JP2007068521A - DNA encoding xylanase - Google Patents

Abstract

A novel xylanase DNA, a xylanase encoded by the DNA, a recombinant vector into which the DNA is inserted, and a transformant having the recombinant vector are obtained.
SOLUTION: A. The pullulans ATCC 20524 strain was cultured in a liquid medium in which the pH during the culture was maintained at 6 with a buffer containing xylan as a carbon source. A novel xylanase having a molecular weight of 39,000 and an isoelectric point of 8.9 was obtained by subjecting to various chromatographic purifications by electrophoresis. Using the partial amino acid sequence obtained from this protein, the gene encoding this enzyme was cloned. From this cloned DNA base sequence, an open reading frame (ORF) consisting of amino acid 361 residues including a 26-amino acid extracellular secretion signal was estimated. This homology search of the deduced amino acid sequence suggested that this enzyme is a xylanase belonging to GH family 10.
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Description

  The present invention includes a DNA encoding a novel xylanase, an amino acid encoded by the novel nucleotide sequence of the DNA, a recombinant vector into which the novel nucleotide sequence of the DNA has been inserted, and further transformed by the recombinant vector. Relates to a host cell such as yeast.

  Aureobasidium pullulans is an incomplete fungus that lives in raw sugar, fruit surfaces, breweries and soil. It is known that some of these bacteria produce a polysaccharide pullulan having an α-1,6 bond, a gluconic acid, and a xylan-degrading enzyme. Xylan occupies most of hemicellulose and is expected to be effectively used in the future as one of the biomass resources. Therefore, the appearance of xylan-degrading enzymes with new functionality is expected. Based on this expectation, many studies have been made so far, including the inventors, on unused hemicellulose-degrading enzymes containing xylan.

Japanese Patent Publication No. 2003-522086 Kazuyoshi Ohta, Satoshi Moriyama, Hideori Tanaka, Takato Shige, and Hidetoshi Akimoto, Journal of Bioscience and Bioengineering, 92, 262-270 Yoko Sato, Yoshihito Niimura, Kei Yura, and Mitiko Go, Gene, 238, 93-101, 1999

  From the Aureobasidium pullulans ATCC 20524 strain which is a conserved strain owned by the inventor, an acidophilic xylanase belonging to the glycosyl hydrolase (GH) family 11 having an optimum pH of 2.0 has already been isolated and purified, The gene that it encodes is cloned. Therefore, if a new xylanase having an optimum pH in the neutral range can be obtained in addition to the acidic range, xylo-oligosaccharides and the like can be obtained under milder conditions. In order to decompose as much hemicellulose as possible, xylanase belonging to GH family 10 having a wider substrate specificity is desired rather than GH family 11 in which the range of substrates that can be decomposed is somewhat narrow. Therefore, there is a demand for a GH family 10 xylan-degrading enzyme having an optimum pH in the neutral range and having a new DNA base sequence.

  A specific object of the present invention is to elucidate a novel xylanase DNA having the above functions and pH characteristics, the amino acid sequence encoded by the DNA, and construction of a recombinant vector into which the DNA is inserted. And to obtain a transformant having the recombinant vector.

  In order to achieve the above object, the Aureobasidium pullulans ATCC 20524 strain possessed by the inventor was cultured in a liquid medium in which the pH during culture was maintained at 6 with a buffer containing xylan as a carbon source. A novel xylanase having a molecular weight of 39,000 and an isoelectric point of 8.9 was obtained by subjecting to various chromatographic purifications by electrophoresis. Using the partial amino acid sequence obtained from this protein, the gene encoding this enzyme was cloned. From this cloned DNA base sequence, an open reading frame (ORF) consisting of amino acid 361 residues including a 26-amino acid extracellular secretion signal was estimated. This homology search of the deduced amino acid sequence suggested that this enzyme is a xylanase belonging to GH family 10.

  It was suggested that the obtained promoter of this enzyme gene was a promoter without a TATA sequence [TATA-less promoter]. Further, in the promoter region, consensus sequences to which PacC protein involved in expression control in response to culture pH was bound were present at two positions of base numbers 628 to 633 and 920 to 925 of SEQ ID NO: 1 in the sequence listing. The existence of this sequence was first found in Aureobasidium pullulans. In addition, an insertion sequence that exists only in this enzyme was found from multiple alignment of amino acid sequences with other GH family 10 xylanases. From these facts, the base sequence of the DNA obtained was recognized as novel. That is, the present invention is a xylanase DNA having a new base sequence that has the possibility of having substrate specificity that has not been found so far.

