JPH05219948A - Cyclodextrin glucanotransferase, its production and gene coding the same enzyme - Google Patents

Abstract

PURPOSE:To produce the subject enzyme improved in a relative productivity of gamma-CD to beta-CD in comparison with wild type CGTase by substituting the 188th tyrosine of the sequence No.1 amino acid sequence derived from a specified microorganism with alanine, etc. CONSTITUTION:The 1756th to 1758th TAT in the sequence No.1 base sequence of a DNA of Bacillus ohbensis sp. nov. C-1400 are substituted with GCT, GCC, GCA, GCG, TCT, etc., and the resultant modified cyclodextrin glucanotransferase (CGTase) gene is subsequently introduced into a microorganism. The obtained transformat microorganism is then cultured and the objective CGTase in which the 188th tyrosine of the sequence No.1 amino acid sequence is substituted with alanine, serine or tryptophan can be obtained from the resultant culture solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cyclodextrin glucanotransferase, a method for producing the same, and a gene encoding the cyclodextrin glucanotransferase.

[0002]

BACKGROUND OF THE INVENTION Cyclodextrin (hereinafter referred to as CD) is a non-reducing maltooligosaccharide in which glucose molecules are linked in a ring, and α (6
There are three types, i.e.), β (7) and γ (8).
CD is produced from starch by a CD-producing enzyme produced by many microorganisms including Bacillus .

Due to its molecular structure, CD is known to take in various organic compounds into the molecular cavity to form a stable clathrate compound having excellent physical properties. It is a substance that is widely used for stabilization, protection of oxidative and photodegradable substances, changes in properties such as solubility, color and taste, and emulsification of water-insoluble substances.
Demand for CDs in various fields such as pharmaceuticals and fragrances is rapidly increasing. In particular, γ-CD is excellent in solubility and biodegradability, and has a great utility value in that it can enclose many kinds of substances because it has the largest intramolecular cavity. The smallest production volume.

Cyclodextrin glucanotransferase (hereinafter referred to as CGTase) is a transferase that produces linear dextrin from starch as a substrate and at the same time produces a mixture of the above-mentioned three types of CD by the same reaction. The enzyme molecule is a typical multifunctional enzyme that catalyzes many kinds of reactions. CGTase is shown in Table 1.
It is known that it is produced from many microorganisms including Bacillus as shown in Fig. 2, and these mainly produce α or β-CD in the early stage of the reaction, but as the reaction proceeds, CD other than the main product is produced. Will also be generated.

[0005]

[Table 1]

CGTases are roughly classified into two groups, α-CD producing type and β-CD producing type, depending on the difference in the main products of the enzymatic reaction. For the γ-CD producing enzyme, Bacillus
Only CGTase produced by Bacillus subtilis No. 313 is known, and usually γ-CD
Is only produced in a small amount as a by-product when producing α-CD or β-CD from starch. Bacillus subtilis No.313 CGT
Also in the case of asase, the production amount of γ-CD from starch is only 2-3% in terms of sugar yield, and this value is α
The amount is equal to or less than the amount of γ-CD produced as a by-product by the CD-producing or β-CD-producing enzyme.

On the other hand, CGTase produced from Bacillus ohbensis C-1400 strain, which is one of the alkalophilic bacteria, produces β-CD using starch as a substrate, and actually produces β-CD at an industrial level. It is an enzyme used for the production of CD. The amino acid sequence and the DNA sequence thereof have been elucidated by the present inventors, and it became possible to mass-produce them by introducing the gene into Escherichia coli or the like and using their expression system (Japanese Patent Laid-Open No. 3-21).
No. 6191). This enzyme is α-CD under normal reaction conditions.
Is characterized in that γ-CD is produced in a relatively large amount as compared with other β-CGTases, but when separating a mixture of β-CD and γ-CD, HPLC is used. It has to be used, which is a big problem in terms of cost.

As described above, γ-CD is currently in a small amount of production and expensive although it has the most applications. On the other hand, with the spread of gene manipulation technology, CGT
The asase gene has been cloned one after another, and attempts have been made to improve the enzyme protein by modifying the gene, as well as studies on the correlation between the structure and function of CGTase to date. For example, Kimura et al. Reported that the alkalophilic Bacillus ( Bacill) mainly produces β-CD from starch.
us ) 1011 10 or 1 at the C-terminal side of CGTase
It was shown that deletion of 3 amino acids leads to the production of a considerable amount of glucose, maltooligosaccharides and α-CD in addition to β-CD (Biochemical and Biophysi).
cal Research Communications, 161, 1273 (1989) and Abstracts of the Japan Agricultural Chemistry Conference, 65, 286 (1991)). However, there are no reports to date for the purpose of obtaining a large amount of γ-CD.

[0009]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide Bacillus ohbensis C-1.
It is intended to provide a CGTase excellent in γ-CD productivity, a method for producing the same, and a gene encoding the CGTase by gene manipulation technology of the CGTase produced from 400 strains.

[0010]

The inventors of the present invention have found that CGTase produced from Bacillus ohbensis C-1400 strain produces almost no α-CD under normal reaction conditions. Β-CGTas
Focusing on the characteristic that γ-CD is produced in a relatively large amount compared to e, CGTa which mainly produces α-CD
Comparing the structures of CGTase that mainly produces se and β-CD, it was confirmed that there are large differences in some regions that are considered to be involved in enzyme specificity. Also, Bacillus orvensis ( Bacillus
The present inventors have found that γ-CD can be produced from starch in a higher yield than before by modifying a specific site of CGTase produced by the strain Ohbensis C-1400 with another amino acid.

