JPH0759574A - Novel dna - Google Patents

Abstract

PURPOSE:To obtain a novel DNA coding an endo type inulinase hydrolyzing inuline to give inulooligosaccharide, and capable of producing the inulooligosaccharide in a high yield by inserting the novel DNA into a proper vector, transforming a microorganism, etc., with the vector and subsequently culturing the microorganism, etc. CONSTITUTION:Penicillum purpurogenum var. rubrischerotium (FERM BP-3162) is cultured in a medium containing inuline, and all RNAs are extracted from the cultured cells by a guanicline chloride/phenol method. A mRNA is separated from all the RNAs with an oligo (dT) column, and a cDNA is synthesized with the mRNA as a template. The library of the cDNA is made by a conventional method, and subsequently screened with a probe prepared by synthesizing a part of a DNA coding endo-type inulinase. A positive clone is selected, and the DNA is recovered and subsequently treated with a proper restriction enzyme to obtain the objective novel DNA coding the endo type inulinase capable of hydrolyzing the inuline to produce inulooligosaccharide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding an endo-type inulinase. Endo-type inulinase is an enzyme used to obtain useful inulooligosaccharides by hydrolysis of inulin.

[0002]

Inulin is a polysaccharide in which about 35 fructose are linearly linked by β-1,2 bonds and one molecule of glucose is bonded to the non-reducing end thereof. Extensive in roots and tubers.

The research and utilization of inulinase, an enzyme that hydrolyzes inulin, has been mainly aimed at the production of fructose. For this purpose, exo-type inulinase, which releases fructose in order from the reducing end of inulin, has been mainly studied. However, for the purpose of producing inulooligosaccharide, an endo-type inulinase that finally produces an oligosaccharide consisting of 3 to 6 molecules is desired.

Up to now, reports of detailed investigations on the properties of endo-type inulinase have been carried out by the present inventors on molds of the genus Penicillium (Japanese Patent Laid-Open No. 62-228293, Agric. Biol. Chem., 52 ,. 256
9-2576 (1988), and other molds of the genus Aspergillus (Journal of the Japanese Society of Agricultural Chemistry, 52, 159-16).
6 (1978)) and there are only a few examples. Furthermore, the nucleotide sequence of the inulinase gene has only been reported for exo-type enzymes (FEBS Lett., 289, 6).
4-68 (1991)) endo-type enzyme is not known at all.

[0005]

The object of the present invention is to provide a gene encoding an endo-type inulinase. Exo-type inulinase, which hydrolyzes inulin to the monosaccharide fructose, is not suitable for the production of inulooligosaccharides. However, normally, the endo-type inulinase-producing microorganisms also simultaneously produce exo-type inulinase, which causes a decrease in the yield of inulooligosaccharide from inulin. Obtaining the endo-type inulinase gene leads to the creation of a microorganism that produces only the endo-type enzyme.

[0006]

[Means for Solving the Problems] The present inventors have previously found that molds of the genus Penicillium induce and produce a strong endo-type inulinase. The properties were examined. as a result,
A technique for easily and uniformly purifying endo-type inulinase by electrophoresis was established. Then, we started by determining the partial amino acid sequence of the purified enzyme, succeeded in cloning the endo-type inulinase gene, and completed the present invention.

That is, the present invention provides SEQ ID NO: 1 in the sequence listing.
A DNA encoding the amino acid sequence shown in is provided.

The present invention will be described in detail below.

The DNA of the present invention is obtained roughly as follows. That is, the partial amino acid sequence of the enzyme protein was analyzed using an amino acid sequencer for the peptide fragmented with protease. Based on this sequence, a synthetic DNA probe was prepared by a DNA synthesizer. On the other hand, cDNA was synthesized from mRNA prepared from mold of Penicillium genus producing endo-type inulinase, which was packaged in vitro and infected with E. coli. The target clone was selected by plaque hybridization with the labeled probe. This method is detailed in the examples below.

As the endo-type inulinase-producing bacterium, Penicillium purpurogenum var. Rubrisclerot ( Penicillium purpurogenum var.
, FERM BP-3162) can be used, but it is clear that the type of organism in the present invention does not matter as long as the amino acid sequences constituting the enzyme are the same.
Furthermore, it is generally known that a small number of amino acids in the amino acid sequence may be replaced or deleted with other amino acids, or the enzymatic activity may not be substantially changed even if a small number of amino acids are inserted. Even in such a case, the gene of the enzyme is considered to be included in the scope of the present invention.

