WO2016143873A1 - 改良型β-フルクトフラノシダーゼ - Google Patents
改良型β-フルクトフラノシダーゼ Download PDFInfo
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- WO2016143873A1 WO2016143873A1 PCT/JP2016/057657 JP2016057657W WO2016143873A1 WO 2016143873 A1 WO2016143873 A1 WO 2016143873A1 JP 2016057657 W JP2016057657 W JP 2016057657W WO 2016143873 A1 WO2016143873 A1 WO 2016143873A1
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- fructofuranosidase
- amino acid
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- kawachii
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
Definitions
- the present invention relates to an improved ⁇ -fructofuranosidase, and in particular, an improved ⁇ -fructofuranosidase capable of efficiently producing kestose while suppressing the production of nystose, and a polypeptide comprising the amino acid sequence thereof
- the present invention relates to a production method and a production method of kestose using them.
- ⁇ -fructofuranosidase is an enzyme that recognizes and hydrolyzes sugar fructose containing fructose residues at its ends. Some ⁇ -fructofuranosidases have fructose transfer activity for transferring fructose generated by hydrolysis to a substrate in addition to the hydrolysis activity. According to such ⁇ -fructofuranosidase, kestose, which is a trisaccharide in which one molecule of glucose and two molecules of fructose are bound, can be generated using sucrose as a substrate.
- 1-kestose has a sweetness similar to sucrose (sugar) and produces about one third of the sweetness of sugar, while it is about half the calorie of sugar, It is known that it is a useful oligosaccharide because it is difficult to increase the value and has an allergy suppressing function (Patent Document 1).
- ⁇ -fructofuranosidase producing 1-kestose include, for example, ⁇ -fructofuranosidase derived from Aspergillus niger and ⁇ -fructofuranoside of a mutant in which an amino acid mutation is introduced into its amino acid sequence. Sidases have been disclosed (Patent Document 2 and Patent Document 3). *
- nystose When producing kestose using sucrose as a substrate and ⁇ -fructofuranosidase, tetrasaccharide nystose is usually produced as a by-product.
- Example 1 (1) which will be described later, nystose present in a sugar solution at a certain ratio or more inhibits crystallization of kestose in the crystallization step.
- Example 1 (2) described later nystose is difficult to separate from kestose in chromatography, and it is difficult to reduce the content ratio even after a separation and purification step by chromatography. .
- the present invention has been made to solve such problems, and an improved ⁇ -fructofuranosidase that can efficiently produce kestose while suppressing the production of nystose, and its amino acid sequence
- An object is to provide a method for producing cetofuranosidase and a method for producing kestose.
- an amino acid mutation that replaces the 85th glycine (G) from the amino terminus (N-terminus) with a protein-constituting amino acid other than glycine (G), or a histidine (H) at the 310th terminus from the N terminus is lysine ).
- the improved ⁇ -fructofuranosidase according to the present invention comprises the following amino acid sequence (a) or (b): (A) an amino acid sequence obtained by introducing an amino acid mutation of the following i) and / or ii) with respect to the amino acid sequence shown in SEQ ID NO: 2 (total length: 628 amino acids); i) an amino acid mutation that replaces the 85th glycine (G) from the N-terminus with a protein-constituting amino acid other than glycine (G); ii) an amino acid mutation replacing histidine (H) at position 310 from the N-terminus with lysine (K), arginine (R) or tyrosine (Y); (B) An amino acid sequence consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding one or more amino acids excluding the amino acid into which an amino acid mutation has been introduced and having ⁇ -fructofuranosidase activity in (a) .
- the polypeptide according to the present invention comprises the amino acid sequence of the improved ⁇ -fructofuranosidase described in (1).
- the DNA according to the present invention encodes the improved ⁇ -fructofuranosidase described in (1).
- a recombinant vector according to the present invention comprises the DNA described in (3).
- the transformant according to the present invention is a transformant obtained by introducing the DNA described in (3) or the recombinant vector described in (4) into a host.
- a method for producing an improved ⁇ -fructofuranosidase according to the present invention comprises a step of obtaining an improved ⁇ -fructofuranosidase from a culture obtained by culturing the transformant according to (5).
- the method for producing kestose according to the present invention comprises culturing the improved ⁇ -fructofuranosidase described in (1), the transformant described in (5), or the transformant described in (5). A step of contacting the culture obtained in this manner with sucrose.
- the transformant according to the present invention and the kestose production method according to the present invention, it is possible to efficiently produce kestose while suppressing the production of nystose.
- the polypeptide according to the present invention the DNA according to the present invention, the recombinant vector according to the present invention, the transformant according to the present invention, and the method for producing the improved ⁇ -fructofuranosidase according to the present invention, It is possible to obtain an improved ⁇ -fructofuranosidase that efficiently generates kestose while suppressing the ratio of nystose.
- ⁇ -fructofuranosidase can be replaced with “fructosyltransferase”, “saccharase”, “ ⁇ -D-fructofuranosidase”, “invertase”, “invertase” or “invertin”. May be used.
- the “wild-type ⁇ -fructofuranosidase” in the present invention refers to ⁇ -fructofuranosidase having an amino acid sequence into which no amino acid mutation has been introduced using a genetic engineering technique.
- “Fructofuranosidase” refers to ⁇ -fructofuranosidase consisting of an amino acid sequence in which one or more amino acid mutations are introduced into the amino acid sequence of wild-type ⁇ -fructofuranosidase.
- the improved ⁇ -fructofuranosidase comprises the following amino acid sequence (a) or (b): (A) an amino acid sequence obtained by introducing an amino acid mutation of the following i) and / or ii) with respect to the amino acid sequence shown in SEQ ID NO: 2 (total length: 628 amino acids); i) an amino acid mutation that replaces the 85th glycine (G) from the N-terminus with a protein-constituting amino acid other than glycine (G); ii) an amino acid mutation replacing histidine (H) at position 310 from the N-terminus with lysine (K), arginine (R) or tyrosine (Y); (B) An amino acid sequence consisting of an amino acid sequence obtained by deleting, substituting, inserting or adding one or more amino acids excluding the amino acid into which an amino acid mutation has been introduced and having ⁇ -fructofuranosidase activity in (a) .
- protein-constituting amino acids refers to those listed in Table 1 below (Biochemical Dictionary 4th edition; published by Tokyo Chemical Dojinsha, page 57, December 2007).
- G protein constituting amino acid other than glycine
- W tryptophan
- F phenylalanine
- Y tyrosine
- D glutamic acid
- R arginine
- the above (b) is an amino acid sequence having high sequence identity with (a), having an amino acid mutation corresponding to the amino acid mutation introduced in (a), and having ⁇ -fructofuranosidase activity.
- examples of the value of identity between (a) and (b) include 80 to 85% or more, 85 to 90% or more, and 90 to 95% or more. That is, (b) is, for example, “one or more amino acids excluding the amino acid introduced with the amino acid mutation in (a) within a range where the value of identity with (a) is not less than 80 to 90%” Is a deletion, substitution, insertion, or addition amino acid sequence ”.
- the number of “deleted, substituted, inserted or added amino acids” in (b) is specifically 1 to 125 (identity with (a) is 80% or more), 1 to 113, for example. (Identity with (a) is 82% or more), 1 to 100 (identity with (a) is 84% or more), 1 to 87 (identity with (a) is 86% or more) 1 to 75 (identity with (a) is 88% or more), 1 to 62 (identity with (a) is 90% or more), 1 to 50 (identity with (a) 92% or more) 1 to 37 (identity with (a) is 94% or more), 1 to 25 (identity with (a) is 96% or more), 1 to 12 ((a) , The identity of which is 98% or more).
- the identity of the amino acid sequence can be confirmed according to a conventional method.
