JPH0310688A - Plasmid containing cgtase gene expressed by trp promotor - Google Patents

Abstract

NEW MATERIAL:A recombinant vector having a gene of CGTase(Cyclomaltodextrin glucanotransferase) integrated into a vector containing a trp promoter. USE:For producing CGTase. PREPARATION:For example, alkaline Bacillus sp. #1011 (FERM P-8685) is cultured and collected. A DNA is then extracted and purified by a conventional method to provide a chromosomic DNA, which is subsequently treated with a restriction enzyme to afford a DNA fragment. The resultant fragment is then linked to a phage DNA to infect Escherichia coil therewith. The infected Escherichia coli is subsequently cultured and plaques capable of exhibiting alkaline CGTase activity are selected from many plaques, treated with a restriction enzyme and then linked to a vector to afford the objective plasmid containing the CGTase gene.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to cyclodextrin synthase (Cyclodextrin synthase).
maltodextrin glucanotrans
ferase, EC2,4,1゜19, hereinafter referred to as CGT
It is called ase. ) gene, that is, the CGTase gene, and is related to the avian 1 promoter ( 抛pro
moter, hereinafter referred to as trp promoter. ), and specifically relates to a recombinant vector containing the gene, a transformed strain containing the recombinant vector, and a method for producing CGTase using the transformed strain.

[Prior art] Conventionally, Bacillus has been used as a microorganism that produces CGTase.
However, with the progress of genetic engineering, the CGTase gene was extracted from the chromosomal DNA of these microorganisms into appropriate plasmids. A method was developed by Sakai et al. to produce CGTase using a transformed strain using Bacillus subtilis or Escherichia coli as a host.

However, in general, the enzyme activity produced by these transformed strains carrying the CGTaseilt gene in their plasmids was considerably lower than that of the parent microorganism.

[Problem to be solved by the invention]

The object of the present invention is to efficiently express the cloned CGTase gene in the cells of a transformed strain, and to
The goal is to produce large amounts of CGTase, and to establish an economical production technology for CGTase using genetic recombination technology.

[Means for Solving the Problems] Conventionally, when CGTase was produced by a transformed strain such as E. coli, the enzyme amount of CGTase produced by the parent strain was lower than that of the parent strain. The cause of this was thought to be various effects caused by the use of systems such as ribosomes and gnal peptidase in host cells that are different from those of the parent strain in the transcription, translation, and secretion of genes of the parent strain.
Since the amount of GTase produced is the result of reactions at each stage of transcription, translation, and secretion, it has been difficult to rationally analyze the phenomenon and increase the amount of enzyme produced.

Therefore, as a result of intensive research, the present inventors found that the main reason for the low production amount of CGTase by transformed microorganisms is CGT.
By using the promoter derived from the gene of the parent strain as it is to express the ase gene, we came to the conclusion that it works effectively in the host microorganism (without using a promoter), and we compared and studied various promoters.As a result, The Trizo promoter is most preferred, and in that case, the DNA sequence upstream of the translation start site of the CGTase gene is
As a result of observing the effect on gene expression, CG
By inserting and linking a new CGTase gene containing not only the structural gene region of the Tase gene but also at least a portion of the DNA sequence upstream of its translation start point downstream of the Tolizo promoter (Gendo promoter). The expression efficiency of CGTase is improved, and CGTas
It was discovered that the production amount of e was dramatically increased, and the present invention was completed.

That is, the present invention provides the following features: 1. Trizo promoter (η]! promoter
) into a vector containing CGTase (Cyclomalt
odextrin glucano-transfer
ase, hereinafter referred to as CGTase. ), the recombinant vector according to 1 above, wherein the CGTase gene contains the structural gene of CGTase and at least a part of the DNA sequence upstream of its translation initiation site. Recombinant vector 3, CGTase gene is alkaline Bacit
lus sp.

