JP2008199977A - Novel isoprimeverose-producing oligoxyloglucan hydrolase, gene encoding the same and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an isoprimeverose-producing oligoxyloglucan hydrolase by preparing the enzyme from a microorganism belonging to genus Oerskovia as a prokaryotic microorganism, elucidating its enzymatic characteristics, further taking out its gene, and furthermore a gene technology using the gene. <P>SOLUTION: A prokaryotic microorganism growable by utilizing oligoxyloglucan as a carbon resource has been screened from the natural world and the isolated microorganisms has been examined whether the isoprimeverose-producing oligoxyloglucan hydrolase is produced. As a result, genus Oerskovia Y1 strain belonging to genus Oerskovia being a kind of Actinomyces has been found to produce the enzyme. The enzyme has been purified and its amino acid sequence and a base sequence of its gene has been elucidated. The present invention has been completed by establishing a method for producing a recombinant enzyme in large quantities using the gene. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel isoprimebellose-producing oligoxyloglucan hydrolase, a polypeptide having a specific amino acid sequence having isoprimebellose-producing oligoxyloglucan hydrolyzing activity, its gene, and a recombinant containing the gene The present invention relates to a vector, a transformant transformed with the recombinant vector, a method for producing the polypeptide by culturing the transformant, and the like.

  Xyloglucan is a heteropolysaccharide composed of glucose, xylose, galactose, fucose, arabinose, etc., but the main chain in which glucose is linked by a number of β-1,4-glucoside bonds, xylose is α- It is called xyloglucan because it is based on a structure in which 1,6-xyloside bonds are branched at a high frequency. This xyloglucan plays an important role as a component of the primary cell wall of plants and as a seed storage polysaccharide of tropical legumes represented by tamarind beans, and its structure and function are attracting attention. In particular, xyloglucan in the primary cell wall of plants is considered to be closely related to cellulose, and its relative form with cellulose plays an important role in plant cell elongation and morphological differentiation. It is considered to be responsible. Various techniques have been used to elucidate the role of xyloglucan. Among them, various glycosidases capable of degrading xyloglucan provide an important and powerful analytical means.

  As described above, xyloglucan has glucose, xylose, galactose, fucose, arabinose and the like as a constituent monosaccharide and the structure of the main chain is the same as that of cellulose. 4-β-D-glucanase, β-galactosidase, isoprimeverose-producing oligoxyloglucan hydrolase, α-xylosidase, β-glucosidase, α-fucosidase, etc. are known, and the structure and function of xyloglucan are elucidated. In addition, these enzymes of various origins have been used.

  Among them, isoprimebellose-producing oligoxyloglucan hydrolase has the action of cleaving the β-1,4-glucoside bond of the non-reducing end glucose residue of oligoxyloglucan to produce isoprimeverose. Yes. There are very few reports of the enzyme, and only those derived from eukaryotic microorganisms have been reported (Non-patent Document 1). In addition, the gene is completely unknown.

Yoji Kato et.al. Journal of Biochemistry (1985) 97, 801-810

  An object of the present invention is to produce isoprimiverose-producing oligoxyloglucan hydrolase from a prokaryotic microorganism of the genus Oerskovia, to clarify its enzymatic characteristics, to further extract the gene, and to use the gene Another object of the present invention is to provide a method for producing the enzyme by genetic engineering techniques.

  The present inventors screened prokaryotic microorganisms capable of growing using oligoxyloglucan as a carbon source from the natural world, and further examined the ability of isolated microorganisms to produce isoprimebellose-producing oligoxyloglucan hydrolase. As a result, we found that one of the actinomycetes, one strain of the genus Oerskovia (Oerskovia strain Y1) produces the enzyme, purified the enzyme and elucidated the amino acid sequence and the base sequence of the gene. did. And the manufacturing method of a lot of recombinant enzymes was established using this gene, and it came to complete this invention.