- FASTA http://www.genome.JP/tools/fasta/
- BLAST Basic local align search tool
- PSI-BLAST Position-Specific Iterated BLAST
- identity indicates coincidence and is used interchangeably with “identity”.
- a certain protein has ⁇ -fructofuranosidase activity can be confirmed according to a conventional method.
- the protein is treated with sucrose. After incubating in the reaction solution containing or culturing the transformant expressing the protein in the reaction solution containing sucrose, the content of the reaction solution is confirmed by high performance liquid chromatography (HPLC) or the like.
- HPLC high performance liquid chromatography
- the protein can be determined to have ⁇ -fructofuranosidase activity.
- the improved ⁇ -fructofuranosidase according to the present invention can be obtained according to a conventional method, and examples of such a method include a chemical synthesis method and a method using a gene recombination technique.
- a chemical synthesis method for example, based on the amino acid sequence information of the improved ⁇ -fructofuranosidase according to the present invention, Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method)
- the improved ⁇ -fructofuranosidase according to the present invention can be synthesized according to chemical synthesis methods such as the above, and various commercially available peptide synthesizers can also be used for synthesis.
- the improved ⁇ -fructofuranosidase according to the present invention can be obtained by expressing the improved ⁇ -fructofuranosidase according to the present invention in a suitable expression system. it can. That is, a transformant is obtained by introducing DNA encoding the improved ⁇ -fructofuranosidase according to the present invention into a suitable host. Alternatively, as shown in Example 2 to be described later, after a DNA encoding the improved ⁇ -fructofuranosidase according to the present invention is inserted into an appropriate vector to obtain a recombinant vector, the recombinant vector is A transformant is obtained by introducing it into an appropriate host. Then, the improved transformant according to the present invention can be obtained by culturing the obtained transformant to express the improved ⁇ -fructofuranosidase.
- the DNA encoding the improved ⁇ -fructofuranosidase according to the present invention can be synthesized using various commercially available DNA synthesizers based on the base sequence information, It can be obtained by performing polymerase chain reaction (PCR) using a DNA encoding ⁇ -fructofuranosidase or a DNA encoding improved ⁇ -fructofuranosidase as a template.
- PCR polymerase chain reaction
- a DNA primer encoding the amino acid mutation to be introduced is used.
- a DNA encoding an improved ⁇ -fructofuranosidase having an amino acid sequence into which an amino acid mutation has been introduced can be obtained.
- DNA encoding furanosidase can also be obtained by PCR. That is, first, a DNA primer encoding an amino acid sequence corresponding to a position where an amino acid has been deleted, substituted, inserted or added is designed, and the DNA primer is used to perform PCR using the DNA encoding (a) as a template. By performing, in (a), DNA encoding the amino acid sequence in which the amino acid is deleted, substituted, inserted or added can be obtained.
- the present invention also provides a polypeptide comprising the amino acid sequence of improved ⁇ -fructofuranosidase.
- the description of the same or equivalent configuration as the above-described improved ⁇ -fructofuranosidase according to the present invention is omitted.
- the sequence length is not particularly limited, and the amino acid of the improved ⁇ -fructofuranosidase according to the present invention is not limited.
- the polypeptide according to the present invention can be obtained by the same method as the method for obtaining the improved ⁇ -fructofuranosidase according to the present invention described above.
- the present invention provides a DNA encoding an improved ⁇ -fructofuranosidase.
- the description of the same or equivalent configuration as the above-described improved ⁇ -fructofuranosidase and polypeptide according to the present invention is omitted. .
- the present invention also provides a recombinant vector comprising DNA encoding an improved ⁇ -fructofuranosidase.
- a recombinant vector comprising DNA encoding an improved ⁇ -fructofuranosidase.
- the description of the same or corresponding configuration as the above-described improved ⁇ -fructofuranosidase, polypeptide and DNA according to the present invention is omitted.
- the recombinant vector according to the present invention can be obtained, for example, by inserting a DNA encoding the improved ⁇ -fructofuranosidase according to the present invention into the vector. Insertion of DNA into a vector can be performed according to a conventional method, for example, by ligating DNA with a DNA fragment of a linearized vector.
- examples of the vector include a phage vector, a plasmid vector, a cosmid, and a phagemid, and can be appropriately selected depending on the host, operability, and the like.
- the recombinant vector according to the present invention includes a selection marker gene for a transformant such as a drug resistance marker gene and an auxotrophic marker gene, It may contain a transcription regulatory signal such as a promoter necessary for expression of the improved ⁇ -fructofuranosidase, a transcription initiation signal, a ribosome binding site, a translation termination signal, a transcription termination signal, a translation regulation signal, and the like.
- a selection marker gene for a transformant such as a drug resistance marker gene and an auxotrophic marker gene
- a transcription regulatory signal such as a promoter necessary for expression of the improved ⁇ -fructofuranosidase, a transcription initiation signal, a ribosome binding site, a translation termination signal, a transcription termination signal, a translation regulation signal, and the like.
- the present invention also provides a transformant.
- the description of the same or corresponding configuration as the above-described improved ⁇ -fructofuranosidase, polypeptide, DNA and recombinant vector according to the present invention is omitted.
- the transformant according to the present invention is obtained by introducing into a host a DNA encoding the improved ⁇ -fructofuranosidase or a recombinant vector containing the DNA encoding the improved ⁇ -fructofuranosidase according to the present invention. It is done.
- the host include bacteria such as Escherichia coli and Bacillus subtilis, yeasts, filamentous fungi, and the like, which can be appropriately selected according to the type and operability of the recombinant vector.
- Introduction (transformation) of DNA or a recombinant vector into a host can be performed according to a conventional method. For example, when a recombinant vector using a plasmid is introduced into E.
- the present invention provides a method for producing an improved ⁇ -fructofuranosidase.
- the method for producing an improved ⁇ -fructofuranosidase according to the present invention includes a step of obtaining an improved ⁇ -fructofuranosidase from a culture obtained by culturing the transformant according to the present invention.
- the same or equivalent to the above-described improved ⁇ -fructofuranosidase, polypeptide, DNA, recombinant vector and transformant according to the present invention The description of the configuration to be repeated is omitted.
- the method for obtaining the improved ⁇ -fructofuranosidase includes a mode of the transformant, etc. It can be selected as appropriate according to the conditions.
- the culture obtained by culturing the transformant may be obtained as an improved ⁇ -fructofuranosidase as it is, or obtained by purifying the improved ⁇ -fructofuranosidase from the culture. May be.
- the improved ⁇ -fructofuranosidase is expressed on the cell surface or inside the cell of the transformant. If the DNA or recombinant vector is designed as described above, the culture is subjected to centrifugation, and the transformant is recovered and directly obtained as an improved ⁇ -fructofuranosidase. A method of crushing the body and obtaining it as an improved ⁇ -fructofuranosidase can be mentioned.
- the improved ⁇ -fructofuranosidase As a method for purifying the improved ⁇ -fructofuranosidase from the culture, for example, when a DNA or a recombinant vector is designed so that the improved ⁇ -fructofuranosidase is secreted outside the transformant.
- the improved ⁇ -fructofuranosidase can be purified by subjecting the culture to centrifugation and collecting the culture supernatant.
- the culture is subjected to centrifugation to recover the precipitated transformant, which is suspended in a buffer solution.
- the improved ⁇ -fructofuranosidase can be purified by crushing the transformant by freeze-thawing, sonication or grinding, etc., and collecting the supernatant by centrifugation.
- Other purification methods include heat treatment, salt precipitation, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse Examples of the method include phase chromatography and isoelectric focusing.
- the present invention provides a method for producing kestose.
- the method for producing kestose according to the present invention comprises the improved ⁇ -fructofuranosidase according to the present invention, the transformant according to the present invention or a culture obtained by culturing the transformant according to the present invention and sucrose. A step of contacting.
