JPH02265486A - Dna fragment, slightly acidic and slightly cryotolerant cellulose and production thereof - Google Patents

Abstract

PURPOSE:To easily obtain a large quantities of the subject substance by transforming a cell with a plasmid DNA containing a DNA fragment originated from a specific microorganism and culturing the obtained transformant in a medium. CONSTITUTION:Ruminococcus albus is cultured in a medium containing glucose, etc., and the obtained microbial cell is extracted to obtain a DNA fragment (A) having a base sequence of formula and coding a genetic information of a neutral cellulase. A part of the component A is mutated to obtain a mutated DNA fragment (B) effective in (a) coding a protein corresponding to the matured protein of said neutral cellulase provided that the 15 or 24 amino acids from the amino terminal are deleted, (b) coding a slightly acidic cryotolerant cellulase, etc. A microbial strain (FERM P-10624) transformed with a plasmid DNA containing the component B is cultured in a medium and the cultivation product is extracted and purified to obtain the objective slightly acidic and slightly cryotolerant cellulase having a molecular weight of 35,000+ or -5,000 dalton, exhibiting maximum activity at pH5-7 and 20-35 deg.C and keeping >=80% of the activity after keeping at 37 deg.C for 1hr.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel NA fragment obtained by mutating a DNA fragment, a slightly acidic microtherogenic cellulase, and a method for producing the same.

Specifically, the present invention encodes the amino acid sequence of a neutral (pH 6, 8) mesophilic (37°C) cellulase produced by the obligate anaerobic rumenococcus Ruminococcus alpus that converts cellulose, which is an agricultural and forest product resource, into useful substances. The present invention relates to a DNA fragment obtained by mutating a part of a DNA fragment, a plasmid DNA incorporating this fragment, and a transformed bacterium produced using this plasmid DNA.

The present invention also relates to a cellulase produced by this transformed bacterium and a method for producing the same.

[Conventional technology]

Cellulose, which is an agricultural and forestry resource that exists widely and in large quantities on earth, is hydrolyzed to glucose by cellulase, and then various microorganisms are used to effectively utilize cellulose, such as converting it into energy resources, raw materials for chemical industry, and microbial protein. Research is being conducted to Currently, cellulase has been detected and purified from many microorganisms, but most of them are aerobic bacteria and filamentous bacteria, and cellulases have been detected from anaerobic bacteria, especially rumen bacteria.
There are few reports on the improvement and use of purified cellulases.

The preparations are (Japanese Patent Laid-open No. 62-208283) a recombinant vector containing a chromosomal DNA fragment carrying genetic information involved in cellulase production of a cellulase-producing bacterium belonging to the genus Ruminococcus, and Escherichia transformed with the recombinant vector. The invention has been completed regarding cellulase-producing bacteria belonging to the genus. The optimum pH for enzyme production by the transformed bacteria according to the invention of Yodai et al. is 6.8, and the optimum temperature is around 35°C.

[Problem to be solved by the invention]

Conventionally known cellulase derived from rumen bacteria is 37-40
Many of them have optimal activity at pH around neutrality at ℃1, but cannot express practical activity under the environmental conditions such as those inside plant cells, that is, under slightly acidic and extremely low temperatures (Yota et al.
283), where lower temperatures and lower p
A cellulase with high activity in the H region has been sought. Furthermore, in order to develop cellulose utilization technology using cellulase derived from anaerobic rumen bacteria, it is necessary to elucidate the chemical properties of cellulase enzymes and proteins.

The present inventors mutated a part of the DNA fragment encoding the cellulase gene of Ruminococcus alpine, which belongs to an anaerobic bacterium that generally has excellent cellulose decomposition ability, and developed a cellulase gene that can be used in a slightly acidic and extremely low temperature environment. The cellulase according to the present invention, which produced a cellulase capable of expressing activity, is clearly distinguished from the invention by Yodai et al. in that, for example, some amino acid residues are deleted from the amine terminus of the mature protein. The present invention was completed based on this new knowledge.

[Means to solve the problem]

The present invention consists of the following configuration.

1. Ruminococcus alps
This is a DNA fragment obtained by mutating a portion of the DNA fragment encoding the genetic information of neutral cellulase isolated from the DNA of A. usalbus.

