JPH05244945A - Production of alpha-cyclodextrin - Google Patents

Abstract

PURPOSE:To provide cyclodextrin.glucanotransferase(CGTase) having a high alpha-cyclodextrin(alpha-CD) production rate by modifying the CGTase of Bacillus.stearothermophilus by a genetic industrial method, and to provide a method for producing the alpha-CD by using the CGTase. CONSTITUTION:The CGTase of Bacillus.stearothermophilus in which the phenylalanine unit at the 222th position or the phenylalanine units at the 222th and 286th positions are replaced with tyrosine units, and a method for producing the alpha-CD includes the production of the alpha-CD by treating starch with the CGTase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an improved method for producing α-cyclodextrin (hereinafter abbreviated as αCD). Furthermore, the present invention provides Bacillus s. Bacillus with improved performance.
T. thermophilus) cyclomaltodextrin glucanotransferase.

[0002]

2. Description of the Related Art Cyclomaltodextrin glucanotransferase of Bacillus macerans (hereinafter abbreviated as CGTase) is known as an αCD producing enzyme. Furthermore, apart from this, Bacillus stearothermophili
lus) (hereinafter abbreviated as B. stearothermophilus) is also known as CGTase. B. CGTase of Stearothermophilus is B. Macerance CGTa
It can be used at a starch concentration higher than that of se, and the αCD productivity is high at the beginning of the reaction.

[0003]

However, B.I. CGTase of stearothermophilus has a large amount of β-cyclodextrin (hereinafter abbreviated as βCD) co-produced, and αC
There is a problem that the burden of separating and removing βCD from D is heavy.

Therefore, an object of the present invention is to Α modified by modifying CGTase of stearothermophilus by genetic engineering
CGTase with high CD production rate and this CGTase
An object of the present invention is to provide a method for producing αCD, which has a higher αCD production rate than βCD using.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides Bacillus stearothermophilus (Bacillus stearothophilus) in which phenylalanine at the 222nd position or phenylalanine at the 222nd and 286th positions is substituted with tyrosine.
Cyclomaltodextrin from Ermophilus)
It relates to glucanotransferase.

Furthermore, the present invention provides Bacillus stearothermophilus (Bacillus stearothermophilus) in which the phenylalanine at the 222nd position or the phenylalanine at the 222nd and 286th positions is substituted with tyrosine.
hilus) cyclomaltodextrin glucanotransferase on starch to produce α-cyclodextrin.

The present invention will be described below. B. As stearothermophilus, FERM P-2225
The TC-91 strain which is a known microorganism having a deposit number of

In the present invention, B. The CGTase of Stearothermophilus basically means the CGTase produced by the TC-91 strain. However, TC-91CGT
In the present invention, CGTase obtained by substituting a part of amino acids having no influence on CGTase activity with another amino acid is also referred to as B. Stearothermophilus CGTase
Include in. For example, 10 of CGTase of TC-91 strain
In the present invention, CGTase in which the 8th aspartic acid is replaced with valine and the 460th arginine is replaced with alanine is also described in B.I. Stearothermophilus CGTa
Include in se. CGTase in which some amino acids are substituted as described above can be obtained by genetic engineering by a site-directed mutagenesis method. Or, for example, B.I. Stearothermophilus No. 2 strains of B. It can also be obtained from another strain belonging to Stearothermophilus.

B. Stearothermophilus No. Two
The strain has the following mycological properties. It is a spore bacillus, and is an spore oval type having a diameter of 0.8 μ and a length of 2.5 μ. It decomposes catalase +, aerobic, glucose, arabinose, xylose, mannitol fermentable +, casein, gelatin and starch. The optimum temperature for growth is 55 to 65 ° C. Produces cyclomaltodextrin glucanotransferase.

B. Stearothermophilus No. The two strains were transferred to the Institute of Microbial Industry, Institute of Industrial Technology, on March 3, 1992, under the contract number Micro Engineering Lab. No. 12822 (FERM P
-12822).

