JPS6128383A - Variant escherichia coli - Google Patents

Abstract

PURPOSE:Variant Escherichia coli useful for producing industrially heat-resistant alpha-amylase, by cloning a heat-resistant alpha-amylase structural gene of Bacillus stearothermophilus in a mold of Escherichia coli, so that heat-resistant alpha- mylase can be produced. CONSTITUTION:Chromosome DNA is separated from a culture mold of (Bacillus stearothermophilus) A631, IAM11003, ATCC7954 which is middle thermophilic bacterium to secrete thermophilic-amylase out of the mold, it is partially decomposed with restriction enzyme Sau3A, and a fregment of chromosome is separated. Separately, plasmid pBR322 is scissored with restriction enzyme BamHI, it is blended with the fragment of chromosome, ligase T4DNA is added to them, they are connected, the treated solution is added to suspension of culture mold of Escherichia coli C600, which is transformed to give the aimed variant Escherichia coli having plasmid pTUE107.

Description

[Detailed description of the invention] The present invention is a method for producing thermostable α-amylase.

It concerns Schierichia coli (E. coli).

More specifically, the present invention relates to Escherichia coli which is made capable of producing thermostable α-amylase by cloning the structural gene of Bacillus stearothermophilus thermostable α-amylase into Escherichia coli cells. I can buy things.

In general, Escherichia coli has been used for a long time as a bacterium related to genetic manipulation, and since it was quickly approved for use in the laboratory as a genetic manipulation model, much research has been conducted and attempts have been made to produce various useful substances. It's here.

However, in Escherichia coli.

Even if the productivity of useful vIJ members was confirmed through genetic manipulation, they were only accumulated within the bacterial body and no useful substances were secreted outside the bacterial body. If you attempt to separate useful substances accumulated in the bacterial cells by lysing the bacteria or grinding the bacterial cells, cell membrane components and intracellular components will be mixed together with the useful substances, and the useful substances will be separated. separation of,
This made refining extremely difficult.

The present inventors conducted intensive research in search of Escherichia coli that secretes useful substances outside the bacterial body. By cloning, we succeeded in secreting heat-stable α-amylase outside the bacterial cell.

The present invention is Escherichia coli that produces thermostable α-amylase.

Escherichia coli that produces the thermostable α-amylase of the present invention can be created as follows.
hia, coli) C600-pTUE 107
It has been deposited with the Institute of Fine Technology under the name FERMIP-7670.

Bacillus stearothermophilus
stearothermophilus) A 63
1. IAM110D3, ATCC'7954 has heat resistance α
- Known as a moderately thermophilic bacterium that secretes amylase outside the bacterial body.

Chromosomal DNA is isolated from the cultured cells of this strain, partially digested with restriction enzyme 8au 3 A, and the digested product is subjected to sucrose density gradient ultracentrifugation to separate chromosome fragments of approximately 2 kb or more.

Separately, a commercially available plasmid pBR322 (Takara Shuzo Co., Ltd. product) (Fig. 1) was cut with the restriction enzyme BamHI, the previous chromosome fragments were mixed therein, TA DNA ligase was added, and ligation was carried out. ·Ko! jc6
Le
derberg, E. M., and S.

Cohen, N. (1974) Bacter, J.
iol, -υβ-91072-1074).

The transformed strain is isolated as a strain that is resistant to ampicillin and produces α-amylase. This strain is cultured, and plasmid pTUE107 is obtained from the cultured cells. The physical map (restriction enzyme cleavage map) of plasmid pTUE 107 is shown in Figure 2, where the white part is derived from vector pBR322, and the black dotted part is a chromosomal fragment, and the size of this cloned fragment is The length is about 7.6
It is kb.

The transformant obtained here is an excellent mutant E. coli that secretes heat-stable α-amylase well outside the cell.

This mutant Escherichia coli is completely new in terms of productivity of thermostable α-amylase, and is extremely useful for industrial production of thermostable α-amylase.

This mutant Escherichia coli is Escherichia coli IJ C600-pT.
Named UB 107, FFRM P-7 was sent to the Microtech Institute.
It has been deposited as 670.

Next, examples and manufacturing examples of the present invention will be shown.

Example Creation of mutant Escherichia coli Bacillus stearothermophilus A6!11゜IAM
1100! l, ATCC7954 in LG medium (Bactodryptone 1011% yeast extract 5.p, NaC 15g
Glucose 2y was dissolved in 11 water and adjusted to pH 75) and cultured overnight at 60°C, collected by centrifugation, washed, and the resulting bacterial cells were cultured using the 8aito + Miura method (5ai
to + H,, and K, Miura, (1964
) Biochim, Biopys, Acta, 7
2+ 619-629) was used to separate the chromosomal DNA, dissolve it in Tris-HCl/EDTA buffer, add restriction enzyme 8au 5 A (Takara Shuzo Co., Ltd. product), and incubate at 37°C.
fJ2k and the degraded product was separated by sucrose density gradient ultracentrifugation.
Chromosome fragments larger than b were isolated and obtained.

