JPS59198978A - Recombinant plasmid containing xylanase gene and a microorganism in escherichia transformed therewith - Google Patents

Abstract

PURPOSE:A recombinant plasmid having xylanase gene is introduced into a microorganism in Escherichia to permit the easy mass-production of xylanase by means of a microorganism in Escherichia. CONSTITUTION:The DNA of xylanase gene originating from Bacillus pumilus is introduced into the vector plasmid pBR322 in Escherichia coli to prepare the recombinant plasmid containing xylanase gene DNA. The above xylanase gene includes xylanase gene and beta-xylositase gene. The resultant recombinant plasmid is introduced into Escherichia coli C600 and the microorganism is cultured to produce xylanase.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a silkworm plasmid containing a xylan degrading enzyme gene, and an Escher transformed with the silkworm plasmid.
A method for producing a xylan-degrading enzyme using a strain of the genus Ichia and this bacterium is described.

Conventional technology Effective use of agricultural and forestry waste σ Savings and the taste of kidneys are also extremely cheap. This agricultural and forestry waste contains a large amount of hemicellulose as well as cellulose.

It is composed of hemicellulose, nyxylan, mannan, and galactan-based polysaccharides, but the content of silane is particularly high. If an enzyme system that decomposes xylan could be easily obtained in large quantities, it would be possible to produce xylan sweeteners, use it as a 1-part hydrate in fermented xylan and F-, or add it to animal feed. It is possible to improve the efficiency of digestion by decomposing the present silane, and to clarify the bitterness derived from xylan in fruit juices and alcoholic beverages. Ethanol can also be obtained by isomerizing the xylose obtained from Xylan Sword 1 to xylulose, and fermenting this with yeast. Microorganisms that produce this silane-degrading enzyme, which decomposes xylan into xylose, are known to include filamentous fungi such as Aspergillus and bacteria such as Bacillus liana, but these have not yet been industrialized.

OBJECTS OF THE INVENTION An object of the present invention is to provide a recombinant plasmid 2 having a xylan degrading enzyme gene and a bacterium of the genus Escherichia 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 Escher-iehia bacteria into which it has been introduced.

Summary of the Invention The silk recombinant plasmid of the present invention is a Bacillus
Xylan degrading enzyme system gene of Bacillus genus bacteria such as P. pumilus 1) Esct+erich N A
ia coli nohector plasmid pBR322, and this was transformed into Escheriehia c.
By introducing the xylan degrading enzyme system gene into Escherichia @ bacteria such as Oli C600, the above object is achieved. The chromosome 1]NA fragment contained in the recombinant plasmid has directionality with respect to vector plasmid pBR322. When the β-xylosidase gene on chromosome 1) is connected to the promoter side of the β-lactamase gene on p1 and p322 with respect to the xylanase gene, the transgenic plasmid can be transformed. The transmuted Cherichia coli can produce highly active β-supercilosinase. The same holds true for xylanase. In addition, the method for producing the xylan degrading enzyme of the present invention includes Esche
It includes cultivating a bacterium belonging to the genus Richia and collecting xylan degrading enzymes accumulated within the bacterium or in the medium,
As a result, the above purpose is achieved. The production of β single silosyl-ase and xylanase is from the parent strain Bacillus.
While it is inducible in S. pumilus, it is $-contractive in Escherichia coli.

1+:5cheriehia of the xylan degrading enzyme system gene of Bacillus pumilus in the present invention
Cloning into E. coli is performed as follows: 13acillus pumilus I PO (7)
5aito-Miura method (K
-Miura, Methods in Engym-
ology, vol, 12A (L, Grossman
and K+Moldaveeds, ) pp543.
A, academic, new Yrok
(1967)) chromosome L) NA is prepared. This was completely digested with the restriction enzyme Pstl, resulting in two i(Q
I) Bless the NA fragment. Pstl is ↓ TGCAG 'l' in GAC. )) and cut at the point indicated by the arrow, this l)NA fragment has the nucleotide sequence shown in FIG. 1 at its end. Tsukuda, vector plasmid p'B
I (322 is ampicillin (A
p) The resistance gene has a sequence that can be cut at only one place.

Cut the conovector plasmid p'BR322 into a straight line. Then, the terminal phosphoric acid is cut off with alkaline phosphrotase. This will result in the addition of Shubanzyme T'41, which will be added later.
) NA ligase pBl (322 is the original circular I
) Prevents return to NA. Next, since the end of the chromosome 1) NA has a sequence that can form an A/r or G/C pair, a hybrid I) NA of the chromosome 1) NA fragment and PBK322 is formed. Acquire this enzyme T4-77-
The target clue recombinant plasmid is completed by connecting the broken phosphate bonds with DNA ligase. this.

Next, foreign rice is treated with CaC4 in advance]) N
Escheri of Shuo Weisei and proboscis that made it easier to accept A.
Put it in ehiacol i C6QQ. Because A immediately enters chromosome 1) into Apr of the recombinant plasmid pRR322.

Escherichia coli C600@ampicillin resistance gene was isolated and ampicillin sensitivity (A
P S ).

