), and M203W / Q216E / G259E / R298I (quadruple mutation) are preferable from the viewpoint of glucose tolerance.

  The mutant isomaltase of the present invention is an isomaltase consisting of the amino acid sequence represented by SEQ ID NO: 1, or one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1. The isomaltase can be produced using a gene constructed by substituting one or more amino acids of amino acid numbers 200 to 300 with an amino acid selected from isoleucine, tryptophan and glutamic acid.

  The gene for producing the mutant isomaltase of the present invention is a gene having a base sequence encoding the above-mentioned mutant isomaltase. For example, in the gene encoding the amino acid sequence shown in SEQ ID NO: 1, It can be constructed by substituting a base sequence encoding an amino acid sequence with a base encoding a desired amino acid residue. Various methods for such site-specific base sequence substitution are well known in the art, and can be performed, for example, by PCR using appropriately designed primers. Alternatively, a gene encoding a modified amino acid sequence may be totally synthesized.

  The gene thus obtained is inserted into an appropriate expression vector, and this is transformed into an appropriate host (for example, E. coli). Many vector / host systems for expressing foreign proteins are known in the art. As an expression vector for incorporating a mutant isomaltase gene, a plasmid vector can be mentioned. For example, pET-14b, pBR322 and the like can be mentioned for E. coli. Examples of Bacillus subtilis include pUB110. Examples of filamentous fungi include pPTRI. Moreover, pRS403 etc. are mentioned for yeast use.

  The obtained recombinant plasmid is transformed into microorganisms such as Escherichia coli, Bacillus subtilis, filamentous fungi, yeast and the like, and the mutant isomaltase of the present invention is obtained by culturing the transformant.

The mutant isomaltase of the present invention has an action of decomposing isomaltose into glucose and acts even in the presence of a high concentration of glucose.
Therefore, glucose can be produced in high yield by allowing an enzyme selected from α-amylase, glucoamylase, isoamylase and pullulanase and the isomaltase of the present invention to act on starch. Here, as α-amylase, for example, Christase T10 (Daiwa Kasei Co., Ltd.) can be used. As the glucoamylase, for example, GODO-ANGH (Godo Sakesei Co., Ltd.) can be used. As the isoamylase, for example, GODO-FIA (Godoshusei Co., Ltd.) can be used. As the pullulanase, for example, pullulanase “Amano” 3 (Amano Enzyme Inc.) can be used.

  The reaction is performed, for example, by adding the enzyme to a starch saccharifying enzyme such as starch or amylase, and mixing and stirring at a pH and temperature conditions at which the enzyme acts. According to the method of the present invention, since the reaction proceeds even when the glucose concentration is improved, glucose can be produced in a high yield.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this at all.

Example 1
(Cloning and expression of isomaltase gene from Geobacillus thermoglucosidasius)
For expression of the isomaltase gene, plasmid pHY300PLK (manufactured by Yakult) was used as an expression vector, and Bacillus subtilis ISW1214 strain (manufactured by Yakult) was used as an expression host.
Geobacillus thermoglucosidasius NCIMB 11955 strain was cultured, and the whole genome was extracted. The isomaltase gene (gtagl) contained in the genome was amplified by PCR. It was designed so that restriction enzyme sites were introduced during PCR.
A Bacillus subtilis protein high expression promoter was introduced into the multicloning site of pHY300PLK, and a gtagl sequence amplified by PCR was further introduced downstream thereof to prepare a plasmid pHY300PLK-GTAL for expression of isomaltase. The prepared expression plasmid pHY300PKL-GTAGL was introduced into Bacillus subtilis ISW1214 strain by electroporation to construct a recombinant GTAGL production system.
The base sequence of the obtained isomaltase gene and the corresponding amino acid sequence are shown in SEQ ID NO: 1.

Example 2
(Mutation introduction to isomaltase gene)
Mutation was introduced using PCR method using PrimeSTAR ^™ Mutagenesis Kit (TAKARA). Table 1 shows primer sequences for site-directed mutagenesis used for PCR.