The recombinant vector 4 described in 2 above, characterized in that it is derived from the plasmid pDR720.
Recombinant vector 5 according to claim 1, characterized in that it is Alternatively, Escherichia having a recombinant vector according to any one of 4.
ta) Microorganisms belonging to the genus.

6. 5 above, where the microorganism belonging to the genus Escherichia is Escherichia coli (ε5cherichia coli)
Microorganisms mentioned.

7. The microorganism according to 6 above, wherein the microorganism belonging to Escherichia coli is Escherichia coli C600p.

8. A method for producing CGTase, which comprises culturing the microorganism according to any one of 5.6 or 7 above in a medium, accumulating CGTase in the medium, and collecting the CGTase from the culture.

It is related to.

The present invention will be explained in detail below.

The CGTase gene used in the present invention may be any gene for cyclodextrin synthase (hereinafter referred to as CGTase), which is an enzyme that produces cyclodextrin from starch.
β-CGTase of Cillus sp, 11011
genes, or Bacillus macerans
Examples include the α-CGTase gene.

The former gene has already been cloned into the BamHI site of E. coli plasmid pBR322 and isolated as a 10 kb plasmid ρTUE217 or pTUE202 (Mokudo et al., JP-A-62-208286 and imura, K.).

et al, Appl Microbiol Bto
tect+r+ol 26: 149-153 (198
7)), its DNA base sequence and deduced amino acid sequence consisting of 713 amino acid residues have been determined.

(Kimura, K, et al, J Bact
eriol 169:4399-4402 (1987)
). These recombinant vectors contain the CGTase gene with the original promoter and are now available.

E. coli obtained by transforming E. coli HBIOI stably retains these vectors.

Regarding the latter gene, Bacillus subtilis plasmid ptlB
110 (Takano et al., JP-A-59-254)
215), plasmid pDs1 consisting of 6.8 kb
0, and its DNA@base sequence and deduced amino acid sequence consisting of 714 amino acid residues have been determined (Takano, T, et al, J
Bacteriol 166:1118-1122(1
986)).

In the present invention, the CGTase gene desirably includes not only the structural gene region but also at least a portion of the DNA sequence upstream of its translation initiation site. Regarding the number of base pairs of the DNA sequence upstream of this translation start point, in Examples and other examples, CGT starting from 18 base pairs upstream of the translation start point or 48 base pairs upstream of the translation start point
Although the ase gene is used, the present invention is not particularly limited to this, and any number of genes may be used.

Trizo promoter (trp
For example, E. coli tr
Examples include the p promoter and its improved trp promoter, and examples of host microorganisms that can be used include Escherichia coli C600m11, C600, and HBIOl.

The recombinant vector, the method for preparing the transformant, and its CGTase production ability in the present invention will be described in detail below, taking as an example the case where Escherichia coli is used as the host microorganism.

E. coli trizo promoter (trp promoter)
Er.

Hereinafter, it will be referred to as Lrp promoter. For example, plasmid pDR720 (P
(manufactured by Harmacia) can be used. The Kono promoter vector contains an ampicillin resistance gene, and a BaII site is inserted downstream of the promoter as a DNA fragment insertion site.
) II It has an I site. The method of inducing the gene of interest is a conventional method (Maniatis, T., eLal, M.
o1ecular Cloning, Co1d Sp
ring Harbor Laboratory198
2), l P T G (Isopropyl-
β-D-thiogalactoside), IA
By adding A (3-4ndolylacetic acid) and increasing the culture temperature (from 37°C to 42°C).

As an example of CGTase gene, alkaline Bacillus
The CGTase gene (hereinafter referred to as the original gene) of llussp. This gene is on the plasmid pT
Included in UE217.