  Expression quantification using a real-time PCR (polymerase chain reaction) method suggested that the expression of this xylanase gene was controlled by the pH during culture. This appears to be pH control due to the presence of the PacC binding region in the promoter sequence. In addition, the molecular phylogenetic tree based on the amino acid sequence suggests that this xylanase forms a cluster different from others. Therefore, the xylanase of the present invention is characterized in that it is encoded by the DNA represented by the nucleotide sequence shown in SEQ ID NO: 1.

  Furthermore, the recombinant vector of the present invention is a recombinant vector into which a DNA encoding the above xylanase is inserted.

  Furthermore, the present invention is a host cell which is a transformant having the recombinant vector. Yeast is preferred as the host cell of the present invention.

  As is well known, functional xylo-oligosaccharides can be expected to have an effect as a sweetener, intestinal adjuster, or calcium absorption enhancer having a low calorie and refreshing sweetness. The novel xylanase of the present invention is a new species having a molecular weight of 39,000 and an isoelectric point of 8.9 and having a sequence that has not been known so far. Unlike a conventionally known xylanase, this is a new species having a DNA base insertion sequence that has not been known so far, that is, an amino acid insertion sequence encoded by this base sequence. Furthermore, since it belongs to GH family 10, it has a wider range of substrates that can be decomposed compared to xylanases belonging to GH family 11, and therefore has an optimum pH in the neutral range due to the presence of the insertion sequence, and decomposes a wider range of xylan. It has already been found to have possible properties. In addition, there is a possibility that the resolution of unused substrates will be further expanded.

  The present invention is described in detail below. As described above, the xylanase gene of the present invention is derived from the preferred strain Aureobasidium pullulans ATCC 20524, which belongs to the microorganism of the genus Aureobasidium and has a high ability to produce xylanase. Therefore, the amino acid sequence encoded by the base sequence of the DNA is also novel as will be described later, and particularly has an insertion sequence not possessed by other xylanases.

  The xylanase gene of the present invention can be obtained from the above Aureobasidium microorganism as follows. First, culturing is carried out under such conditions that the above Aureobasidium microbial strain is sufficiently grown and produces the target enzyme. The cells are removed from the obtained culture solution and centrifuged, and then the supernatant is collected. The supernatant is used for purification by the method shown in Table 1. The confirmation of the purification is confirmed to be single by sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE) method and isoelectric focusing. It is desirable to purify by this method from the viewpoint of smoothly performing the following matters.

  The molecular weight of the xylanase is 39 kDa, the isoelectric point is 8.9, and the specific activity is 29.5 U / mg. The optimum reaction conditions for xylanase activity are at pH 6.0 and 70 ° C. In the range of pH 4.0 to 10, more than 80% of the original activity remains, and the temperature is stable up to 70 ° C. Further, the xylanase activity is not affected by EDTA, guanidine hydrochloride, and p-chloromercuribenzoate under conditions of a final concentration of 1 mM.

The base and amino acid sequence of the DNA of the present invention will be described below. SEQ ID NOs: 1 to 11 in the sequence listing are as follows.
SEQ ID NO: 1: Base sequence of the xylanase DNA obtained from chromosomal DNA SEQ ID NO: 2: Base sequence of the xylanase DNA obtained from cDNA SEQ ID NO: 3: Amino acid sequence of the xylanase SEQ ID NO: 4: Prepared from the amino acid sequence DNA sequence of the degenerate primer DNA 1
SEQ ID NO: 5: DNA sequence 2 of degenerate primer prepared from amino acid sequence
SEQ ID NO: 6: nucleotide sequence 1 of primer DNA for use in cDNA terminal high-speed amplification method
SEQ ID NO: 7: nucleotide sequence 2 of primer DNA for use in cDNA terminal high-speed amplification method
SEQ ID NO: 8: Base sequence 1 of primer DNA for quantification of expression
SEQ ID NO: 9: DNA sequence 2 of primer for quantification of expression
SEQ ID NO: 10: Base sequence 1 of primer DNA for preparing a recombinant vector
SEQ ID NO: 11: DNA sequence 2 of a primer for preparing a recombinant vector

(Amino acid sequence)
As a result of identifying the N-terminal and internal amino acid sequences of the xylanase, 18 sequences from the 27th Ser to the 44th Gln in the amino acid sequence represented by SEQ ID NO: 3 in the Sequence Listing were revealed. It was. In addition, in the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, 10 sequences from Asn at amino acid number 75 to Pro at 84th amino acid were revealed. Furthermore, in the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, 10 sequences from amino acid 277th Leu to 286th Thr were revealed.