  For double to quadruple mutants, pHY300PLK-GTAL is used as a template, mutation is first introduced using a primer that adds G259E mutation, pHY300PLK-GTAL-G259E plasmid is prepared, and then pHY300PLK-GTAL-G259E is used as a template. A second mutation, R298I mutation, was introduced. In the same manner, M203W and Q216E mutations were introduced in sequence to prepare a plasmid for obtaining double to quadruple mutants.

  The obtained plasmid was similarly introduced into Bacillus subtilis ISW1214 strain by electroporation to obtain mutant GTAGL protein.

The nucleotide sequence of the M203W gene and the corresponding amino acid sequence are shown in SEQ ID NO: 2.
The nucleotide sequence of the Q216E gene and the corresponding amino acid sequence are shown in SEQ ID NO: 3. The base sequence of the G259E gene and the corresponding amino acid sequence are shown in SEQ ID NO: 4. The base sequence of the R298I gene and the corresponding amino acid sequence are shown in SEQ ID NO: 5. The base sequence of the G259E / R298I gene and the corresponding amino acid sequence are shown in SEQ ID NO: 6. The nucleotide sequence of the M203W / G259E / R298I gene and the corresponding amino acid sequence are shown in SEQ ID NO: 7. SEQ ID NO: 8 shows the nucleotide sequence of the M203W / Q216E / G259E / R298I gene and the corresponding amino acid sequence.

(Isomaltase activity measurement method: GOD method)
The isomaltase activity was measured by the GOD method. A 20 mM phosphate buffer solution (pH 7.0) was added and mixed so that the final concentrations were 0.041% GOD, 0.01% POD, and 0.5% O-dianisidine to obtain a GOD reagent. 5 μl of enzyme solution was added to a mixed solution of 10 μl of 20 mg / mL isomaltose and 5 μl of 200 mM phosphate buffer (pH 7.0) to obtain a reaction solution (total amount: 20 μl). Further, a solution in which the enzyme solution was distilled water was used as a control. The enzyme reaction was incubated at 60 ° C. for 10 minutes. After the enzyme reaction solution was treated at 100 ° C. for 3 minutes, 200 μl of GOD reagent was added and incubated at 30 ° C. for 10 minutes. After adding 4 μl of 4N HCl, the activity was measured from the absorbance at 400 nm. (Hereinafter, “GOD method”). In addition, the pH of the reaction solution and the reaction temperature were changed by each test. The principle of the GOD method is as follows.

  1 U of α-glucosidase activity was defined as an activity that produces 1 μmol of glucose per minute under these conditions, and the enzyme activity was calculated from the following equation.

Example 3
(Characteristics of mutant isomaltase)
(1) Heat resistance 20 mg / mL isomaltose 10 μl, 200 mM phosphate buffer solution (pH 7.0) 5 μl, heated for 10 minutes at each temperature (4, 30, 40, 50, 60, 70, 80 ° C.) 5 μl of the treated enzyme solution was added to prepare a reaction solution, and isomaltase activity was measured by the GOD method. (Total volume 20 μl).
As a result, as shown in FIG. 1, GTAGL and its quadruple mutant maintained approximately 60% activity at 70 ° C.

(2) Optimal temperature 5 μl of enzyme solution was added to a mixed solution of 10 μl of 20 mg / mL isomaltose and 5 μl of 200 mM phosphate buffer (pH 7.0) to obtain a reaction solution (total amount 20 μl). The reaction solution was incubated at a temperature of 4 to 80 ° C. for 10 minutes, and the activity was measured by the GOD method.
As a result, as shown in FIG. 2, GTAGL and its quadruple mutant showed the highest activity at 60 ° C.