This CGTase gene is linked to the downstream side of the trp promoter, but it is desirable that the linked CGTase gene contains not only the structural gene region but also at least a part of the DNA sequence upstream of its translation start point. This includes, for example, 1 upstream of the translation start site of the original gene.
A DNA fragment starting from 8 base pairs and containing the structural gene (hereinafter referred to as p-'ia), and a DNA fragment starting from 48 base pairs upstream of the translation start point of the original gene and containing the structural gene (hereinafter referred to as F-'ia). 48), etc.
Prepare and use the fragments. Primer extensicon method using synthetic NA (Schreir, P, tl
, et al. (1979) J Mo1Bio11
29:169-172), BamHI linker-DNA is added to both ends of the inserted DNA fragment to match the insertion site of the promoter vector.

Then, insert the above insert D into the above promoter vector.
A recombinant plasmid was constructed by ligating the NA fragments, and Led
Competent cell transformation method by Erberg & Cohen (J Bacteriol (1974)
119:10721074) to obtain the desired novel E. coli transformed strain.

This E. coli transformed strain is then cultured in liquid to produce these enzymes. For liquid culture, ampicillin 50 μg
5 containing 100rd L-broth medium containing /rtdl
Inoculate a 2% preculture solution into a 00 d volume Sakalo flask and inoculate 3
It was carried out under the conditions of 7°C and 125 spm, and the above
Induction is applied by adding . Escherichia coli production J・'1
Enzyme activity was determined by dividing the culture solution into extracellular fraction, periplasmic fraction, and intracellular fraction using Fuwa blue.
e value method (J Bioches+
(1954) 41: 583-603) using soluble starch as a substrate.

The E. coli transformed strain obtained by the present invention is CGTas
More than 90% of e activity was observed in the periplasmic fraction, and the activity reached its maximum value at 24 hours of culture.

Other promoters, such as the tack promoter (t
ac promoter) and PL promoters, but showed the highest activity when expressed by the Lrp promoter.
In the case of a DNA fragment that starts from a base pair and contains a structural gene, and in the case of a DNA fragment that starts from 48 base pairs upstream of the translation start point of the original gene and contains a structural gene, it is much more difficult than a DNA fragment that contains only a structural gene. showed high activity.

〔Effect of the invention〕

According to the present invention, the CGTase gene could be efficiently expressed in a host microorganism, and the production amount of CGTase could be dramatically improved. This has made it possible to economically produce CGTase by applying genetic engineering technology.

(Example) Plasmid pTI+ containing CGTase gene DNA
Cloning of 217 was performed using the following method. The CGTase gene was developed by the inventor of the present application in the above-mentioned Japanese Patent Application Laid-open No.
No. 208286, or the said Appl Micr
Biol Biotec-hnol 26: 149
-153 (1987) and J Bacteri, supra.
o1169: 4399-4402 (1987). That is, plasmid pTLIE202 described in JP-A No. 62-208286 and ppl Mic
Robiol Biotechnol 26:149-
153 (1987) and J Bacterio supra.
169:4399-4402 (1987), plasmids ρTIIE202 and pT[JE217 were both obtained by the same method. Both plasmids are E. coli vector pBR322 Ram)
II To I site alkaline Bacillus sp.

CGTase from Sha 1011 (Feikoken Bibori No. 8685)
The genetic DNA of the 1011 strain was cloned, and the difference between the two is that
The only difference is the length of the region near the se gene. Therefore, pT[IE202 is 9.1kb, and pTLIE217
was 10.0kb.

FIG. 3 shows a physical map of plasmid pTtlE217.

Although any plasmid can be used to implement this application,
In the examples, plasmid pTUE217 was used.

1. Cloning of CGTase gene (1) Alkaline Bacillus sp.
5g, peptone 10g, yeast extract 5g, liver liver 0
41g, Mg5Oa.7) 1z00.2g containing sodium carbonate l.

[composition adjusted to pH 10.0] at 40°C.
After shaking culture for 5 hours and collecting bacterial cells in the late phase of logarithmic growth,
Saito, Miura method (Saito, H., Miura method)
ura, K.:Biochem, Biophys,
^cta, 72.619. (1964))
Extract and purify DNA using the NA extraction method to obtain chromosomal DNA.
Approximately 5 ■ A was obtained.