The CGTase of the present invention is the same as the above-mentioned B. In CGTase of Stearothermophilus, the phenylalanine at the 222nd position or the phenylalanine at the 222nd and 286th positions are substituted with tyrosine. With these substitutions, B. Stearothermophilus CG
While maintaining high αCD initial productivity of Tase, βCD
The production ratio of αCD can be increased.

The substitution of phenylalanine with tyrosine can be carried out by a known site-directed mutagenesis method.

In FIG. 1, FIG. 2, FIG. 3 and FIG. Stearothermophilus TC-91 strain and No. 2 shares of CGT
The amino acid sequence of as and the amino acid sequence of CGTase of the present invention are shown.

The method for producing αCD of the present invention is based on CGTas.
As e, the tyrosine-substituted CGTase of the present invention is used. Except that tyrosine-substituted CGTase was used as the enzyme. It can be carried out in the same manner as the method for producing αCD by CGTase of Stearothermophilus.

For example, within 8 hours, preferably 12% or more of starch, at 60 to 80 ° C., the production amount of αCD is larger than the production amount of βCD, for example, 1 to 10 hours, the CGT of the present invention.
It can be carried out by applying ase. still,
There is no upper limit to the starch concentration, but in practice it is 20% or less, preferably 15% or less. This method can be performed not only in a batch system but also in a continuous system using an immobilized enzyme.
Incidentally, αCD can be separated from other βCD and the like by a conventional method.

[0016]

According to the present invention, B. It is possible to provide CGTase in which the production ratio of αCD is increased by suppressing the production of βCD while maintaining the high αCD initial productivity of CGTase of Stearothermophilus. Furthermore, according to the production method of the present invention, a high concentration of starch can be used as a raw material, and CD having a high αCD content can be produced, which facilitates the separation and purification of αCD.

[0017]

EXAMPLES The present invention will be described below with reference to examples.

Materials and Methods (1) Strains, plasmids and phages used CGTase thermophilic bacterium B. Stearothermophilus No. Two strains (FERM P-12822) were isolated from soil. B. Macerance IF03490 was donated by the Osaka Fermentation Research Institute. Bacillus
s subtilis) NA-1 (arg-15 hs
^{mM hsmR Amy - Npr - (Kuriki} ,
T. , S. Okada, and T.M. Imanaka.
(1988) J. Bacteriol. 170,155
4-1559) and Escherichia coli (Escherichia)
coli) TG1 (supE hsd 5 thi (l
ac-proAB) F '[traD36 proA ⁺ B
⁺ lacIq lacZM15] (Gibson,
T. J. (1984) Studies on theE
pstain-Barr virus genomo.
Ph. D. thesis, Cambridge Un
(iversity, UK) was used as a host when performing recombinant DNA experiments.

As a plasmid vector, pBR322 (Bolivar, F., RL Ro in the case of Escherichia coli)
drivez, P.D. J. Greene, M .; C. Be
tlac, H .; L. Heyneker, H .; W. Bo
yer, J. et al. H. Crosa, and S.C. Falko
w. (1977) Gene 2, 95), pUC118.
(Messing, J., and J. Vieira.
(1983) Methods Enzymol. 10
1, 20-78) and pTB523 for Bacillus subtilis. pTB523 is a low copy plasmid of Bacillus subtilis pT
B522 (Imanaka, T., T. Himeno,
and S. Aiba. (1985) J. Gen. Mi
crobiol. 131, 1753-1763) cloning site HindIII was changed to EcoRI.

M13mp18 and mp19 as phage vectors (Messing, J., and J. Vi)
eira. (1983) Methods Enzymo
l. 101, 20-78).

(2) Medium For culture of E. coli, 2xYT medium, for Bacillus subtilis L medium, C
When the S1 medium was used as a solid medium, 1.5% (wt / V) of agar was added. Antibiotics were added at the following final concentrations. In the culture of E. coli, in the case of tetracycline (Tc), 20 ug / ml, ampicillin (A
In p), 100 μg / ml was added. Tc for culture of Bacillus subtilis
Was added at 15 μg / ml. The composition of each medium is as shown below.

