JPS6075286A - Recombinant plasmid, bacteria belonging to bacillus genus transformed thereby, and production of xylanase using the same - Google Patents

Abstract

PURPOSE:To produce a large amount of xylan-decomposition enzyme, by culturing a bacterial strain belonging to Bacillus genus and obtained by the transformation with a recombinant plasmid prepared by bonding a xylanase gene DNA originated from Bacillus pumilus IPO to a vector plasmid pUB110. CONSTITUTION:A recombinant plasmid is prepared by introducing the xylanase gene DNA originated from Bacillus pumilus IPO to the vector plasmid pUB of Bacillus subtilis MI113. The recombinant plasmid is introduced into the cell of a bacterial strain belonging to Bacillus genus, e.g. Bacillus subtilis MI111, etc. to effect the manifestation of the xylan-decomposition enzyme gene, especially xylanase gene. Xylanase can be produced easily, in large amount, by culturing the strain of Bacillus genus.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention is a recombinant plasmid containing a xylanase gene in 1% of the xylan degrading enzyme system gene.

The present invention relates to a Bacillus bacterium transformed thereby and a method for producing a xylan degrading enzyme using this bacterium.

BACKGROUND OF THE INVENTION Effective use of agricultural and forestry waste is extremely important from the perspective of resource conservation. This agricultural and forestry waste contains a large amount of hemicellulose as well as cellulose.

Hemicellulose is composed of xylan, mannan, and galactan polysaccharides, and has a particularly high content of xylan. xylanta (If the enzyme system that decomposes it can be easily obtained in large quantities, this xylan can be used to produce sweeteners, to be used as a carbohydrate as a fermentation raw material, and to decompose xylan contained in animal feed and raise digestible young plants. Furthermore, it is possible to clarify the cloudiness derived from xylan in fruit juices and alcoholic beverages.The xylose obtained from xylan is isomerized to xylulose, and this can be fermented with yeast to obtain ethanol. Filamentous fungi such as Aspergillus and bacteria such as Bacillus are known as microorganisms that produce xylan-degrading enzymes that decompose xylan into xylose, but their activity is extremely low and they have not yet been industrialized.

OBJECTS OF THE INVENTION An object of the present invention is to provide a recombinant plasmid having a xylan-degrading enzyme gene and a Bacillus bacterium into which this recombinant plasmid has been introduced. An object of the present invention is to provide a method for easily producing a xylan degrading enzyme in large quantities using this recombinant plasmid or a Bacillus bacterium into which it has been introduced.

Summary of the Invention The recombinant plasmid of the present invention is a Bacillus plasmid.
The xylan-degrading enzyme system gene DNA of Bacillus pumilus IPO Yumai was transferred to Bacillus 5ubtilis Ml.
Bacillus 5ubtilis
By introducing the present invention into Bacillus @ bacteria such as Ml 111, the xylan degrading enzyme system gene, particularly the xylanase gene, is expressed, thereby achieving the above object. The method for producing a xylan-degrading enzyme of the present invention includes culturing a Bacillus bacterium containing the above-mentioned recombinant plasmid and collecting the xylan-degrading enzyme accumulated within the bacterium or in the medium, thereby achieving the above-mentioned purpose. is achieved. The production of xylanase is due to the parent strain Bac
It is inducible in B. lluspumilus, whereas it is constitutive in the transformed strain Bacillus 5ubtiformes.

Bacillus 5ubt of the xylan degrading enzyme system gene of Bacillus pumilus in the present invention
Cloning into B. ilis is carried out as follows: Bacillus pumilus l PO cells are cloned using the 5aito-Miura method (KoM
iura, Methods in Engym = o1
ogy, vol+12 A (L, Gros sm
an and K+Mol daveeds,) pp5
43. Academic Press, new Y
Rok (1967)) to prepare chromosomal DNA. In order to cut this with a restriction enzyme↑, this DNA fragment has a base sequence as shown in Figure 1 at the end. On the other hand, vector plasmid p
BN222 was converted to ampicillin (,
Ap) It has a sequence that can be cut at only one place on the resistance gene.