(2) Insertion of chromosomal DNA fragment into phage DNA Take 300 μg of the chromosomal DNA obtained in (1) above, add restriction endonuclease Saq 3AI (manufactured by Takara Shuzo), and react at 0°C for 30 minutes to partially cleave it. did. Furthermore,
Approximately 7 to 12 kb of chromosome D was obtained by sucrose density gradient ultracentrifugation.
The NA fragment was separated and purified to prepare a DNA fragment. 20 gg of bacteriophage λD69 DNA (provided by the Institute of Medical Science, University of Tokyo) was added to restriction endonuclease B.
am1l I (manufactured by Takara Shuzo), a part of it was mixed with the above DNA fragment, and the mixture was incubated at 10°C at 124°C with DNA ligase derived from T4 phage (manufactured by Takara Shuzo).
The ligation reaction of the DNA strands was carried out using an in vitro packaging kit (Amersham International
iona1) to prepare transducing phage particles. # Lambda medium containing % starch (10 g Batatotryptone (Dirco) per Iffi, NaC12,5
[Composition containing g] Escherichia coli strain 5M32 (distributed by the Institute of Medical Science, University of Tokyo, genetic trait: 5A500.
His■d, 1onsA1.00.1.5trA) was grown at 37°C and infected with the transducing phage particles. Among the many plaques (lytic plaques), those having alkaline CGTase activity were selected. Transducing phage λD69-TUIIO particles were obtained. The transducing phage particles were cultured with Escherichia coli strain 5M32 in lambda medium of #ffi followed by PEG 6000 method (K,R).

Yamamoto, B., Malberto, R.Be.
gziner, T., Lowhorne & G., Tre.
iber:Virology+ 40+ 734 (
Transducing phage DNA was extracted and purified according to the method (1970)) to obtain about 150 με of about 50 kb DNA.

(3) Insertion of transducing phage DNA fragment into plasmid hector DNA50.0 obtained in (2) above. Take czg and Sau
3A I was added and reacted at 0°C for 30 minutes to partially cleave. Furthermore, a transducing phage DNA fragment of about 2 to 9 kb was separated and purified by sucrose density gradient ultracentrifugation to prepare DNA fragment (2). On the other hand, pBR322 plasmid D, which has tetracycline resistance (Tet) and ampicillin resistance (Amp'), is used as a plasmid hector.
N A (Bethesda Re5earchLa
boratories (USA)) was completely decomposed with BamHI and heat-treated at 65°C for 110 minutes to obtain BAP (
Bacterial Alkaline Phospha
tase) After treatment, mix with the DNA fragment
The DNA strands were ligated using A ligase at 10°C for 124 hours to construct plasmid DNA incorporating the DNA and fragments.

(4) Transformation with a plasmid carrying the extracellular production gene of CGTase E. coli HB 101 strain was grown in L medium (10 g of tryptone (Dirco) per 11 cells, yeast extract (Dirc)).
o) 5 g, containing 15 g of NaC and adjusted to pH 7.0) was inoculated at 50 d, cultured with shaking at 37°C, grown to late logarithmic growth, and then harvested. This was cooled with water at a final concentration of 0. The cells were sequentially suspended in a solution of IM CaC1z to obtain competent cells. The plasmid DNA solution obtained in (3) was added to this cell suspension solution, allowed to react for 30 minutes under water cooling, and then heat shocked at 42°C for 12 minutes to prevent the plasmid DNA from being incorporated into the cells. Next, this cell suspension was separately inoculated into the above-mentioned medium and cultured with shaking at 37°C for 160 minutes to perform a transformation reaction (Lederberg, E, M, +Cohen,
S, N,; J, Bacteriol, 119.107
2 (1974)), and the resulting transformed strain was transformed into LS.
Amp agar plate (II!) per tryptone (Dirco
) 10g, yeast extract (Difco) 5g
, Starch Log, , NaCl 5 g.

