JPH04126080A - Production of heat-resistant xylose isomerase - Google Patents

Abstract

PURPOSE:To enable the mass-production of a heat-resistant xylose isomerase useful for the production of a high-fructose corn syrup by culturing a xylose isomerase gene of Clostridium thermohydrosulfuricum and a specific microorganism. CONSTITUTION:(A) A xylose isomerase gene of Clostridium thermohydrosulifluricum (ATCC 33223) (producible by cloning to E.coli) and (B) Bacillus brevis (transformed with a plasmid of Bacillus brevis containing cell wall protein gene promoter) are cultured and the objective xylose isomerase is separated from the cultured product. The production yield of xylose isomerase of the present process is several hundred times as high as the yield attained by the use of unmodified C. thermohydrosulphuricam.

Description

[Detailed description of the invention] [Industrial application field] (Hereinafter abbreviated as C, thermohydrosulfuricum)
The present invention relates to a method for producing heat-stable xylose isomerase with high productivity.

[Prior art and purpose of the invention]

Xylose isomerase is an enzyme that catalyzes the interconversion of D-xylose and D-xylulose, and at the same time has the ability to convert glucose to fructose, and is industrially important.

On the other hand, C. thermohydrosulfuricum is a type of hyperthermic bacterium and has the deposit number ATCC 33223.

It has long been known that C. thermohydrosulfuricum produces xylose isomerase [J.B.
acteriol, 164.1146-1152 (1
[985)] However, the xylose isomerase gene of C. thermohydrosulfuricum is unknown;
The present inventors determined the sequence and previously filed a patent application [Japanese Patent Application No. 2-44716].

By the way, isomerase is used to produce high fructose corn syrup (HFC5). The conventional production temperature of HFC5 is 6 due to the heat resistance of isomerase.
It is set at 0°C, and the concentration of fructose in the product at 60°C is 42%. However, in practical terms,
If the fructose concentration is increased to 55%, it can be used as is as a sweetener for foods such as soft drinks. Therefore, if the production temperature can be increased to 85°C, the chemical equilibrium will shift to the fructose side, making it possible to increase the fructose concentration to 55%.

Xylose isomerase of C. thermohydrosulfuricum has an optimum temperature around 85°C, and if this xylose isomerase can be obtained in large quantities, it will be possible to produce HFC5 with a fructose concentration of 55%. However, the productivity of xylose isomerase by C. thermohydrosulfuricum is low, and it is not practical to produce xylose isomerase using C. thermohydrosulfuricum itself.

Therefore, an object of the present invention is to provide a method for producing a large amount of thermostable xylose isomerase using the xylose isomerase gene of C. thermohydrosulfuricum.

[Structure of the invention]

The present invention relates to Bacillus plebis containing the xylose isomerase gene and cell wall protein gene promoter of C. thermohydrosulfuricum.
The present invention relates to a method for producing thermostable xylose isomerase, which comprises culturing B. brevis transformed with a plasmid of B. brevis (hereinafter abbreviated as B. plevis) and collecting the produced xylose isomerase.

The xylose isomerase gene of C. thermohydrosulfuricum used in the present invention has, for example, the base sequence shown in FIG. The xylose isomerase gene of C. thermohydrosulfuricum can be prepared by cloning into E. coli.

The xylose isomerase gene contains a cell wall protein gene promoter, e.g.
et al., Proc, Natl, Ac, Sci, 19
89; 86 (3589-3593)],
Furthermore, a transformant transformed with the plasmid obtained in B. Previs is used in the production method of the present invention. B. Transformation of Plevis was performed using the method of Takahashi et al.
W, et al., J. Biol, Chew, 1987;
262°21 (10035-10038)).

In addition, 47 stocks (FERM P-7
224), 47 stocks (FERM BP-2308),
47-5 Q strain (Udaka, Agr, Biol, Che
m, 1976; 40.3 (523-528).

In addition, plasmid pNU210 is a multicopy plasmid containing Bacillus subtilis plasmids pUB 110 and B, and the control region at position 5 of the Plebis cell wall protein gene.
87) and T, Kawazu et al., J, Bac.
teriol, 169.1564 (1987).
Yamagata H, et al., Proc, Na
tl, Ac.

Sci, 1989; 86 (3589-3593
) can be obtained by the method.