That is, the present invention is: 1) Bacillus obensis SP Knob C-1
In the amino acid sequence of SEQ ID NO: 1 derived from 400 (Bacillus ohbensis sp. Nov. C-1400), the cyclodextrin glucanotransferase in which the tyrosine at 188 is modified to alanine, serine or tryptophan, 2) Bacillus obensis sp. Knob C-1
DN of 400 (Bacillus ohbensis sp. Nov. C-1400)
In the gene containing the nucleotide sequence of SEQ ID NO: 1 in A, 1756 to 1758th "TAT" is "GCT",
"GCC", "GCA", GCG "," TCT "," T "
CC ”, TCA”, “TCG”, “AGT”, “AG
A cyclodextrin glucanotransferase gene modified to C "or" TGG ", and 3) culturing a transformed microorganism into which the gene described in 2 above is introduced, and obtaining a cyclodextrin glucanotransferase from the culture solution. The present invention provides a method for producing cyclodextrin glucanotransferase.

The present invention will be described in detail below. The present inventors first of all, as a representative enzyme of α-CD producing type, Bacillus macerans CGTase,
Bacillus stearothermophilus C as a representative enzyme of β-CD producing type
Using GTase, Bacillus orvensis ( Bacill
us ohbensis ) C-1400 CGTase.

Bacillus stearothermophilus ( Baci
llus stearothermophilus ) CGTase is β-C
It is known as a representative D-producing enzyme, and its three-dimensional structure has been elucidated by X-ray crystallography (Kubota, M., Mikam
i, B., Tsujisaka, Y., & Morita, Y.,: J.Biochem., 104,
12 (1988) and Nobuo Kubota, Yoshiki Matsuura, Shuzo Sakai, Koki Katsube: Abstracts of the Japan Agricultural Chemistry Conference, 65 , 671 (1991)) It is one of the few CGTases. In the vicinity of the active center in the three-dimensional structure, structural features considered to be involved in determining the type of CD to be produced are recognized.
In particular, phenylalanine 19 is present in the cavity of the generated CD.
1 is presumed to exist, and if the amino acid residue at this site becomes spatially large, it may be difficult to generate a small CD. The equivalent portion of phenylalanine 191 of Bacillus stearothermophilus CGTase is Bacillus stearothermophilus.
Macerans (Bacillus macerans) CGTase in tyrosine 195, Bacillus Obenshisu (Bacillus
ohbensis ) Tyrosine 188 in C-1400 CGTase
It was found to be a residue. So, Bacillus ohbensis C-1400 CGTase
In addition to tryptophan, which is spatially larger than Bacillus stearothermophilus phenylalanine, the tyrosine 188 residue in Escherichia coli was replaced with histidine, alanine, and serine, and the enzymatic properties were found to be alanine and serine. It was confirmed that β-CD and γ-CD were produced at almost the same rate in the substitution product, and that the yield of γ-CD was 15% in terms of sugar yield when substituted with tryptophan. It was the invention.

Any method may be used as the method for causing the substitution of the amino acid or the base used in the present invention, as long as it can cause the substitution of the target amino acid or the base. Site-specific mutagenesis is one of the methods for causing mutations at a high frequency.
This is a method of using a gene incorporated in a double-stranded DNA plasmid as a template and an oligonucleotide having a substitution base at a target mutation site as a mutation source (MJZoller, MS
mith, "Method in Enzymology", vol.100, R.Wu, L.Gro
ssmann, K. Moldave ed., P468, Academic Press (198
3)), the Kunkel method (Kunkel, TA (1985) Proc. Natl. Ac
ad. Sci. USA., 82 , 488.) and Grapped duplex method (Karmer,
There are several improved methods such as W. et al. (1984) Nucl. Acids Res., 12 , 9441), and any of them may be used for modification.

The method of causing amino acid substitution is not limited to the site-directed mutagenesis method, but a method using polymerase chain reaction (PCR) (Mullis, K., Falo
ona, F., Scharf, S., Saiki, R / .Horn, G., and Erlich,
H., (1986) Cold Spring Harber Symp. 51: 263) and cassette mutagenesis method (JHRichards, Nature, 3
23 , 187 (1986)), and these methods can also be used.

The wild-type CGTase gene used as a mutation template in the present invention is Bacillus obensis sp. Nob C-1400 (Bacillus ohbensi).
s sp. nov. C-1400). Cloning is a known method, for example, JP-A-3-21.
It can be carried out by the method shown in No. 6191, but other methods may be used. As long as this DNA fragment contains at least a portion encoding a structural gene containing a prepro body portion, there is no particular limitation on the length of the DNA or the restriction enzyme cleavage sites at both ends, and any DNA fragment can be used. Good. Also, when actually performing the mutation operation,
It is not always necessary to use all of the DNA fragments, and only a part of the DNA fragments containing the part to be mutated may be used for the mutation operation, then linked to other parts, and used for the subsequent operation. The modified CGTase according to the present invention is produced by introducing a mutated gene into a microorganism and culturing the microorganism in a medium to produce CG from the culture solution.
It can be obtained by collecting Tase.

At this time, the DNA to be used is Bacillus obensis SP Knob C-1400 (Baci
llus ohbensis sp. nov. C-1400) -derived CGTase gene containing all the parts involved in expression and production may be used, or another promoter region, signal sequence, terminator region or the like may be used in the structural gene part of CGTase. It is also possible to use a DNA fragment in which The promoter region, signal sequence, terminator region, etc. used at this time are CGTa in the host strain into which the gene is introduced.
Any substance may be used as long as it can express and produce se.

As the host bacterium, any bacterium capable of introducing the modified CGTase gene and capable of expressing and producing can be used. However, since the produced modified CGTase is secreted out of the cells, a bacterium belonging to the genus Bacillus is used for industrial production. Is preferred. In addition, Bacillus su
btilis ) 207-21 (Otozaki, K., et. al., J. Gen.
Appl. Microbial., 30 , 15 (1984)) or a mutant strain thereof can be more preferably used.