- the improved ⁇ -fructofuranosidase, polypeptide, DNA, recombinant vector, transformant and improved ⁇ -fructofuranosidase according to the present invention described above are produced. A description of the same or corresponding configuration as the method is omitted.
- kestose is usually produced by binding fructose to sucrose, and three types of 1-kestose, 6-kestose and neokestose can be generated depending on the position where fructose is bound. That is, 1-kestose is produced by fructose binding ⁇ (2 ⁇ 1) to fructose units in sucrose, and 6-kestose is produced by fructose binding ⁇ (2 ⁇ 6) to fructose units in sucrose.
- Neokestose is produced by fructose binding ⁇ (2 ⁇ 6) to glucose units in sucrose.
- Nistose is a tetrasaccharide produced by fructose binding ⁇ (2 ⁇ 1) to a fructose unit in 1-kestose.
- kestose means a trisaccharide in which one molecule of glucose and two molecules of fructose are bound, and includes 1-kestose, 6-kestose and neokestose.
- the improved ⁇ -fructofuranosidase is added to a solution containing sucrose and then heated at 30 ° C. to 50 ° C. for about 20 hours. The method of leaving still can be mentioned.
- the transformant according to the present invention is added to a solution containing sucrose and shaken at 50 ° C. for several days. A method for culturing can be mentioned.
- the culture obtained by culturing the transformant according to the present invention is put into a solution containing sucrose.
- the method include adding and allowing to stand at 30 ° C. to 50 ° C. for about 20 hours or shaking.
- the culture is crushed, ground, suspended in buffer, freeze-thawed, sonication, centrifugation, heat treatment, salt precipitation, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide It may be subjected to some treatment such as gel electrophoresis, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, isoelectric focusing, etc., or may not be subjected to treatment.
- some treatment such as gel electrophoresis, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, isoelectric focusing, etc.
- the method for producing kestose according to the present invention may have other steps as long as the characteristics of the method for producing kestose according to the present invention are not impaired, for example, a kestose separation step by chromatography or sucrose A crystallization process, a drying process, a washing process, a filtration process, a sterilization process, a process of adding food additives, and the like may be included.
- improved ⁇ -fructofuranosidase, polypeptide, DNA, recombinant vector, transformant, improved ⁇ -fructofuranosidase production method, kestose production method, and kestose production method according to the present invention This will be described based on each example. Note that the technical scope of the present invention is not limited to the features shown by these examples.
- nystose content ratio ((w / w)% in the saccharide contained in the aqueous solution; hereinafter referred to as “nystose content ratio”) is about 5% and The effect of the nystose content ratio at the time of crystal production of kestose was examined by producing a kestose crystal using a raw sugar solution of about 10% and confirming the recovery rate and crystal size of the kestose. The specific procedure is shown below.
- the content ratio of kestose ((w / w)%; hereinafter referred to as “kestose content ratio”) in the sugar contained in the aqueous solution is 80% or more and the sugar concentration is about 60 (w / w)%.
- Kestose crystals and fructooligosaccharide powder (Mayoligo P; Meiji Food Materia) were dissolved in water. 1 to 4.
- no. Nos. 1 and 2 had a nystose content of 5.2%, 3 and 4 were prepared so that the nystose content was 10%.
- a kestose crystal was ground and passed through a mesh sieve to obtain a uniform grain size.
- the Brix value and weight of the sugar solution were measured, and then subjected to a small centrifuge (H-112; Kokusan Co., Ltd.) to recover crystals.
- the recovered crystals were left to stand in a drier set at 80 ° C. for about 1 hour and dried.
- the recovery rate of kestose is no. In Nos. 1 and 2, it was 40% and 45%, respectively. 3 was 15%.
- the crystal size is No. In both Nos. 1 and 2, the crystal size was large. In 3, the crystal size was small. Furthermore, no. In No. 4, the produced crystal was small and the crystal could not be recovered.
- the time required for crystallization when the seed addition amount was 2.5 ppm was No. No. 1 was 8 hours, whereas no. 3 was 23 hours.
- the nystose content ratio in the raw sugar liquid is 5.2%, the recovery rate of kestose is high, the crystal size is large, and the time required to obtain the crystals is short, whereas in the raw sugar liquid
- the nystose content was 10%, it was found that the recovery rate of kestose was low, the crystal size was small, and the time required to obtain the crystal was long. From these results, in order to efficiently produce kestose crystals, it was found that the nystose content in the raw sugar solution is preferably less than 10%.
- the kestose content ratios of No. 1 to 8 in the reaction solution were 45.1%, 45.8%, 52.9%, 53.7%, 52.8%, 46.0, respectively. %, 45.8%, and 48.4%, whereas in Kestose high-purity liquid, No. 1 to 8 were 76.6%, 78.1%, 86.4%, and 88.3%, respectively. 88.0%, 78.7%, 79.2% and 78.6%. That is, no. In any of 1 to 8, the kestose high-purity liquid had a high kestose content to the extent sufficient for crystal production (about 80%).
- the nystose content ratios of No. 1 to No. 8 in the reaction solution were 11.6%, 10.6%, 6.0%, 4.7%, 4.7%, 10.2%, 9.7, respectively. % And 11.3%, whereas in Kestose high-purity liquid, No. 1 to 8 were 18.6%, 17.0%, 9.2%, 7.3%, 7.4%, respectively. 16.4%, 15.8% and 17.3%. That is, no. In any of 1 to 8, the kistose high-purity solution had a nystose content ratio about 1.5 times higher than that of the reaction solution. Specifically, when the content ratio of nystose is about 5% in the reaction solution (No.
- Example 1 (2) in order to efficiently produce kestose crystals, the reaction after the enzyme reaction with ⁇ -fructofuranosidase It was revealed that the nystose content in the liquid needs to be suppressed to about 5%.
- H310K Aspartic acid
- D aspartic acid
- R arginine
- Y tyrosine
- G glycine
- W tryptophan
- the PgsA anchor protein (GenBank: AB016245.1) of Bacillus subtilis (IAM1026, ATCC 9466) is encoded by performing polymerase chain reaction (PCR) under the following conditions. DNA to be amplified was amplified. The obtained PCR product was digested with restriction enzymes NdeI and BglII according to a conventional method to obtain a PgsA-DNA fragment. Further, the PgsA-DNA fragment was sequenced according to a conventional method, and its base sequence was confirmed. The nucleotide sequence of the DNA encoding the confirmed PgsA anchor protein is shown in SEQ ID NO: 3, and the amino acid sequence of the PgsA anchor protein encoded thereby is shown in SEQ ID NO: 4, respectively.
- pCDFDuet-1 plasmid (hereinafter abbreviated as “pCDF plasmid”; Merck) was digested with restriction enzymes NdeI and BglII according to a conventional method to obtain a pCDF plasmid fragment. Subsequently, DNA Ligation Kit Ver. Using 2.1 (Takara Bio Inc.), the pCDF plasmid fragment and the PgsA-DNA fragment were ligated according to the attached instruction, and a pCDF-PgsA recombinant vector was constructed.
- a primer is designed to remove the signal sequence, and PCR is performed under the following conditions.
- a DNA encoding kawachii-derived wild-type ⁇ -fructofuranosidase was amplified and used as a kawachii-DNA fragment.
- A. Conditions for PCR for amplification of DNA encoding kawachii-derived ⁇ -fructofuranosidase >> This template: A.
- Example 2 [1-1] DNA encoding wild-type ⁇ -fructofuranosidase derived from kawachii Forward primer; 5'-AAATCTAAAAGATCCTCCGTGGTCCATCGACTAC-3 '(SEQ ID NO: 7) Reverse primer; 5′-TTTACCAGATCTGAGTCAACATGACGGATCCGGC-3 ′ (SEQ ID NO: 8) Enzyme for PCR; KOD-Plus-Neo (Toyobo)
- PCR was performed using the pCDF-PgsA recombinant vector as a template under the following conditions, and the obtained PCR product was used as a pCDF-PgsA-DNA fragment.