Cut this vector plasmid pBR322 into a linear form. Then, the terminal phosphoric acid is cut off with alkaline phosphatase. This allows the acquired enzymes T and DN to be added later.
A ligase prevents pBN222 from returning to its original circular DNA. Then 9 cars 1) The end of N A is A/T
Because it has a sequence that can form a G/C pair,
Hybrid DNA between chromosomal DNA wfr and pBR322 is formed. This is repaired by a repair enzyme) - the DNA of the phage
Recombinant plasmid pOXN29 is obtained by connecting the broken phosphate bonds with IJ gauze. This is 1
Next, a host microorganism that has been pre-treated with CaCJ to make it easier to accept foreign rice DNA0:) Escheri
Put it into ehia coli C3QQ. plasmid p
Because chromosomal DNA enters the A pr of BR322, the ampicillin resistance gene is separated in Escberichia coli C3QQ, making it susceptible to ampicillin (A p' ). Another tetracycline resistance gene (Tcr) in pBR322 is preserved.

Escberichia coli C6QQ is tetracycline resistant (Tcr).

Bacillus pumilus I PO free rice β
- To select transformed strains expressing xylosidase and xylanase genes, ApS and TCr E
Colonies of ich-erichia coli C600 are selected, and strains having β-xylosidase and xylanase genes are screened among them. A DNA plasmid was again extracted from this transformed Escherichia coli and confirmed to be plasmid pOXN29 expressing the xylanase gene and β-xylosidase gene. This plasmid p
OXN29 is partially digested with the restriction enzyme EcoR1 and then recombined to obtain Ap''-Tcr with a molecular weight of 8.2 and having xylanase activity and β-xylosidase activity.
Mdal deletion plasmid pOXN293 is obtained. This was partially digested again with the restriction enzyme EcolLI, and the same (Ec
Vector plasmid pUB110 completely digested with oRl
Connect with.

The recombinant plasmid thus obtained allows E. coli
Transform C600. Among these convertible stocks.

A plasmid carried by a strain having both xylanase activity and β-xylosidase activity is designated as pOXWl. This plasmid pOX~v1 is tetracycline resistant (T
c") and kanamycin resistance (Kmr), and possesses xylanase gene and β-xylosidase gene. Molecular weight is 11.2 Md@l.

9. This plasmid pOXW1. subtilim
The Kmr transformed strain is obtained by introducing it into protoplasts of the Mlll1 strain. Among these transformed strains, the plasmids possessed by two strains with particularly high xylanase activity are the target recombinant plasmids poxwtt and POXW.
It is 12. Plasmid pOXW 11 is a vector plasmid pLIBllQ and B of 1.7 Mdal, put
nilus DNA fragment. plasmid poxw
12 is the vector plasmid pUB110 and 1. Q5Md
B of al, pumilus DNA fragment, both contain xylanase gene and lack β-xylosidase gene.

Recombinant plasmids pOXWl and POXW1
B. 5ubtilis, which each carry 2, secretes 2 to 3 times as much xylanase into the medium as the parent strain B. pum-41ua IpO. The xylanase produced by both transformed strains is immunologically identical to that produced by the parent strain.

Example The present invention will be explained in detail below based on examples.

Example 1 (Preparation of recombinant plasmids pOXWl and pOXWl2 and determination of groove structure) Preparation of A0 recombinant plasmid POXN29 A-■ Parent strain: Parent strain Bacillus pumilus I PO (
Accession number: Microtechnical Research Institute No. 6994 (FE Ai M P-6
994>) was isolated and identified as a high-producing xylan-degrading enzyme strain from a soil crush in Thailand. The identification data is shown in Table 1. This strain is.

From this identification data, R, E, Bucbanan an
dN, E.

Gibbons, Bergey's Manual o
f Determinative Bacceriol
ogy, 3rd edition, Tbe
Wi 11 jams & Wi-rinkins Co
It was identified as Bacillus pumilus by B. Mpany, Baltimore, 1974. This strain secretes xylan content (21,000) containing xylanase and β-xylosidase into the extracellular space, which hydrolyzes xylan to xylooligosaccharides. This xylooligosaccharide is processed by the intracellular enzyme β-xylosidase (
There are two types, but the main enzyme (consisting of two SAG units with a molecular weight of 70,000) hydrolyzes it into xylose. Xylanase does not produce xylose, and β-xylosidase does not act on xylan.

Table 1 8-■ Preparation of chromosomal DNA: lQQmJ I, B medium (
Bacillus pu to
One platinum loop of milua IPO was planted on an agar slant and precultured overnight.

Next, 202μ of the cells were planted in one volume of the same medium, cultured for about 3 hours, and the resulting cells were collected using a centrifugal trowel.

Wash this twice with P) 17.5 of 5mM EDTA/20mM Tris-HCl. Wash cells with 50% of the same Tris-E.
Resuspend lysozyme 9 in DTA (Sigma M)
was added to a final concentration of l rng/mno, and heated at 30°C.
Hold for 5 minutes. Then sodium dodecyl sulfate (
Add 0.5% (final concentration) of SDS) and stir slowly for about 5 minutes.