Transformation of the host bacterium can be performed by a usual method. For example, in the case of Bacillus bacteria, the competent cell method (J. Spizizen, Proc. Natl. Sci. USA, 44 , 1072 (195
8)), protoplast method (Chang, S., SNCohen, Molec.
Gen. Genet. 168 , 111 (1979)), etc., but not limited to these methods. The introduced mutant CGTase gene may be stably maintained by a method of existing and replicating extrachromosomally using a plasmid or the like, or may be incorporated into a chromosome (integration). In the case of the method of existing and replicating extrachromosomally, a vector capable of replicating in the host bacterium is used. For example, when Bacillus bacterium is used as a host, pUB110, pUP110, pC19
4, known plasmids for Bacillus subtilis such as pE194, pTP5, pSD3501 and shuttle vectors of the above Bacillus subtilis plasmid and E. coli plasmid can be used. Absent.

On the other hand, in the method of integrating into the chromosome, it is desirable to use no vector or one that does not replicate in the host bacterium. For example, E. coli plasmid p
BR322, pUC18, pHSG396, pSD31
65, pACYC184, etc. can be used. The transformant can be selected by the expression of a gene marker linked to the transgene.

The medium for culturing the transformant obtained as described above is not particularly limited, and any medium can be used as long as the transformant can grow. Separation and purification of CGTase from the culture solution can be performed according to a general method for separating and purifying proteins. For example, adding starch such as corn starch
This can be done by precipitating and then eluting the enzyme in a buffer solution, but the method is not limited to this, and other methods are also possible.

In the present invention, the activity of CGTase was measured by the blue value method as shown below. That is, soluble starch (potato starch, manufactured by Sigma) was dissolved in 50 mM Na ₂ CO ₃ -NaHCO ₃ buffer solution (pH 10.0) so that soluble starch (potato starch, manufactured by Sigma) was 0.025%. The thing was used as the substrate solution. 0.2 m of enzyme solution for 2.5 ml of substrate solution
1 was added and reacted at 40 ° C. for 10 minutes, then 0.6 N
The reaction was stopped by adding 2 ml of HCl, and 0.1% KI
The -0.01% I ₂ 1 ml was added, and the absorbance was measured at 660 nm. One unit of enzyme was used as an amount that reduces the absorbance at 660 nm by 1.0 under the above conditions.

[0023]

The present invention will be described in more detail below with reference to typical examples. However, these are merely examples, and the present invention is not limited to these. Example 1: Construction of modified CGTase gene A modified CGTase gene was constructed as shown in FIGS. 1 (1) to (5).

Bacillus ohbensis sp. Nov. C-1400
The fragment of the wild type CGTase gene cloned from (1) was partially digested with SphI and HindIII to excise a pUC19CGT5A fragment of about 4.8 Kb, which was excised from the plasmid pUP110BSCsacQ (Uozumi,
T., et.al., Agric. Biol. Chem., 55 (9), 2367-2374 (19
91)), mixed with T4 phage DNA ligase and ligated. This plasmid was designated as pUP110Ce-CGT.
And Furthermore, in order to increase the expression level of the CGTase gene, the Bacillus subtilis sacQ gene, which is known to increase the expression level of the Bacillus subtilis cellulase gene, was added to BamHI on a plasmid.
-Incorporated between SphI. The plasmid thus obtained is designated as pUP110CeCGTsacQ. This D
E. coli MV1184 was transformed with NA,
Transformants resistant to ampicillin were selected. From this transformant, plasmid DNA was extracted by a conventional method, purified, and analyzed to find that wild-type plasmid DNA pUP1
Had 10CeCGTsacQ.

The site-directed mutation is Kunk as follows.
It was performed according to the EL method. Plasmid pUP110Ce
CGTsacQ was transformed into Escherichia coli CJ236 and E. coli C harboring the plasmid pUP110CeCGTsacQ
Get J236. From this E. coli, the method of Messing et al.
ieira, J. and Messing, J. (1987) Methodin Enzymolog
Single-stranded DNA was prepared according to y, 153 , 3), and a part of deoxythiamine (dT) in the DNA was deoxyuracil (dT).
A single-stranded DNA replacing U) is obtained. Sequence number 2
Each of the oligonucleotides shown in 6 was chemically synthesized, and T4
The 5'end is phosphorylated by polynucleotide kinase.

Using this oligonucleotide, the above 1
Annealing buffer (200 mM T
ris-HCl, pH 8.0, 100 mM MgCl ₂ ,
The mixture was mixed in 500 mM NaCl, 10 mM DTT), left standing at 65 ° C. for 15 minutes, and further left at 37 ° C. for 15 minutes to anneal the oligonucleotide to the target site of mutation. Next, add 2.5 times as much extension buffer (50 mM Tris-HC
1, pH 8.0, 60 mM ammonium acetate, 5 mM
MgCl ₂ , 5 mM DTT, 1 mM NAD, 0.5 m
MdNTP (A, C, G, T)) was added, and E.
E. coli DNA ligase and T4 DNA polymerase were added and the mixture was allowed to stand at 25 ° C. for 2 hours to synthesize a complementary strand. Then, this DNA was used for Escherichia coli BMH71-18 m
utS was transformed by a conventional method to select an ampicillin-resistant transformant. Single-stranded DNA was prepared from some of the obtained transformants according to a conventional method, and the nucleotide sequence was determined by the dideoxy method by Sanger et al., And the base "TAT" of the gene encoding tyrosine 188 was "TGG.""PCT,""GCT","TCT","TTT" and "CAT"
110CeCGTsacQ-Y188W, pUP110
CeCGTsacQ-Y188A, pUP110CeC
GTsacQ-Y188S, pUP110CeCGTs
acQ-Y188F and pUP110CeCGTsa
cQ-Y188H was obtained.