(Acquisition of probe sequence)
The base sequence of DNA is decoded from these amino acid sequences, and a set of primers (SEQ ID NOS: 4 and 5 in the sequence listing) is prepared based on the sequence. A polymerase chain reaction (PCR) method is performed according to a conventional method using these one set of primers and using genomic DNA extracted from the microorganism as a template. As a result, a clear band of about 150 bp can be obtained.

(Analysis of probe sequence)
As a result of cloning the band (PCR product) of about 150 bp and decoding the base sequence of the DNA, the present inventor has determined the base sequence of the DNA consisting of 152 bp shown in base numbers 1612 to 1863 of SEQ ID NO: 1 in the sequence listing. It was found to have Furthermore, when the base sequence of the obtained 152 bp DNA was translated into amino acids, the amino acid sequence of the previously obtained peptide fragment (amino acid numbers 27 to 44 and 75 to 84 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing) was obtained. ) Was observed. From this, it was confirmed that the base sequence of this 152 bp DNA is a part of the xylanase gene.

(Hybridization with the xylanase on the chromosome)
Subsequently, Southern hybridization is performed using the genomic DNA of the microorganism with the base sequence of DNA consisting of 152 bp shown in base numbers 1612 to 1863 of SEQ ID NO: 1 in the sequence listing as a probe. First, a probe obtained by chemically modifying a DNA base sequence consisting of 152 bp shown in base numbers 1612 to 1863 of SEQ ID NO: 1 in the sequence listing by the random prime method is used. Next, a probe containing a part of the enzyme gene sequence prepared earlier is hybridized to the genome of the microorganism cut by the restriction enzyme EcoRI and transferred to a nylon membrane. Hybridization was carried out for 16 hours in the presence of 0.5 M chartilic acid buffer containing 7% SDS and 1 mM EDTA and kept at 65 ° C.

(DNA base sequence of the xylanase on the chromosome)
As a result, an EcoRI fragment of about 9,000 bp for which a signal was confirmed was inserted into the EcoRI site of pUC18 to produce plasmid pXYN204. The approximately 3,800 bp XbaI and KpnI fragment contained in pXYN204 is further subcloned into pUC18 to create pXYN205. From this fragment, the sequence of 3293 bp can be determined from the KpnI site side to obtain the base sequence of the DNA containing the xylanase (SEQ ID NO: 1 in the sequence listing).

(DNA base sequence of the xylanase DNA)
In order to determine the base sequence of cDNA, the microorganism was cultured under the same conditions as in the enzyme production, the cells were collected, and total RNA was extracted using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.). From this total RNA, polyadenylated messenger RNA was purified. Using this messenger RNA as a template, a primer was prepared from the base sequence of the 152 bp DNA shown in base numbers 1612 to 1763 of SEQ ID NO: 1 in the previously obtained sequence listing (SEQ ID NO: 6 and 7 in the sequence listing) Starting from the sequence, the base sequence of the cDNA containing the xylanase gene can be obtained by 5 ′ cDNA end rapid amplification (RACE) method and 3 ′ RACE method (SEQ ID NO: 2 in the sequence listing).

(Estimation of secretory signal location)
By comparing SEQ ID Nos. 1 and 2 in the sequence listing, the DNA base sequence of xylanase precursor containing two introns and its transcriptional regulatory site were identified. From this and the previously identified N-terminal amino acid sequence (amino acid numbers 27 to 44 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing), the mature protein coding region of xylanase from base number 1594 of SEQ ID NO: 1 in the sequence listing It was revealed that. From these facts, it was presumed that amino acid Nos. 1 to 26 in the amino acid sequence of SEQ ID No. 3 in the sequence listing are secretion signals necessary for secretion outside the cells.

(Characteristics of DNA base sequence at promoter site)
The transcriptional regulatory site of the xylanase gene contained in SEQ ID NO: 1 in the sequence listing includes the CreA repressor binding region involved in carbon catabolite repression in Aspergillus nidulans from nucleotide numbers 1166 to There is one place in 1171. Furthermore, in Aspergillus nidulans, there are two zinc finger transcription factor PacC binding sites whose gene expression is regulated by the surrounding pH, in base numbers 628 to 633 and 920 to 925 of SEQ ID NO: 1 in the sequence listing. .