Then, keep at 60°C for 10 minutes to lyse the bacteria. Add an equal volume of phenol (saturated with 0.05M Na, PO4) to this and mix slowly to form a homogeneous emulsion.

This is centrifuged at 10,000×g for 15 minutes.

Take the aqueous layer and add the same phenol again for extraction.

Add 2 times the volume of ethanol to the obtained aqueous layer, and scatter the resulting filamentous precipitate onto a glass rod. This is 701.80 section and 9
After soaking in 0% ethanol and washing, dry with a dencator. This is 0. lX5SC (S SC: 15
(ly+MNac] and 15mM sodium citrate). This is RNase (S i gm
Manufactured by company a, DINase freeJ 50 jig/I
After treating with n-' at 37°C for 30 minutes to decompose the contaminating RNA, the ethanol precipitate was collected in the same manner as in the above treatment. Finally, it is dissolved in 0.1XSSC and dialyzed with 0.1XSSC to obtain a chromosomal DNA specimen.

Preparation of 8-0 chromosome DNA fragment: Chromosome DNA obtained above
A IOQμA (8μg) was added to the restriction enzyme Pstl (
Takara Shuzo ■) 5-fold concentration buffer (20mM)
SO, +, 1100fi/mJl-BovineS
erum Albumin, pH7,5) 25 f
i! and 5 µm of psLl solution at 37°C for 2 hours to obtain PstL chromosomal DNA fragments.

A-■ Vector plasmid pBR322: This vector plasmid has a small molecular weight of 2.7810'dal; it has two selection markers: ampicillin resistance (Apr) and tetracycline rigidity (Tcr); it contains restriction enzymes EcoRI, Hind1. BamHl,
5a11. The number of cutting points by pstl is one each;
And when a foreign 1) NA is inserted into the above antibiotic rigidity gene, it loses resistance; for example, when it is inserted into the Pstl breakpoint, it loses the property of Ap, and BamHI,
It has the advantage that it loses its 'rer properties when inserted at the Sal 1 or Hind 1 break point. Vector plasmid 322 is Bethe-sdaRese
It is commercially available from Arcb Laboratories Inc. (Maryland, USA 20877), and in this example, it was extracted from E. coli C600 strain. To obtain vector plasmid pBR322,
First, the E. coli C600 strain (a well-known strain distributed all over the world, stored, for example, at Osaka University's Faculty of Science, Faculty of Engineering, and Institute of Scientific and Industrial Research) was placed in 1 volume of LB medium ( The cells were cultured at 37° C. to 10′ cells/m in a medium containing 5 g of yeast extract, Bactopeptone Log, 45 g of NaC, and 1)) 50 μV of ampicillin. 200 μg/ml chloramphenicol was added to the culture solution, and the culture was further cultured for 16 hours.

cleired lysa from the bacterial cells obtained by centrifugation.
te method (Y.

Kuperstock and Y, Hel 1nsk
i, Biophys, Res, Com-mun,,
vol, 54. PP1451 (1973)) to obtain a crude plasmid fraction, and Bazaral and 1 (el
Lnski's method (M, Bazaral and D,
RoHel 1nski, J., Mol, Bi.
ol,, vol.

36, PP185 (1968)) by (:, s (
: l-ethidium bromide equilibrium density gradient centrifugation (Hitachi separation ultracentrifuge 80p, rotor 65T, 8 people 000
Xg, 40 hours.

After removing ethidium bromide by extracting the plasmid fraction fractionated at 20°C with isoamyl alcohol.

Purified plasmid DNA was obtained by dialysis overnight against SSC (150 mM NaCl, 15 mM thorium citrate).

A-■ Cleavage of vector plasmid pBR322; the above purified vector plasmid DNA pBR322120
μj! (~17μg DNA) was added to a 5x concentration of restrictive P
React with st■ buffer 24β and 5μ of Pscl solution at 37°C for 2 hours. After reaction, p) 25m of Is, o
M) Dialyze against Lis-HCl. Next, alkaline phosphatase c~1 unit) (manufactured by Zenshuzo ■) was added.

React for 30 minutes at 65°C. After the reaction, the chromosome D
As in the case of NA, phenol extraction was performed, and the aqueous layer was extracted three times with ethynyl ether (1:IV/V).

Phenol was excluded. The ether remaining in the aqueous layer was removed in a desiccator and dialyzed with Q, lX5sc.
It was cut into a straight line. Alkaline phosphatase treated pBR322 was obtained.