That is, the present invention relates to the following (1) to (19).
(1) A protein derived from a microorganism belonging to the genus Oerskovia, which has the action of cleaving the β-1,4-glucoside bond of the non-reducing end glucose residue of oligoxyloglucan to produce isoprimeverose. Isoprimebellose-producing oligoxyloglucan hydrolase.
(2) a polypeptide having the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence in which one or several amino acid residues in the polypeptide are deleted, added, inserted or substituted, and Polypeptide having primeverose-generating oligoxyloglucan hydrolyzing activity.
(3) The polypeptide according to (2) above, wherein methionine is added to the N-terminus.
(4) The polypeptide according to (2) above, to which a signal sequence is added.
(5) a polypeptide having the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide having an amino acid sequence in which one or several amino acids in the polypeptide are deleted, added, inserted or substituted, A polypeptide that is a precursor of a polypeptide having hydrolyzing activity of isoprimebellose-producing oligoxyloglucan.
(6) A polynucleotide encoding the polypeptide according to any one of (2) to (5) above.
(7) A polynucleotide having the base sequence shown in SEQ ID NO: 9 or a base sequence in which one or several nucleic acids in the polynucleotide are deleted, added, inserted or substituted, A polynucleotide expressing a polypeptide having hydrolyzing activity of bellows-producing oligoxyloglucan.
(8) The polynucleotide according to (7) above, to which an initiation codon has been added.
(9) The polynucleotide according to (7) above, to which a base sequence corresponding to the signal peptide sequence is added.
(10) A polynucleotide having the base sequence shown in SEQ ID NO: 7, or a polynucleotide having a base sequence in which one or several nucleic acids in the polynucleotide are deleted, added, inserted or substituted, A polynucleotide expressing a precursor of a polypeptide having primeverose-producing oligoxyloglucan hydrolyzing activity.
(11) A polynucleotide that hybridizes under stringent conditions to the polynucleotide according to any one of (7) to (10) above.
(12) A polynucleotide having homology to the polynucleotide according to any one of (7) to (11) above.
(13) A polynucleotide that degenerates to the polynucleotide according to any one of (7) to (11) above.
(14) A recombinant vector comprising the polynucleotide according to any one of (6) to (13) above.
(15) A transformant comprising the recombinant vector according to (14) above.
(16) A method for producing a polypeptide, comprising culturing the transformant according to (15) above, and collecting a polypeptide corresponding to the recombinant polynucleotide from the culture.
(17) The method for producing a polypeptide according to the above (16), wherein the polypeptide has an activity of hydrolyzing isoprimiverose producing oligoxyloglucan.
(18) A microorganism belonging to the genus Oerskovia is cultured in a medium, and isoprimiverose-producing oligoxyloglucan hydrolase is collected from the culture. Production method.
(19) The method for producing an isoprimiverose-producing oligoxyloglucan hydrolase according to (18) above, wherein the microorganism belonging to the genus Oerskovia is Oerskovia sp. Y1 strain (FERM P-21157).
(20) Oerskovia sp. Y1 strain (FERM P-21157).

  In the present invention, the term “polypeptide having an amino acid sequence” or “polynucleotide having a base sequence” is not limited to those consisting of the amino acid sequence or base sequence pointed out in the description, and these sequences are used as part thereof. The polypeptide and polynucleotide contained are also included.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel and useful eucaryotic microorganism-derived isoprimiverose-producing oligoxyloglucan hydrolase can be provided, and its amino acid sequence and gene structure have been clarified. A large amount of recombinant enzyme can be produced using this gene.

The isoprimebellose-producing oligoxyloglucan hydrolase in the present invention is an enzyme derived from a microorganism belonging to the genus Oerskovia, and is a β-1,4-glucoside bond of a glucose residue at the non-reducing end of oligoxyloglucan. Has the effect of producing isoprimiverose.
More specifically, this enzyme is produced by the Oerskovia sp. Y1 strain, and the Oerskovia sp. Y1 strain is the industrial technology as Oerskovia sp. Y1 strain, FERM P-21157 Deposited at the Research Institute and the Patent Microorganism Deposit Center.

  In order to produce the enzyme of the present invention from the microorganism belonging to the genus Orskovia, oligoxyloglucan is usually used as a carbon source, and as a nitrogen source, nitrate, ammonium salt or peptone, yeast extract, etc. Using a liquid or solid medium containing an inorganic or organic nitrogen source and a small amount of metal salt, the cells are aerobically cultured at 20-30 ° C. for about 4-10 days. Since the enzyme of the present invention is an enzyme produced outside the cells, after culturing in the case of a liquid medium, the filtered or centrifuged supernatant is used. In the case of solid culture, after culturing, water or a suitable inorganic substance is used. The liquid extracted with salts can be used as a crude enzyme solution. The crude enzyme solution contains various enzyme activities that degrade xyloglucan in addition to the enzyme of the present invention. Therefore, it is necessary to remove this, but the known enzyme coexists other than the enzyme of the present invention. Glycosidase activity can be removed.

  The enzyme (purified product) of the present invention obtained as described above has the following properties.

(1) Molecular weight: The purified enzyme of the present invention has a molecular weight of about 105,000.

(2) Action: The enzyme of the present invention degrades the β-glucoside bond of the glucose residue at the non-reducing end of oligoxyloglucan to produce isoprimeverose.

(3) Optimum pH: When oligoxyloglucan is used as a degradation substrate, the optimum pH of the enzyme of the present invention is around pH 4-5.

(4) Stable pH range: The enzyme of the present invention is allowed to stand in a citrate-phosphate buffer solution at 35 ° C. for 20 minutes, and the stable pH range in view of the residual activity is about pH 3.5 to 7.5. .

(5) Optimal action temperature: When oligoxyloglucan is used as a degradation substrate, the optimum action temperature of the enzyme of the present invention is 55 ° C.

(6) Thermal stability: The residual activity of the enzyme of the present invention after 20 minutes of heat treatment at 20 ° C. under 20 mM phosphate buffer (pH 6.0) hardly deactivates up to 45 ° C.