- ⁇ Conditions for PCR for DNA amplification derived from pCDF plasmid inserted with DNA encoding PgsA protein >> Template: pCDF-PgsA recombinant vector forward primer; 5′-CTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAA-3 ′ (SEQ ID NO: 9) Reverse primer; 5′-GGATCTTTTAGATTTTTAGTTTGTCACTATGATCAA-3 ′ (SEQ ID NO: 10) Enzyme for PCR; KOD-Plus-Neo (Toyobo)
- the kawachii-DNA fragment and the pCDF-PgsA-DNA fragment were ligated according to the attached instructions, and this was used as the kawachii (wild type) recombinant vector. .
- the restriction enzyme DpnI was added to the PCR product and digested at 37 ° C. for 1 hour, and then subjected to agarose gel electrophoresis to cut out the gel and purify the DNA fragment.
- Ligation high ver. 2 Toyobo Co., Ltd.
- T4 Polynucleotide Kinase Toyobo Co., Ltd.
- a recombinant vector was created.
- Recombinant vector kawachii (G85E) recombinant vector, kawachii (G85R) recombinant vector, kawachii (H310K) recombinant vector, kawachii (H310D) recombinant vector, kawachii (H310Y) Recombinant vector, kawachii (H310R) recombinant vector was kawachii (H310G) recombinant vectors and kawachii (H310W) recombinant vector.
- ⁇ Conditions for PCR for DNA amplification encoding improved ⁇ -fructofuranosidase >> Template: Kawachii (H310K) recombination vector of Example 2 (2) [2-1] Forward primer; TGGAGCGGGCATCTCCAGTGCCACCA-3 ′ (SEQ ID NO: 11) Reverse primer; 5'-ATCGGTGAAGGAAGCCGACGTGGAAGAGG-3 '(SEQ ID NO: 12) Enzyme for PCR; KOD-Plus-NEO (Toyobo)
- telomere sequence was synthesized by the method described in Example 2 (2) [2-1], and the double mutant DNA encoding improved ⁇ -fructofuranosidase was inserted.
- a recombinant vector thus prepared was prepared and used as a kawachii (G85W / H310K) recombinant vector.
- M9 SEED medium total 100 mL; water 72 mL, 5 ⁇ M9 salt 20 mL, 20% casamino acid 5 mL, 20% D-glucose 2 mL, 2 mg / mL thymine 1 mL, 50 mM CaCl 2 0.2 mL, 2.5 M MgCl 2 40 ⁇ L, 100 mg / mL FeSO 4 28 ⁇ L, antibiotics (final concentration 50 ⁇ g / mL streptomycin sulfate).
- M9 Main medium (total 100 mL); water 67 mL, 5 ⁇ M9 salt 20 mL, 20% casamino acid 5 mL, 2 mg / mL thymine 1 mL, 50 mM CaCl 2 0.2 mL, 100 mg / mL FeSO 4 28 ⁇ L, Overnight Expression Automation 1 N.E .; Merck) Sol. 12 mL, O.I. N. E. Sol. 2 5 mL, O.D. N. E. Sol. 3 100 ⁇ L, antibiotic (final concentration 50 ⁇ g / mL streptomycin sulfate).
- Example 3 Confirmation of activity of improved ⁇ -fructofuranosidase (1)
- Enzymatic reaction of ⁇ -fructofuranosidase 0.04M sodium phosphate buffer (pH 7.) containing 30 (w / w)% sucrose. 0) was prepared and used as a 30% sucrose solution.
- 0.5 mL of the recombinant E. coli culture of Example 2 (3) was collected by centrifugation and the wet weight (wet cell weight) was measured.
- 350 ⁇ L of a 30% sucrose solution was added and suspended, and then the enzyme reaction of ⁇ -fructofuranosidase was performed by shaking at 30 ° C. and 200 rpm for a certain period of time.
- Enzyme reaction times were 3, 9, 32 and 48 hours.
- reaction solution was diluted by adding 950 ⁇ L of water to 50 ⁇ L of the reaction solution, and heated at 100 ° C. for 10 minutes. This was centrifuged at 4 ° C. and 15000 ⁇ g for 10 minutes to collect the supernatant, and then filtered through a 0.45 ⁇ m pore filter, and the resulting filtrate was used as an HPLC sample.
- HPLC sample was subjected to HPLC under the conditions described in Example 1 (1), and the content ratio of each saccharide contained in the reaction solution was confirmed.
- the amount of kestose and nystose contained in the reaction solution is calculated by multiplying the mass of sucrose contained in the reaction solution at the start of the enzyme reaction by the respective content ratio of kestose and nystose, and dividing by the weight of wet cells. It was expressed as a value. The results are shown in Table 4. In Table 4, “-” indicates that it was below the detection limit.
- the amount of kestose per wet cell weight is the reaction solution of the recombinant Escherichia coli into which the kawachii (wild type) recombinant vector has been introduced.
- the amount of nystose per wet cell weight was 1.26 mg in the control, whereas the Kawachii (G85W) recombinant vector, the Kawachii (G85F) recombinant vector, the Kawachii (G85Y) recombinant vector, In the reaction solution of recombinant Escherichia coli introduced with the Kawachii (G85D) recombinant vector, the Kawachii (G85E) recombinant vector, and the Kawachii (G85R) recombinant vector, 0.85 mg, 0.23 mg, 0.57 mg, and 0.29 mg, respectively. 0.46 mg and 0.79 mg.
- the nystose content was 29.64% in the control, whereas the Kawachii (G85W) recombinant vector, the Kawachii (G85F) recombinant vector, the Kawachii (G85Y) recombinant vector, and the Kawachii (G85D) group
- Kawachii (G85E) recombinant vector and kawachii (G85R) recombinant vector 4.51%, 3.46%, 13.36%, 7.36%, 6.65% and 12.78%.
- a Kawachii (G85W) recombinant vector a Kawachii (G85F) recombinant vector, a Kawachii (G85Y) recombinant vector, a Kawachii (G85D) recombinant vector, a Kawachii (G85E) recombinant vector, and a Kawachii (G85R) recombinant vector
- the amount of kestose and the kestose content ratio increased, and the amount of nystose and the content ratio of nystose significantly decreased compared to the control.
- the amount of nystose per wet cell weight was 0.15 mg in the control, whereas Kawachii (H310K) recombinant vector, Kawachii (H310D) recombinant vector, Kawachii (H310R) recombinant vector, In the recombinant E. coli introduced with the kawachii (H310Y) recombinant vector, the kawachii (H310G) recombinant vector, and the kawachii (H310W) recombinant vector, 0.37 mg, below detection limit, 0.95 mg, 0.01 mg, detection limit, respectively. And 0.003 mg.
- the nystose content was 1.23% in the control, whereas the Kawachii (H310K) recombinant vector, the Kawachii (H310D) recombinant vector, the Kawachii (H310R) recombinant vector, and the Kawachii (H310Y) group Recombinant vector, Kawachii (H310G) recombinant vector and recombinant Escherichia coli introduced with Kawachii (H310W) recombinant vector were 2.76%, below detection limit, 4.96%, 0.11%, below detection limit and 0.02%.
- the amount of kestose per wet cell weight was 1.99 mg in the control, whereas Kawachii It was 3.86 mg in the recombinant Escherichia coli introduced with the (H310Y) recombinant vector.
- the amount of nystose per wet cell weight was 3.89 mg in the control, and 0.16 mg in the recombinant Escherichia coli introduced with the kawachii (H310Y) recombinant vector.