A-■ Preparation of recombinant plasmid POXN29 2 100 m of Pstr-cleaved chromosomal DNA (~5
tLtDNA) and the above Pstl-cleaved and alkaline phosphatase-treated pBlt322404 (
~5M DNA), IM) Lis 5M and 100mM Mg
Cl, 204 and 13mM ATP 15.44 and 130
mM DT-r 15.4 ILl and 0.1 x 5SC
Recombinant plasmid pOXN29 was obtained by reacting with 4, 2 and T DNA ligase (manufactured by Zenshuzo ■) overnight at 16°C.

Introduction of the enzyme-degrading enzyme gene into host microorganisms E and coli B-■Host microorganism: Escher-i as a host microorganism
The chia coli strain C600 (accession number: FERM P-6995) was used. Its genotype is “JC-, rn, l tbr-1+
1eu-5+tht-l.

sup E 44. lac Yl, ton A21
It is.

B-■ Pretreatment of host strain: E cultured overnight in LB medium,
The same medium was inoculated with 2% culture of coli 0600, and
Incubate for hours. Cells were collected in a centrifuge, centrifuged and washed with 100mM cooled MgCl feces, +100 rrlM Ca
Suspend in Clt. Then keep at 0°C for 20 minutes. and.

Bacterial cells are collected by centrifugation, resuspended in a 15-cycle glycerol solution containing 100 mM CaCl, and stored frozen at -80°C.

B- ■ Host [Recombinant plasmid pOX to 'i4 strain
Introduction of N29: the recombinant plasmid POXN29
180 ya CaC1, the above treated E, Co11
Hold at 0°C for 1 hour together with C600 strain. Then 4
Leave at 2°C for 2 minutes. Add 10 times the volume of LB medium to this and let stand at 37°C for 30 minutes. Then, shake for 90 minutes at 37°C. Add this to tetracycline 15It
Spread on LB agar plate containing g/mat (200 lLt
/plate) Culture overnight at 37°C to obtain colonies.

B-0 recombinant 1) NA plasmid POXN 29
Search for F. coli transformants having the following: E. coli transformed with recombinant plasmid POXN29
, E. coli C600 strain is Ap sensitive and TC resistant. Therefore, the above-mentioned TC Taira Kamino Colony (TCr
) woAP (5eug, Muri and TC (15μg/rr
4), and those that do not grow on Ap-Tc plates remain as recombinant plasmid pOXN29-carrying strains (E).

coli C600 (POXN29) (Accession number: FERM P-6996)
).

B-■E#coli with xylan-degrading enzyme system gene
Search for transformed strains: one in each well of two microplates
Add 100μ of P-nitrophenyl-β-D-xyloside solution (20mM IJ awakening buffer, pH 7.0) at mg/m
Add 1 of each of the above APs-[Cr colonies to this.
Add a platinum looper, incubate at 30°C, and look for anything that turns yellow. This results in 1 having β-xylosidase activity.
I chose stocks. Add this to 100 m) of LB medium (containing Tc)
The cells that had been cultured overnight were disrupted by ultrasonic waves, and the cell extract was collected. As a result of quantifying β-xylosidase and xylanase in this extract, activity of both enzymes was observed. β−
The xylanase gene was also included in the xylosidase screen because the nine genes were close to each other.

B-■ Identification of recombinant plasmid POXN29 containing β-xylosidase and xylanase genes: The above-mentioned transformed strain is cultured in liquid, and a plasmid is prepared from the cells in the same manner as PBR322. Add this again to E coli
C600 strain IC introduced (!: AP' -rCr
All strains have both β-xylosidase and xylanase activities. This recombinant plasmid is
Name it OXN29. FIG. 2 shows the results obtained by digesting p0XN29 with various restriction enzymes and staining the DNA with ethidium bromide after agarose electrophoresis. DNA
The number of cutting points for each restriction enzyme can be determined from the number of bands (white spots). The molecular weight of each band can be determined from the position of the band. The left end is a marker with a known molecular weight, which is obtained by cutting the λF phage DNA with 1 (indl).As is clear from Figure 2, there is only one cut site with BamHI and Kpml, so it becomes a straight line. The molecular weight of DNA is 1
It is 0.4M d a l. 7.7 and 2 in Pstl
．． A fragment of 7 Mdal is generated. Since the molecular weight of pBR322 is 2.7 Mdal, 7.7 Mdal is the inserted chromosome fragment. The total IQ is 4Mdal, which agrees well with the molecular weight of the recombinant plasmid pOXM29. Therefore, POXN29 is confirmed to be the recombinant plasmid of interest.