─────────────────────────────────── 2 × YT (/ 1) ───── ─────────────────────────────── Bactrypton 16g Yeast Extract 10g NaCL 5g ─────────── ───────────────────────── L-broth (/ 1) ─────────────────── ────────────────── Bactrypton 10g Yeast Extract 5g NaCL 5g ─────────────────────── ──────────── SC1 (/ 1) ────────────────────────────────── ── Soluble starch 10g Polypeptone 5g Yeast Sutokuto _{5g K 2 HPO 4 0.5g MgSO 4} · 7H 2 O 0.1g ──────────────────────────────── In the agar medium, starch azul was used instead of soluble starch.

(3) Purification of CGTase Since CGTase is secreted outside the cells as a secretory enzyme, purification was carried out from the culture medium without the cells. The cells were cultured in CS1 medium (11) at 37 ° C. for 17 hours, and the cells were removed by centrifugation (8,000 × g, 15 minutes) to obtain a crude enzyme solution.
Fractions saturated with 30% to 70% were collected by ammonium sulfate salting out and dissolved in 0.05M phosphate buffer (pH 6.0). Ion exchange column (Sephadex
After adsorption to DE52, φ4 cm × 15 cm), elution was performed by applying a 0 M to 0.6 M NaCl concentration gradient.
After concentration by ultrafiltration, a gel filtration column (Sephad
ExG-100, φ1 cm x 50 cm) fractionated and SD
Purified to unity by S-PAGE analysis.

(4) Enzymatic activity of CGTase The activity of CGTase is determined by the method of Lejeune et al. (Lejeune, A., K. Sakaguch).
i, and T.I. Imanaka. (1989) Ana
l. Biochem. 181, 6-11), and performed αCD quantification using methyl orange (MeOr).
0.6 ml of 5% soluble starch, 0.105 ml of 0.05M phosphate buffer (50 mM, pH 6.0), enzyme solution 0.1.
After adding 0.105 ml of 1 mM MeOr solution to 05 ml and reacting for 10 minutes, 0.15 ml of 6N HCl is added to stop the reaction, and the mixture is left at 16 ° C. for 30 minutes. Fifty
Absorption at 5 nm was measured to examine αCD synthesis.

The examination of the product by HPLC was carried out by the method of Kaneko et al. (Kanaeko, T., T. Kudo, and K.
Horikoshi. (1990) Agri. Bio
l. chem. 54, 197-201).

(5) Enzymatic activity of amylase Bernfeld's dinitrosalicylic acid (DNS) method (Bernfeld, P. (195)
5) Methods Enzymol. 1,149-1
58) or the immutable method (Fuwa, H. (1954)
J. Biochem. 41, 538-603).

(6) Protein concentration measuring method BCA protein analysis reagent (PIERCE) was used.

(7) Determination of nucleotide sequence A DNA fragment having an appropriate size was cloned into Escherichia coli phage M13 based on the restriction enzyme map to prepare ssDNA, and then the dideoxy method was used (Sanger, F.,
and S. Nichlen, and A .; R. Cou
lson. (1977) Proc. Ntil. Aca
l. Sci. USA 74, 5463-5467).

(8) Gene mutation introduction Gene mutation introduction is carried out by Amersham.
This was carried out according to the site-specific mutagenesis kit of the same company. The KpnI-SacI fragment of the cgt1 gene of plasmid pKB1 (FIG. 5) was introduced into M13 phage mp18, and base substitution was performed. The sequences of the synthetic oligonucleotides used for the construction of F222Y and F286Y are 5'-AAAT, respectively.
CTGTATGACTTGGGCG-3 ', 5'-GGG
GAGTGGTATTTTGTCAGAA-3 '.
The introduction of the mutation was confirmed by determining the nucleotide sequence. RF (replicatio) of the mutagenized phage
nform) DNA, a KpnI-SacI fragment was extracted and replaced with this region of pKB1 to obtain pKB1-
F222Y and pKB1-F222Y-F286Y were constructed.

(9) Others All recombinant DNA experiments were conducted in the literature (Sambrook,
J. , E. F. Fritsch, and T.S. Mani
atis. (1989) Molecular Clon
ing, a laboratory manual, 2n
d edition. Cold Spring Har
Bor Laboratory, New York).