  In order to examine whether the xylanase gene is controlled by the surrounding pH during culture, a pair of primers (SEQ ID NOs: 6 and 7 in the sequence listing) was prepared, and relative quantification was performed by quantitative real-time PCR. As a result, the amount of transcription increased 8 times and 22 times in those in which the pH was adjusted to 6.0 and 8.0, compared to the culture in which the pH was not adjusted. Therefore, SEQ ID NO: 1 in the sequence listing includes a transcriptional regulatory site in which a cis element whose expression is controlled by the surrounding pH is present.

(Novelty of this sequence)
As a result of homology search of the amino acid sequence of the xylanase gene of the present invention, it is estimated that the xylanase belongs to GH family 10. In addition, as a result of multiple alignment with amino acid sequences of similar xylanases, it was revealed that there were insertion sequences with unknown functions not included in other similar xylanases.

  Based on the results of the multiple alignment, a rootless phylogenetic tree was created with the TREECON software (see FIG. 1). FIG. 1 shows the phylogenetic relationship at the catalytic site of the amino acid sequence of a known xylanase having a relatively high homology with the amino acid sequence encoded by the gene sequence derived from Aureobasidium pullulans as a rootless phylogenetic tree. The numerical value displayed on the branch is a percentage of the bootstrap value based on the one calculated 1000 times. However, 50% or less is not shown. The upper left scale bar corresponds to a substitution of 0.1 amino acid in one place. As a result, the amino acid sequence encoded by the xylanase gene of the present invention was a novel one not belonging to any of the two groups (I or II) classified in Non-Patent Document 2.

  The plasmid containing the xylanase gene of the present invention can be obtained by introducing the above xylanase gene into the plasmid by a conventional method, an example of which is shown below.

(Preparation of recombinant vector)
A recombinant vector, preferably pPIC3.5 (manufactured by Invitrogen) is previously digested with restriction enzymes. Based on the base sequence of the xylanase gene of the present invention, a pair of primers (SEQ ID NOs: 10 and 11 in the sequence listing) is chemically synthesized so that the restriction enzyme-cutting plasmid matches the base sequence of DNA. Using the cDNA containing the xylanase gene of the present invention prepared previously as a template, the DNA encoding the full length of the xylanase gene is amplified by PCR using the above both primers. A restriction enzyme is allowed to act on the obtained amplification product to obtain a restriction enzyme-cleaved DNA fragment, and the above-mentioned restriction enzyme-cleavage plasmid pPIC3.5 is joined to this to prepare a recombinant vector.

(Acquisition of host cells)
In order to integrate into a host cell, preferably the chromosome of yeast Pichia pastoris, the above recombinant vector is linearized with the restriction enzyme SacI and introduced by electroporation to obtain a transformant. it can.

  The expression of the xylanase gene can be confirmed by culturing the above-mentioned transformant yeast and measuring the enzyme activity in the culture supernatant. Further, the xylanase can be obtained by purifying the culture supernatant by a conventional method.

Hereinafter, determination of the DNA base sequence of the xylanase gene of the present invention, and the deduced amino acid sequence and the enzyme activity of the xynII gene product will be described with reference to examples.
Example 1
(Step 1: Culture solution preparation / purification / electrophoresis)
Aureobasidium pullulans ATCC 20524 strain containing oat-derived xylan 1%, yeast nitrogen base 0.67%, and maintaining the pH in the medium at 6.0 with a final concentration of 0.1 M phosphate buffer After culturing in the prepared medium, the cells were removed by centrifugation and the culture supernatant was collected. The supernatant was concentrated and desalted and adsorbed onto a column packed with a carrier for cation exchange chromatography CM-Cellulofine (manufactured by Seikagaku Corporation). When the adsorbed protein was eluted with a sodium chloride concentration gradient, one xylanase peak could be obtained when the activity was measured at pH 6.0. On the other hand, most of the family 11 xylanase (XynI) reported by Ota et al. In Non-Patent Document 1 for purification and various properties thereof was not adsorbed on the carrier under these conditions. Next, elution was performed in a state where activity and protein peaks coincided with a column packed with Superdex 75 pg (manufactured by Amersham Biosciences) as a gel filtration carrier. By this procedure, the xylanase was purified from the culture supernatant 48 times by 27% recovery. This enzyme was confirmed to be single by SDS-PAGE and isoelectric focusing. The molecular weight was 39 kDa and the isoelectric point was 8.9. The specific activity of the purified enzyme was 29.5 U / mg.