(7) Purification method: The enzyme of the present invention is obtained by concentrating and desalting the culture supernatant by ultrafiltration, followed by anion exchange columns HiPrep DEAE, HiPrep Q and ResourceQ, cation exchange column ResourceS, hydrophobic column Resource PHE ( Chromatography using GE Healthcare Biosciences) is repeated, the fractions that retain activity are concentrated, and gel filtration is performed using a HiLoad Superdex 200 pg column (GE Healthcare Biosciences). By carrying out the above, it was possible to separate and purify until SDS electrophoretic uniformity.

(8) Activity measurement method: Since the enzyme of the present invention specifically degrades oligoxyloglucan, activity measurement using oligoxyloglucan heptasaccharide (Tokyo Chemical Industry) reduced by sodium borohydride treatment as a substrate Went. An appropriate amount of enzyme was added to the 0.1% aqueous solution (pH 4.5) of the substrate and reacted at 50 ° C. for 10 minutes, and the resulting reducing sugar was measured by the bicinchoninic acid method to determine the activity. Under this condition, the amount of enzyme that produces a reducing power corresponding to 1 micromole of glucose per minute was defined as 1 unit.

  The novel isoprimebellose-producing oligoxyloglucan hydrolase of the present invention is a secreted protein and has the amino acid sequence shown in SEQ ID NO: 8, in which residues 1 to 28 are presumed to be signal sequences. The The mature protein of this enzyme is predicted to have the residues 1 to 28 removed, and the amino acid sequence is shown in SEQ ID NO: 10.

Accordingly, the preferred polypeptide of the present invention has at least the amino acid sequence of SEQ ID NO: 10, and the polypeptide and one or several amino acid residues deleted, added, or inserted. Alternatively, even if the amino acid sequence differs depending on the substitution, it is included in the present invention as long as it has the activity of hydrolyzing isoprimiverose-producing oligoxyloglucan.
The polypeptide of the present invention includes a precursor polypeptide having the above signal sequence as shown in SEQ ID NO: 8, or a deletion or addition of one or several amino acid residues from the polypeptide. Even those having different amino acid sequences due to insertion or substitution can include those capable of producing a polypeptide having isoprimebellose-producing oligoxyloglucan hydrolyzing activity by splicing.

  Furthermore, the polypeptide of the present invention can be produced using genetic recombination means. In this case, for example, a methionine derived from a start codon may be added to the N terminus of the amino acid sequence shown in SEQ ID NO: 10, and for example, isoprimebellose production is performed for the polypeptide shown in SEQ ID NO: 14. It has been confirmed to have oligoxyloglucan hydrolyzing activity.

The gene encoding the polypeptide having hydrolyzing activity of the isoprimebellose producing oligoxyloglucan of the present invention includes a gene that can be obtained by cloning the gene from a microorganism that produces the enzyme, and homology to the gene. The gene which has is mention | raise | lifted. The homology includes a gene having at least 60% homology, preferably a gene having 80% or more homology, more preferably a gene having 95% or more homology.
As a gene encoding a polypeptide having an isoprimebellose-producing oligoxyloglucan hydrolyzing activity of the present invention, for example, the following polynucleotides (DNA or RNA) are preferable.

(1) A polynucleotide having the base sequence shown in SEQ ID NO: 9, or a polynucleotide having a base sequence in which one or several nucleic acids of the polypeptide have been deleted, added, inserted or substituted, A polynucleotide encoding a polypeptide having hydrolyzing activity of bellose-generating oligoxyloglucan.
(2) A polynucleotide in which a start codon is added to the polynucleotide of (1) above.
(3) A polynucleotide in which a nucleotide sequence corresponding to a signal sequence is added to the polynucleotide shown in (1) above, or one or several nucleic acids in the polynucleotide are deleted, added, inserted or substituted. A polynucleotide having a base sequence and capable of expressing a matured product by splicing, specifically, a polynucleotide having the base sequence shown in SEQ ID NO: 7.

In addition, a gene that hybridizes under stringent conditions to the polynucleotides (1) to (3) above, a polynucleotide having homology to the polynucleotide, and a polynucleotide degenerate to the polynucleotide So long as the polypeptides they encode have hydrolyzing activity for isoprimiverose producing oligoxyloglucan, they are included in the present invention.
Here, “stringent conditions” refers to the following conditions, for example. That is, 6 × SSC (1) containing 0.5% SDS, 5 × Denhartz [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 μg / ml salmon sperm DNA X SSC refers to the condition of incubation at 50 ° C to 65 ° C for 4 hours to overnight in 0.15 mol / l NaCl, 0.015 mol / l sodium citrate, pH 7.0).

  The polynucleotide encoding the polypeptide having the isoprimebellose-producing oligoxyloglucan hydrolyzing activity of the present invention is, for example, as described below from a microorganism that produces the above-described isoprimebellose-producing oligoxyloglucan hydrolase. It can be obtained by cloning the gene by a simple method. First, a novel isoprimiverose-producing oligoxyloglucan hydrolase of the present invention is isolated and purified from a microorganism that produces a novel isoprimebellose-producing oligoxyloglucan hydrolase by the method described above, and its partial amino acid is purified. Get information about sequences.