In addition, Sal I, Bgl I and Aval are 2
EcoRl has 7 locations (minimum fragment 0.09Mdal
is not visible in Figure 2. ) Pvul and Hindf
There are 8 locations. Furthermore, the cleavage position of the restriction enzyme can be determined by degrading the protein using a combination of various enzymes and analyzing the fragments. FIG. 3 is a restriction enzyme cleavage map of the recombinant plasmid POXN29. pOXN
29 has 7.7Mdal at the Psc1 site of pBN222
Contains chromosomal DNA. B, K, S J and P are respectively Bgl I, Kpn 1, Sal
The positions of cleavage by I and Pmtl are shown, and x shows the position of cleavage by restriction enzyme Xbal.

XN and XD represent xylanase and β-xylosidase, respectively.

Chi recombinant plasmids pOXWll and pOXW1
Preparation of 2 C-1 Preparation of plasmid DNA pOXN293 The recombinant plasmid pOXN29 described above has 7 EcoR1 sites as shown in FIG. This POXN
29 is the Pst1 site in the Ap resistance gene PBR322
(7) Because of the insertion, it is Ap sensitive and Tc resistant. This POXN29 is partially digested with restriction enzyme EcoRI.

It contains plasmid POXN2996ttJl (3
ttg DNA ) wo.

Restriction enzyme EcoRI 9 units and H-buffer (H-buffer: 10QmM NaCl, 5QmM Tris-HCl, pH 7.5, 10mM MgCl, and l
mM di-chistreicol (DTT))
The mixture is reacted at 37° C. for 30 minutes to cause partial decomposition.

Next, the temperature of the 9 reaction system was raised to 60° C. and maintained for 10 minutes to stop the reaction. Obtained partially degraded plasmid pOXN2g
ilo μt (2,75 pg DNA), I
M l-IJ x 2μ of hydrochloric acid, 100mM MgG
1.4 pno, l 3mM ATPl 1.541L1
．． 130mM DTT 11.54m, water 10.92/
Recombination was achieved by reacting overnight at 16C with 1 g and 5 units of T4 ligase. Obtained p OXN 29
The partial decomposition and recombination reaction solution was transformed into E. coli c600, which had been previously treated with CaCls, in the same manner as in B-■ above to obtain Tc-resistant colonies. Among the 40 Tc-resistant strains, those with both β-xylosidase and xylanase activities were examined for plasmid size using a simple method, and those smaller than pOXN29 were found to be smaller than pOXN29.
I chose XN293. pOXN293 C, as shown in Figure 4, EcoR1 site E, ~E of POXN29,
, and E, ~E6 is deleted, and E! ~E, is entered in the opposite direction.

C-■ Preparation of vector plasmid PUBIIO Niblasmid pLJBllQ is 5taphylococcui
It is a kanamycin-resistant plasmid of P. aureus (Gryczan).

T, JlContente, S, , Dubna
u+ 1), : J, Bacteriol.

134, 318 (1978)). Strain B that holds this
, 5ubtilis.

For details on replication in Ml 113 (PUBIIO), see J a
1 anko, A.

Pa1va, 1. ,58derl@nd,H,:
Gene, IC325 (1981). This B, 5ubtil is Ml 113 (P
UB11O) containing 10 mg/no kanamycin
No Pen-assay broth (Difco)
Culture at 30°C for 11 nights.

The cells collected in the centrifugation group were once diluted with TBS buffer (20PMM
Tris JM acid, P) (7,5,5mM ED
Wash with TA, 100mM NaCl) and rinse with
- lysozyme buffer (3Q mM Tris-HCl,
P) (8,0,50y+M EDTA, 50mM
NaC1゜25% 5ucrose, 0.5 mg
/m! suspended in lysozyme).

React for 30 minutes at 37°C. Next, add SDS buffer (20qb SDS 6mL 500mMEDT) to this.
A6 m).

12m no rEs, l-1,024-) 48m1
- was added, and the mixture was further reacted at 37°C for 30 minutes. Add 115 m of 5M Nac to this and soak in water for 20 hours. Centrifuge at 15.00 rpm for 30 minutes to remove cell lysate. To this, 50% polyethylene glycol #60001
Add 5F71 and leave in water for 3 to 5 hours. Then,
Centrifuge for 1θ min at 1°C, ooorpm, and io°C, and dissolve the resulting precipitate in 6 m TBS. Next, RNase (Sigma) was added at 501 g/m-1.
Add. This was subjected to CsC1 ultracentrifugation and plasmid p[J
Collect Bloo's band. This plasmid p[I
BIIO is Km resistant and has a molecular weight of 3.0 Mdal. and.