2. A DNA fragment obtained by mutating a portion of a DNA fragment encoding the genetic information of neutral cellulase, which encodes a protein in which a part of the amino acid sequence is deleted from the amino terminus of the mature protein of neutral cellulase. D as described in l above, which is
NA fragment.

3. 15 from the amino terminus of the mature protein of neutral cellulase
Alternatively, the DNA fragment described in 2 above, which encodes a protein with 24 amino acids deleted.

4. DNA encoding the amino acid sequence of neutral cellulase
2. The DNA fragment according to 1 above, wherein the partially mutated DNA fragment coats a protein in which a part of the amino acid sequence is deleted from the carboxyl terminus of the mature protein of the neutral cellulase.

5. The DNA fragment described in 1 above, which encodes a slightly acidic low temperature cellulase.

6. Plasmid DNA containing the DNA fragment described in 1 to 5 above
A.

7. A transformant transformed with the plasmid DNA described in 6 above.

8. Rusinococcus alps
This protein is obtained by translating a partially mutated DNA fragment that encodes the amino acid sequence of neutral cellulase isolated from the DNA of A. usalbus.

9. A slightly acidic microcold cellulase that exhibits maximum activity at pH 5-7.20-35°C.

10. It has a molecular weight of approximately 35,000 ± 5.000 Daltons as determined by 5O5-polyacrylamide gel electrophoresis, exhibits maximum activity at pH 5-7, 20-35°C, and 80 % or more of residual activity.

11. A method for producing a slightly acidic low-temperature cellulase, which comprises culturing the transformant described in 7 above and collecting the slightly acidic low-temperature cellulase from the resulting culture.

The present invention will be explained in more detail below.

The slightly acidic micropsychrophilic cellulase according to the present invention is mesophilic, neutral p.
Escherichia coli (FERN P-106) in aerobic conditions at
24), there are few economic constraints. The present inventor has determined that part of the amino acid sequence of the enzyme protein is
For example, we succeeded in obtaining a DNA fragment that encodes the amino acid sequence of a novel slightly acidic microthermic cellulase deleted from the amino or carboxyl terminus.

As a result, it has become possible to carry out industrial mass production by incorporating it into a high copy plasmid such as a runaway plasmid. Ruminococcus alps used in the present invention is an obligate anaerobic bacterium belonging to the genus Ruminococcus, and has an optimal growth temperature of 37°C, a growth temperature of 25 to 50"C, an optimal growth pH of 6.8, a growth potential of 5 to 8, Durham staining, and Durham staining. Positive guanine + cytosine content is 42.6-45.8%.

The medium used for extracting DNA encoding the amino acid sequence of cellulase is not particularly limited. Ruminococcus・
Any medium that can grow Alps can be used.For example, glucose (7) can be used as a carbon source, and cellobiose can be used instead.
A medium containing ball milled cellulose, water hyacinth, rice straw, etc. can also be used. Use of glucose facilitates subsequent operations in that it does not contain insoluble substances.

When constructing and preparing a recombinant plasmid, the DNA cutting method is not particularly limited. It may be physically cut using a homogenizer or the like. However, it is preferred to use the same restriction enzymes for later ligation into the vector. Efficiency can be increased by treatment with alkaline phosphatase. The ligation reaction is usually carried out at 12°C to 16°C.
Complete in 50 minutes.

Competent cells are not limited to E. coli, but Bacillus subtilis, yeast, plant cells, etc. can be used. The method for preparing competent cells involves calcium chloride treatment. Furthermore, any conventionally known methods such as protoplastization and DNA transfection (gene introduction) methods can be used.

The method for detecting recombinant bacteria that produces cellulase is not particularly limited. For example - membrane force ruboxymethylcellulose (hereinafter referred to as
A method called the multilayer Congo red method (denoted as CMC) can be used. The culture time is preferably 10 to 14 hours, and the holding time after CMC overlay is not limited to 2 hours. The staining time may also be 5 to several tens of minutes.

Plasmid pURA I-di and other deletion mutant DNAs
The plasmid vector used for cloning the fragment is also not particularly limited. The insertion site may be any site that can express cellulase activity.