The transformant of B. plebis is preferably grown at around 37°C, which is the optimal temperature for B. plebis, in a culture solution containing glucose, yeast extract, polypeptone, endophyte, uracil, erythromycin, etc. It is appropriate to culture under strong agitation.

After culturing, the bacteria are collected by centrifugation, the resulting bacteria are crushed using ultrasound, and the debris is removed by centrifugation. Furthermore, 8
Heat-treated at 5℃ to denature unnecessary proteins,
Remove denatured proteins by centrifugation,
Further, if necessary, the desired heat-stable xylose isomerase can be obtained by purification using a conventional method.

As in the method of the present invention, if B. plevis transformed with a plasmid of B. plebis containing a cell wall protein gene promoter is used, the yield will be hundreds of times higher than when using C. thermohydrosulfuricum itself. C. Thermohydrosulfuricum xylose isomerase can be produced. Furthermore, according to the method of the present invention, mutations and alteration do not occur during cloning and expression of cloned genes, and the resulting xylose isomerase is the same as that obtained using C. thermohydrosulfuricum itself. You will get the same thing as .

The present invention will be further explained below with reference to Examples.

〔Example〕

Section 1: Clostridium thermohydrosulfuricum: AT
CC33223 Escherichia coli MV1184: ara, Δ(lac-pro),
5trA, thi, (φ80△1acIZ△M15
), Δ(srl-recA)306: :TnlO(t
et');F';traD36゜proAB, 1a
cI'Z△M15 (1) Bacillus plevis 47 strain (F
ERM P-7224) Growth conditions C, Thermohydrosulfuricum can be grown using Melasniemi's method CMelasniemi, J, Gen, Mi
cro, Biol.

1987, 133 (883-890)] using xylose as a carbon source and NH,CI Ig/j
l',C.

The cells were grown at 65° C. in a medium supplemented with 3 g/l of C120.0.

E. coli cells were grown at 37°C in 2x yeast extract tryptone medium (Dirco).

Cloning and sea-fencing engineered genomic DNA was performed using the method of Saito (Saito, H., and Miura et al.
Biochim, Biophys, Acta 19
63.72 (619-629)] from C. thermohydrosulfuricum. Frozen pellets (0,
34g) with 200mM NaCl and 10% EDTA.
It was dissolved in 4.5Wl of 50mM Tris (pH 8,0) containing 0mM. 0.5-10% SDS was added and the mixture was rotated slowly for 5 min at 60 °C, then incubated at 60 °C.
30 minutes RNA5e A (0,05+ng/-)
Processed with. Next, proteinase K (
0.5■/-). The lysate was washed three times with phenol/Tris buffer and once with phenol/chloroform.
Extracted twice. Centrifuge the residue (10 minutes, 15,000
rpm). After adding 0.8 volumes of isopropanol, the DNA was unwound and dissolved in 10mM Tris (pH 8,0) 1,5- containing 1mM EDTA.

Finally, the DNA was transferred to a DEAE column (washing buffer: Tris 50mM, pH 8,0°EDTA 1mM,
NaClO, 15M; Elution buffer: Tris 50
mM, pH 8.0, EDTA lmM, NaC
1IM) on an anion exchange resin.

Genomic DNA was obtained from EcoRI, Hindll and P
Digested using stI. Fragments were separated by 0.4% agarose gel electrophoresis. DNA was denatured by soaking the gel in 1.5 M NaCl, 0.5 M NaOH for 25 minutes. The gel was neutralized with 3M sodium acetate (pH 5.5). DNA was transferred to a nylon membrane (Biodyne) by electroblotting (3 h, 2 mA/cm) and baked at 80 °C for 1 h.

This plot was plotted against Bacillus subtilis (Bacillus subtilis).
32P-labeled 240 nucleotides B obtained from the xylose isomerase gene
hybridized with the be[Ddel fragment (10'cpm/ml) at 58°C. This fragment contains two regions of relatively high homology as seen when compared to the Swiss isomerase amino acid sequence.

After hybridization, EcoRI is 8.7
Kb.

2.2Kb for Hindll and 3.6 for Pstl
A single band appeared near Kb.