- the nystose content was 31.50% in the control, and 1.36% in the recombinant Escherichia coli introduced with the kawachii (H310Y) recombinant vector.
- the amount of kestose per wet cell weight was 0.82 mg for the control, and the group into which the Kawachii (G85W) recombinant vector was introduced.
- the recombinant E. coli was 1.30 mg and the recombinant E. coli introduced with the Kawachii (H310K) recombinant vector was 5.79 mg, whereas the recombinant E. coli introduced with the Kawachii (G85W / H310K) recombinant vector was 6. It was 46 mg.
- the amount of nystose per wet cell weight was 1.08 mg for the control, 1.29 mg for the recombinant Escherichia coli introduced with the Kawachii (G85W) recombinant vector, and the recombinant Escherichia coli introduced with the Kawachi (H310K) recombinant vector.
- the recombinant Escherichia coli introduced with the kawachii (G85W / H310K) recombinant vector had 0.40 mg.
- the nystose content was 31.78% in the control, 25.14% in the recombinant E.
- ⁇ -fructofuranosidase homologous to wild-type ⁇ -fructofuranosidase derived from Kawachii and confirmation of its activity
- Preparation of improved ⁇ -fructofuranosidase [1-1 Alignment
- As a ⁇ -fructofuranosidase consisting of an amino acid sequence having 68% identity with the wild type ⁇ -fructofuranosidase derived from Kawachii The following (A) was extracted as ⁇ -fructofuranosidase consisting of an amino acid sequence having 60% identity with kawachii-derived wild-type ⁇ -fructofuranosidase.
- A 68% identity: ⁇ -fructofuranosidase (XP_003190558) of Aspergillus oryzae RIB40 (hereinafter abbreviated as “A. oryzae”)
- A 60% identity: ⁇ -fructofuranosidase (XP — 001214174) of Aspergillus terreus NIH2624 (hereinafter abbreviated as “A. terreus”)
- G78W amino acid mutation
- a single mutant ⁇ -fructofuranosidase consisting of the amino acid sequence was prepared and used as an improved ⁇ -fructofuranosidase. Specific procedures are shown in the following Example 3 (1) [1-2] and [1-3].
- the nucleotide sequence of DNA encoding wild-type ⁇ -fructofuranosidase derived from oryzae is represented by SEQ ID NO: 35
- the amino acid sequence of wild-type ⁇ -fructofuranosidase derived from oryzae is represented by SEQ ID NO: 36
- the base sequence of DNA encoding wild-type ⁇ -fructofuranosidase derived from terreus is represented by SEQ ID NO: 37
- the amino acid sequence of terreus-derived wild-type ⁇ -fructofuranosidase is shown in SEQ ID NO: 38, respectively.
- PCR conditions for amplifying oryzae wild-type DNA fragment-1 >> Forward primer; 5′-ACATCACAGATAACATATGAAGCTCTCAACCCGCGAGTCCT-3 ′ (SEQ ID NO: 39) Reverse primer; 5'-CCGAGCCCCAAGTACACTAGGGGCAAAAGTCC-3 '(SEQ ID NO: 40) Enzyme for PCR; KOD-Plus- (Toyobo) ⁇ A.
- PCR conditions for amplifying oryzae wild-type DNA fragment-2 >> Forward primer; 5'-AGTACTTGGGCTCGGTCCCTGGTACAAGAAACTCGACTGACATCAAG-3 '(SEQ ID NO: 41) Reverse primer; 5′-GAGCAAGCTTCTCGAGTTAGACACCGCTCAGGCCAGGCTTCA-3 ′ (SEQ ID NO: 42) Enzyme for PCR; KOD-Plus- (Toyobo) ⁇ A.
- A. oryzae wild-type DNA fragments-1 and 2 were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.), respectively. oryzae wild-type DNA fragment-3 Terreus wild-type DNA fragment-3 was designated.
- a primer is designed to remove the signal sequence, and PCR is performed under the following conditions. DNA encoding wild-type ⁇ -fructofuranosidase from Oryzae and A. terreus-derived wild-type ⁇ -fructofuranosidase-encoding DNA was amplified, and the resulting PCR products were respectively A. oryzae wild-type DNA fragment-4 and A.I. Terreus wild-type DNA fragment-4 was designated.
- PCR conditions for amplifying oryzae wild-type DNA fragment-4 >> A mold; oryzae wild type DNA fragment-3 Forward primer 5'-AAATCTAAAAGATCTCCCGCCATCGATTACACG-3 '(SEQ ID NO: 47) Reverse primer 5'-TTTACCAGATCTGAGTTTAGACACGCTCCAGGCCA-3 '(SEQ ID NO: 48) Enzyme for PCR; KOD-Plus- (Toyobo) ⁇ A.
- PCR was performed under the following conditions to amplify the DNA of the pCDF plasmid into which the DNA encoding the PgsA anchor protein was inserted, and the obtained PCR product was used as a pCDF-PgsA-DNA fragment.
- A. oryzae wild-type DNA fragment-4 and pCDF-PgsA-DNA fragment; terreus wild-type DNA fragment-4 and pCDF-PgsA-DNA fragment were ligated using In-Fusion HD HD Cloning Kit (Takara Bio Inc.), respectively, to obtain recombinant vectors.
- a replacement vector was used, and the latter was a terreus (wild type) recombinant vector.
- DNA fragment was purified and self-ligated by the method described in Example 2 (2) [2-1].
- Recombinant vectors into which DNAs encoding improved terreus-derived ⁇ -fructofuranosidase were inserted were prepared, the former being the oryzae (G78W) recombinant vector and the latter being the terreus (G78W) recombinant vector.
- Example 4 (1) Activity confirmation Using the recombinant Escherichia coli of Example 4 (1) [1-4], the enzyme reaction of ⁇ -fructofuranosidase and the method described in Example 3 (1) and (2) The enzyme reaction product was confirmed. However, the enzyme reaction time was 3 hours. The results are shown in Table 5. For comparison, Table 5 also shows the results of recombinant Escherichia coli into which the kawachii (wild type) recombinant vector and the kawachii (G85W) recombinant vector have been introduced. In Table 5, “-” indicates that it was below the detection limit.
- the amount of kestose per wet cell weight was determined by recombinant Escherichia coli introduced with kawachii (wild type), oryzae (wild type) and terreus (wild type) recombinant vectors.
- Kawachii (G85W) recombinant vector, oryzae (G78W) recombinant vector and terreus (G78W) recombinant vector were introduced.
- the amounts were 9.62 mg, 2.84 mg, and 2.96 mg, respectively.
- the nystose content is 29.64% in the reaction solution of recombinant Escherichia coli introduced with kawachii (wild type) recombinant vector, oryzae (wild type) recombinant vector and terreus (wild type) recombinant vector, Compared to 31.68% and 15.42%, in the reaction solution of recombinant Escherichia coli introduced with kawachii (G85W), oryzae (G78W) and terreus (G78W) recombinant vectors Respectively, 4.51%, 20.86% and 16.91%.
- the amount of kestose increased in the reaction solution of recombinant E. coli introduced with the kawachii (G85W) recombinant vector compared to the reaction solution of recombinant Escherichia coli introduced with the kawachii (wild type) recombinant vector.