Composition of ampicillin 50■, agar 15g) at 37°C
The E. coli transformed strain pTIJE21?, which is ampicillin resistant and has CGTase activity, was cultured in pTIJE21? /E, c
odanHB 101 and pTLIE202/llB 10
1 was isolated. These strains were treated with 50 μg of ampicillin.
Using a 5 L Erlenmeyer flask containing 500 d of L-broth medium containing /d, culture was carried out at 37°C, 120 Orp, and cultured using the alkaline method (Rapid alkalin
e method, Birnboim & Doly
(1979) Nucleic Ac1ds Res
7: 1513-1523), each plasmid 1
．． I got 8 ■.

A restriction enzyme map of plasmid pTUE217 is shown.

2. Construction of a recombinant vector and creation of a transformed strain containing the same A method for preparing an insert DNA fragment for constructing a desired recombinant vector using plasmid pTUE217 DNA is shown in a flow sheet in FIG.

In order to express this gene with an E. coli promoter,
A DNA fragment that starts from the translation start site of the original gene and includes the structural gene (hereinafter referred to as F+1), a DNA fragment that starts from 18 base pairs upstream of the translation start site of the original gene and includes the structural gene (hereinafter referred to as F-18). (hereinafter referred to as F-48), and a DNA fragment that starts from 48 base pairs upstream of the translation start site of the original gene and includes the structural gene (hereinafter referred to as F-48).
Three types of inserted DNA fragments were prepared.

Bam)I linker DNA was added to both ends of the inserted DNA fragment prepared by the primer extension method to match the insertion site of the promoter vector. The DNA fragments were separated and prepared by 1.2% agarose gel electrophoresis.
After separating the A fragment and detecting it with ethidium bromide, the desired hand was adsorbed onto DE814 paper, collected with a solvent solution, and purified by ethanol precipitation.

(1) To prepare the inserted DNA fragment, use the primer DNA
Three types of 17n+ shown by thick solid lines in Figure 1 as A
er synthesized NA to DNA synthesizer (AppliedBio
4 fb each using systems (380A type)
/g, and then polynucleotide kinase (manufactured by Takara Shuzo) was added to 100 μl containing 4 μg of a portion thereof, and reacted at 37°C for 3 hours.
Phosphorylate the 5' end of mer-synthesized NA to 8, K12
and completed 13. In Figure 1, which shows the vicinity of the translation start point of the original CGTase gene, three types of synthetic NAs are represented by K8, K12, and K13, and the one starting from the translation start point and the 18th base upstream of the translation start point, respectively. Those starting from , and those starting from the 48th base upstream of the translation start point are shown.

(2) Structural gene part of this original gene (second
The black part of plasmid pTUE217 in the figure is CGTas
(showing the structural gene of e) contains the BamHI site and the Ec
Since each oRI site is included (in Figure 2, B
and E. ), when preparing three types of inserted DNA fragments, the fragment before this EcoRI site (hereinafter f
8, f12 and f13. ) and the fragment after the EcoRE site (hereinafter referred to as fR). FIG. 4 illustrates f8, f12, f13 and [R.

(3) Preparation of Temperate DNA for f8, [12 and f13100 preparation Sense single of the original CGTase gene
Strand DNA (anti-strand of the DNA sequence shown in Figure 1) was prepared, and this was used as template DNA to synthesize three types of synthetic DNA shown as 8, K12, and 13.
After annealing, double-stranded DNA was synthesized by enzymatic reaction using Klenow fragment and dNTP.