  As a partial amino acid sequence determination method, for example, the purified present enzyme protein may be directly subjected to amino acid sequence analysis by the Edman degradation method, or obtained by subjecting a protein hydrolase to action to perform limited hydrolysis. It is effective to separate and purify peptide fragments and perform amino acid sequence analysis on the obtained purified peptide fragments.

  Based on the partial amino acid sequence information thus obtained, a novel isoprimebellose-producing oligoxyloglucan hydrolase gene is cloned. In general, cloning can be performed using a PCR method or a hybridization method.

  When the hybridization method is used, for example, the method described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third edition, Cold Spring Habor Laboratory Press (2001) can be used.

  Moreover, when using PCR method, the following methods can be used. First, using a synthetic oligonucleotide primer designed on the basis of partial amino acid sequence information, using a genomic DNA or cDNA of a microorganism that produces a novel isoprimiverose-producing oligoxyloglucan hydrolase as a template, a PCR reaction is performed, Obtain the desired gene fragment.

  The PCR method is carried out according to the method described in PCR Technology [PCR Technology, edited by Erlich HA, Stocktonpress, 1989]. Further, when the base sequence is determined by a method usually used for this amplified DNA fragment, for example, the dideoxy chain terminator method, in addition to the sequence of the synthetic oligonucleotide primer in the determined sequence, a novel isoprimebellose-generated oligoxyloglucan hydrolase is added. A sequence corresponding to the partial amino acid sequence of the degrading enzyme is found, and a part of the target enzyme gene can be obtained. Furthermore, by performing the hybridization method using the obtained gene fragment as a probe, the RACE (Rapid Amplification of cDNA ends) method, the inverse PCR method, etc., a novel isoprimeverose-producing oligoxyloglucan hydrolase full-length enzyme is encoded. The gene to be cloned can be cloned.

The present inventor determined the entire nucleotide sequence of a gene encoding an isoprimebellose-producing oligoxyloglucan hydrolase using PCR using the Oerskovia sp. Strain Y1. This base sequence is shown in SEQ ID NO: 7.
The amino acid sequence encoded thereby is shown in SEQ ID NO: 8. Among these amino acid sequences, amino acid residues 1 to 28 at the N-terminal are presumed to be signal sequences, and the mature polypeptide has an amino acid sequence starting from the 29th alanine (SEQ ID NO: 10). The nucleotide sequence of the polynucleotide encoding the peptide is as shown in SEQ ID NO: 9.
In addition, it is obvious that the base sequence corresponding to the amino acid sequence described in SEQ ID NO: 8 or 10 is not only the polynucleotide described in SEQ ID NO: 7 or 9, and as described above, in the present invention, SEQ ID NO: 8 , 10 are included in the gene having a base sequence encoding the amino acid sequence shown in FIG.

  Moreover, based on the amino acid sequence set forth in SEQ ID NO: 8 or 10 and the base sequence information set forth in SEQ ID NO: 7 or 9, the target gene polynucleotide can also be obtained by chemical synthesis (Reference: Gene, 60 (1), 115-127 (1987)).

  Furthermore, using the whole or part of a novel isoprimebellose-producing oligoxyloglucan hydrolase gene whose entire nucleotide sequence has been clarified using Oerskovia sp. Y1 strain as a probe for hybridization, other isoprime Homologous to a novel isoprimebellose-producing oligoxyloglucan hydrolase gene shown in SEQ ID NO: 7 from the genomic DNA library or cDNA library of microorganisms producing a bellows-producing oligoxyloglucan hydrolase High DNA can be selected.

Hybridization can be performed under the stringent conditions described above. For example, a nylon membrane to which a genomic DNA library or cDNA library obtained from a microorganism that produces a novel isoprimebellose-producing oligoxyloglucan hydrolase is immobilized is prepared, and 6 × SSC, 0.5% SDS, 5 × Block the nylon membrane at 65 ° C. in a prehybridization solution containing Denharz, 100 μg / ml salmon sperm DNA. Then add each probe labeled with ³² P and incubate at 65 ° C. overnight. This nylon membrane is in 6 × SSC for 10 minutes at room temperature, in 2 × SSC containing 0.1% SDS for 10 minutes at room temperature, in 0.2 × SSC containing 0.1% SDS for 30 minutes at 45 ° C. After washing, autoradiography can be taken to detect DNA that specifically hybridizes with the probe. In addition, genes having various homologies can be obtained by changing conditions such as washing.

  On the other hand, a primer for PCR reaction can be designed from the base sequence of the gene of the present invention. By performing a PCR reaction using this primer, a gene fragment having high homology with the gene of the present invention can be detected, or the entire gene can be obtained.