In the production of a novel slightly acidic microcold cellulase using pURA I-di, the composition of the medium is not particularly limited, and any medium can be used as long as competent cells can retain and grow ptlRA I-dl. . The pH of the medium is preferably 6 to 7, but even pH 5.5 to 8.0 is 1.
/3 amount of enzyme can be produced. The culture time is preferably 10 to 14 hours, but may vary depending on the growth rate.

The purification method is not particularly limited, and can be performed by combining conventionally known methods. For example, it can be purified by a combination of methods such as osmoti ink shock and ion exchange chromatography.

The physicochemical properties of the novel slightly acidic micropsychrogenic cellulase obtained by the present invention are as follows.

1. Action: Decomposes cellulose such as CMC, resulting in a decrease in viscosity.At the same time, a small but significant amount of reducing sugar is also produced.

2. Substrate specificity: Acts specifically on cellulose such as CMC.

3. Optimum pH: The effect on CMC is optimal around pH 5.5 to 6.0.

4. Stable pH range: When treated at 37°C for 10 minutes, p
CHC decomposition activity shows residual activity of 80% or more in H5-7.

5. Optimum temperature: around 20-30''C when CMC is used as a substrate at pH 6.8.

6. Thermal stability: When treated at 40° C. for 11 hours at pH 6.8, it shows residual activity of 80% or more against CMC.

7. Molecular weight: It has a molecular weight of 35.000±5.000 daltons when measured by SDS-polyacrylamide gel electrophoresis.

〔Effect of the invention〕

The truncated cellulase obtained by the present invention is simple and can be produced in large quantities, and has a lower p value than non-truncated cellulase.
It is constructed so that optimal activity can be expressed at high temperatures and low temperatures, and it also controls cellulose synthesis reactions. Therefore, the present invention can be widely used in industries that decompose and utilize cellulose and industries that aim to synthesize cellulose, such as forest tree breeding, and the economic effects brought about by it are extremely large.

The present invention will be explained in more detail below with reference to Examples.

〔Example〕

(1) Cellulase assay The enzymatic activity of cellulase in this example was determined by measuring the activity due to viscosity reduction when CMC was used as a substrate.

That is, 1.0% CMC (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.) solution 5 - and enzyme solution 1 jlt were mixed and held at 37°C for 5 minutes in a cone-plate rotational viscometer (E-type viscometer manufactured by Tokyo Keiki Co., Ltd.). The viscosity at that time is vI. Note that the blank uses the viscosity V when 1 d of water is used instead of 1 d of the enzyme solution.

1 unit - (1/V, -1/vs) The Somogyi-Nelson method was used to quantify the reducing sugar produced by Xl15.

(2) Preparation of DNA encoding the genetic information of cellulase
When preparing DNA from S. albus ATCC 27210), a known anaerobic synthetic medium shown below using glucose as the main carbon source was used.

Composition of anaerobic synthetic medium 1100a! Medium 7.5 ml 7.5 d O, 311 d O, 1 adt O, Ig 1.0 g 0.5 g 1.0 I11 Adult mineral I Mineral ■ FA 0.1 χ Resazurin solution Yeast extract glucose Na, CO3 2.5 χ Cysteine solution Mineral ■ Composition of JPO4 H, 0 min 500 3.0 g 97 d Mineral Acid n-valeric acid 1so-valeric acid 0L-2-methylbutyric acid 3.0 g in 500 mf 6.0 g 6.0 g 0.6 g 0.6 g 483.8 d 17 d in 31 d! d adl d d d IIi Of the above medium composition After adding all the components except NazcOs and cysteine solution, the pH was adjusted to 7.0 and N
a1CO, and cysteine solution were added. And C
After passing Ot gas through it for 30 minutes, it was autoclaved at 110° C. for 10 minutes and used.