3.6Kb± of Pstl digested Crosslidium DNA
The 0.6 Kb fragment was obtained from glass milk (glas milk).
The vector pUc119 was isolated by extraction using PstI-killed E. coli vector pUc119. Competent E. coli MV1184 cells were transformed with the ligated DNA. Desired Clostridium DN
Colonies with A were detected by hybridization of transformant colonies with B subtilis probes. Plasmid DNA was extracted from colonies giving a positive signal by the alkaline lysis method, digested with restriction enzymes, and analyzed by Southern hybridization. 3, which strongly hybridizes with the probe obtained from the Bacillus subtilis xylose isomerase gene;
A 55 Kb PStI fragment was obtained.

This 3.55 Kb fragment was digested with the restriction enzyme Alul
, HaellI, Ndel and old ndIII into small fragments. These fragments were transformed into E. coli MV using pUC118 and pUc119.
1184. Single-stranded DNA was prepared according to the method of Vieira and sequenced using T7 DNA polymerase (Siefens ver2.O: trade name) and 7-deaza-dGTP.

The base sequence and amino acid sequence of the xylose isomerase gene of C. thermohydrosulfuricum are shown in FIG.

B. Expression in P. plebis. C. Complete sequence of xylose isomerase gene of Thermohydrosulfuricum and 2°5 K Pstl containing 100 upstream nucleotides and 1000 downstream nucleotides.
The -HindIII fragment was ligated into the B, previs expression vector pNL'210 (restriction enzyme map shown in Figure 2) that had been digested with EcoRI and Pstl. In the resulting vector, the xylose isomerase gene is controlled by the cell wall protein gene promoter previously present in the vector.

Using the obtained vector, the method of Takahashi et al.
ashi W, et al., J., Biol, Che.
m, 1987; 262. 21 (10035-1
B. plevis was transformed by 0038)). That is, B. plebis bacteria was suspended in a 50mM phosphate buffer (pH 8.5), then DNA was added, polyethylene glycol was added to a concentration of 30%, and the plasmid was incubated at 37°C for 2 minutes. A transformant was performed.

B. The transformant of Plebis was cultured at 37° C. in the following culture solution with strong stirring.

Glucose 10g/f 8 yeast extracts 2g/l Polypeptone Log/f Internal secretions 5g/12 Uracil
0.1 g/p erythromycin l
After Omg/1 incubation, centrifugation (5 to 10 min;
Bacteria were collected as a stationary phase at 15°C.

The obtained bacterial pellet was soaked in phosphate buffer (50mM, pH
7). The washed pellet was treated with triethanolamine 100mM, pH 7, fructose 400mM,
It was dissolved in 1mM PMSF (protease inhibitor) containing 10mM MnC. Bacteria were disrupted by ultrasonication for 30 seconds, and debris was removed by centrifugation (15°C, 10 min, 4°C). Additionally, the supernatant was heat treated by incubating at 85°C for 10 minutes. After cooling, the denatured proteins that cannot be recovered are centrifuged (15
Then, the crude xylose isomerase was obtained by removing it for 10 minutes at 4°C. Next, 500 times the weight of EDTA 5m
3 times and 500 times the weight of ED against buffer containing M
Dialysis was performed twice each against a TA-free buffer. It was further purified by FPLC anion exchange chromatography and gel filtration on Superose I2.

The productivity of xylose isomerase using the method of the present invention using B. plebis as a host is compared with the productivity of xylose isomerase using C. thermohydrosulfuricum and Escherichia coli as hosts, respectively, as shown in Table 1. show.

Table 1 The culture conditions for C. thermohydrosulfuricum and E. coli are as follows.

C. Nutrient conditions for Thermohydrosulfuricum: 65℃, anaerobic culture, 2 days Xylose 10g/z Yeast extract 5g/l Tryptone Log/l Meat extract 5g/I! Phosphate buffer 50mM, pH 6.8FeSO
,0,02g/J MgS○,-7H,OO,1g/j7 Ca Cl 2.2 H2O0,l g/INH,C
I 1.0g/IC0Cj72
0.03g#' Escherichia coli culture conditions 37°C, 16 hours Yeast extract 10 g/l Tryptone 16 g/f NaC 15 g/l pH 6,8 Tibial 1yLt hill enzyme activity: 0.1-0.4 units of xylose isomerase Hepes 100mM, pH 7, IL, '7
It was carried out at 85°C in 400mM of Tose and 10mM of MgCl. Glucose production was performed using glucose oxidase analysis (Glucose B-Test, Wako) with 20 μl
I followed the sample. In addition, the buffer used for the temperature profile was Hepes 100mM, pH 7, and fructose 4O.

mM, MgC! :50mM, CoC1+0.5
mM was used.