[0027] Example 2: Bacillus subtilis (Bacill
production of modified CGTase by us subtilis ) 207-21 The wild-type plasmid DNA pUP11 obtained in Example 1
0CeCGTsacQ and modified plasmid DNApU
P110CeCGTsacQ-Y188W, pUP11
0CeCGTsacQ-Y188A, pUP110Ce
CGTsacQ-Y188S, pUP110CeCGT
sacQ-Y188F, and pUP110CeCGT
using sacQ-Y188H, Bacillus subtilis and (Bacillus subtilis) 207-21 was transformed by the protoplast method, and transformants were selected for kanamycin resistance. From these transformants, plasmid DNA was extracted, purified, and analyzed by a conventional method.
Wild-type plasmid DNA pUP110CeCGTsac
Q and modified plasmid DNA pUP110CeCGT
sacQ-Y188W, pUP110CeCGTsac
Q-Y188A, pUP110CeCGTsacQ-Y
188S, pUP110CeCGTsacQ-Y188
F, and pUP110CeCGTsacQ-Y188
Had H. 10 μg of the transformant at a final concentration
L-broth containing 100 mg / ml kanamycin
The cells were cultivated with shaking at 37 ° C. for 36 hours to produce the modified enzyme in the medium. This culture solution was centrifuged at 4 ° C. and 15000 rpm to remove bacterial cells, and the supernatant was taken to prepare a crude enzyme solution. In addition, plasmid DNApUP110CeCGTs
Bacillus which is a microorganism incorporating acQ-Y188W
Subtilis (Bacillus subtilis) 207-21 (pU
P110CeCGTasesacQ (Y188W)) has been deposited at the Institute of Microbial Science and Technology on Jan. 22, 1992 under the deposit number, Microindustrial Research Institute No. 12720 (FERM P-12720).

Example 3: Purification of modified CGTase Starch was added to the various crude enzyme solutions obtained in Example 2 in an amount of 2% to stir at 4 ° C. for 1 hour to adsorb the enzyme. Collect starch by centrifugation (4 ℃, 7000rpm), 50mM sodium phosphate buffer (pH 6.8)
After washing with, the cells were suspended in the same buffer and stirred at 40 ° C. for 2 hours to elute the enzyme. The starch was removed by centrifugation, and the supernatant was used as a purified enzyme preparation. If necessary, Centriprep-30 (manufactured by Amicon) is used for ultrafiltration to concentrate the enzyme solution. The purification process was confirmed by SDS-PAGE.

Example 4: Enzymatic properties of modified CGTase Using the enzyme solution obtained in Example 3, the properties of each mutant enzyme were examined by comparing with the wild-type enzyme. (1) Measurement of molecular weight The molecular weights of various mutant enzymes obtained in Example 3 were measured by SDS-P.
When examined by AGE and compared with wild type CGTase, it was found that the molecular weights were all about 80,000 daltons. The appearance of bands on the gel by SDS-PAGE is shown in FIG.

(2) Measurement of optimum pH The soluble starch as a substrate was reacted at 40 ° C. for 10 minutes, and the activity of various mutant enzymes depending on pH was examined. The measurement results of relative activity with respect to maximum activity are shown in FIG. Wild type CG
While Tase has two active regions of pH 5.0 and pH 10.0, the optimum pH of alanine-, histidine-, and serine-substitute mutant CGTases was changed to around pH 6.0.

(3) Analysis of enzymatic reaction products Using starch as a substrate, the hydrolyzing ability of various mutant enzymes was examined with time. The product CD was analyzed by HPLC. 0.5 ml each of 15 units of enzyme solution was added to 2.5 ml of a substrate solution prepared by dissolving 6% soluble starch in 100 mM McIlvaine buffer (pH 6.0) and reacted at 50 ° C. for 1 to 24 hours. After that, the enzymatic reaction was stopped by boiling for 10 minutes and used for analysis. 20 μl of the reaction solution was added to an NH ₂ 1251-N (4.6 × 250 mm, Senshu Scientific Co., Ltd.) column, which was eluted with 65% acetonitrile at a flow rate of 1.5 ml / min, and a refractive index detector (ERC-7510) was used. , Erma Opt
The product, CD, was detected by using ICAL WORKS.

The time-dependent changes in the reaction products of each mutant enzyme are shown in FIG. As can be seen from FIG. 4, wild-type CGTas
In the tyrosine 188 substitution mutant enzyme in e, the alanine, serine and tryptophan substitution mutant enzyme has a γ-to β-CD compared to the wild-type CGTase.
It can be seen that the relative production of CD is increasing. In particular,
Alanine and serine substitution mutant enzymes have β-CD and γ-C
D was produced at approximately the same rate. In addition, the tryptophan-substituted mutant enzyme produced a γ-CD production amount of 15% in terms of sugar yield.

[0033]

EFFECT OF THE INVENTION Bacillus ohbensis sp. Nov. C-1400 (Bacillus ohbensis sp. Nov. C-140)
0) in the amino acid sequence of SEQ ID NO: 1
By substituting the tyrosine at the other with another amino acid,
When the tyrosine 188 residue is replaced with another spatially larger amino acid, tryptophan, alanine, or serine, it has a great influence on the properties of the produced enzyme and its interaction with the substrate. Mutant CGTa
The CD produced from the starch of Se is wild-type CGTas.
The relative production amount of γ-CD with respect to β-CD is increased as compared with e. Β-CD for alanine and serine substitution mutant enzymes
And γ-CD were produced at almost the same rate, and the tryptophan-substituted mutant enzyme produced γ-CD at a yield of 15% in terms of sugar yield. The above can be used for mass production of γ-CD.