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Abstract
Description
(a)配列番号2に示すアミノ酸配列(全長628アミノ酸)に対して、以下のi)および/またはii)のアミノ酸変異を導入したアミノ酸配列;
i)N末端から85番目のグリシン(G)をグリシン(G)以外のタンパク質構成アミノ酸に置換するアミノ酸変異、
ii)N末端から310番目のヒスチジン(H)をリシン(K)、アルギニン(R)またはチロシン(Y)に置換するアミノ酸変異、
(b)(a)において、アミノ酸変異が導入されたアミノ酸を除く1もしくは複数個のアミノ酸を欠失、置換、挿入もしくは付加したアミノ酸配列からなり、かつβ-フルクトフラノシダーゼ活性を有するアミノ酸配列。
(a)配列番号2に示すアミノ酸配列(全長628アミノ酸)に対して、以下のi)および/またはii)のアミノ酸変異を導入したアミノ酸配列;
i)N末端から85番目のグリシン(G)をグリシン(G)以外のタンパク質構成アミノ酸に置換するアミノ酸変異、
ii)N末端から310番目のヒスチジン(H)をリシン(K)、アルギニン(R)またはチロシン(Y)に置換するアミノ酸変異、
(b)(a)において、アミノ酸変異が導入されたアミノ酸を除く1もしくは複数個のアミノ酸を欠失、置換、挿入もしくは付加したアミノ酸配列からなり、かつβ-フルクトフラノシダーゼ活性を有するアミノ酸配列。
ここで、(a)と(b)との同一性の値としては、例えば、80~85%以上、85~90%以上、90~95%以上などを挙げることができる。
すなわち、(b)は、例えば、「(a)との同一性の値が80~90%未満とならない範囲で、(a)において、アミノ酸変異が導入されたアミノ酸を除く1もしくは複数個のアミノ酸を欠失、置換、挿入もしくは付加したアミノ酸配列」からなる。
したがって、(b)の「欠失、置換、挿入もしくは付加したアミノ酸」の個数として、具体的には、例えば、1~125個((a)との同一性が80%以上)、1~113個((a)との同一性が82%以上)、1~100個((a)との同一性が84%以上)、1~87個((a)との同一性が86%以上)、1~75個((a)との同一性が88%以上)、1~62個((a)との同一性が90%以上)、1~50個((a)との同一性が92%以上)、1~37個((a)との同一性が94%以上)、1~25個((a)との同一性が96%以上)、1~12個((a)との同一性が98%以上)などを挙げることができる。
(1)結晶化試験
水溶液に含まれる糖におけるニストースの含有割合((w/w)%;以下、「ニストース含有割合」という)が5%程度および10%程度である原料糖液を用いてケストースの結晶を製造し、ケストースの回収率および結晶サイズの確認を行うことにより、ケストースの結晶製造時におけるニストース含有割合の影響を検討した。具体的な手順を以下に示す。
式1;ケストースの回収率={結晶重量/(小型遠心分離機に供した糖液の重量×糖液のBrix値/100)}×100
また、顕微鏡を用いて結晶サイズを観察した。また、結晶および原料糖液を下記の条件により高速液体クロマトグラフィー(HPLC)に供して、各糖(単糖;フルクトース、単糖;グルコース、2糖;スクロース、3糖;ケストース、4糖;ニストース、およびその他の糖)の含有割合を確認した。各糖の含有割合は、検出された全ピークの面積の総和に対する各ピークの面積の割合として、面積百分率で算出した。その結果を表2に示す。なお、表1中、「-」は測定不可であったことを示す。
《HPLCの条件》
カラム;SHODEX KS 802(8.0φ×300mm) 2本
移動相;水
流速 ;1.0mL/分
注入量;20μL
温度 ;50℃
検出 ;示差屈折率検出器(RID;昭和電工社)(ピーク面積を用いた面積百分率)
スクロースを基質としてβ-フルクトフラノシダーゼによる酵素反応を行い、種々の割合でニストース、ケストース、スクロース、グルコースおよびフルクトースを含む反応液を得て、No.1~8とした。これらについて、結晶製造に足る程度までケストース含有割合を高める(ケストース含有割合(ケストース純度)が約80%程度)ために、イオン交換樹脂を用いたクロマトグラフィーに供してケストースの分離精製を行い、ケストース高純度液を得た。なお、クロマトグラフィーの条件はNo.1~8のサンプル間で、同一条件とした。反応液およびケストース高純度液における各糖の含有割合を表3に示す。
A.kawachii由来の野生型β-フルクトフラノシダーゼのアミノ酸配列(配列番号2)に対して、N末端から85番目のグリシン(G)を、トリプトファン(W)、フェニルアラニン(F)、チロシン(Y)、アスパラギン酸(D)、グルタミン酸(E)またはアルギニン(R)に置換するアミノ酸変異(以下、それぞれのアミノ酸変異を「G85W」「G85F」「G85Y」「G85D」「G85E」「G85R」と略記する。)、および、N末端から310番目のヒスチジン(H)を、リシン(K)、アスパラギン酸(D)、アルギニン(R)、チロシン(Y)、グリシン(G)またはトリプトファン(W)に置換するアミノ酸変異(以下、それぞれのアミノ酸変異を「H310K」「H310D」「H310R」「H310Y」「H310G」「H310W」と略記する。)を導入したアミノ酸配列からなる一重変異体および二重変異体のβ-フルクトフラノシダーゼを作成し、これらを改良型β-フルクトフラノシダーゼとした。具体的な手順を以下に示す。
[1-1]DNAの取得
A.kawachii由来野生型β-フルクトフラノシダーゼ(GenBank:GAA88101.1)をコードするDNAをジェンスクリプト社に依頼して人工合成して取得した。A.kawachii由来野生型β-フルクトフラノシダーゼをコードするDNAの全長の塩基配列を配列番号1に、それにコードされるA.kawachii由来野生型β-フルクトフラノシダーゼのアミノ酸配列を配列番号2に、それぞれ示す。なお、配列番号2において、シグナル配列は1~24番目に相当する。
下記の条件でポリメラーゼ連鎖反応(PCR)を行うことにより、Bacillus subtilis(IAM1026、ATCC9466)のPgsAアンカータンパク質(GenBank:AB016245.1)をコードするDNAを増幅した。得られたPCR産物を常法に従って制限酵素NdeIおよびBglIIで消化し、これをPgsA-DNA断片とした。また、PgsA-DNA断片について、常法に従ってシークエンスを行い、その塩基配列を確認した。確認したPgsAアンカータンパク質をコードするDNAの塩基配列を配列番号3に、それにコードされるPgsAアンカータンパク質のアミノ酸配列を配列番号4にそれぞれ示す。
鋳型;Bacillus subtilis(IAM1026、ATCC9466)のゲノムDNA
フォワードプライマー(下線はNdeIサイトを示す);5’-AAACATATGAAAAAAGAACTGAGCTTTCATG-3’(配列番号5)
リバースプライマー(下線はBglIIサイトを示す);5’-AAAAGATCTTTTAGATTTTAGTTTGTCACTATG-3’(配列番号6)
PCR用酵素;KOD-Plus-(東洋紡社)
《A.kawachii由来β-フルクトフラノシダーゼをコードするDNA増幅用PCRの条件》
本鋳型;実施例2(1)[1-1]のA.kawachii由来野生型β-フルクトフラノシダーゼをコードするDNA
フォワードプライマー;5’-AAATCTAAAAGATCCTCCGTGGTCATCGACTAC-3’(配列番号7)
リバースプライマー;5’-TTTACCAGACTCGAGTCAATACTGACGATCCGGC-3’(配列番号8)
PCR用酵素;KOD-Plus-Neo(東洋紡社)
《PgsAタンパク質をコードするDNAが挿入されたpCDFプラスミド由来のDNA増幅用PCRの条件》
鋳型;pCDF-PgsA組換えベクター
フォワードプライマー;5’-CTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAA-3’(配列番号9)
リバースプライマー;5’-GGATCTTTTAGATTTTAGTTTGTCACTATGATCAA-3’(配列番号10)
PCR用酵素;KOD-Plus-Neo(東洋紡社)
[2-1]一重変異体
本実施例2(1)[1-2]のkawachii(野生型)組換えベクターを鋳型とし、KOD-Plus-NEO(東洋紡社)をPCR用酵素として用いて、下記のプライマーおよび反応条件でPCRを行うことにより、改良型β-フルクトフラノシダーゼをコードするDNAを含んだDNA断片を増幅した。
「G85W」