Results (1) B. Stearothermophilus No. 2, and B.
Cloning of macerance IF03490 CGTase gene Cloning of the gene was performed by the shotgun method.
B. Stearothermophilus No. 2, and B. After culturing the Macerans IF03490 strain, the chromosomal DNA was extracted and cleaved with the restriction enzyme EcoRI, and then Bacillus subtilis plasmid p
It was inserted into the EcoRI site of TB523. Bacillus subtilis NA
One strain was subjected to the competent method (Imanaka, T., TT
anaka, H .; Tsunekawa, and S.M. A
iba. (1981) J. Bacteriol. 14
7, 776-786d) and transformed with CS1 Azure agar medium (Tc 15 μg / ml) to select Tc resistant and amylase halo positive candidate strains.

B. Stearothermophilus No. Two different DNA fragments were harvested from 2. As shown in FIG. 5, the respective CGTase genes were designated as cgt1 and cg.
t5 and cgt232 were named, and the recombinant plasmid was designated as pK.
They were B1, pKB5, and pKB232. B. One type of DNA fragment was cloned from Macerans IFO3490. When the obtained DNA fragment was subcloned into pUC118 using the restriction enzyme Sau3AI,
It was cloned as a 3.6 kb DNA fragment. pU
C using PstI and EcoRI existing on C118
The GTase gene was isolated, PstI of pTB523,
PKBM1 was obtained by inserting it into the EcoRI site.
B. The CGTase gene of macerans IFO3490 was named cgtM.

(2) Properties of cloned CGTase 4 cloned CGTase genes (cgt
1, Cgt5, cgt232, cgtM) was purified from the culture solution of Bacillus subtilis NA-1 and its properties were investigated.

B. Macerance IFO3490 CGTa
For se, the properties of the enzyme have already been reported (Kitahata, S., and S. Okad.
a. (1982) J. Am. Jpn. Soc. Starch
Sci. 29, 13-18), and the properties of CgtM were in agreement with the previously reported results. The main product from starch was αCD at an optimum reaction temperature of 1 ° C at 55 ° C.

On the other hand, B. Stearothermophilus No.
2 (FERM P-12822) is a strain newly isolated from soil. cgt1, cgt5, cgt23
2 were independently purified, and the properties of the enzymes were examined. As a result, all of the enzymes had an optimum reaction temperature of 65 ° C. and an optimum pH of 6.
0, the main product from starch was βCD.

(3) Determination of base sequence of CGTase gene The base sequence of each CGTase gene was determined by the dideoxy method. B. Stearothermophilus No. 21 derived from cgt1, cgt5, and cgt232 are all 21
It was encoded by the ORF consisting of 33 bases and was composed of 711 amino acids. It was considered that the three genes had mutations at 5 positions, all of which were concentrated in the third letter of the codon, and had the same amino acid sequence. The base sequence of cgt1 is shown in FIGS. 6 and 7. The mutation sites of the three cgt genes are as shown in FIG. From the deduced amino acid sequence, CGTase (cgt1, cgt5, cgt
The molecular weight of 232) was estimated to be 78.918.

These base sequences are described in B. It differed from the amino acid sequence of CGTase produced by Stearothermophilus TC-91 (Sak
ai, S.A. , M .; Kubota, K .; Yamamot
o, T. Nakada, K .; Torigoe, O .; An
do, and T.D. Sugimoto (1987) J.
Jpn. Soc. Starch Sci. 34,140
-147). When the N-terminal side sequence of the purified CGTase was determined, Ala-Gly-Asn-Leu-A was determined.
It is sn-Lys-, and from this it was revealed that the 31 amino acids on the N-terminal side functioned as a secretory signal sequence.

(4) Amino Acid Homology with Known CGTases The amino acid sequence of CGTases reported so far is shown in B. Stearothermophilus No. Two strains of B. Compared with those of Macerance IF03490,
It became like FIG. 3 and FIG. The amino acid sequence of CGTase showed very high homology despite being of different origin.