[0034]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 4785 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin: Bacillus obensis SP Knob C-14
00 (Bacillus ohbensissp. Nov. C-1400) Sequence features Characteristic symbols: mat peptide Location: 1195. ． 3219 method to determine the characteristics: S sequence GAGCTCGCCG CCTTCGTAAG CGCCTTCATT ATTTAAACCA ACATCCTCCG TATCATATTC 60 CCCTACATCA CCGCCAAAAA TATAGGCACC GTAATTTTTG AGGAAAGGAA AAGCAAAATA 120 GAGATCATTA GGTCTCATCA GAAATCCATA TTGATCAGCT GATGCATCCG TATTTTCTTC 180 AAGAATCGTT CTTAAATCTT CGATCGTCTC TGGTGCTTCT GGTACAATTT CTTTATTGTA 240 GAATATTCCA TATGTTTCGA TAACAGCTGG AACTCCATAA ATATCCGTTT CTCCTTCGAA 300 CTCATAGGTG ACCGCATCTA TAGAGGAACT AGCATAATTG CTTAACTCGT CATCTGATAG 360 AGTAAGAGGA TCAGCTAATC CTTGTGCCAC AATATTTCCG ATCTGGTCAT GTGGCTGGAA 420 AAATAAATCG GGACCATTCC CTTCTGGACC TGCTAATGAC AATTCCTGCA ATTGGTCCAT 480 CATCGGCTTT GTCTCAACCT TCACGTTAAT TCCCGTTTGC TCTGTATAAT CGTTGCTATT 540 TTTTCAATCG CCTCTAATTG CTCTTCTCTA TCATTTGCCC AGATGGGTTA ATTCTTCAGG 600 TTGATCCGCT TCCCCACCAT TTGTATCCGT GGTTACTGCC TCTTCTTCCC GTTCAGGTGC 660 ACATGCCGCT AAGAGAATAA TCATTAAAAA CATGGTAATC AGTGATAATA ATGAAAAACC 720 TTTTTTCATT TCATTTCCCC CCTGAAAAAA TTATATTAAT CTAGTAAATG TTGAGCTACC 780 TTTACATAAA GGAAAGTAAC GCAAACGTTT GCACTAGATT A GATACAACA ATCATACATG 840 GCAATTCAAT TTTTTGCAAT CGTTTACAGT AAAGAATTTT TTGAATTTTT ATTTAGTTAC 900 AATTACCTAT TTACAAATGA AATGTCTATG AGATAAAAAT AAATTAACAA CTATGAATGT 960 TCATATTCTC AATACGAGGG CAATGCTTTT GTTTTTTACA AACAACCCCG TCTTTTATTC 1020 CATTAAAAGG CGGGGTTATT TTATTGGATA AGGAGGGTTG TATTGCTTGT TCATACGATT 1080 CATTTTTAAT GTATAGGGGG AGTATTT TTG AAA AAT TTA ACG GTT TTA TTG AAA 1134 Met Lys Asn Leu Thr Val Leu Leu Lys -25 ACG ATT CCA TTA GCT CTG TTA CTT TTC ATC CTT CTT TCT TTA CCA ACA 1182 Thr Ile Pro Leu Ala Leu Leu Leu Phe Ile Leu Leu Ser Leu Pro Thr -20 -15 -10 -5 GCG GCT CAG GCT GAC GTT ACA AAT AAG GTT AAT TAT ACA AGA GAT GTT 1230 Ala Ala Gln Ala Asp Val Thr Asn Lys Val Asn Tyr Thr Arg Asp Val 1 5 10 ATT TAT CAA ATT GTG ACC GAC CGT TTT TCT GAT GGA GAT CCA TCC AAC 1278 Ile Tyr Gln Ile Val Thr Asp Arg Phe Ser Asp Gly Asp Pro Ser Asn 15 20 25 AAC CCA ACG GGG GCG ATT TAT AGT CAG GAT TGT AGC GAC CTT CAT AAA 1326 Asn Pro Thr Gly Ala Ile Tyr Ser Gln Asp Cys Ser Asp Leu His Lys 30 35 40 TAT TGT GG G GGC GAC TGG CAA GGA ATT ATC GAC AAA ATC AAT GAC GGG 1374 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly 45 50 55 60 TAT TTA ACC GAT TTA GGA ATT ACA GCG ATT TGG ATT TCA CAG CCT GTA 1422 Tyr Leu Thr Asp Leu Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val 65 70 75 GAA AAT GTC TAT GCT CTT CAT CCG AGT GGT TAT ACC TCT TAT CAC GGG 1470 Glu Asn Val Tyr Ala Leu His Pro Ser Gly Tyr Thr Ser Tyr His Gly 80 85 90 TAT TGG GCA AGA GAT TAT AAA AGA ACG AAC CCT TTT TAT GGC GAT TTC 1518 Tyr Trp Ala Arg Asp Tyr Lys Arg Thr Asn Pro Phe Tyr Gly Asp Phe 95 100 105 TCT GAC TTT GAC CGG TTA ATG GAT ACT GCA CAT AGT AAT GGC ATT AAG 1566 Ser Asp Phe Asp Arg Leu Met Asp Thr Ala His Ser Asn Gly Ile Lys 110 115 120 GTA ATC ATG GAC TTT ACG CCC AAC CAT TCA TCA CCA GCT CTT GAA ACC 1614 Val Ile Met Asp Phe Thr Pro Asn His Ser Ser Pro Ala Leu Glu Thr 125 130 135 140 GAT CCA AGC TAT GCC GAG AAC GGA GCG GTT TAT AAT GAT GGC GTA TTA 1662 Asp Pro Ser Tyr Ala Glu Asn Gly Ala Val Tyr Asn Asp