フォワードプライマー;5’-TGGAGCGGCATCTCCAGTGCCACCA-3’(配列番号11)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号12)
「G85F」
フォワードプライマー;5’-TTCAGCGGCATCTCCAGTGCCACCA-3’(配列番号13)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号14)
「G85Y」
フォワードプライマー;5’-TATAGCGGCATCTCCAGTGCCACCA-3’(配列番号15)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号16)
「G85D」
フォワードプライマー;5’-GATAGCGGCATCTCCAGTGCCACCA-3’(配列番号17)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号18)
「G85E」
フォワードプライマー;5’-GAAAGCGGCATCTCCAGTGCCACCA-3’(配列番号19)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号20)
「G85R」
フォワードプライマー;5’-CGTAGCGGCATCTCCAGTGCCACCA-3’(配列番号21)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号22)
「H310K」
フォワードプライマー;5’-AAAGACATGCTCTGGGTGTCCGGTACAGTC-3’(配列番号23)
リバースプライマー;5’-GATGCTGGTGAGCTGGGGCACGACGGGCA-3’(配列番号24)
「H310D」
フォワードプライマー;5’-GATGACATGCTCTGGGTGTCCGGTACAGTC-3’(配列番号25)
リバースプライマー;5’-GATGCTGGTGAGCTGGGGCACGACGGGCA-3’(配列番号26)
「H310Y」
フォワードプライマー;5’-TATGACATGCTCTGGGTGTCCGGTACAGTC-3’(配列番号27)
リバースプライマー;5’-GATGCTGGTGAGCTGGGGCACGACGGGCA-3’(配列番号28)
「H310R」
フォワードプライマー;5’-CGTGACATGCTCTGGGTGTCCGGTACAGTC-3’(配列番号29)
リバースプライマー;5’-GATGCTGGTGAGCTGGGGCACGACGGGCA-3’(配列番号30)
「H310G」
フォワードプライマー;5’-GGCGACATGCTCTGGGTGTCCGGTACAGTC-3’(配列番号31)
リバースプライマー;5’-GATGCTGGTGAGCTGGGGCACGACGGGCA-3’(配列番号32)
「H310W」
フォワードプライマー;5’-TGGGACATGCTCTGGGTGTCCGGTACA GTC-3’(配列番号33)
リバースプライマー;5’-GATGCTGGTGAGCTGGGGCACGACGGGCA-3’(配列番号34)
下記の条件でPCRを行うことにより、「G85W」および「H310K」を導入したアミノ酸配列からなる二重変異体の改良型β-フルクトフラノシダーゼをコードするDNAを増幅した。
《改良型β-フルクトフラノシダーゼをコードするDNA増幅用PCRの条件》
鋳型;本実施例2(2)[2-1]のkawachii(H310K)組換えベクター
フォワードプライマー;TGGAGCGGCATCTCCAGTGCCACCA-3’(配列番号11)
リバースプライマー;5’-ATCGTGAAGGAAGCCGACGTGGAAGAGG-3’(配列番号12)
PCR用酵素;KOD-Plus-NEO(東洋紡社)
本実施例2(1)[1-2]、(2)[2-1]および(2)[2-2]の各組換えベクターを、E.coli JM109コンピテントセル(ニッポンジーン社)に導入して、組換え大腸菌を回収した。続いて、組換え大腸菌から組換えベクターを回収し、これを、E.coli BL21(DE3)コンピテントセル(コスモバイオ社)に導入して、形質転換体として組換え大腸菌を得た。これを30℃で一晩プレート培養した後、組換え大腸菌のコロニーをピックアップしてM9 SEED培地0.5mLに植菌し、30℃、220rpmで20時間振盪培養した。続いて、このうち5μLをM9 Main培地5mLに植え継ぎ、25℃、220rpmで24時間振盪培養して、培養物を得た。M9 SEED培地およびM9 Main培地の組成を以下に示す。
M9 Main培地(計100mL);水 67mL、5×M9塩 20mL、20% カザミノ酸 5mL、2mg/mL チミン 1mL、50mM CaCl2 0.2mL、100mg/mL FeSO4 28μL、Overnight Express Autoinduction System 1(O.N.E.;Merck社)Sol.1 2mL、O.N.E.Sol.2 5mL、O.N.E.Sol.3 100μL、抗生物質(終濃度50μg/mL ストレプトマイシン硫酸塩)。
(1)β-フルクトフラノシダーゼの酵素反応
30(w/w)%のスクロースを含む0.04Mのリン酸ナトリウムバッファー(pH7.0)を調製し、これを30%スクロース溶液とした。実施例2(3)の組換え大腸菌の培養物0.5mLを遠心分離に供して集菌し、湿重量(湿菌体重量)を測定した。ここに、30%スクロース溶液350μLを加えて懸濁した後、30℃、200rpmで一定時間振盪することによりβ-フルクトフラノシダーゼの酵素反応を行い、これを反応液とした。酵素反応の時間は、3、9、32および48時間とした。
続いて、反応液50μLに水950μLを加えることにより希釈し、100℃で10分間加熱した。これを4℃、15000×gで10分間遠心分離に供して上清を回収した後、0.45μm孔フィルターで濾過して、得られた濾液をHPLCサンプルとした。HPLCサンプルを実施例1(1)に記載の条件でHPLCに供して、反応液に含まれる各糖の含有割合を確認した。また、反応液に含まれるケストースおよびニストースの量を、酵素反応開始時の反応液に含まれていたスクロースの質量にケストースおよびニストースのそれぞれの含有割合を乗じて算出し、湿菌体重量で除した値で表した。その結果を表4に示す。なお、表4中、「-」は検出限界以下であったことを示す。
(1)改良型β-フルクトフラノシダーゼの作成
[1-1]アラインメント
A.kawachii由来野生型β-フルクトフラノシダーゼと68%の同一性を有するアミノ酸配列からなるβ-フルクトフラノシダーゼとして下記(ア)を、A.kawachii由来野生型β-フルクトフラノシダーゼと60%の同一性を有するアミノ酸配列からなるβ-フルクトフラノシダーゼとして下記(イ)を、それぞれ抽出した。
(ア)68%の同一性:Aspergillus oryzae RIB40(以下「A.oryzae」と略記する。)のβ-フルクトフラノシダーゼ(XP_003190558)
(イ)60%の同一性:Aspergillus terreus NIH2624(以下「A.terreus」と略記する。)のβ-フルクトフラノシダーゼ(XP_001214174)
A.oryzaeおよびA.terreusのゲノムDNAをそれぞれ鋳型として下記の条件によりPCRを行い、A.oryzae由来野生型β-フルクトフラノシダーゼをコードするDNAおよびA.terreus由来野生型β-フルクトフラノシダーゼをコードするDNAを増幅した。なお、鋳型において、β-フルクトフラノシダーゼをコードする領域にイントロンが存在しており、これを除くためにPCRは2回に分けて行った。このPCRにより得られたPCR産物を、それぞれA.oryzae野生型DNA断片-1、2およびA.terreus野生型DNA断片-1、2とした。
フォワードプライマー;5’-ACATCACAGATAACATATGAAGCTCTCAACCGCGAGTGCCT-3’(配列番号39)
リバースプライマー;5’-CCGAGCCCAAGTACTCAGGGCAAAACGTCC-3’(配列番号40)
PCR用酵素;KOD-Plus-(東洋紡社)
《A.oryzae野生型DNA断片-2を増幅するPCRの条件》
フォワードプライマー;5’-AGTACTTGGGCTCGGTCCTGGTACAAGAACTCGACTGACATCAAG-3’(配列番号41)
リバースプライマー;5’-GAGCAAGCTTCTCGAGTTAGACACGCTCAGGCCAGGCTTCA-3’(配列番号42)
PCR用酵素;KOD-Plus-(東洋紡社)