FIG. 3 shows the activity versus temperature profile of the obtained thermostable xylose isomerase. This figure shows that the obtained thermostable xylose isomerase has an optimum temperature around 858C.

Isomerization of glucose to fructose Glucose was isomerized to fructose using the thermostable xylose isomerase obtained by the method of the present invention.

xylose isomerase o, 1-0.40/1nl,
100mM Hepes buffer pH 7, 400mM fructose, 10
The reaction was carried out in mMMgCf at 70°C or 85°C, and the sugar concentration in the reaction solution was measured by high performance liquid chromatography 13 and 46 hours after the start of the reaction, and expressed as area percentage of the chromatogram. The results are shown in Table 2. The theoretical equilibrium concentration of fructose at 70'C is 52%, and the theoretical equilibrium concentration of fructose at 85°C is 54.8%.

Table 2 ■ Table 2-2 HFC3=i semi-high fruct scone syrup Unit: Percent

[Brief explanation of drawings]

FIG. 1 shows the base sequence of the xylose isomerase gene of C. thermohydrosulfuricum used in the method of the present invention. FIG. 2 shows B, a restriction enzyme map of the Previs expression vector pNU210. FIG. 3 shows a profile of the activity and temperature of the thermostable xylose isomerase obtained by the method of the present invention. Fig. 1 CTGCAGGATA CCACACGGAG CTTTCAATC TGGAGCAAAG 8o TTTTAAATAA 9C TTATATTTC TTTTACATC CAGATGAAAT AATAGACGGA GTAGCGTACT GGCA CACATT TACAGCCAAT CAAAGGCCAT GGGACCATTT TAC:TGACCCT GCCTTTGAAC TATTTGAAAA ACTCGACGTA GCTCCGGAAG GAGAGACATT AAGGGAGACG ATAAAAG ACT ACTTAAAGAC GAGCAAAACA So ■ + 40 50
60″; AGACAATGGA
AGCATATATT GCATGGCACT+
100 110
120: AAAAAGGAGG AAAA
TGACAT GGAATACTTCCCAAAT
CAA ACAATCCATA TGCATTTAAA AAACCTTTAA AAGAACACTT GCGTTTTTCA GGGACAGATC CATTTGGAGC ACCCACATG ATGGATATTG CCAAAGCGAG AGTAGAAGC A CCATTTTTCT GTTTCCATGA CAGAGATATA AACAAAAATT TAGATACAAT AGTTGCAATG AAAGTATTAT GGGGCACAGC GAACCTTTTTT Figure 1 → TCAAATCCGA GATTTGTA CA TGGAGCAGCT GCAGCAGCGC AAGTAAAAAA AGCACTTGAG GTATTTTGGG GTGGAAGAGA AGGATATGAA CTTGATAACT TAGCAAAGATT TTTGCACATG GAAGGGCAGT T TTTAATTGA GCCAAAACCT GATGCAGCAA GTGTACATGC ATTTTAAAG AACATAGAAG CGAACCATGC GACACTTGCA GCAAGGATTA ACAATATGTT AGGCTCTATA TGGGATACAG ACCAATATCC AACAGACATA Snake 2 ACTTCCTGTA ATGCAGATGT ATTTGCATAT ATAACAAAAG AACTTGGAGG ACAGAACTAT ACATTACTTA ACACAGA TAT GGAATTGGAA GCAGTAGAAT ATGCACAAGA GATAGGATTT AAAGAACCGA CAAAACACCA ATATGATTTT AAGTACGACC TTGATAAATA CTTCAAATTA GGACATGACT TTCAACATGA ATTAAGATAT GATGCAAATA TGGGAGATAT GCTTTTAGGA CGAATGACA CCCTTGCGAT GTACGAAGTC

Claims (2)

[Claims] (1) Production of thermostable xylose isomerase, which involves culturing Bacillus brevis transformed with a plasmid of Bacillus subrevis containing the Clostridium thermohydrosulfuricum xylose isomerase gene and cell wall protein gene promoter, and collecting the produced xylose isomerase. Method. (2) The production method according to claim 1, wherein the Clostridium thermohydrosulfuricum is ATCC33223 and the cell wall protein gene promoter is derived from Bacillus brevis.