There is one EcoR1 site. Plasmid pLIBl
10 are commercially available, 9 e.g. Bethesda R
e5earch Laboratories Inc.
Available from IJSA, Maryland.

C-■ Preparation of recombinant plasmid pOXWl: above n
F2 FuraX Mit' pOXN293140. gJ (
9 μg) was reacted with 35 μg of a 5-fold concentration H-buffer containing restriction enzyme EcoR12 units at 37° C. for 20 minutes to cause partial decomposition. Obtained partially degraded pOXN2931
75 μg of DNA) and pUBl digested with EC0RI and then treated with alkaline phosphatase.
Mix lo 25μ, +2 (~4μg). This mixture is combined using a system similar to the recombination system for pOXN29 in C-① above. However, in this system, 10 units of T4 ligase were used. This was transformed into E. coli C600 in the same manner as in B-① above to obtain a transformed strain that was Km resistant and Tc resistant. The β-xylosidase and xylanase activities of the 20 strains were measured, and the size of the plasmid was also examined. Then, the recombinant plasmid pOXWl possessed by one strain was selected.

This plasmid pOXW1 is as shown in Section 4.

POXN293 (D EcoRI site Ex Ic p
L] B110 is included. This is pBR322
and p[JBllo, it exists in both E.coli and B.5ubtilis.

C-■ Recombinant plasmid pOXW1 B, 5ul)
Introduction to the market: host strain B, 5ubtilis M
I strain 111 (Accession number: 725y
(FEkLM P-ri 2nd 5th)) 100m P
Culture in enassay broth until mid-log phase. The obtained bacterial cells were collected by centrifugation, and then transferred to 10FF.
1 of SMMP solution (4x concentration Penassay b
Mix roth and double concentration SMM at a volume ratio of 1:1. SMM=0.5M 5unrise, 0
．． 02Mmaleate.

0.02M M, gCI, pH 6,5), add 20 mg of lysozyme, and shake slowly at 37°C for 2 hours.

The resulting protoplasts were collected by centrifugation at 26,008 g for 15 minutes, washed once with 10 m of SMMP, and then purified with SMMP.
Resuspend in 10ml. In this protoplast suspension,
POXWI (1-5 μg) in 50μ TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8,
Mix 0) and add 50μ of double concentration SMM. Add 40% polyethylene glycol #60003- dissolved in 2x SSM, and after 2 minutes add 10m of SMM.
Add P. Centrifuge this at 2.600Xg for 10 minutes.

The resulting protoplast precipitate is suspended in 2 m of SMMP and slowly shaken at 30°C for 1.5 hours.

This was mixed with the following DM- containing 100 μg/m nokanamycin.
3 Place on medium. The resulting colony is replicated onto an L-agar medium (LB medium with agar added) containing 20 μg/m of nocaseisin to obtain a +Kmr transformed strain.

Agar 4 g/l 00 mJ IM Sodium succinate 250m/5th Casamino acid 50m/10th yeast extract 25m/3.54 KHIPOs + 15% K-Hl P
Oa 50 m 20 Tiglucose 12.5 m IM MgCl, 10. , NoC-■ Recombinant plasmid pOXW11 and POX
Preparation of W12 and determination of its structure: in the same manner as in the preparation of the vector plasmid pLIBl 10 described above in B.

Recombinant plasmids pOXWll and POXW12 are prepared from the S. ublilis transformed strain. The obtained plasmid was treated with restriction enzymes Hindl, BglM,
Single and/or double digestions are carried out with l'vul and Eco R1r. The structures of these plasmids pOXW11 and POXW12 were determined by examining the sizes of the degraded fragments by agarose electrophoresis. This is shown in FIG. pOXWll and pOXW
12 both have the Pvu It fragment of pOXWl. B.

In general, such deletions are likely to occur in S. 5ubtilii. Both plasmids lack PBR322 and therefore are not replicated, ie, not retained, in E. coli. The molecular weight of pOXWll is 4.74 Mdal.

The molecular weight of pLIBllQ linked thereto is 3.0 Md.
After all, this plasmid p OXW 1
1 is pUBllo and 1.74 Mdal B, pumil
Consists of us DNA fragment. The molecular weight of pOXWl2(7) is 4.06Mdal.

Conoplasmid POxW12 ended up with PUB110 and 1.06Mdal of 13. pumilus DNA fragment.