Gly Val Leu 145 150 15 5 ATA GGC AAT TAT TCT AAC GAC CCT AAC AAC CTC TTT CAC CAT AAT GGT 1710 Ile Gly Asn Tyr Ser Asn Asp Pro Asn Asn Leu Phe His His Asn Gly 160 165 170 GGG ACA GAT TTT TCA TCC TAT GAA GAT AGC ATT TAT CGA AAT CTA TAT 1758 Gly Thr Asp Phe Ser Ser Tyr Glu Asp Ser Ile Tyr Arg Asn Leu Tyr 175 180 185 GAT TTA GCC GAC TAT GAT TTA AAC AAT ACT GTA ATG GAC CAA TAT TTA 1806 Asp Leu Ala Asp Tyr Asp Leu Asn Asn Thr Val Met Asp Gln Tyr Leu 190 195 200 AAA GAA AGT ATA AAG CTT TGG TTA GAT AAA GGA ATT GAT GGG ATT CGA 1854 Lys Glu Ser Ile Lys Leu Trp Leu Asp Lys Gly Ile Asp Gly Ile Arg 205 210 215 220 GTC GAT GCG GTC AAA CAT ATG TCC GAG GGC TGG CAA ACC TCC TTA ATG 1902 Val Asp Ala Val Lys His Met Ser Glu Gly Trp Gln Thr Ser Leu Met 225 230 235 AGT GAT ATT TAT GCA CAC GAG CCT GTC TTT ACA TTT GGC GAA TGG TTT 1950 Ser Asp Ile Tyr Ala His Glu Pro Val Phe Thr Phe Gly Glu Trp Phe 240 245 250 TTA GGA TCA GGG GAA GTC GAC CCA CAA AAT CAT CAC TTT GCC AAT GAA 1998 Leu Gly Ser Gly Glu Val Asp Pro Gln Asn His His Phe Ala Asn Glu 255 260 265 AGT GGT ATG AGC TTA CTA GAC TTT CAG TTT GGT CAA ACG ATT AGA GAT 2046 Ser Gly Met Ser Leu Leu Asp Phe Gln Phe Gly Gln Thr Ile Arg Asp 270 275 280 GTA TTA ATG GAC GGT AGC AGC AAT TGG TAT GAC TTT AAT GAG ATG ATT 2094 Val Leu Met Asp Gly Ser Ser Asn Trp Tyr Asp Phe Asn Glu Met Ile 285 290 295 300 GCA AGC ACC GAA GAG GAT TAT GAC GAA GTG ATT GAT CAG GTT ACG TTT 2142 Ala Ser Thr Glu Glu Asp Tyr Asp Glu Val Ile Asp Gln Val Thr Phe 305 310 315 ATT GAT AAT CAT GAC ATG AGC CGT TTT TCC TTT GAA CAA TCT TCA AAC 2190 Ile Asp Asn His Asp Met Ser Arg Phe Ser Phe Glu Gln Ser Ser Asn 320 325 330 CGC CAT ACA GAC ATT GCT TTA GCT GTC CTG TTA ACC TCT CGT GGA GTT 2238 Arg His Thr Asp Ile Ala Leu Ala Val Leu Leu Thr Ser Arg Gly Val 335 340 345 CCG ACT ATA TAT TAC GGT ACA GAG CAA TAT TTA ACA GGA GGC AAC GAC 2286 Pro Thr Ile Tyr Tyr Gly Thr Glu Gln Tyr Leu Thr Gly Gly Asn Asp 350 355 360 CCT GAA AAC CGC AAG CCG ATG AGC GAT TTT GAC CGT ACG ACA AAT TCT 2334 Pro Glu Asn Arg Lys Pro Met Ser Asp Phe Asp Arg Thr Thr Asn Ser 365 370 375 380 TAT CAA ATT ATT AGT ACG CTT GCT TCC TTA AGA CAA AAC AAC CCG GCA 2382 Tyr Gln Ile Ile Ser Thr Leu Ala Ser Leu Arg Gln Asn Asn Pro Ala 385 390 395 TTA GGC TAT GGA AAT ACA AGC GAA CGA TGG ATT AAC TCC GAC GTT TAT 2430 Leu Gly Tyr Gly Asn Thr Ser Glu Arg Trp Ile Asn Ser Asp Val Tyr 400 405 410 ATT TAT GAA CGA TCC TTC GGA GAC AGT GTC GTG CTT ACT GCT GTT AAC 2478 Ile Tyr Glu Arg Ser Phe Gly Asp Ser Val Val Leu Thr Ala Val Asn 415 420 425 AGT GGC GAT ACT AGT TAC ACG ATT AAT AAC TTA AAT ACC TCG TTA CCT 2526 Ser Gly Asp Thr Ser Tyr Thr Ile Asn Asn Leu Asn Thr Ser Leu Pro 430 435 440 CAA GGT CAA TAT ACA GAT GAG CTT CAG CAG CTT TTG GAT GGA AAT GAG 2574 Gln Gly Gln Tyr Thr Asp Glu Leu Gln Gln Leu Leu Asp Gly Asn Glu 445 450 455 460 ATT ACA GTC AAT AGC AAT GGA GCT GTA GAC TCG TTC CAG CTA AGT GCA 2622 Ile Thr Val Asn Ser Asn Gly Ala Val Asp Ser Phe Gln Leu Ser Ala 465 470 475 AAT GGA GTA TCC GTG TGG CAA ATA ACC GAA GAG CAT GCT TCT CCT CTA 2670 Asn Gly Val S er Val Trp Gln Ile Thr Glu Glu His Ala Ser Pro Leu 480 485 490 ATC GGC CAT GTA GGA CCA ATG ATG GGT AAA CAC GGA AAT ACT GTC ACT 2718 Ile Gly His Val Gly Pro Met Met Gly Lys His Gly Asn Thr Val Thr 495 500 505 ATA ACG GGA GAA GGC TTT GGT