That is, 5 μg of M13 phage mpH (manufactured by Takara Shuzo)
Digest the DNA with the restriction enzyme EcoRI.1. The BAP treatment shown in (3) was performed. 100 μi containing 0.3 μg of this vector DNA and pTUE217 were digested with Liao R1, and 3 μg of the Liao R1-1.7 kb fragment was added, inserted and ligated using DNA ligase, and the ligation solution was added. 1. to E. coli JM105. Transform the target plasmid pTUE by the method shown in (4).
E. coli containing 217E was isolated. This strain was cultured in 2XTY medium containing 50 μg/− of ampicillin [containing 16 g of tryptone, log yeast extract, and 15 g of NaC per IL. ) Cultured in a Sakaro flask containing 100 mfl,
200 μg of single strand DNA was obtained.

(4) Preparation of f8, f12 and [13 fragments 50 μg (
Single strand DNA prepared in step 3), 4 μg of synthetic NA prepared in step (1), and KE1200μ! The synthesized NA was heated to 60'C in a Klenow buffer solution and then gradually cooled to room temperature to anneal the synthesized NA to the single-strand DNA. Next, annealed synthetic resin NA
Using the primer extension method as a primer, add lenow to the annealed reaction solution.
fragment (DNA polymerase)
I, manufactured by Takara Shuzo ■) and dNTP (manufactured by Takara Shuzo ■)
was added and reacted for 20 minutes at room temperature, partially forming double-stranded DNA.
produced A. Then, Mung Bean Nuclease (manufactured by Takara Shuzo) was added to digest the remaining single-stranded DNA and blunt both ends of the double-stranded DNA.
Hl linker DNA (purchased from Takara Shuzo) was added and Bam1 was added to both ends of the double-stranded DNA using DNA ligase.
A l l linker was ligated. Finally, the DNA was digested using Bam) + 1 and EcoR1 to prepare 12 μg of a 470 bp f8 fragment. In the same manner, 10 μg each of the f12 fragment of 1 tsubo old-αo RI-1,488 bp and the [13 fragment of 518 bp of 1 tsubo old-αo RI-L] were prepared from synthetic NAK12 and ni-13.

(5) Preparation of fR fragment On the other hand, in order to prepare fR, pTUE21 shown in FIG.
7 old ndI [[-shear d lll-4,5kb DN
Bam! 50μg of A fragment! l I methylase (Takara Shuzo■
The Bam1l 1 site contained in this DNA fragment (as shown above, this site is contained in the structural gene portion of the CGTase gene) was removed by slightly modifying the DNA fragment. Then, both ends were blunted using mung bean nuclease, and DNA was added to both ends.
Bam11 I linker-DNA was ligated using IJ gauze. Finally, restriction enzymes EcoRI and Bam1l
BamHI-EcoR1 which is r8 after digestion with I
15 μg of the 2.2 kb fragment was prepared.

(6) Preparation of inserted DNA fragments F+l, F-18 and F-48 Next, 3 μg of f8-DNA fragment and 3 μg of fR-DNA fragment were ligated using DNA ligase, and then Bam1
2 μg of inserted DNA fragment F+1 (3
, 7 kb) was prepared. f12 and f1 in a similar manner
3, inserted DNA fragments F-18 (3,7 kb) and F
-48 (3,7 kb) was prepared in an amount of 2 μg each.

(7) Construction of recombinant vector E. coli promoter, tac promoter, and t used to express the newly constructed CGTase gene.
Plasmid pDR54 is a three-promoter vector containing the rp promoter and the PL promoter.
0 (4,1 kb), pDR720 (4,1 kb) and p
3u each of MY12-6Amp-1 (7,2kb)
g was prepared, the BoμII site located downstream of the promoter was cleaved with a restriction enzyme, and further treated with BAP.

(8) CGTas to tac promoter vector
Ligation of e gene and isolation of transformed strain Plasmid pDR54 with Bam1l l site cleaved
0.5 μg of insert DNA fragment F+1 was added to 10 μf containing 0.1 μg of DNA fragment F+1, and inserted and ligated using DNA ligase. ＃． By the transformation method shown in (4), E. coli Moon 103
ampicillin resistance and CGTase activity (
CGTas by thin layer chromatography by Kobayashi et al.
Based on the detection method of e activity. Kobayashi et al.
Carbohydr Res 61: 229-23
8 (1978)) as an index, the transformed strain pA80
7/JM103 was isolated.