The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing shows the sequence of the mature protein portion having endo-type inulinase activity, but it has several amino acids and peptides at the N-terminal side, which are endo-type. It is also clear that the gene of the enzyme having inulinase activity is included in the present invention.

The above-mentioned DNA of the present invention can be obtained by the method detailed in the following examples. Further, according to the present invention, the nucleotide sequence of the endo-type inulinase gene has been clarified, so that Penicillium purpurogenum val lubriskrerotium ( Penicillium purpurogen)
um var. rubrisclerotium, FERM BP-316
The DNA of the present invention can be easily prepared by the PCR method using the genomic gene of 2) as a template.

By incorporating the DNA of the present invention into an appropriate expression vector and expressing it in a host such as Escherichia coli,
An endo type inulinase can be obtained. The endo-type inulinase produced by such a transformed microorganism has an advantage that it does not contain exo-type inulinase, unlike natural inulinase. The expression vector and the transformation method using the expression vector are well known in the art, and various host vector systems are commercially available. The target endo-type inulinase can be obtained by expressing the gene of the present invention using such a commercially available vector and host.

[0014]

INDUSTRIAL APPLICABILITY According to the present invention, a DNA encoding an endo type inulinase was cloned for the first time. By incorporating the DNA of the present invention into an appropriate vector, it can be expected that the endo-type inulinase activity is expressed in a microorganism having no exo-type inulinase activity. This allows inulin to be efficiently produced from inulin.

[0015]

EXAMPLES The present invention will be described in detail below based on examples. Unless otherwise specified, the operating procedure is Molecular.
Cloning (J. Sambrook et al., Cold
Spring Harbor Laboratory,
(1989)).

(1) Purification of enzyme Penicillium purpurogenum var. Rubriscler
otium, FERM BP-3162) culture of endo-type inulinase was obtained from Agric. Biol. C
hem. , 52, 2569-2576 (1988) to obtain electrophoretically uniform preparations. However, among the items described in the literature, the molecular weight was about 5400 as a result of reexamination.
It was estimated to be zero.

(2) Analysis of amino acid partial sequence The purified enzyme was digested with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries), and the resulting peptide was analyzed by HPLC (JASCO, TRI).
It was collected by ROTAR-VI). The column is made by JASCO F
inepak SIL C8-P50 was used. The amino acid sequence of each of the separated peptides was analyzed by the protein sequencer ABI477A (Applied Biosystem).
(manufactured by ms). The determined amino acid partial sequences at 9 positions are shown in SEQ ID NOS: 3 to 11 of the sequence listing.

On the other hand, when the N-terminal of the enzyme protein before being digested with lysyl endopeptidase was analyzed by using a protein sequencer in the same manner, it was in agreement with the sequence of SEQ ID NO: 8 in the sequence listing. From this, it was found that the N-terminal of this enzyme starts from aspartic acid.

(3) Synthesis of DNA probe The following 2 corresponding to the amino acid sequence of SEQ ID NO: 3 in the sequence listing
0 mer DNA to DNA synthesizer ABI380
B (manufactured by Applied Biosystems) was used for the synthesis. Synthetic DNA: AA (A / G) GTITT (T / C) TT (T / C) CA (T / C) GA (A / G)
CC () indicates that it is a mixture of two kinds, and I indicates inosine.

(4) Acquisition of mRNA Penicillium purpurogenum var. Rubriscler
otium, FERM BP-3162) inulin (purified from chicory root) 2%, maltose 0.5%,
NH ₄ H ₂ PO ₄ 0.5%, KH ₂ PO ₄ 0.1%,
_{MgSO 4 · 7H 2 O 0.05%} , Tween80
(Brand name) Sterile medium containing 0.02% (pH 7.
0) was inoculated with 1 platinum loop. 100 mL of the medium was put in each of four 500 mL Sakaguchi flasks. 4 at 28 ℃
Culture with shaking for 8 hours, from 400 mL of culture solution to 20 g of wet cells
Got From this microbial cell, 4.8 mg of total RNA was extracted by the guanidine hydrochloride / phenol method. 100 μg of this was applied to an Oligotex-dT30 column (manufactured by Takara Shuzo) to obtain 2 μg of mRNA.