GAC AAT GAG GGA AGT GTT CTC TTT GAT 2766 Ile Thr Gly Glu Gly Phe Gly Asp Asn Glu Gly Ser Val Leu Phe Asp 510 515 520 TCT GAT TTC TCT GAT GTC CTT TCT TGG TCT GAC ACG AAA ATA GAA GTA 2814 Ser Asp Phe Ser Asp Val Leu Ser Trp Ser Asp Thr Lys Ile Glu Val 525 530 535 540 AGC GTT CCG GAT GTA ACA GCT GGT CAC TAT GAT ATT AGC GTT GTA AAT 2862 Ser Val Pro Asp Val Thr Ala Gly His Tyr Asp Ile Ser Val Val Asn 545 550 555 GCT GGA GAC AGC CAA AGT CCA ACG TAT GAT AAG TTT GAA GTA TTA ACC 2910 Ala Gly Asp Ser Gln Ser Pro Thr Tyr Asp Lys Phe Glu Val Leu Thr 560 565 570 GGT GAC CAA GTT AGT ATC CGT TTT GCT GTT AAT AAT GCG ACC ACT AGC 2958 Gly Asp Gln Val Ser Ile Arg Phe Ala Val Asn Asn Ala Thr Thr Ser 575 580 585 CTT GGT ACC AAT CTG TAT ATG GTT GGA AAT GTG AAT GAG CTT GGA AAT 3006 Leu Gly Thr Asn Leu Tyr Met Val Gly Asn Val Asn Glu Leu Gly Asn 590 595 600 TGG GAC CCT GAT CAA GCG ATT GGG CCG ATG TTT AAT CAA GTG ATG TAT 3054 Trp Asp Pro Asp Gln Ala Ile Gly Pro Met Phe Asn Gln Val Met Tyr 605 610 615 620 CAA TAC CCA ACC TGG TAC TAT GAT ATA AGT GTT CCT GCC GAG GAA AAT 3102 Gln Tyr Pro Thr Trp Tyr Tyr Asp Ile Ser Val Pro Ala Glu Glu Asn 625 630 635 CTT GAA TAT AAA TTT ATT AAA AAG GAC AGC AGC GGA AAC GTC GTT TGG 3150 Leu Glu Tyr Lys Phe Ile Lys Lys Asp Ser Ser Gly Asn Val Val Trp 640 645 650 GAA AGT GGA AAT AAC CAT ACG TAT ACA ACA CCT GCA ACT GGA ACC GAT 3198 Glu Ser Gly Asn Asn His Thr Tyr Thr Thr Pro Ala Thr Gly Thr Asp 655 660 665 ACA GTC CTC GTC GAC TGG CAA TAACACAAAA TACACATCAA CTTAAGGGCT 3249 Thr Val Leu Val Asp Trp Gln 670 675 ATGCATTGTGAATCATATCATCGAG TATCATACACAGAAT TGGAATTAG 3G CCTATTCAAA GGATACGCTA CACATTCGTC TACGCACCAA AAAGAATGAT 3429 CTCACGCAAG TCGAGCTT CT CTATGCTGAT CCTTACAATT GGAATGAAGA TGGTTGGCTC 3489 TATGAGCAAA AAGCGATGCG TTTAGAGGCT TCTGACCACT TATTTGACTA TTGGATGATT 3549 GATGTTACCG TCCCATACCG TCGACTCCGC TATGGCTTTA AGCTTACAAG TGATGATGAA 3609 ACGTTATATT ATACGGAAAA AGGCTTTTAT GAAACAGCAC CTACCGATGA TACGGCTTAC 3669 TATTTTTGTT TTCCCTTTAT CAATCCTGTT GATATCTTTC AAGCTCCTGA ATGGGTCAAA 3729 AAGACCGTTT GGTATCAAAT CTTCCCGGAG CGTTTTGCCA ATGGTGACTC GAGCATTAAT 3789 CCAGCCTCAA CCCTCCCTTG GGGAAGTACA GAAGCTACAC CAACTAATTT TTTCGGTGGA 3849 GATTTCGAGG GAATACTTAA TCATTTAGAT TATTTAGTTG ATTAGGCATT AATGGCATTT 3909 ACTTTACTCG ATTTTTAAAG CTAAATCAAA CCATAAGTAC GATACGATTG ATTATATGGA 3969 AATGATCCAC AGTTTGGAGA TAAAGAAACC TTCCGGAAGC TCGTTAATGC CTGTCATGAA 4029 AAAGGGATAA AAATCATGCT TGATGCTGTA TTTAATCATA GTGGCTATTA TTTCGAAGCG 4089 TTTCAGGATG TCCTGAAGCA TCAGGAGCAA TCGAAGTATA AAGATTGGTT TCATATACGC 4149 GACTTTCCAG TCACTCCTGG TCCTAAGCCT AATTATGATA CATTTGGGTT TGTTGAATAT 4209 ATGCCGAAGT TAAATACGGA AAACCAAGAG GTGAAGGATT ATTTATTAAA AGTGGCGCGC 4269 TACTGGATCG AGGAATTTAA TAT CGATGGC TGGCGTCTTG ATGTCGCCAA TGAAGTCGAC 4329 CATCAATTTT GGAGAGATTT TAGAAGAGAA GTAAAAGCGA TTAACCCCGA TGTTTATATC 4389 TTAGGTGAGA TATGGCATGA CTCGATGCCG TGGCTGCAGG GCGATCAGTT CGATGCCGTC 4449 ATGAATTATC CTTTTACGGT AGCGGCTCTC GATTATATTG CGAAGGATAA GATTAATGCC 4509 GAAGAGTTTG CTCATCAGCT GACCGATGTT CTTTGCTCGT ATCCAGCCAA TATTCATGAA 4569 GTGACCTTTA ACCTGCTTGG CAGTCATGAC ACTGCACGAG TCCTGACCGT ATGTAAGGAC 4629 AACAAAGAAA AAACGAAGCT ACTTTATTTA CTATTGTTAT CATCTAAAGG AAGTCCTTGC 4689 ATTTTTTATG GCGAGGAAAT CGGCATGGCC GGTGAAAACG ATCCAGGCTG CAGAGACTGC 4749 ATGATTTGGG AGGAAGACCA ACAAGACCTC GAATTC 4785