Example 2 - (B, Production of xylanase by 5ubtilis transformed strain) ■Culture conditions of B. 5ubtilis Ml 111 transformed strain: Section 1 LB medium prepared by adding xylose was mixed with 3j! B, 5ubtilis ml, which was placed in a Yosaka flask and cultured on a slanted agar medium in advance.
1. Plant 3 loops of the transformed strain. This was heated to 30℃ for 20 minutes.
Culture with shaking (120 rpm) for hours. Parent stock B, pu
milus IPO and host strain, 5ubtLl
is Ml 111 is similarly cultured. The xylanase contained in the supernatant obtained by centrifuging the culture solution was defined as extracellular xylanase, and the xylanase in the cell extract obtained by ultrasonic disruption of the precipitated cells was defined as intracellular xylanase.

Table 2 shows the xylanase production of each strain.

B, 5ubtN transformed with the recombinant plasmid pOXW11 or POXW12 of the present invention
is Mill (poxwil) (Accession number: Microtechnology Research Institute λ5'6 (1"ERMP-7,:t/,)
) or B, 5ubtillii Mill (pOX
Wl2) (Accession number: 725'ri
No. (F' E RM l'-ri 2 dak)) both have a concentration of 1.57-2.
Produces 5 times more extracellular xylanase. This xylanase.

As described below, the parent strain B is immunologically identical to that of P. pumilus.

(untts /nff1) (units shoulder) B,
pumilus IPOo, s s.

13.5ubt City sM1111 0.09, OB,
5ubt city s Ml 111 (pOXWll) 2.1
6 0.01 ■ Production form of xylanase m: LB medium contains glycerol, malic acid, arabinose, xylose, crucose, and xylobiose shown in Table 3.

Maltose, cellobiose, and xylan carbon sources: 0
．． 1 was added, each culture was cultured for 40 hours, and the enzyme activity was measured. As shown in Table 3, xylanase is produced inducibly in B. pumilus, whereas xylanase is produced in the transformed strain B. subtilis Ml.
111 (POXWII) is produced constitutively.

B.

In P. pumilui, xylanase is most highly induced and produced by xylose, followed by xylan and then xylobiose. How to use the transformed strain is capable of producing xylanase without using a relatively expensive xylanase inducer such as xylose as a carbon source. Similar results are obtained for B. 5ubcilis (POXW12). The xylanase activities in the table are expressed as relative values, with the production of B. pumilus I PO in xylose medium taken as 100.

-0211 Glycerol 0203 Linchoic acid 0166 Arabinose 0 257 Xylose 100 154 Glucose 0200 Xylobiose 16 160 Maltose 0169 Cellobiose 0 166 ■ Purification of xylanase: B, pumilus 3
Centrifuge the culture solution for 6 hours and add 2 volumes of supernatant to 0.2~
Precipitate with 0.6 saturated ammonium sulfate. This precipitate fraction was collected by centrifugation and added to 5QmM phosphate buffer with a pH of 5.
Dissolve to 0m. This is dialyzed against the same buffer. The resulting precipitate is removed by centrifugation. DEAE-Sephadex 8-5 in which dialysis oxygen solution (210F71) was equilibrated with the same buffer.
0 column (3 x 50 ctn) and eluted with the same buffer, the active fraction will elute in the non-adsorbed section. Active fraction (2
60 m)) is applied to a CM-Sephadex C-50 column (3 x 50 crn) equilibrated with the same buffer and eluted with a concentration gradient of 0-0.3 M NaCl in phosphate buffer.

Activity eluted with 0.22 M NaCl. This was refined into a single protein. Its purity was confirmed by polyacrylamide electrophoresis, 5DS-polyacrylamide electrophoresis, and equilibrium density ultracentrifugation. Measurement of xylanase activity is performed as follows.

1 mJ of 1'l xylan (Sigma) solution (pJ
(6,5 50mM J acid buffer) and 0.5mM enzyme solution were reacted at 40℃ for 10 minutes to remove the reducing sugars that formed a living film.
Quantitate using the omgyi method. 1 unit is 1
This is the amount of enzyme that produces reducing sugar equivalent to 1 μmOl xylose per minute.

■ Immunological evidence of xylanase produced by plasmid-carrying strain B, 5ubtilis: B, pumilus l
The xylanase enzyme of PO is purified and injected into rabbits to prepare xylanase antiserum. For its preparation, first, about 2 mg of purified enzyme was added to Freund's
A total of 3 m of Complete Adjuvλnt was mixed in an equal volume to form an emulsion, and then subcutaneously injected into rabbits in three doses every week. Whole blood was collected orally for 4 weeks, incubated at 37°C for 1 hour, and then 10.0008 g
Centrifuge for 10 minutes. The supernatant was used as crude antiserum.