(5) Preparation of cDNA library cDNA was synthesized from mRNA using a cDNA synthesis kit based on the Gubler-Hoffman method (manufactured by Amersham). An adapter (EcoRI-NotI-BamHI Adapto) was added to the obtained cDNA.
r, manufactured by Takara Shuzo Co., Ltd., and EcoRI at both ends, and Ec of phage vector λgt11 (manufactured by Amersham)
It was ligated to the oRI site. In this
ro Packaging Kit GIGAPACK Gold
(Strategene) and packaged, and infected with E. coli NM514 strain to obtain cD
A library of NA was created.

(6) Acquisition of target clone Using a synthetic DNA probe labeled with ³² P at the 5'end (using MEGALABEL manufactured by Takara Shuzo Co., Ltd.), 11 out of 1.5 × 10 ⁵ were obtained by plaque hybridization. We obtained positive clones. Phage DNA was prepared, excised with EcoRI, the size of the insert fragment was examined, and clones having an insert fragment of at least 1.5 kb or more were selected to obtain 4 clones.

(7) Determination of nucleotide sequence The inserted EcoRI fragment 1.7 kb was subjected to the dideoxy method (Messing, Methods in
Enzymol. , 101, 20-78 (1983))
The base sequence was determined by. That is, pBluesc
The subdivided fragment was subcloned into riptII KS (manufactured by Strategene), and this was subcloned into Bca.
It processed using the BEST sequencing kit (made by Takara Shuzo). Nucleotide sequence analysis is performed by the automatic sequencer ABI3
70A (manufactured by Applied Biosystems)
Was performed using.

As a result, it was possible to determine the base sequence of the region 1470 base which encodes an extremely appropriate 490 amino acid residue in view of the molecular weight of the enzyme protein of 54000, including the base sequence corresponding to the N-terminal amino acid sequence.
This base sequence is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence deduced from this base sequence is shown in SEQ ID NO: 1. All of the amino acid sequences that had already been identified by the protein sequencer could be confirmed from the base sequence. That is, the amino acid sequences shown in SEQ ID NOS: 3 to 11 were present at the positions shown in Table 1 below in the amino acid sequence shown in SEQ ID NO: 1.

[0025]

[Table 1]

The 1470 base pair sequence was present at a position of about 150 base pairs from the upstream in the 1.7 kb EcoRI fragment.

[Sequence list]