  In order to confirm whether or not the obtained gene is a gene encoding a polypeptide having the desired isoprimiverose-producing oligoxyloglucan hydrolyzing activity, the determined nucleotide sequence is used for the novel isoprimevelope of the present invention. It can also be deduced from its gene structure and homology by comparing it with the base sequence or amino acid sequence of a sulpho-producing oligoxyloglucan hydrolase. It is also possible to produce a polypeptide of the obtained gene and determine whether it is a gene encoding a polypeptide having the desired enzyme activity by measuring the hydrolysis activity of isoprimebellose-producing oligoxyloglucan. it can.

  The following method is convenient for producing a polypeptide having an isoprimebellose-producing oligoxyloglucan hydrolyzing activity gene using the isoprimebellose-producing oligoxyloglucan hydrolase gene of the present invention. First, recombination of the expression vector with the target enzyme gene, transformation of the host using the recombinant vector containing the gene, and subsequent culturing of the transformant under normal conditions result in a novel isoprime. A polypeptide having hydrolyzing activity of bellose-producing oligoxyloglucan is produced. At this time, in order to obtain a mature polypeptide having hydrolyzing activity of isoprimebellose-producing oligoxyloglucan, no methionine residue is attached to the N-terminus of the polypeptide. A start codon (atg) is added in the sequence, and a stop codon is added if necessary (taa / tag / tga).

  As the host, microorganisms, animal cells, plant cells and the like can be used. Examples of the microorganism include bacteria such as Escherichia coli, Bacillus genus, Streptomyces genus, and Lactococcus genus, yeasts such as Saccharomyces genus, Pichia genus, and Kluyveromyces genus, and filamentous fungi such as Aspergillus genus, Penicillium genus, and Trichoderma genus. And baculovirus strains.

  Confirmation of expression and confirmation of the expression product can be easily performed using an antibody against a novel isoprimebellose-producing oligoxyloglucan hydrolase, but expression can also be confirmed by measuring enzyme activity. it can.

  To purify a novel isoprimebellose-producing oligoxyloglucan hydrolyzing polypeptide from the transformant culture, as described above, various chromatographies such as centrifugation, UF concentration, salting out, ion exchange resin, etc. It is possible to perform a combination of graphy appropriately.

  On the other hand, since the primary structure and gene structure of isoprimebellose-producing oligoxyloglucan hydrolase were clarified by the present invention, random mutation or site-specific mutation was introduced using the gene of the present invention, It is possible to obtain a gene having at least one of deletion, addition, insertion or substitution of one or several amino acid residues in the amino acid sequence of isoprimebellose-producing oligoxyloglucan hydrolase is there. As a result, it has isoprimebellose-producing oligoxyloglucan hydrolyzing activity, but has properties such as optimum temperature, stable temperature, optimum pH, stable pH, and substrate specificity that are slightly different. A gene encoding a glucan hydrolase can be obtained, and a polypeptide having these enzyme activities can be produced by genetic engineering.

  Methods for introducing random mutation include, for example, a method of chemically treating DNA, a method in which sodium bisulfite is reacted to cause a transition mutation that converts cytosine base to uracil base [Proceedings of the National Academy of Sciences of the USA, Vol. 79, pp. 1408-1412 (1982)], as a biochemical method, a method of generating base substitution in the process of synthesizing a double strand in the presence of [α-S] dNTP [ Gene, Vol. 64, pp. 313-319 (1988)] As a method of using PCR, a method in which manganese is added to the reaction system and PCR is carried out to reduce the accuracy of nucleotide incorporation [Analytical Bio Chemistry (Analytical Biochemistry), Vol. 224, pp. 347-353 (1995)] and the like can be used.

  As a method for introducing site-specific mutation, for example, a method using an amber mutation [gapped duplex method, Nucleic Acids Research, Vol. 12, No. 24, 9441- 9456 (1984)], a method using a restriction enzyme recognition site [Analytical Biochemistry, Vol. 200, 81-88 (1992), Gene, 102, 67-70 (1991)] , Method using the dut (dUTPase) and ung (uracil DNA glycosylase) mutations (Kunkel method, proceedings of the national of sciences of the USA, Vol. 82, pp. 488-492 (1985)) , A method utilizing amber mutation using DNA polymerase and DNA ligase [Oligonucleotide-directed Dual Amber (ODA) method, 152, pp. 271-275 (1995), JP-A-7-289262], a method using a host in which a DNA repair system is induced (JP-A-8-70874), DNA strand A method using a protein that catalyzes an exchange reaction (Japanese Patent Laid-Open No. 8-140685), a PCR method using two types of mutagenesis primers with restriction enzyme recognition sites (USP 5,512,463), an inactivating agent PCR method using a double-stranded DNA vector having a resistance gene and two kinds of primers [Gen, 103, 73-77 (1991)], PCR method using amber mutation [International Publication WO98 / No. 02535] can be used.