On the other hand, the gel was mixed with 10 mM potassium phosphate buffer at pH 7.Q, 150 mM NaCl and 1 thiagarose (
(type R manufactured by Sigma@), as shown in Fig. 5.

A total of 7 wells are provided. The above antiserum in the center well, B in well 1 in the upper center row, purified xylanase of pumilus l PO, B in well 2 in the upper right row, 5ubt
Iris (pOXW 11 ) culture supernatant, B, 5ubtil is (POXW 12
) culture supernatant, fL 5ubt in well 4 in the lower center row.
Ilis culture supernatant, lower left row (7) 'i/L/
5 NiB, 5ubcilis (Poxwll)
culture supernatant, and B, 5ub in well 6 in the upper left row.
tilii (pOxw12) culture supernatant was added to each
Add 0μ increments.

When this was left overnight at 4°C, as is clear from Fig. 5, the xylanase antiserum
The purified xylanase of us IPO and the culture supernatant of the transformed strain create a merging line of sedimentation. Host strain B, 5u
The culture supernatant of B. btilis was concentrated, and B. pumi
Even if the enzyme amount is approximately the same as the xylanase activity of lusIPO, no precipitation line is formed with the xylanase antiserum.

Above lf4 to 31M convertible stock B, 5ubc i l
i s (pOXWl 1) and B, 5ubtil
is (POXW12) are all parent stocks B.

It is concluded that P. pumilus IPO produces the enzyme xylanase immunologically identical to P. pumilus IPO.

Effects of the invention Recombinant plasmid POxW1 obtained by the present invention
1 Oyohi POXW12. Oyohikoreniyosu transformed B, 5ubtilis MI 111 (pOXWl
l) and B, 1ubtil is Mlllll(p
OXW 12 ) contains the xylanase gene required for the degradation of xylan. The vine produces xylanase twice as much as the parent strain.

This transformed strain constitutively produces xylanase and does not require xylose, a relatively expensive xylanase-inducing substance, as a carbon source.

The vine is extremely easy and inexpensive to culture.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram illustrating the preparation of recombinant plasmids pOXW11 and pOXW 12 of the present invention and transformants carrying them. FIG. 2 is a diagram showing agarose electrophoresis analysis of recombinant plasmid POXN29 treated with restriction enzymes. Mapping of cutting points, 2g3 diagram is PO
Restriction enzyme cleavage map of XN29, Figure 4 is
pOXN 29. POXN 293. poxw
1+pOXW 11 Oyobi P OXW 12 (7)
till 'JR enzyme amount 11 t (h figure. Figure 5 is B, 5ubtslis M 111 (pOX
Wll) and B. Xylanase produced by 5ubtilis Ml 111 (POXW12) and parent strain B, pumilus I
FIG. 3 shows the immunological identity of xylanase produced by PO. Agent: Hide Yamamoto, Patent Attorney Figure 2 Figure 3

Claims (1)

[Claims] 1. A recombinant plasmid in which the xylan degrading enzyme gene DNA derived from Bacillus puritilus IPO is ligated to the vector plasmid p[JBllo. 2. The recombinant plasmid according to claim 1, wherein the xylan degrading enzyme gene is a xylanase gene. 3. The xylan degrading enzyme gene has a molecular weight of 1.7 Md.
al or 1. The recombinant plasmid according to claim 1, which is Q5Mdal. 4. A microorganism belonging to the genus Bacillus transformed with a recombinant plasmid in which the xylan degrading enzyme gene DNA derived from Bacillus pumilus IPO was ligated to the vector plasmid pUB110. 5. The microorganism according to claim 4, wherein the xylan degrading enzyme gene is a xylanase gene. 6. The xylan degrading enzyme gene has a molecular weight of 1.7 Md.
The microorganism according to claim 4, which is al or 1,06 Mda summer. 7. B that has the ability to produce xylan degrading enzyme by being transformed with a recombinant plasmid in which Bacillus pumilus l PO free rice xylan degrading enzyme system gene DNA is linked to vector plasmid pLIBllQ
A method for producing a xylan-degrading enzyme, which comprises culturing a microorganism belonging to the genus Acillus in a medium, producing and accumulating the enzyme within the microorganism, the medium, or the ground, and collecting the enzyme from the microorganism or the culture solution. 8. The method according to claim 7, wherein the xylan degrading enzyme gene is a xylanase gene. 9. The xylan degrading enzyme gene has a molecular weight of 1.7 Md.
al or 1. 9. The method of claim 8, wherein Q5Mdal.