Similarly, using 0.5 μg each of inserted DNA fragments F-18 and F-48, transformant strain pA1202/
JM103 and the transformed strain p^130B/JM103 were isolated.

(9) CGTas to trp promoter vector
Ligation of e gene and isolation of transformed strain Bam) Plasmid pDR72 with cleaved l site
Insert DNA fragment F+1 into 10μl containing 0.1ug of
0.5 μg was added and inserted and ligated using DNA ligase. Using the transformation method shown in (8), 600 g of E. coli C was obtained.
ampicillin resistance and t:GTa
Using se activity as an indicator, the transformed strain pH850/C (l
ioOgalK- was isolated. Similarly, insert DNA
Using 0.5 μg each of fragments F-18 and F-48, the transformant pR1221/C600galK- and the transformant pR1303/C600gal- were isolated.

(10) CGTas to PL promoter vector
Ligation of e gene and isolation of transformed strain Current use) IIl site cleaved plasmid pMY12-6
10 μg containing 0.1 μg of Amp-1! insert DN into
0.5 μg of A fragment F+1 was added and inserted and ligated using DNA ligase. E. coli C600 was transformed using the transformation method shown in (8) to improve ampicillin resistance and CGTa.
Using se activity as an indicator, the transformed strain ρP878/C60
Q was isolated.

Similarly, insert DNA fragments F-18 and F-48 were
．． Using 5 μg each, transformant strain ppt251/
C600 and the transformed strain pP1325/C600 were isolated.

(11) The above nine isolated strains were cultured using the method shown in 114), the contained plasmids were extracted and a physical map was created, and the DNA sequence of the junction portion was analyzed to determine the contained plasmids. CGT constructed with the target gene as
It was confirmed that it was the ase gene.

3. Production of CGTase by E. coli transformed strain Ampicillin 50μg/ml! 100 m of L broth medium containing
Transfer the preculture solution to a 500 d volume Sakalo flask containing 1 ml of preculture solution.
% inoculation at 37°C and 125 spm.
The strain was cultured.

When the OD at 660 nm of the culture solution reached 2.5, E. coli transformed strains pA807/JM103, pA1
In the case of 202/JM103 and pA1308/JM103, two coats of IPTG were added, and the E. coli transformed strain p
R850/C600F,! ±Jin, 9R1221/C60
In the case of 0L field (and pR1303/C600v feathers),
20 μg/rtdl of IAA was added, and the culture temperature was increased to 37° for E. coli transformed strains pP878/C600, pP1251/C600, and pP1325/C600.
The temperature was raised from 42°C to 42°C for induction.

Enzyme activity was determined by dividing the culture solution into extracellular fraction, periplasmic fraction, and intracellular fraction.
Measured according to method. As a result, the enzyme activity of all E. coli bacteria showed the maximum value after 24 hours of culture, and more than 90% of the enzyme activity was present in the periplasmic fraction. Table 1 shows the measured enzyme activity of the periplasmic fraction during 24 hours of culture. Furthermore, as a control, the enzyme activity of the periplasmic fraction of E. coli pTUE217/IIBIOI was 75 units/unit.

The results showed that the highest enzyme activity was obtained when the inserted DNA fragment F-18 or F-48 was inserted into the Bamll site of plasmid ρDR720. These values are 5.2 times and 5.5 times higher, respectively, compared to plasmid pTIJE217 containing the original CGTase gene.
It has increased five times.

That is, CGTa is added to the vector containing the trp promoter.
CG containing the structural gene of se and at least a part of the DNA sequence upstream of the translation start site of the original gene
Contains a recombinant vector incorporating the Tase gene,
A transformed strain of a microorganism belonging to the genus Escherichia efficiently produces CGTase and accumulates a large amount of the enzyme in its periplasmic fraction.