  In addition, site-specific mutation can be easily introduced by using a commercially available kit. Commercially available kits include, for example, Mutan (registered trademark) -G (manufactured by Takara Bio Inc.) using the gaped duplex method, Mutan (registered trademark) -K (manufactured by Takara Bio Inc.) using the Kunkel method, ODA method QuikChangeTM Site-Directed Mutagenesis Kit [Stratagene (Stratagene) Co., Ltd.] using Mutan (registered trademark) -Express Km (manufactured by Takara Bio Inc.), a primer for mutagenesis and DNA polymerase derived from Pyrococcus furiosus As a kit using the PCR method, TaKaRa LA PCR in vitro Mutagenesis Kit (manufactured by Takara Bio Inc.), Mutan (registered trademark) -Super Express Km (manufactured by Takara Bio Inc.), etc. should be used. Can do.

  Thus, according to the present invention, the primary structure and gene structure of isoprimebellose-producing oligoxyloglucan hydrolase are provided, so that the cost of a polypeptide having isoprimebellose-producing oligoxyloglucan hydrolase activity can be reduced. High-purity genetic engineering production is possible. In the present specification, various documents and the like have been cited, all of which are incorporated herein by reference. EXAMPLES Hereinafter, although this invention is explained in full detail using an Example, this invention is not limited to these. Hereinafter, unless otherwise specified, in the present specification,% is expressed as W / V%.

Example 1
Oligoxyloglucan 7 sugar (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.5%, peptone 0.8%, magnesium sulfate 0.05%, monopotassium phosphate 0.2% and yeast extract 0.05% Was sterilized by a conventional method, then inoculated with Oerskovia sp. Y1 strain (FERM P-21157) and cultured at 30 ° C. for 7 days with aeration. Thereafter, the culture supernatant was obtained by centrifugation. After concentrating and desalting the culture supernatant by ultrafiltration, use anion exchange columns HiPrep DEAE, HiPrep Q, ResourceQ, cation exchange column ResourceS, and hydrophobic column Resource PHE (all manufactured by GE Healthcare Biosciences) The fractions that retained the activity were concentrated, and gel filtration through a HiLoad Superdex 200 pg column (GE Healthcare Biosciences) was performed. The purified enzyme was homogeneous by SDS electrophoresis (FIG. 1).
Using the oligoxyloglucan heptasaccharide (Tokyo Chemical Industry) reduced by sodium borohydride treatment as a substrate, the activity of the purified enzyme was measured. To 10 μl of this substrate 0.1% aqueous solution (pH 4.5), 30 ng of purified enzyme was added and reacted at 50 ° C. for 10 minutes, and the resulting reducing sugar was measured by the bicinchoninic acid method to determine the activity. Under this condition, the amount of enzyme that produces a reducing power corresponding to 1 micromole of glucose per minute was defined as 1 unit.
The activity per 1 mg of purified enzyme protein was 85 units.

Example 2
<Isolation of a gene encoding a novel isoprimebellose-producing oligoxyloglucan hydrolase from Oerskovia sp. Strain Y1>

  In this specification, unless otherwise specified, gene manipulation techniques were performed according to the written manual (for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third edition, Cold Spring Habor Laboratory Press, 2001).

[Determination of partial amino acid sequence]
The purified preparation of the isoprimebellose-producing oligoxyloglucan hydrolase obtained in Example 1 was decomposed with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The resulting degradation product was subjected to reverse phase liquid chromatography, and two of the separated peptide fractions were subjected to a protein sequencer, and the internal amino acid sequences shown in SEQ ID NOs: 1 and 2 were determined.

  Preparation of genomic DNA Culture was carried out in the same manner as in Example 1, and genomic DNA was prepared by a conventional method using a genomic DNA isolation kit (FastDNA Kit, manufactured by BIO 101) according to the attached instructions.

  Amplification by PCR Based on the internal amino acid sequences of SEQ ID NOs: 1 and 2, four mixed oligonucleotides of sense primers shown in SEQ ID NOs: 3 and 4 and antisense primers shown in SEQ ID NOs: 5 and 6 were DNA synthesizers Synthesized by [Applied Biosystems] and used as PCR primers.

  Using the sense primer shown in SEQ ID NO: 3 and the antisense primer shown in SEQ ID NO: 5, using the genomic DNA of Oerskovia sp. Y1 strain as a template, GeneAmp PCR system 9700 [Applied] PCR reaction was performed using Biosystems (Applied Biosystems).

<PCR reaction solution> 10 x PCR reaction buffer (manufactured by Takara Bio Inc.) 10 μl, dNTP mixed solution (2.5 mmol / l, manufactured by Takara Bio Inc.) 8 μ, 100 μmol / l sense primer 5 μl, 100 μmol / l anti Sense primer 5 μl, distilled water 71 μl, genomic DNA solution (100 μg / ml) 0.5 μl, EX-Taq DNA polymerase (manufactured by Takara Bio Inc.) 0.5 μl

<PCR reaction conditions> Stage 1: Denaturation (94 ° C., 2 minutes) 1 cycle
Stage 2: Denaturation (94 ° C, 30 seconds) Annealing (48 ° C, 30 seconds) Extension (72 ° C, 30 seconds) 45 cycles Stage 3: Extension (72 ° C, 2 minutes) 1 cycle