SEQ ID NO: 1 Sequence length: 490 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin: Organism name: Penicillium perprogenum val lubulisclerotium ( Penicillium purpurogenum v
ar. rubrisclerotium) Cell type: Liquid cultured cells Sequence features: Characteristic symbols: mat peptide Location: 1..490 Method of determining features: E sequence Asp Asp Tyr Arg Pro Thr Phe His Phe Cys Pro Ala Glu Asn Trp Met 1 5 10 15 Asn Glu Pro Asn Gly Leu Ile Lys Ile Asp Ser Thr Trp His Leu Phe 20 25 30 Tyr Gln Ala Asp Pro Thr Ala Asn Val Trp Gly Asn Glu Cys Trp Gly 35 40 45 His Ala Thr Ser Ser Asp Leu Leu His Trp Asp His Leu Pro Val Ala 50 55 60 Ile Pro Val Glu Asn Gly Ile Glu Ser Phe Thr Gly Thr Ser Tyr Tyr 65 70 75 80 Asp Ala Asn Asn Thr Ser Ser Leu Gly Thr Ser Thr Asn Pro Pro Tyr 85 90 95 Leu Ala Phe Phe Thr Gly Tyr Thr Ser Ser Asn Gly Thr Gln Asp Gln 100 105 110 Arg Leu Ala Tyr Ser Thr Asp Leu Gly Thr Thr Trp Leu Lys Phe Ser 115 120 125 Gly Asn Pro Ile Ile Ser Ala Ala Leu Glu Ala Pro His Asp Val Thr 130 135 140 Gly Gly Leu Glu Ser Arg Asp Pro Lys Val Phe Phe His Glu Pro Ser 145 150 155 160 Gly Lys Trp Val Met Val Leu Ala His Gly Gly Gln Asp Lys Leu Thr 165 170 175 Phe Trp Thr Ser Leu Asp Ala Lys Ser Trp Thr Trp Met Ser Asp Leu 180 185 190 Leu Ala Ser Gln Ile Glu Gly Phe Pro Ser Ser Val Thr Gly Trp Glu 195 200 205 Val Pro Asp Met Phe Gln Leu Pro Ile Gln Gly Thr Asn Glu Thr Thr 210 215 220 Trp Val Ile Ile Phe Thr Pro Ala Gln Gly Ser Pro Ala Gly Gly Asn 225 230 235 240 Gly Val Val Ala Leu Thr Gly Ser Phe Asp Gly Glu Thr Phe Leu Ala 245 250 255 Asn Pro Val Asp Ser Ser Thr Leu Trp Leu Asp Tyr Gly Arg Asp Phe 260 265 270 Asp Gly Ala Met Ser Trp Glu Asn Val Pro Ala Ser Asp Gly Arg Leu 275 280 285 Ile Ile Ala Ala Val Met Asn Ser Tyr Gly Ser Asn Pro Pro Thr Asn 290 295 300 Thr Trp Lys Gly Met Leu Ser Phe Pro Arg Thr Leu Thr Leu Glu Lys 305 310 315 320 Ile Gly Ser Lys Gln Tyr Phe Leu Gln Gln Pro Ile Ala Glu Leu Ser 325 330 335 Thr Val Asp Asn Ala Leu Ala Ser Ile Gln Asn Gln Thr Ile Ala Pro 340 345 350 Lys Gln Thr Leu Leu Ser Ser Ile His Gly Ser Ser Leu Asp Val Arg 355 360 365 Ile Ala Phe Ser Val Asp Ser Gly Ala Thr Leu Ser Leu Ala Val Arg 370 375 380 Lys Gly Gly Ser Glu Gln Thr Val Ile Arg Tyr Ser Gln Ser Asn Ser 385 390 395 400 Thr Leu Ser Val Asp Arg Thr Ala Ser Gly Asp Ile Ser Tyr Asp Pro 405 410 415 Ala Ala Gly Gly Ile His Ser Ala Gln Leu Ala Arg Asp Asn Thr Glu 420 425 430 Leu Val Tyr Leu Arg Val Leu Val Asp Thr Cys Ser Val Glu Val Phe 435 440 445 Gly Gly Gln Gly Glu Ala Val Ile Ser Asp Leu Ile Phe Pro Ser Asn 450 455 460 Ser Ser Asp Gly Leu Ser Leu Glu Val Ile Gly Gly Thr Ala Thr Leu 465 470 475 480 Gln Ser Val Glu Val Phe Ser Val Ser Leu 485 490 SEQ ID NO: 2 Sequence length: 1470 Sequence type: Nucleic acid Number of strands: double stranded Topology: Linear Sequence type: DNA Origin: Organism name: Penicillium purpurogenum var. Ru
brisclerotium) Cell type: Liquid cultured cells Sequence characteristics: Characteristic symbols: mat peptide Location: 1..1470 Method of determining features: E sequence GAT GAC TAT CGT CCA ACC TTT CAT TTC TGC CCG GCG GAG AAT TGG ATG 48 AAT GAG CCC AAT GGG CTG ATT AAG ATT GAT TCA ACA TGG CAC TTA TTT 96 TAT CAG GCT GAC CCA ACA GCA AAT GTA TGG GGT AAT GAG TGC TGG GGC 144 CAT GCA ACC AGC TCC GAC TTA CTC CAT TGG GAT CAT CTG CCT GTC GCA 192 ATT CCA GTC GAA AAC GGA ATT GAA TCT TTC ACT GGC ACC TCT TAT TAT 240 GAT GCT AAT AAT ACA TCG AGC TTA GGC ACG TCT