The obtained bacterial cells were treated with 5aline-EDTA (0.5M
After washing with NaCl (1,0,1 MEDTA, pH 8,0), suspended in 5aline-EDTA for 0.5 d,
After adding 1 part of Norizozyme and stirring, the mixture was allowed to stand at 37°C for 10 minutes. Then add 10jd of Tris-5OS buffer (0,
1M NaCl, pH 9,0) was added and kept at 60°C for 5 minutes to lyse the bacteria. To this, 10°5I11 of Tris-5OS-phenol (redistilled phenol saturated with Tris-5os-81 buffer) was added, allowed to stand at 0°C for 20 minutes, centrifuged at 1.500×g for 10 minutes, and then lysed. I got the liquid. After adding 21 m of cold ethanol to this lysate, the filamentous DNA was wound up with a sterilized glass rod and dissolved in sterilized water. For 500 μg/d of this DNA, 1
Heat at 00°C for 15 minutes to remove deoxyribonuclease (DN).
to inactivate bonuclease (RNase) or bonuclease (RNase).
A was added to a final concentration of 50 ug/d, and the mixture was kept at 37°C for 30 minutes to degrade coexisting RNA.

Tris-5O5- to stop the ribonuclea A reaction
After the phenol treatment, the precipitate is vacuum dried and then dissolved in an appropriate amount of sterilized water to obtain pure NA.

Figure 1 shows the base sequence of this DNA.

In the figure, the underlined parts of -35 and -IO represent the RNA polymerase binding site and recognition site, respectively, SD is the ribosome binding site, ↓ is the transcription initiation site, and Mu is the signal sequence cleavage site, horizontally The →← represents a palindromic sequence, respectively, and the stop codon is indicated using three asterisks.

(3) Preparation of recombinant plasmid The DNA obtained in (2) above was incubated at 37°C with old ndl [[
Complete decomposition was carried out for 1 hour. DNA) fin dI[[
The complete decomposition product was subjected to 0.8% agarose gel electrophoresis, and a 4 to 10 kbp fragment was cut out, eluted in a dialysis tube, and then treated with phenol and precipitated with ethanol.

Separately, plasmid pBI1322 was also completely digested with old ndul. After treating this with alkaline phosphatase, phenol treatment, ether treatment, and ethanol precipitation were performed in the same manner. Ligation was performed using T41J gauze at a temperature of 16° C. for 1 hour. Complete ligation was confirmed by agarose gel electrophoresis. Excise the DNA fragment encoding the desired cellulase from plasmid pURA I and use it as pUc.
19, pURA I was constructed and the copy number was significantly increased.

(4) Preparation of deletion mutant DNA gene DNA with genetic information of cellulase with amino terminus deleted
The Deyley-silane method was used to construct the A fragment. That is, a 1.8 kbp fragment containing the cellulase gene was prepared by completely digesting pURA I with the restriction enzyme Accl. This was treated with SI nuclease to make the ends blunt,
It was recloned into the Sea I site of puc 119. This is Kpn I and old ncll! A 1.6 kbp fragment containing the cellulase gene was prepared. P this
Partially disassembled with vu U, 5vaa l of puc 119
Religated to the site. Among the colonies formed by transformation, those with activity are Pvu ll of 423b.
A -1656 bp old ncII 1.2 kdp fragment was linked in frame to the lac promoter. Let this be p[IRA-Pv.

To delete the amino terminus of the cellulase, pURA-
Pν is cut with sph I and Sal I in the multi-cloning site where there is no restriction enzyme site in the insert fragment,
A 3' overhanging end (Sρhl site) is formed on the promoter side, and a 5' overhanging end (Sal I site) is formed on the structural gene side. After decomposing this 5' protruding end with exonuclease ②, the single-stranded part was deleted with mung bean nuclease, the end was made completely blunt with Klenow enzyme, and then ligated with T4 ligase. JM 103 was transformed.

In addition, before transformation, the transformation efficiency was increased by acting with 5alI to cut the shortened and unreacted circular plasmid. As a result, approximately 10
0 strains were obtained, and 5 of them had shortened DNA fragments. As a result of DNA sequencing, depending on the degree of delay in the DNA fragment, the length of the DNA fragment is 15 to 60 minutes from the amine end.
D with genetic information for cellulase lacking amino acids
The NA fragment was linked to the lac promoter with matching reading frames. In addition, in order to prepare a DNA fragment encoding cellulase with an amino acid deleted from the carboxyl terminus, a DNA fragment was constructed in which an Ω fragment with stop codons at both ends was inserted into the Ban Hl site and the EcoRI site, and 4 to 4 to A cellulase with a deletion of 75 amino acids was prepared. There is no limit to the degree of delay.