Using this transformed strain, CGTase could be economically produced from the periplasm fraction by liquid culture.

(Margins below this page) Table 1 pA807/JM103 pA1202/JM103 pA1308/JM103 pR878/C600 total ↓(- pR1221/C600fLi! L pR1303/C600 Note 2 p　P　878　/　C600 pP1251/C600 pP1325/C600 contrast pTUE217/1 (Blot F+I -18 -48 F+I -18 -48 F+I -18 ・F-48 tac　゛ tac・ tac.

trp・ trp・ trp・ PL・ P.L. P.L. 5

[Brief Description of the Drawings] Figure 1 shows alkaline Bacillus sp. 11
DNA near the translation start site of the CGTase gene of 011
The sequence and deduced amino acid sequence are shown. The thick solid lines denoted by K8, K12 and 13 are II) NA fragments f8, [1
Three types of 171 used to prepare 2 and f13
11er synthetic NA is shown. The boxes marked -35 and -10 indicate the putative promoter region of the gene, and the thin solid line marked RBS indicates the putative ribosomal region.
Indicates a binding site. The arrow indicates the cleavage point by the signal peptidase of the enzyme, and the frame adjacent to it indicates the confirmed amino-terminal amino acid sequence of the mature protein of the enzyme. Figure 2 shows CGTas for constructing recombinant vectors.
This is a flow sheet showing a method for preparing an inserted DNA fragment of the e gene. B, , E and H represent the restriction enzyme sites of RHI, R1 and H4ndlI. In the figure, the black part of the plasmid shows the CGTase structural gene, the white part shows the vector pBR322 DNA, and the diagonal line part shows the vicinity of the CGTase structural gene derived from the chromosomal DNA of B, sp11011. FIG. 3 shows a restriction enzyme map of plasmid pTUE217. In the figure, the black part of the plasmid shows the structural gene of CGTase, and the white part shows the vector pBR322D N
A is shown, and the shaded area shows the vicinity of the CGTase structural gene derived from the chromosomal DNA of sp 11011. Figure 4 shows inserted DNA fragments F+1, F-18 and F48.
DNA fragments f8, f12, f1 prepared to construct
3 and fR are illustrated. The black part in the diagram is C
The structural gene of GTase is shown, and the white part is shaded sp t
The DNA near the 3' side of the structural gene of CGTase derived from the chromosomal DNA of sp #1011 is shown, and the diagonally shaded area shows the DNA sequence upstream of the translation start site of CGTase of sp #1011. E is EcoRI site, C
B) indicates a Bamtl I site modified by methylase, and S indicates a ribosome binding site present in the DNA sequence upstream of the translation start point.

Claims (1)

[Claims] 1. trp promoter
) into a vector containing CGTase (Cyclomalt
odextrin glucano-transfer
ase, hereinafter referred to as CGTase. ) recombinant vector. 2. The recombinant vector according to claim 1, wherein the CGTase gene contains the structural gene of CGTase and at least a part of the DNA sequence upstream of its translation initiation site. 3. The CGTase gene is found in alkaline Bacillus.
us sp. The recombinant vector according to claim 2, wherein the recombinant vector is derived from #1011 (Keikoken Bibori No. 8685). 4. The recombinant vector according to claim 1, wherein the vector containing the tryp promoter is plasmid pDR720. 5. Escherichia spp. having the recombinant vector according to any one of claims 1, 2, 3, or 4.
A microorganism belonging to the genus Hia). 6. The microorganism according to claim 5, wherein the microorganism belonging to the genus Escherichia is Escherichia coli. 7. The microorganism according to claim 6, wherein the microorganism belonging to Escherichia coli is Escherichia coli C600galK^-. 8. CGTase, characterized in that the microorganism according to any one of claims 5, 6, or 7 is cultured in a medium, CGTase is accumulated in the medium, and the CGTase is collected from the culture.
manufacturing method.