《A.terreus野生型DNA断片-1を増幅するPCRの条件》
フォワードプライマー;5’-ACATCACAGATAACATATGAAATCCTCAGTGACACGGATGG-3’(配列番号43)
リバースプライマー;5’-CGAACCCAAGTAGAGAGCGCAAATCGCGAA-3’(配列番号44)
PCR用酵素;KOD-Plus-Neo(東洋紡社)
《A.terreus野生型DNA断片-2を増幅するPCRの条件》
フォワードプライマー;5’-CTCTACTTGGGTTCGGCCTTGGTACAGTTATTCGAATGAGATTAG-3’(配列番号45)
リバースプライマー;5’-GAGCAAGCTTCTCGAGTTACCTCTCGCGCTCGGGGTAAGCA-3’(配列番号46)
PCR用酵素;KOD-Plus-Neo(東洋紡社)
鋳型;A.oryzae野生型DNA断片-3
フォワードプライマー5’-AAATCTAAAAGATCCTCCGCCATCGATTACAACG-3’(配列番号47)
リバースプライマー5’-TTTACCAGACTCGAGTTAGACACGCTCAGGCCA-3’(配列番号48)
PCR用酵素;KOD-Plus-(東洋紡社)
《A.terreus野生型DNA断片-4を増幅するPCRの条件》
鋳型;A.terreus野生型DNA断片-3
フォワードプライマー5’-AAATCTAAAAGATCCGCAGCGCAGGACTACAAT-3’(配列番号49)
リバースプライマー5’-TTTACCAGACTCGAGTTACCTCTCGCGCTCGGG-3’(配列番号50)
PCR用酵素;KOD-Plus-(東洋紡社)
《PgsAアンカータンパク質をコードするDNAを挿入したpCDFプラスミドのDNA増幅用PCRの条件》
鋳型;実施例2(1)[1-1]のpCDF-PgsA組換えベクター
フォワードプライマー;5’-CTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAA-3’(配列番号51)
リバースプライマー;5’-GGATCTTTTAGATTTTAGTTTGTCACTATGATCAA-3’(配列番号52)
PCR用酵素;KOD-Plus-(東洋紡社)
下記の条件でPCRを行うことにより、改良型β-フルクトフラノシダーゼをコードするDNAを含んだDNA断片を増幅した。
《A.oryzae由来の改良型β-フルクトフラノシダーゼをコードするDNA増幅用PCRの条件》
鋳型;本実施例4(1)[1-2]のoryzae(野生型)組換えベクター
フォワードプライマー;5’-TGGAGTGGTATCGCAGGCGCTAC-3’(配列番号53)
リバースプライマー;5’-ATTGTACAGGAAGCCAACGTGGAAC-3’(配列番号54)
PCR用酵素;KOD-Plus-(東洋紡社)
《A.terreus由来の改良型β-フルクトフラノシダーゼをコードするDNA増幅用PCRの条件》
鋳型;本実施例4(1)[1-2]のterreus(野生型)組換えベクター
フォワードプライマー;5’-TGGACTGGGATTTCGGCTGTC-3’(配列番号55)
リバースプライマー;5’-ATTGTGTAGGAACCCGACATG-3’(配列番号56)
PCR用酵素;KOD-Plus-(東洋紡社)
本実施例4(1)[1-2]および[1-3]の各組換えベクターについて、実施例2(3)に記載の方法により形質転換および形質転換体の培養と回収を行い、組換え大腸菌を得た。
本実施例4(1)[1-4]の組換え大腸菌を用いて、実施例3(1)および(2)に記載の方法によりβ-フルクトフラノシダーゼの酵素反応および酵素反応生成物の確認を行った。ただし、酵素反応の時間は3時間とした。その結果を表5に示す。なお、表5には、比較のために、表4に示す結果のうちkawachii(野生型)組換えベクターおよびkawachii(G85W)組換えベクターを導入した組換え大腸菌における結果を併せて示す。表5中、「-」は検出限界以下であったことを示す。
Claims (7)
- 下記(a)または(b)のアミノ酸配列からなる改良型β-フルクトフラノシダーゼ;
(a)配列番号2に示すアミノ酸配列に対して、以下のi)および/またはii)のアミノ酸変異を導入したアミノ酸配列;
i)N末端から85番目のグリシン(G)をグリシン(G)以外のタンパク質構成アミノ酸に置換するアミノ酸変異、
ii)N末端から310番目のヒスチジン(H)をリシン(K)、アルギニン(R)またはチロシン(Y)に置換するアミノ酸変異、
(b)(a)において、アミノ酸変異が導入されたアミノ酸を除く1もしくは複数個のアミノ酸を欠失、置換、挿入もしくは付加したアミノ酸配列からなり、かつβ-フルクトフラノシダーゼ活性を有するアミノ酸配列。 - 請求項1に記載の改良型β-フルクトフラノシダーゼのアミノ酸配列を含むポリペプチド。
- 請求項1に記載の改良型β-フルクトフラノシダーゼをコードするDNA。
- 請求項3に記載のDNAを含む組換えベクター。
- 請求項3に記載のDNAまたは請求項4に記載の組換えベクターを宿主に導入して得られる、形質転換体。
- 請求項5に記載の形質転換体を培養して得られる培養物から改良型β-フルクトフラノシダーゼを取得する工程を有する改良型β-フルクトフラノシダーゼの製造方法。
- 請求項1に記載の改良型β-フルクトフラノシダーゼ、請求項5に記載の形質転換体または請求項5に記載の形質転換体を培養して得られる培養物とスクロースとを接触させる工程を有するケストースの製造方法。
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KR1020177027177A KR20170139002A (ko) | 2015-03-11 | 2016-03-10 | 개량형 β-프룩토프라노시다아제 |
JP2017505409A JP6657177B2 (ja) | 2015-03-11 | 2016-03-10 | 改良型β−フルクトフラノシダーゼ |
US15/557,152 US10240136B2 (en) | 2015-03-11 | 2016-03-10 | β-fructofuranosidase |
EP16761839.6A EP3269808A4 (en) | 2015-03-11 | 2016-03-10 | Improved beta-fructofuranosidase |
AU2016228331A AU2016228331A1 (en) | 2015-03-11 | 2016-03-10 | Improved beta-fructofuranosidase |
CN201680015141.8A CN107429243A (zh) | 2015-03-11 | 2016-03-10 | 改良型β‑呋喃果糖苷酶 |
BR112017019202-0A BR112017019202A2 (ja) | 2015-03-11 | 2016-03-10 | Advanced beta- Fructofuranosidase |
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WO2018117198A1 (ja) * | 2016-12-20 | 2018-06-28 | 物産フードサイエンス株式会社 | 糖液ならびにこれを用いる液体甘味料およびハナバチ用飼料 |
JPWO2018117198A1 (ja) * | 2017-04-14 | 2020-01-23 | 物産フードサイエンス株式会社 | 糖液ならびにこれを用いる液体甘味料およびハナバチ用飼料 |
WO2023032268A1 (ja) * | 2021-08-31 | 2023-03-09 | 株式会社島津製作所 | 情報提供方法、分析システム、およびプログラム |
WO2023058637A1 (ja) * | 2021-10-04 | 2023-04-13 | 京都府公立大学法人 | 改良型β-フルクトフラノシダーゼ |
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WO2018117198A1 (ja) * | 2016-12-20 | 2018-06-28 | 物産フードサイエンス株式会社 | 糖液ならびにこれを用いる液体甘味料およびハナバチ用飼料 |
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US10240136B2 (en) | 2019-03-26 |
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