A schematic cleavage map of the commercially available plasmid pBR522 (Takara Shuzo Co., Ltd. product) is shown in Figure 1;
522 is 4.4 kb and is ampicillin resistant (Amp'
) and tetracycline resistance (Tc'), and can be cleaved at one site by the restriction enzyme Bam HI.

pBR322 cut at one site with BamHI and about 2k obtained from the above Bacillus stearothermophilus A631
Chromosome fragments larger than b were mixed and ligated by adding T4DN-containing gauze. (Weia8+L+5ab
lon, A, J-r LIVe+ T, 几-+ Fa
reed, G.

C9, 几1chardson, C, C- (1968) J
, Biol.

Chem.
M, +and 8. Cohen, N. (1974)
J, Bacteriol, 119y1072-1
The plasmid was introduced by 074).

The obtained transformed bacterial cells were used as a selective medium with soluble number #
L medium (10g Bactodrypton, 5g yeast extract)
, Nzu1c15 ,! Dissolve 9 in 11 water and make pi17.
5), cultured at 37°C, and sprayed an iodine solution onto the ampicillin-resistant strains that appeared. Strains that did not react with iodine could be isolated by secreting α-amylase. Ta.

The resulting α-amylase producing strain was isolated and cultured overnight at 57°C in L medium containing 100μi ampicillin/ITLI, the culture solution was collected by centrifugation, washed, and the isolated bacterial bodies were extracted with alkali. Law (Birnboirn + H
, C. and J. Doly (1979) Nucl.
A novel plasmid was obtained by isolating and searching the plasmid using the following method (eic Acld Res, 7 r 1513).

This plasmid was named plasmid pTUE107.

Plasmid pTUE 107 is 12. The physical map (restriction enzyme cleavage map) of Okb is shown in Figure 2. Here, the white part is vector pBR32
2, the black dotted part is a chromosome fragment, and each abbreviation is a restriction enzyme.

This plasmid pTUE 107 was labeled with 32F,
Bacillus stearothermophilus A631 strain chromosome D
NA, Escherichia coli strain C600 chromosomal DNA and α-amylase high producing strain Bacillus subtilis (Ba
cillus 5ubtilis) NA EcoRI digestion and Southern hybridization of each of the 64 chromosomal DNAs L 5outhern, E, M,
(1975) J, Mo1. Biol,, 9th,
503-517), Escherichia
What is Bacillus subtilis chromosomal DNA?
Although it did not hybridize, it specifically hybridized with the chromosomal DNA of Bacillus stearothermophilus A661 strain at 6 locations, confirming that the cloned gene was derived from Bacillus stearothermophilus A661. It was done.

Furthermore, the obtained plasmid pTUB, 107 was added to a bacterial cell suspension of Escherichia co.
nd S, N, Cohen (1974) J, B
acteriol (1 unit 9° 1072-1074) was introduced into the bacterial cells.

Add the transformant obtained here to the above-mentioned selection medium,
The strain was cultured at 67°C, and a strain with a high α-amylase value was selected from the bacterial strains produced.

Production example Production of thermostable α-amylase Escherichia coli C600='pTUE107, FE
BMP-7670 with ampicillin 100μV 1rL
Using L medium containing 1001 rL, 1001 rL was dispensed into a soomz volume Sakaro flask, and cultured with shaking at 37°C for 48 hours.

During that time, the culture was collected for 1 d each, and the osmotic shock method C.
Chan, S, J, + J, '%'i'eiss
, M., Konrad, T.

荊百e, C, Babl, S, -D, Tu+ D
, Marks+ and D.

F, 8teener (1981) Proc, N
atl, acad, sci.

U, S, A, Mu 5401-5405) and extracellular fraction (Extrdcellular fr
ac, tion), periplasmic fraction (Pe
riplasmic fraction, intracellular fraction
on) and the activity of α-amylase was measured. At the same time, alkaline phosphatase (J'ase) activity, which is a representative enzyme localized in the periplasm, was similarly measured.

FIG. 3 shows changes over time in α-amylase activity, APage, and growth. Here A is Extra
lular fraction, B is Perip
lasmic fraction, C is Intrace
lular fraction, G indicates growth.

As is clear from Figure 6, α-amylase activity is
It was first recognized in tracelular fraction. After that, it showed almost constant activity. What appeared next was Periplasmic fraction. The activity increased rapidly in the late phase of logarithmic growth, but the activity gradually decreased thereafter. Extrac a little late
Activity appeared in the ularfraction, and a constant increase in activity was observed in subsequent cultures. 4
At the time of 8 hours of culture, the extracellular fraction of the total amylase activity had reached 70%. APas as a control
When the e activity was investigated, Periplasmic fra
Most of the activity was measured in the
The trend of APase activity over asmic fraction was similar to that of α-amylase. However, Extracel Iularfract
ion also had activity, and it was thought that a portion of APase leaked out of the bacterial cells. However, APas
The majority of e is Periplasmic fracti
On the other hand, the majority (about 70%) of α-amylase is present in the Extracellular fraction.
exists on.