  GeneAmp PCR system 9700 [Applied Biosystems Co., Ltd.] using the reaction solution after the PCR reaction as a template and the sense primer shown in SEQ ID NO: 4 and the antisense primer shown in SEQ ID NO: 6 under the following conditions PCR reaction was performed using

<PCR reaction solution> 10 x PCR reaction buffer (manufactured by Takara Bio Inc.) 10 μl, dNTP mixed solution (2.5 mmol / l, manufactured by Takara Bio Inc.) 8 μ, 100 μmol / l sense primer 5 μl, 100 μmol / l anti 5 μl of sense primer, 71 μl of distilled water, 0.5 μl of reaction solution after PCR reaction, 0.5 μl of EX-Taq DNA polymerase (manufactured by Takara Bio Inc.)

<PCR reaction conditions> Stage 1: Denaturation (94 ° C., 2 minutes) 1 cycle
Stage 2: Denaturation (94 ° C., 30 seconds) Annealing (52 ° C., 30 seconds) Extension (72 ° C., 30 seconds) 40 cycles Stage 3: Extension (72 ° C., 2 minutes) 1 cycle

  The obtained DNA fragment of about 500 bp was cloned into pGEM-T Easy (manufactured by Promega), and the base sequence was determined.

[Determining the full-length gene sequence of isoprimebellose-producing oligoxyloglucan hydrolase]
Based on the base sequence of the DNA, 5 'and 3' gene fragments are amplified by performing an inverse PCR method, the base sequence is determined, and isoprimeverose-producing oligoxyloglucan hydrolase The entire base sequence of the gene was determined. The inverse PCR method is described in “Cell engineering separate volume PCR Tips (Shujunsha)” Chapter 1 “Basics” 1-8 “Inverse PCR” 61-64.

SEQ ID NO: 7 shows the base sequence of the DNA encoding the determined isoprimebellose-producing oligoxyloglucan hydrolase. The obtained DNA has a total length of 3054 base pairs and encodes a protein consisting of 1018 amino acids. An internal amino acid sequence (SEQ ID NOs: 1 and 2) was found in this amino acid sequence. The sequence from the first amino acid to the 28th amino acid is presumed to be a signal sequence, suggesting that the enzyme of the present invention is a secreted protein.
The amino acid sequence of the mature protein presumed to start from the 29th amino acid residue is shown in SEQ ID NO: 10, and the nucleotide sequence of the polynucleotide encoding the mature protein is shown in SEQ ID NO: 9.

  The present invention is not particularly limited to the polypeptide having the above-described sequence having isoprimebellose-producing oligoxyloglucan hydrolyzing activity and the nucleotide encoding the same, and has isoprimiverose-producing oligoxyloglucan hydrolyzing activity. It includes longer polypeptides consisting of polypeptides and nucleotides that encode them.

Example 3
<Construction of an expression plasmid in Escherichia coli for a novel isoprimebellose-producing oligoxyloglucan hydrolase>
Based on the nucleotide sequence of the DNA encoding the N-terminal region amino acid sequence and the C-terminal region amino acid sequence of the mature body, a sense primer shown in SEQ ID NO: 11 and an antisense primer shown in SEQ ID NO: 12 were prepared. At this time, the restriction enzyme Nde I recognition sequence (catatg) is placed on the 5 ′ side of the sense primer on the N-terminal region side, and the restriction enzyme EcoRI recognition sequence (catatg) is placed on the 5 ′ side of the antisense primer on the C-terminal region side. gaattc) was added. The plurality of bases added to the further 5 ′ side of the restriction enzyme recognition sequence is for increasing the restriction enzyme cleavage efficiency.

  Using these primers and genomic DNA of Oerskovia sp. Y1 strain as a template, PCR reaction was performed using GeneAmp PCR system 9700 (Applied Biosystems) under the following conditions.

<PCR reaction solution> 5 × PrimeStar Buffer (manufactured by Takara Bio Inc.) 10 μl, dNTP mixed solution (each 2.5 mmol / l, manufactured by Takara Bio Inc.) 4 μ, 10 μmol / l sense primer 1 μl, 10 μmol / l antisense Primer 1 μl, distilled water 33 μl, genomic DNA solution (100 μg / ml) 0.5 μl, PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) 0.5 μl

<PCR reaction conditions> Stage 1: Denaturation (98 ° C., 1 minute) 1 cycle
Stage 2: Denaturation (98 ° C, 10 seconds) Annealing (55 ° C, 5 seconds) Extension (72 ° C, 3 minutes) 30 cycles Stage 3: Extension (72 ° C, 10 minutes) 1 cycle

  The obtained DNA fragment of about 3 kbp was cleaved with restriction enzymes Nde I and EcoRI and cloned into pET29a (+) (manufactured by Novagen) cut with restriction enzymes Nde I and EcoRI. After confirming that the nucleotide sequence was correct, it was introduced into E. coli BL21-CodonPlus (DE3) -RP (Stratagene). The obtained transformant was subjected to shaking culture at 37 ° C. in an LB medium containing 30 μg / ml kanamycin and 50 μg / ml chloramphenicol. When production was induced by adding isopropyl-β-D-thiogalactopyranoside to the medium, the target gene product accumulated in the cells as protein inclusion bodies.