FIG. 2 shows the structures of various truncated cellulases and their enzymatic activities. In the figure, CMCI represents the untruncated cellulase containing the signal sequence; CMCI-N
+16, C? ICI-N+25 and CMCI-N+60 are each 15.
CMCI-C+05, CMCI-C+7, representing a truncated cellulase with 24.59 amino acids deleted.
6 represents a truncated cellulase in which 4.75 amino acids were each deleted from the C-terminus of the cellulase.

(5) Preparation and transformation of competent cells The Escherichia coli HB 101 strain used as competent cells was LB (Lur
ia-Bertani) medium, pH 7.5, overnight. From this, ld was taken and inoculated into a new 100-LB medium. When the turbidity of the culture solution reached OD&6g+=0.4, the bacteria were collected and treated with calcium. Add the recombinant plasmid lO■ to competent cells and incubate in ice water for 3
It was held for 0 minutes. Thereafter, the temperature was maintained at 42° C. for 1 minute, and the mixture was further maintained in ice water for 2 minutes. LB medium III was added to this and cultured with shaking at 37°C for 1 hour.

(6) Method for detecting recombinant bacteria that produce cellulase (4)
), 100 μg7 of the transformed strain prepared in
Spread on LB agar medium containing xdl of ampicillin.37
The cells were cultured at ℃ for 12 hours. This agar medium was overlaid with 1% agar containing 1% CMC and maintained at 37°C for 2 hours.

After staining this agar medium with 1% Congo red for 5 minutes, IM
After washing with NaCl, bacteria forming a halo were selected. As a result, recombinant bacteria forming about 100 halos were obtained. This recombinant bacterium contains the amino terminal amino acid 1
The patient had a plasmid containing a DNA fragment containing genetic information to produce a cellulase with five deletions (CMCI-N+16). This plasmid was transformed into pURA I
-di.

(7) Novel cellulase production method using pURA I-dl Escherichia coli JM 10 using plasmid pURA I-di
3 was transformed. This transformed bacterium was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology as B. coli (JM 103/pU).
RA 1-1) (Ff!RM P-10624). This transformed bacterium (FERM P-10
624) in the following medium to pH 6,
5 and cultured.

Cellulase production medium Bactodrypton yeast extract 0g 5g Production uses a 1.51 medium, 21! 37℃ in a fermenter
, Volume of air 1 vv+++ (Volume of air
per volume ofa+ediuo+pe
The stirring was carried out for 7 hours under the conditions of rs+1nute) and stirring speed of 750rpm+a. 10,000 centrifuge the culture solution in a refrigerated centrifuge.
Xg.

After centrifugation for 10 minutes, the enzyme was extracted from the obtained bacterial cells using an osmotic pressure extraction method to obtain a crude enzyme solution. The yield of cellulase obtained at this time was 110 units.

(8) Method for purifying novel cellulase produced by recombinant bacteria The cellulase obtained in (7) was purified by the following method.

The crude enzyme solution extracted by the osmotic shock method was treated with DEAE-Bio Gel A (
Anion exchange chromatography (manufactured by Bioland) was performed. 10mM sodium phosphate buffer pH 6,
8, a concentration gradient of sodium chloride was applied for elution. A concentrator equipped with a YM2 membrane with a molecular weight of 1.000 daltons (manufactured by Amicon) that collects both components with cellulase activity.
It was concentrated to about 1-2 d.

This was subjected to gel filtration chromatography using 5ephacryl S-200HR (manufactured by Pharmacia Fine Chemicals) and eluted with 10 mM sodium phosphate buffer containing 0.5 M sodium chloride, pH 6.8. The fraction having cellulase activity was collected and diluted 10 times or more with 10-M sodium phosphate buffer pH 6.8, and then subjected to anion exchange chromatography using a Mono Q column (manufactured by Pharmacia). Elution was performed using a 10 mM sodium phosphate buffer pH 6.8 using a sodium chloride concentration gradient. The recovered cellulase is 5OS
- It was purified to a single protein by polyacrylamide gel electrophoresis.

Table 1 shows transformed bacteria E, coli (JM 103/pU
Shortened cellulase (CM
The degree of purification and yield in each purification process of CI-N + 25) are shown.