(5) Construction of mutant CGTase Cg in which the 222nd Phe of Cgt1 is replaced with Tyr
t1-F222Y and 286th Phe are also Ty
A mutant Cgt1-F222Y-F286Y in which r was substituted was constructed. Each gene is cgt1-F222Y,
cgt1-F222Y-F286Y and plasmid p
KB1-F222Y, pKB1-F222Y-F286
It was set to Y (refer to FIG. 9).

(6) Cgt1-F222Y, Cgt1-
Properties of F222Y-F286Y Cgt1-F222Y, Cgt1-F222Y-F28
6Y is Cgt, which is a wild-type enzyme in both optimum temperature and optimum pH
It was the same as 1 (65 ° C., 6.0). Cgt1, Cg
tM, Cgt1-F222Y, Cgt1-F222Y-
F286Y was independently purified, 900 U was allowed to act on a 1% starch solution (20 ml), sampled over time, and the product was examined by HPLC. The results are shown in Fig. 10. Cgt1-F222Y, Cgt1-F222Y-F
All of 286Y had a higher αCD ratio than Cgt1, and this tendency was remarkable particularly in the early stage of the reaction. CGTa
a specific amino acid (222-Ph) in domain A of se
e, 286-Phe) could alter the CD production characteristics.

[Brief description of drawings]

FIG. 1B. Stearothermophilus TC-91 strain (B
ST TC-91) and No. 2 shares (BST NO2)
Amino acid sequence of CGTase of the invention, CGTase of the present invention
Amino acid sequence of (F222Y, F222Y-F286
Y) and B. CG of Maserasun (BMA 3490)
The amino acid sequence of Tase is shown.

FIG. 2B. Stearothermophilus TC-91 strain (B
ST TC-91) and No. 2 shares (BST NO2)
Amino acid sequence of CGTase of the present invention, CGTase of the present invention
Amino acid sequence of (F222Y, F222Y-F286
Y) and B. CG of Maserasun (BMA 3490)
The amino acid sequence of Tase is shown.

FIG. 3B. Stearothermophilus TC-91 strain (B
ST TC-91) and No. 2 shares (BST NO2)
Amino acid sequence of CGTase of the present invention, CGTase of the present invention
Amino acid sequence of (F222Y, F222Y-F286
Y) and B. CG of Maserasun (BMA 3490)
The amino acid sequence of Tase is shown.

FIG. 4B. Stearothermophilus TC-91 strain (B
ST TC-91) and No. 2 shares (BST NO2)
Amino acid sequence of CGTase of the invention, CGTase of the present invention
Amino acid sequence of (F222Y, F222Y-F286
Y) and B. CG of Maserasun (BMA 3490)
The amino acid sequence of Tase is shown.

FIG. 5: Recombinant plasmids pKB1, pKB5, pK
Restriction map of B232.

FIG. 6 shows the nucleotide sequence of CGTase gene cgt1.

FIG. 7 shows the nucleotide sequence of CGTase gene cgt1.

FIG. 8: CGTase genes cgt1, cgt5, cg
The mutation site of t232 is shown.

FIG. 9: Plasmids pKB1, pKB1-F222Y,
3 shows the structure of pKB1-F222Y-F286Y.

FIG. 10: αCD productivity ((a): CgtM, (b):
Cgt1, (c): Cgt1-F222Y, (d): C
gt1-F222Y-F286F) with time.
▲, ●, and ■ represent αCD, βCD, and γCD, respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12N 15/54 C12R 1:07)

Claims (3)

[Claims] 1. The 222nd phenylalanine or 22.
Bacillus stearothermophilus (Baci) in which phenylalanine at the 2nd and 286th positions was replaced by tyrosine
and a method for producing α-cyclodextrin, which comprises reacting the starch with cyclomaltodextrin glucanotransferase of L. stearothermophilus) to produce α-cyclodextrin. 2. The method according to claim 1, wherein cyclomaltodextrin glucanotransferase is allowed to act on starch having a concentration of 8% or more. 3. The 222nd phenylalanine or 22.
Bacillus stearothermophilus (Baci) in which phenylalanine at the 2nd and 286th positions was replaced by tyrosine
illus stearothermophilus) cyclomaltodextrin glucanotransferase.