(3) Optimum pH
5 μl of the enzyme solution was added to a mixed solution of 10 μl of 20 mg / mL isomaltose and 5 μl of a buffer solution having various pHs of 200 mM to obtain a reaction solution (total amount: 20 μl). The reaction solution was incubated at 60 ° C. for 10 minutes, and the activity was measured by the GOD method.
As a result, GTAGL showed the highest activity at pH 7.0. The quadruple mutant showed the highest activity at pH 6.0.

(4) Substrate specificity The substrate specificity and transfer reaction of GTAGL and GTAGL-4 double mutants were confirmed by TLC. GTAGL and its quadruple mutants were allowed to act on 3 types of 2% substrates at 60 ° C. for 24 hours. The developing solvent was isopropanol / n-butanol / water (10/5/4), and after developing, air-dried, sprayed with an anthrone sulfuric acid solution, and heated at 105 ° C. to cause color development. As a result, as shown in FIG. 3, neither GTAGL nor the quadruple mutant showed a conspicuous transglycosylation reaction.

(5) Glucose tolerance The reactivity of GTAGL and its mutants at 60 ° C. was examined under substrate conditions where the isomaltose concentration was 1% and the glucose concentration was 2%. The enzyme activity in the reaction solution was reacted at 0.5 U / mL. Sampling was performed over time, and the decomposition of isomaltose was confirmed by liquid chromatography (˜20 hours). The glucose concentration when the decomposition reaction of isomaltase stopped progressing was confirmed, and the value was taken as the glucose tolerance value.
As a result, as shown in Table 2 and FIG. 4, it was shown that the reaction in the presence of a maximum of 2.7% glucose was possible in the quadruple mutant.

Example 4
(Application example to saccharification process sample)
Soluble starch was dissolved by heating in 0.1 M acetic acid buffer (pH 4.2) to a concentration of 30% (w / w). 40 μL of commercially available saccharification enzyme OPTIMAX 4060 (glucoamylase / pullulanase mixed enzyme) was added to 50 mL of soluble starch solution, and the enzyme reaction was performed at 60 ° C. for 16 hours. After the reaction, the sample was heated at 100 ° C. to stop the enzymatic reaction (starch degradation product).
After the starch degradation product was diluted with 0.1 M phosphate buffer (pH 6.8) so that the glucose concentration was 2.5%, the GTAGL obtained in Example 1 or Example 2 and its quadruple The mutant was added and reacted at 60 ° C. for 20 hours. After the reaction, it was subjected to silica gel TLC and analyzed. The developing solvent was isopropanol / n-butanol / water (10/5/4), and after developing, air-dried, sprayed with an anthrone sulfuric acid solution, and heated at 105 ° C. to cause color development.
As a result, it was possible to degrade isomaltase found in the starch degradation product only with the GTAGL-4 double mutant that can react even in the presence of 2.7% glucose (FIG. 5).

  The isomaltase of the present invention makes it possible to dramatically improve the yield of glucose in an industrial glucose production process from starch.

Claims (8)

  In the isomaltase consisting of the amino acid sequence represented by SEQ ID NO: 1 or the isomaltase in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1, the amino acid number 200 An isomaltase in which one or more of ˜300 amino acids are mutated to an amino acid selected from isoleucine, tryptophan and glutamic acid.   2. The amino acid mutation according to claim 1, wherein one or more amino acids selected from methionine, glutamine, glycine and arginine among amino acids of amino acid numbers 200 to 300 are mutated to amino acids selected from isoleucine, tryptophan and glutamic acid. Isomaltase.   The isomaltase according to claim 1 or 2, wherein the amino acid mutation is 1-4 selected from M203W, Q216E, G259E and R298I.   The gene which codes the isomaltase in any one of Claims 1-3.   A recombinant vector having the gene according to claim 4.   A transformant transformed with the recombinant vector according to claim 5.   A method for producing isomaltase, comprising culturing the transformant according to claim 6 and collecting isomaltase from the culture.   A method for producing glucose, wherein an enzyme selected from α-amylase, glucoamylase, isoamylase, and pullulanase is reacted with starch and the isomaltase according to any one of claims 1 to 3.