SEQ ID NO: 2 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCGAAATCTA TGGGATTTAG CCGA 24

SEQ ID NO: 3 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence ATCGAAATCT AGCTGATTTA GCCG 24

SEQ ID NO: 4 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GAAATCTATC TGATTTAGC 19

SEQ ID NO: 5 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GAAATCTATT TGATTTAGC 19

SEQ ID NO: 6 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CGAAATCTAC ATGATTTAG 19

[Brief description of drawings]

FIG. 1 (1) to (5) show the construction of the modified CGTase gene of the present invention.

FIG. 2 Wild type CGTase and various tyrosine 188
It is a figure which shows the band on the gel in SDS-PAGE of mutant CGTase.

FIG. 3 Wild-type CGTase and various tyrosine 188
It is a graph which shows the change of relative activity of mutant CGTase with pH.

FIG. 4 Wild-type CGTase and various tyrosine 188
It is a graph which shows the time-dependent change of CD which is a reaction product in the case of using starch as a substrate in mutant CGTase.

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[Procedure amendment]

[Submission date] March 12, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0024

[Correction method] Change

[Correction content]

Bacillus obensis SP Knob C-1400 (Bacillus ohbensi
s sp. nov. P-1C19) carrying a wild-type CGTase gene cloned from C-1400)
GT5A (Sin, K.-A. et. Al., App
l. Microbiol. Biotechnol. , 3
5,600-605 (1991)) with SphI and Hin
Approximately 2.5 Kb DN by partial digestion with dIII
The A fragment was cut out and used as a plasmid pUP110BS.
CsacQ (Nakamura A. et. Al., A
gric. Biol. Chem. , 55 (9), 236
7-2374 (1991) SphI and HindIII
It was mixed with the digest, treated with T4 phage DNA ligase and ligated. This plasmid was designated as pUP110Ce-CGT.
And Furthermore, in order to increase the expression level of the CGTase gene, the sacQ gene known to increase the expression level of the Bacillus subtilis cellulase gene was incorporated between HindIII- SphI on the plasmid. The thus obtained plasmid was designated as pUP11.
0CeCGTsacQ. Using this DNA, bee
Bacillus subtilis
s) Transformation of 207-21 was performed, and transformants resistant to kanamycin were selected. From this transformant, plasmid DNA was extracted, purified, and analyzed by a conventional method. As a result, wild-type plasmid DNA pUP110CeCGT was obtained.
Had sacQ.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

Example 3: Purification of modified CGTase Starch was added to the various crude enzyme solutions obtained in Example 2 in an amount of 2% to stir at 4 ° C. for 1 hour to adsorb the enzyme. Centrifugation (4 ℃, 7000rpm) to
The starch was collected, washed with 50 mM sodium phosphate buffer (pH 6.8), suspended in the same buffer, and
The enzyme was eluted by stirring for 2 hours at ° C. The starch was removed by centrifugation, and the supernatant was used as a purified enzyme preparation. If necessary, Centriprep-30 (manufactured by Amicon) is used for ultrafiltration to concentrate the enzyme solution. The purification process was confirmed by SDS-PAGE.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction content]

[0033]

[Effects of the Invention] Bacillus obensis SP
Knob C-1400 (Bacillus ohbens
is sp. nov. C-1400) -derived SEQ ID NO: 1
In the amino acid sequence of, the substitution of the 188th tyrosine with another amino acid has a great influence on the properties of the produced enzyme and the interaction between the enzyme and the substrate, and makes the tyrosine 188 residue more spatial. When substituted with alanine or serine in addition to tryptophan, which is another large amino acid, the CD produced from starch of the substitution mutant CGTase is β-CD as compared with wild-type CGTase.
Relative production of γ-CD with respect to Alanine,
The serine-substituting mutant enzyme produces β-CD and γ-CD at almost the same rate, and the tryptophan-substituting mutant enzyme produces γ-C.
Since the production amount of D reached 15% in terms of sugar yield, it was not limited to the application to a known field, and γ-C which had been difficult in the past.
It can be used for mass production of D.

Claims (3)

[Claims] 1. Bacillus ohbensis sp. Nov. C-140
0) in the amino acid sequence of SEQ ID NO: 1
Cyclodextrin glucanotransferase in which the second tyrosine is modified to alanine, serine or tryptophan. 2. Bacillus ohbensis sp. Nov. C-140
In the gene containing the base sequence of SEQ ID NO: 1 in the DNA of (0), the 1756th to 1758th "TAT" s are "GCT", "GCC", "GCA", GCG ", and" T ".
CT "," TCC ", TCA", "TCG", "AG"
A cyclodextrin glucanotransferase gene modified to T "," AGC "or" TGG ". 3. A method for producing a cyclodextrin glucanotransferase, which comprises culturing a transformed microorganism into which the gene according to claim 2 is introduced and obtaining cyclodextrin glucanotransferase from the culture solution.