(Step 2: Substrate specificity)
The substrate specificity of the xylanase was examined. As shown in Table 1, this enzyme hydrolyzed birch-derived xylan to the same extent as oat-derived xylan. The activity was weak against pNP-β-D-cellbioside and pNP-β-D-xypyranoside, and no activity was detected against carboxymethylcellulose and pNP-α-L-arabinofuranoside.

(Process 3: Confirmation of decomposition products)
Analysis using thin layer chromatography showed that the initial hydrolysis of birch-derived xylan with this xylanase yielded oligosaccharides with a degree of polymerization of 2 or more. Xylotriose is produced as an intermediate product, which is finally cleaved into xylobiose and xylose. This enzyme hydrolyzes xylotriose, xylotetraose and xylopentaose, but did not degrade xylobiose. From these properties, the xylanase is endo-1,4-β-xylanase.

(Step 4: Determination of partial amino acid sequence)
Using this purified enzyme, the N-terminal amino acid sequence of 18 residues (amino acid numbers 27 to 44 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing) was determined by Procise 492 Protein Sequencer (Applied Biosystems). . Furthermore, the purified enzyme was partially digested with a protease derived from Staphylococcus aureus V8, and the amino acid sequences of the two fragments were each 10 residues (amino acid numbers 75 to 84 and 277 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing). ˜286). Of these, one region with less codon degeneracy was selected from each of amino acid numbers 27 to 44 and 75 to 84 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing, and forward and reverse degenerate primers ( Sequence numbers 4 and 5) in the sequence listing were chemically synthesized.

(Step 5: Acquisition of probe sequence)
Genomic DNA was extracted from Aureobasidium pullulans ATCC 20524 using ISOPLANT (manufactured by Wako Pure Chemical Industries, Ltd.). Using this genomic DNA as a template, amplification was performed by PCR reaction using the above two primers (SEQ ID NOs: 4 and 5 in the sequence listing). As a result, a clear band of about 150 bp was obtained.

(Step 6: Analysis of probe sequence)
The obtained band was cloned, and the DNA base sequence was decoded with PRISM310 (Applied Biosystems) using a Big Die Terminator Cycle Sequencing Kit (Applied Biosystems). As a result, it was found that the DNA has a base sequence of 152 bp represented by base numbers 1612 to 1763 of SEQ ID NO: 1 in the sequence listing. Furthermore, when the base sequence of the obtained 152 bp DNA was translated into amino acids, the amino acid sequence of the previously obtained peptide fragment (amino acid numbers 27 to 44 and 75 to 84 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing) was obtained. ) Was observed. Thus, the sequence was found to be part of the xylanase gene. Further, this set of primers (SEQ ID NOs: 4 and 5 in the sequence listing) specifically amplifies a part of the xylanase when the genomic DNA of the microorganism is used as a template.

(Step 7: Labeling the probe sequence)
Therefore, this PCR product, that is, a base sequence of 152 bp DNA (base numbers 1612 to 1763 of SEQ ID NO: 1 in the sequence listing) is obtained by a random prime method according to a digoxigenin chemiluminescent nucleic acid detection system (Roche Diagnostics). Digoxigenin-11-dUTP was labeled and used as a probe for cloning the full length of the xylanase gene.

(Step 8: hybridization of probe sequence)
On the other hand, the genomic DNA of Aureobasidium pullulans ATCC 20524 used as a template in the previous PCR was completely digested with the restriction enzyme EcoRI. The obtained restriction enzyme degradation product was separated by agarose gel electrophoresis, the nucleic acid was transferred to a positively charged nylon membrane, and Southern hybridization was performed using a labeled probe. Hybridization was carried out for 16 hours while maintaining at 65 ° C. in the presence of 0.5 M chartilic acid buffer containing 7% SDS and 1 mM EDTA. As a result, the probe hybridized to a DNA fragment of about 9.0 kbp. This indicates that the target xylanase gene is contained in an approximately 9.0 kbp fragment having EcoRI recognition sequences at both ends.