Since the other tetracycline resistance gene ('I'cr) of pli K 322 is kept alive,
Escherichiacol i Cf1OO is tetracycline resistant (=I'cr).

13 Acillus pumilus I PO-derived β-earth silositase and xylanase genes were expressed in order to clarify the transgenic strain that was APS and Tc.
F of r, 5cberi-chia colt C6
00 colonies were selected, among which β-xylose
-Screen for strains having the xylanase gene.

Escherich with the β-supercilianase gene
iacot i C600 search K n , f f
, 1ni2 APS, "r(7)" Take 1 platinum q: cells from the colony and react them with p-nitrophenyl-β-D-superciloside, a substrate of β-xylosidase. The substrate is decomposed and p-
becomes nitrophenol. Since this strain exhibits a unique yellow color, a strain containing β-xylosidase can be obtained by visual observation. Escher with xylanase gene
For example, a radioimmunoassay method may be used to search for C600 of the order Ichiacales, but in the present invention, a strain having β-xylositase also had a xylanase gene.

The transformed strain Es thus obtained in the present invention
Cherichia coli has both a β-xylosiguse gene and a xylanase gene.

By removing the DNA plasmid again from this transformed Escherichia coli strain and cutting it with various restriction enzymes, the β-
The locations of the xylosidase blue gene and xylanase gene are estimated. And β-car silosiguse gene wo pl (
By placing the β-lactamase gene promoter on R322 under the control of the transformed strain E.

The β-xylosidase activity of E. coli C600 was determined from parent strain B.
The activity can be increased to 7 times that of ac-111us pumilus I PO. Similarly, by arranging the xylanase gene near the promoter of the β-lactamase gene, the xylanase activity can be directed.

Transformed strain 5 cherichia coli C
Both β-xylosidase and xylanase produced by 6QQ are immunologically similar to those produced by Miscellaneous.

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

Example 1 (Preparation of Bacillus pum-i plasmid) Bacillus pum-i used in the present invention
lus I PO (Accession number: FEIKEN BIKOYO, g 699
No. 4 (FERM P-6994)) + is xylan from Thai soil! Il as a high enzyme production strain! If identified. The identification data is shown in Table 1. This strain was derived from this identification data by E. Buchanan et al.
to and]? :+Gibbons.

Bergey's Manual of 1. )ete
rminative Bacteriology.

8th Edition, The Williarrl
S & Wilkins Company.

Bac by Ha It imore, l 974
It was identified as Illus pumilus. This beak strain has xylanase genes including xylanase h and β single silosinase. Xylanase (molecular weight approximately 21,000) is secreted extracellularly and hydrolyzes xylan to xylooligosaccharides. This xylooligosaccharide is hydrolyzed into xylose by the intracellular enzyme β-xylosylase (there are two varieties, but the main fermentation cord consists of two Zaku units with a molecular weight of 70,000). The present silanase Hi silose, β-xylosylase, does not act on xylan.

Table 1 ■ Preparation of chromosomal DNA: 100 m of LB medium (1
% Bactotributone, 0.5% Bactoist Extract, (
,0,54Na(J) to Bacillus pumil
One platinum loop of us I PO was planted on an agar slant and precultured overnight.

Next, the cells are planted at 20°C in one volume of the same medium, cultured for about 3 hours, and the obtained cells are collected by centrifugation.

This was added to pH 7.5 (D5mMEDTA/20mM
) Wash twice with Liss hydrochloric acid. The washed cells were resuspended in the same Tris-EDTA at 50°C, lysozyme (Sigma) was added to a final concentration of 1 rH/mi, and the cells were incubated 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 Nag POa) to this and mix slowly to form a homogeneous emulsion.

Centrifuge this at 10,000+/ 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 sequentially soaked in 70%, 80%, and 90% ethanol, washed, and then dried in a desiccator. Add this to 0.1XSSC (SSC: 150mM NaC)
And 15? nM sodium citrate). This was treated with RNase (manufactured by Sigma).

1) Treat with Naase free) 5071!1mi at 37°C for 30 minutes, and after decomposing Contamination 1 (NA), proceed with ethanol precipitate in the same manner as the above treatment.
Dissolve in SC and dialyze with 0.1xssc to remove chromosome 1) N
Obtain A specimen.

■ Chromosome IJ NA 1st record: Chromosome 1 obtained above) NA 100μ! (8) Is l, p) 5 times the restriction enzyme)'5t) (manufactured by Zenshuzo ■)? Silent buffer (20
mM) Lis-HCl, 10mM MgCno2, 50mM
(to I(+)2 SOa, 1001d/mj2
BovineSerum Albumin, pH
7,5) When kept at 37°C for 2 hours with 5μ of PstI solution, chromosomes due to PstI)
N A Fragment will be asked.