Furthermore, when we investigated the activity of intracellular enzyme β-galactosidase, we found that some
Activity was observed in the lar fraction, suggesting that some of the bacteria were lysed.

However, since the decrease in bacterial cell production was small, it was thought that cocoon dissolution occurred only in a small portion.

Test example Enzymological study of thermostable α-amylase Escherichia coli C600-'pTUE107, FgRM P
-7670 was cultured in the same manner as in the production example, and the chemical properties of the obtained α-amylase were examined.

Bacillus stearothermophilus A661 as a control.
, IAM11003, ATCC7953 and Bacillus
Each α-amylase produced by S. subtilis NA64 was used.

First, the optimum temperature for action was determined using Fuwa's method (Fuwa, H-(
1954) J, Biochem, Dan, 583-6
05] and is shown in FIG. Here, D is Escherichia coli C600-pTUE 107, FER
α-amylase produced by MP-7670, E is Bacillus stearothermophilus A631, IAM1100
3. Thermostable α-amylase produced by ATCC7954, F indicates α-amylase produced by Bacillus subtilis NA64. From Figure 4 Escherichia coli C60
α-amylase produced by 0-p'rUE 107, F'ERMF'-7670 and Bacillus stearothermophilus A661, IAMl 1003, ATCC795
The thermostable α-amylase of No. 4 has the same optimal temperature of action, and the α-amylase of Bacillus subtilis NA64
It is clear that it is different from amylase.

Next, after heat-treating these enzyme preparations at each temperature for 10 minutes, the α-amylase F of Bacillus subtilis NA64 was 4.
At 0°C, thermostable α-amylases D and E were subjected to enzymatic reaction at 60°C, and residual activity was determined. The results are shown in FIG. Here D, E.

F indicates the same enzyme as above. Figure 5 shows that the α-amylase of F starts to be inactivated after 40°C and is completely inactivated after 70°C, whereas the α-amylase of E is 85°C.
It is found that it is stable up to about ℃.

From this, Escherichia coli C60D-pTUE
107, α-amylase produced by FBRMP-7670 and Bacillus stearothermophilus A631,
It can be seen that the thermostable α-amylases of IAM11003 and ATCC7954 have the same thermostable properties.

Next, the influence of metal ions on the thermal stability of this amphithermic α-amylase preparation was investigated. 9 each of both enzyme preparations
Heat treatment was performed in the presence of each ion at 0° C. for the time shown in FIGS. 6 and 7, and the residual activity was determined. NaCl is 10
CaCl was used at 2 mM.

It can be seen that when Na+ coexists, the thermal stability is clearly improved compared to when both are the same, when Ca++ is present alone, or when nothing is present. Here, D and E represent the same enzymes as above. Next, we again compared the thermostability of B. subtilis α-amylase (phantom and double-needle fever α-amylase (C and D) in the presence of Na+ and Ca″, and found that Bacillus subtilis NA64 α-amylase is 90'
C All inactivation occurs in about 13 minutes, whereas double-needle fever α
-Amylase is reduced by about 50% by heat treatment at 90℃ for 60 minutes, 6
Even after treatment for 0 minutes, residual activity of about 30% was shown. In FIG. 8, E, 4'' indicates the same enzyme as above.

that's all? In summary, α-amylase, which is the heat-stable α-amylase gene that has been successfully cloned, is derived from the progenitor bacteria Bacillus stearothermophilus A631 and TAM1.
Similar to the thermostable α-amylase of 1005, A'rCC7955 white rice, it showed significantly higher thermostability than the α-amylase of Bacillus subtilis NA64, and this stability was further increased by Na'-.

[Brief explanation of drawings]

Figure 1 shows the physical map of plasmid pBR322 (
Figure 2 shows the plasmid pT.
The physical map of UB ID 7 is shown. Figure 6 shows α-amylase activity, APa
Figure 4 shows the optimal temperature for each enzyme's action, Figure 5 shows the residual activity after heat treatment, and Figure 6 shows the changes in s e activity and growth over time. 7 is a diagram showing the residual activity of each enzyme in the presence of each ion after heating, and FIG. 8 is a diagram showing the residual activity of each enzyme after heating at 90° C. in the presence of Na+ and Ca++. Agent Masurishi Door 1) Kan Male Calculation 3 Monza Mukashi Yoji Time C Section) Bu 4 Figure 5 l

Claims (1)

[Claims] Escherichia coli produces thermostable α-amylase