(Step 9: Determination of the DNA base sequence of the xylanase on the chromosome)
Next, an approximately 9.0 kbp EcoRI fragment was inserted into the EcoRI site of pUC18 to create plasmid pXYN204. pXYN205 was prepared by subcloning the XbaI and KpnI fragments of about 3.8 kbp contained in pXYN204, and the 3293 bp sequence (SEQ ID NO: 1 in the sequence listing) was determined from the KpnI site side.

(Step 10: Determination of the base sequence of the xylanase of cDNA)
In order to determine the base sequence of cDNA, the microorganism was cultured under the same conditions as in the enzyme production, the cells were collected, and total RNA was extracted using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.). From this total RNA, polyadenylated messenger RNA was purified using PolyATact messenger RNA isolation system IV (Promega). Using this messenger RNA as a template, a primer was prepared from the base sequence of the 152 bp DNA shown in base numbers 1612 to 1763 of SEQ ID NO: 1 in the previously obtained sequence listing (SEQ ID NO: 6 and 7 in the sequence listing) Starting from the sequence, 5′RACE and 3′RACE methods were performed using a SMART RACE cDNA amplification kit (Clontech). As a result, the base sequence of the cDNA containing the xylanase gene (SEQ ID NO: 2 in the sequence listing) was obtained, and that there were two transcription start sites at -60 and -54, and 114 bp, 119 bp and 138 bp downstream from the stop codon. It was revealed that polyadenylation was observed at these three points. The first ATG downstream from the transcription start point was presumed to be an initiation codon, and there was a conserved A 3 residues before this A.

(Step 11: Determination of intron position)
Comparison of the base sequence of DNA obtained from genomic DNA (SEQ ID NO: 1 in the sequence listing) and the base sequence of cDNA obtained by the RACE method (SEQ ID NO: 2 in the sequence listing) reveals a xylanase precursor containing two introns. The base sequence of DNA and its transcriptional regulatory site were identified. These two introns existed in the secretory signal and the coding region of the mature protein with base lengths of 56 bp and 73 bp, respectively, and both introns were in accordance with the GT / AG rule.

(Step 12: Estimation of amino acid sequence)
The amino acid sequence translated from the nucleotide sequence of the DNA of this xylanase precursor (SEQ ID NO: 3 in the sequence listing) is converted into the amino acid sequence of the N-terminal of xylanase first identified (amino acid numbers 27 to 27 in the amino acid sequence of SEQ ID NO: 3 in the sequence listing). 44), it was revealed that the base number 1594 and subsequent positions of SEQ ID NO: 1 in the sequence listing are the mature protein coding region of xylanase. From these facts, it was presumed that amino acid Nos. 1 to 26 in the amino acid sequence of SEQ ID No. 3 in the sequence listing are secretion signals necessary for secretion outside the cells. Further, three partial amino acid sequences purified and determined from Aureobasidium pullulans in SEQ ID NO: 3 in the sequence listing (amino acid numbers 27-44, 75-84, 277- in the amino acid sequence of SEQ ID NO: 3 in the sequence listing) No. 286) was confirmed to exist, and it was confirmed that the gene sequence of xylanase obtained by purification was obtained.

(Step 13: Analysis of transcriptional regulatory site)
The TATAAA and CCAAT motifs did not exist in the transcriptional regulatory site of the xylanase gene contained in SEQ ID NO: 1 in the Sequence Listing. The CreA repressor (5′-SYGGRG-3 ′) binding region involved in carbon catabolite suppression in Aspergillus nidulans was present at nucleotide numbers 1166 to 1171 (GTGGGG) in SEQ ID NO: 1 in the sequence listing. Furthermore, the zinc finger transcription factor PacC binding site (5′-GCCARG-3 ′), whose gene expression is regulated by the surrounding pH in Aspergillus nidulans, is represented by base numbers 628 to 633 (complete match) of SEQ ID NO: 1 in the sequence listing. In the form of GCCAAG) and 920-925 (GCCATG in the form of a partial match to the complementary strand). However, 5′-GGCTAAA-3 ′, which is a binding motif of the xylan-related transcriptional activator XlnR in Aspergillus nidulans, did not exist.