Example 4
<Isolation / solubilization / rewinding of protein inclusion bodies>
After collecting the cells cultured as described above, the cells were disrupted and the protein inclusion bodies were purified using a protein extraction kit (BugBuster, Novagen). The purified protein inclusion body was dissolved in 8M urea-50 mM Tris / hydrochloric acid-1 mM ethylenediaminetetraacetic acid solution (pH 8.0) so that the protein concentration was about 1 mg / ml.

  The solubilized supernatant was dialyzed against 50 mM sodium acetate buffer (pH 5.5) and unwound by removing urea.

  The amino acid sequence of the obtained polypeptide is shown in SEQ ID NO: 14, and the base sequence corresponding to the amino acid sequence is shown in SEQ ID NO: 13. The polypeptide is obtained by adding a methionine derived from an initiation codon to the N-terminus of the amino acid sequence of the mature polypeptide described in SEQ ID NO: 10. The xyloglucan oligosaccharide degrading activity of the polypeptide was measured in the same manner as in Example 1 using oligoxyloglucan heptasaccharide as a substrate. As a result, the activity per 1 mg of protein was 32 units, and the activity pattern was the same as that of the enzyme obtained from Oerskovia sp. Y1 strain. That is, this recombinant enzyme had isoprimiverose-producing oligoxyloglucan hydrolyzing activity.

  From these results, it was confirmed that the recombinant enzyme can be produced using Escherichia coli using the novel isoprimiverose-producing oligoxyloglucan hydrolase gene obtained in the present invention.

It is a photograph which shows the result of having performed SDS-polyacrylamide gel electrophoresis about the refinement | purification enzyme protein obtained in Example 1. FIG.

Claims (20)

  A protein derived from a microorganism belonging to the genus Oerskovia, which has the action of cleaving the β-1,4-glucoside bond of the non-reducing end glucose residue of oligoxyloglucan to produce isoprimeverose. Loose-generating oligoxyloglucan hydrolase.   A polypeptide having the amino acid sequence shown in SEQ ID NO: 10, or having an amino acid sequence in which one or several amino acid residues in the polypeptide are deleted, added, inserted or substituted, and isoprimeverose A polypeptide having hydrolytic activity of the produced oligoxyloglucan.   The polypeptide according to claim 2, wherein methionine is added to the N-terminus.   The polypeptide according to claim 2, wherein a signal sequence is added.   A polypeptide having the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide having an amino acid sequence in which one or several amino acids in the polypeptide are deleted, added, inserted or substituted, A polypeptide serving as a precursor of a polypeptide having a hydrolyzing activity of a sulpho-producing oligoxyloglucan.   A polynucleotide encoding the polypeptide according to any one of claims 2 to 5.   A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 9, or one or several nucleic acids in the polynucleotide having a nucleotide sequence deleted, added, inserted or substituted, and producing isoprimeverose A polynucleotide expressing a polypeptide having oligoxyloglucan hydrolyzing activity.   The polynucleotide of claim 7 to which an initiation codon has been added.   The polynucleotide according to claim 7, wherein a base sequence corresponding to the signal peptide sequence is added.   A polynucleotide having the base sequence shown in SEQ ID NO: 7, or a polynucleotide having a base sequence in which one or several nucleic acids in the polynucleotide are deleted, added, inserted or substituted, and comprising isoprimeverose A polynucleotide that expresses a precursor of a polypeptide having hydrolytic activity of the produced oligoxyloglucan.   A polynucleotide that hybridizes to the polynucleotide according to any one of claims 7 to 10 under stringent conditions.   A polynucleotide having homology to the polynucleotide according to any one of claims 7 to 11.   A polynucleotide degenerate on the polynucleotide of any one of claims 7-11.   A recombinant vector comprising the polynucleotide according to any one of claims 6 to 13.   A transformant comprising the recombinant vector according to claim 14.   A method for producing a polypeptide, comprising culturing the transformant according to claim 15 and collecting a polypeptide corresponding to the recombinant polynucleotide from the culture.   The method for producing a polypeptide according to claim 16, wherein the polypeptide has an activity to hydrolyze isoprimiverose-producing oligoxyloglucan.   A method for producing an isoprimiverose-producing oligoxyloglucan hydrolase, comprising culturing a microorganism belonging to the genus Oerskovia in a medium and collecting isoprimeverose-producing oligoxyloglucan hydrolase from the culture.   The method for producing an isoprimiverose-producing oligoxyloglucan hydrolase according to claim 18, wherein the microorganism belonging to the genus Oerskovia is Oerskovia sp. Y1 strain (FERM P-21157).   Oerskovia sp. Y1 strain (FERM P-21157).