ACC AAT CCA CCG TAC 288 TTA GCC TTT TTC ACT GGA TAT ACC TCT TCC AAC GGA CAG GAT CAG 336 CGC CTT GCT TAC AGC ACA GAT CTT GGG ACA ACA TGG TTG AAA TTC TCC 384 GGT AAT CCA ATA ATA TCG GCA GCT CTG GAA GCA CCT CAT GAT GTG ACT 432 GGA GGC CTC GAA AGT CGT GAT CCT AAA GTG TTC TTC CAT GAA CCG TCA 480 GGA AAA TGG GTT ATG GTC CTT GCG CAT GGT GGC CAG GAC AAA TTA ACA 528 TTC TGG ACG TCA CTA GAT GCG AAA AGC TGG ACG TGG ATG AGT GAC CTG 576 CTA GCT TCC CAA ATC GAG GGA TTC CCA TCT AGT GTC ACT GGA TGG GAA 624 GTG CCG GAT ATG TTT CAA CTT CCC ATC CAA GGC ACC AAT GAG ACC 672 TGG GTT ATA ATC TTT ACA CCA GCT CAG GGA TCT CCG GCT GGT GGT ATT 720 GGT GTG GCA CTT ACG GGG TCC TTT GAT GGG GAA ACA TTC CTG GCT 768 AAT CCT GTG GAC TCT TCA ACG TTA TGG TTA GAT TAC GGT CGT GAT TTT 816 GAT GGT GCT ATG AGT TGG GAA AAC GTA CCT GCA TCA GAT GGT ATC C864 ATC GCC GCT GTG ATG AAC AGT TAT GGT TCA AAC CCT CCT ACC AAC 912 ACC TGG AAA GGG ATG CTT TCC TTT CCG AGG ACA TTA ACC CTC GAG AAA 960 ATC GGC TCA AAG CAA TAC TTT CTC CAG CAG CCA ATC GCT G8 TTA AGC 100 ACA GTC GAT AAC GCT CTC GCA AGC ATT CAG AAT CAA ACA ATT GCG CCA 1056 AAA CAA ACG TTA CTT TCA TCA ATT CAC GGA AGT AGC TTA GAC GTC CGG 1104 ATC GCT TTT AGC GTT GAT TCA GGT GCT ACG CTG TCA CGA GCA GTC GT 1152 AAA GGG GGA TCT GAG CAG ACA GTC ATC CGC TAT TCC CAA TCA AAC TCG 1200 ACG CTC TCC GTT GAT CGA ACC GCG AGT GGG GAT ATC TCC TAT GAC CCA 1248 GCA GCA GGA GGT ATT CAT AGC GCA CAA TTA GCA CGG GAC AA T ACT GAG 1296 CTA GTC TAC TTG CGA GTT TTA GTC GAC ACA TGC TCC GTG GAA GTG TTT 1344 GGA GGA CAA GGA GAA GCA GTC ATA TCT GAT CTT ATA TTC CCT AGC AAC 1392 TCT TCT GAT GGC TTA TCT TTG GAG GTA ATT GGA ACG GCC ACT TTG 1440 CAG TCT GTG GAG GTG TTT TCA GTC TCA CTT 1470 SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment SEQ ID NO: 4 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment SEQ ID NO: 5 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Gly Met Leu Ser Phe Pro Arg Thr Leu Thr Leu Glu Lys 1 5 10 SEQ ID NO: : 6 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment SEQ ID NO: 7 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Phe Ser Gly Asn Pro Ile Ile Ser Ala Ala Leu Glu Ala Pro His Asp 1 5 10 15 Val Thr Gly Gly Leu Glu Ser Arg Asp Pro Lys 20 25 SEQ ID NO: 8 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment SEQ ID NO: 9 Sequence length: 28 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment sequence Gln Tyr Phe Leu Gln Gln Pro Ile Ala Glu Leu Ser Thr Val Asp Asn 1 5 10 15 Ala Leu Ala Ser Ile Gln Asn Gln Thr Ile Ala Pro 20 25 SEQ ID NO: 10 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment sequence Ile Asp Ser Thr Trp His Leu Phe Tyr Gln Ala Asp Pro Thr Ala Asn 1 5 10 15 Val Trp Gly Asn Glu 20 SEQ ID NO: 11 Sequence Length: 30 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate fragment Sequence Ser Trp Thr Trp Met Ser Asp Leu Leu Ala Ser Gln Ile Glu Gly Phe 1 5 10 15 Pro Ser Ser Val Thr Gly Trp Glu Val Pro Asp Met Phe Gln 20 25 30

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:80) (C12N 9/24 C12R 1:80) C12R 1:80) (72) Inventor Fukuoka Akio, 1 Toyonuma-cho, Sunagawa-shi, Hokkaido Mitsui Toatsu Chemical Co., Ltd. (72) Inventor Fuminobu Yoshimi, 1 Toyonuma-cho, Sunagawa-shi, Hokkaido Mitsui Toatsu Chemical Co., Ltd.

Claims (2)

[Claims] 1. A DNA encoding the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. 2. The DNA according to claim 1, which has the base sequence shown in SEQ ID NO: 2 in the sequence listing.