  In order to examine whether the transcriptional regulatory site of the xylanase gene is controlled by the surrounding pH, the microorganism was cultured under the culture conditions in which the xylanase gene was obtained. However, the culture was performed for 72 hours under three conditions where pH was adjusted to 6.0 or 8.0 with 0.1 M phosphate buffer and pH was not adjusted. When the pH was not adjusted, the initial pH 6.0 decreased to 2.7, and sufficient growth of the fungus and blackening due to dark mycelia were observed. When the pH was adjusted with a buffer solution, the pH was maintained at 6.0 and 8.0, and the growth of the bacteria was delayed as compared with the medium not adjusted for pH, and the color was yellow or cream . Total RNA was extracted from each of these using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.), and reverse transcription reaction was performed using oligo dT primers. The single-stranded cDNA thus prepared is amplified using primers for real-time PCR specific to the xylanase gene (SEQ ID NOS: 6 and 7 in the sequence listing), and the amplification reaction is performed by an assay method using SYBR Green. It was detected by PRISM7000 (manufactured by Applied Biosystems). As a result, compared to the culture that reached pH 2.7 without adjusting pH, those whose pH was adjusted to 6.0 and 8.0 increased the transcription amount by 8 times and 22 times, respectively. Therefore, SEQ ID NO: 1 in the sequence listing includes a transcriptional regulatory site where a cis element to which a PacC-like transcriptional regulatory factor whose expression is controlled by the surrounding pH is present is present.

(Step 14: Preparation of recombinant vector and acquisition of transformant)
Using the cDNA containing the xylanase gene of the present invention prepared earlier as a template, a set of primers (SEQ ID NOs: 10 and 11 in the sequence listing) to which a restriction enzyme EcoRI recognition site is added is used to encode a cell exocrine secretion signal by PCR. The xylanase gene containing the region to be expanded is elongated. The recombinant vector pPIC3.5 (manufactured by Invitrogen) in the methanol-utilizing yeast Pichia pastoris that was cleaved with a restriction enzyme that recognizes the site to which this extended sequence was added and also cleaved with the restriction enzyme was used. Insert and make pXYN207. This plasmid is linearized with the restriction enzyme SacI, and incorporated into the chromosome of Pichia pastoris by electroporation to obtain a transformant. When this transformant (pXYN207) was grown for 72 hours, 0.12 U / ml xylanase activity was detected in the culture supernatant. On the other hand, a transformant of only recombinant vector pPIC3.5 was used as a control strain, but no extracellular xylanase injection was detected.

(Step 15: Purification of the xylanase secreted by the transformant)
When this extracellular protein is applied to a cation exchanger SP-Sepharose column (Amersham Biosciences), it is separated into two xylanase peaks (P-I and P-II), and each fraction is collected. The gel filtration carrier Superdex 75pg column was used for individual purification. According to SDS-PAGE, the purified recombinant xylanase PI had a slightly higher molecular weight than P-II. The N-terminal amino acid sequence of P-II was the same as that of the original xylanase, but P-I was contained more from 6 residues upstream than the point to be originally cleaved. The concentrations of recombinant xylanase (P-I and P-II) in the culture were 6.5 mg / L and 36 mg / L, respectively, based on their specific activities. This result indicates that most of the precursor proteins encoded by the xylanase gene are correctly recognized and processed in the Pichia pastoris secretion pathway. From this, it is possible for the Aureobasidium pullulans-derived cell exocrine signal encoded by the xylanase gene to exhibit a function of being secreted outside the cell in the yeast Pichia pastoris.

  The xylanase having a novel base sequence of the present invention belongs to the GH family 10 having a broader substrate specificity and has an optimum pH in the neutral range, so that it can decompose many hemicelluloses and is milder. It is useful as a xylanase under which xylo-oligosaccharide can be obtained. Xylooligosaccharides produced from xylan by xylanase are useful as substances having excellent functionality as sweeteners, intestinal regulating agents, or calcium absorption promoters having a low calorie and a refreshing sweetness.

It is a rootless phylogenetic tree indicating that it does not belong to either I or II.

Claims (5)

  A DNA encoding xylanase represented by the base sequence of SEQ ID NO: 1 in the sequence listing.   A protein having a xylanase enzyme activity that hybridizes under stringent conditions with DNA represented by the base sequence amplified using Aureobasidium pullulans chromosomal DNA as SEQ ID Nos. 4 and 5 in the sequence listing DNA derived from Aureobasidium pullulans ATCC 20524 strain encoding   A xylanase encoded by the DNA according to claim 1 or 2.   A recombinant vector into which the DNA according to claim 1 or 2 is inserted.   A host cell which is a transformant having the recombinant vector according to claim 4.

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