(Margins below this page) (9) Decomposition of cellulose oligomer by purified cellulase Using purified cellulase, pH 6 in sodium phosphate buffer
The cellooligomer was hydrolyzed with
0 kg/d, flow rate: 0.7 d/a+in).

This analysis pattern is shown in FIG. 5, in which (A) is the analysis pattern when hydrolysis was carried out for 20 minutes, and (B) is the analysis pattern when hydrolysis was carried out overnight.

According to this, this enzyme hardly decomposes cellobiose (G), but well decomposes cellotriose (G3), cellotetraose (G4), cellopentaose (G), and cellohexaose (G6), and produces products. Glucose (Gt), G, and G3 are produced in a ratio of 1:2:0.5 mole.Since it has become clear that G is produced during the decomposition process of G4, this purified cellulase contains G
, and a synthetic reaction to form G using G4 as a substrate was observed.

Oo) pH and temperature characteristics of purified cellulase (pH characteristics) Figure 3 shows the transformed bacteria JM 103/p in the present invention.
Figure 2 shows the influence of pH on the enzymatic activities of the truncated cellulase derived from URA I-di and the cellulase derived from the transformed strain JM 103/pURA I.

The shortened cellulase of the present invention is derived from transformed bacteria JM 103/
Compared to the cellulase from pURA I, it has maximum activity at significantly lower pH values.

(Temperature characteristics) Figure 4 shows the above JM 103/pURA in the present invention.
1-derived truncated cellulase and the above JM 103/p
This figure shows the relationship between enzyme activity and stability of URA I-derived cellulase with respect to temperature.

The shortened cellulase of the present invention is the above JM 103/pU
Compared to RAI-derived cellulase, it has lower stability at high temperatures, but the optimum temperature for enzyme activity is lower (and has a wider range).

[Brief explanation of drawings]

FIG. 1 shows the structure of a DNA fragment encoding the genetic information of cellulase. FIG. 2 shows various truncated cellulases lacking the amino or carboxyl terminus. Third
The figure shows a graph representing the pH characteristics of the enzyme activity of a purified truncated cellulase with 15 amino-terminal deletions. FIG. 4 shows a graph showing the temperature characteristics of the enzyme activity, etc. of this purified cellulase. FIG. 5 shows a high performance liquid chromatography analysis pattern showing the decomposition characteristics of the substrate cello-oligomer by purified cellulase.

Claims (1)

[Claims] 1. Ruminococcus albus
This is a PNA fragment obtained by mutating a portion of the DNA fragment shown in FIG. 1, which encodes the genetic information of neutral cellulase isolated from the DNA of A. usalbus. 2. A DNA fragment obtained by mutating a portion of a DNA fragment encoding the genetic information of neutral cellulase, which encodes a protein in which a part of the amino acid sequence is deleted from the amino terminus of the mature protein of neutral cellulase. The DNA fragment according to claim 1. 3. 15 from the amino terminus of the mature protein of neutral cellulase
Alternatively, the DNA fragment according to claim 2, which encodes a protein in which 24 amino acids have been deleted. 4. DNA encoding the amino acid sequence of neutral cellulase
2. The DNA fragment according to claim 1, wherein the partially mutated DNA fragment encodes a protein in which a part of the amino acid sequence is deleted from the carboxyl terminus of the mature protein of the neutral cellulase. 5. The DNA fragment according to claim 1, which encodes a slightly acidic low-temperature cellulase. 6. Plasmid DNA containing the DNA fragment according to claim 1
. 7. A transformant transformed with the plasmid DNA according to claim 2. 8. Ruminococcus albus
A protein obtained by translating a partially mutated DNA fragment that encodes the amino acid sequence of neutral cellulase isolated from the DNA of A. usalbus. 9. A slightly acidic microcold cellulase that exhibits maximum activity at pH 5-7 and 20-35°C. 10. It has a molecular weight of about 35,000 ± 5,000 daltons as measured by SDS-polyacrylamide gel electrophoresis, and exhibits maximum activity at pH 5-7 and 20-35°C,
A slightly acidic microcold cellulase characterized by having a residual activity of 80% or more when kept at 37°C for 1 hour. 11. A method for producing a slightly acidic cryogenic cellulase, which comprises culturing the transformant according to claim 7 and collecting the slightly acidic cryogenic cellulase from the resulting culture.