(2) Vector plasmid: The vector plasmid used in the present invention is p13R322. This vector plasmid has a small molecular weight of 2.7% and 106dal:
Ampicillin resistance (Apr) and tetracycline resistance (
1゛εr); restriction enzyme 1', coRl, Hindill, Ram
There is one cleavage site each for HI, Sal I, and Pst I; and resistance is lost when NA is inserted into the antibiotic-associated gene (7).
For example, when inserted at the PstJ breakpoint, the sex of Apr is 10
)(amp(I, Sal I, Hin
It has the advantage that it loses its Tcr properties when inserted at the dIII91 junction. Vector plasmid PH1
(322 is 13ethedαRe5earch La
It is commercially available from Boratories Inc. (Maryland, USA 20877), and in this example, it was extracted from F. coli C600 strain. In order to obtain the vector plasmid plasmid (322), we first used the J coli C600 strain (a known strain distributed all over the world, preserved at the Faculty of Science, Faculty of Engineering, and Institute of Scientific and Industrial Research, Osaka University). 11 B
Medium (yeast extract 51. Batato peptone 10g,
NaCff15 p, 50 pi/ln in water 11)
i (7) 77 h/37 in medium supplemented with IM
The cells were cultured at ℃ to 10" cells/1njl. To the culture solution, 200μ! 1 volume of chloramphenicol was added.
The cells were further cultured for 16 hours. Cleared lysate method (Y.

Kuperstock and Y, Hel 1nsk
i, 13iochem+Biophys.

Kes, Corrn-nun, vol. 54
, PP1451 (1973)) Nimari phase 7-7 Sumido fraction molecule (44, Hazaral and He1i
nski's method (M, tSazaral and D
-R, Hel 1nski, J, MOI, 13io
+,.

vow, 36. PP185 (1968)) K, J:
') CsCl x Titium Bromide Equilibrium Density Gradient Centrifugation 8# (Hitachi Separation Ultracentrifuge M5OP,
5T, 81,0OOXJ+, 4.0 hours.

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

1 to concentration (7) S SC (150+7+M NaCl
The purified plasmid I) was purified by overnight dialysis against 15 mM sodium citrate).

■ Vector plasmid P old (322 cleavage: above purified bacterium 5 sumi) '1) NA pBI (32212
0μ) (~l 71g 1)NA) at a concentration of 4
111 restriction enzyme PstI buffer 24μJ28 and Pstl
Incubate with 5μ of the solution for 2 hours at 37°C. After the reaction,
p) Dialyze against 25mM)-18,Q of IJS hydrochloric acid. Next, alkaline phosphritase (~1 unit) (Takara Shuzo Type 2) was added and reacted at 65°C for 30 minutes. After the reaction, phenol extraction was performed in the same manner as in the case of chromosome I) NA, and the aqueous layer was extracted with ethynyl ether (121 V/V).
So 3 draws? (-fL, phenol was removed. Ether remaining in the water layer was removed in a desiccator, and 0.1XS
It was dialyzed with SC and cut with PstI to form a planar line. PBR322 treated with alkaline phosphatase was obtained.

■ Preparation of silk recombinant plasmid: Pst obtained in the above ■
l cut 111' tetrachromic DNA 100μ7 (~5119
DNA) and pBR32240tt12 cut with the above 1'stI and treated with alkaline phosphatase.
(~5μffl DNA).

1M) Squirrel 5μ and 1.00rrM Mg C122Q
IM and 13mMAT P 15.4. lii and l
3 Q mM I) TT 15.41ift and 0. I
X SSC4: 2μ and T, I) NA ligase (
The recombinant plasmid was obtained by reacting it with 10 units (manufactured by Zenshuzo ■) at 16°C overnight.

■ Host microorganism: Escherich as a host microorganism
iacoli strain C600 (Accession number: Microtechnological Institute Bacteria No. 6
No. 995 (FERM P-6995)) was used. Its genotype urx, m)(, thr-111e
u-6,thi-1,5IIPE44゜Iac Yi,
ton A2 l.

■ Pretreatment of host strain: I, F cultured overnight in B medium,
, coli C600 culture solution was inoculated into the same medium twice.

Incubate for 2 hours. Cells were collected by centrifugation and diluted with 100mM
Centrifuged and cleaned with cold MgCl2, 1.00mM Ca
Suspend in Cl2. Then keep at 0°C for 20 minutes. Then, collect the bacterial cells by centrifugation and iQQrmn CaCl2 (7
) Resuspend in 15% glycerol solution and store frozen at -80°C.

■ Introduction of the recombinant plasmid into the host strain: 180μ of the recombinant plasmid was incubated at 0°C with the CaC1□-treated F, Col i C600a.
Hold time. Then, place at 42°C for 2 minutes. 1 for this
Add 0x LR medium 1 and let stand at 37°C for 30 minutes. Then, shake for 90 minutes at 37°C. This was treated with tetracycline at 15 μp/ml. Spread (200μi/plate) on LB agar plates containing 37°C late culture.

Get a colony.

■ E, col carrying the Itomeyanagi body DNA plasmid
Search for i-transformed strain: The F:, Coccinales C600 strain introduced with the transformant plasmid and transformed is Ap sensitive and Tc resistant. Therefore, the coo on the Tc plate above is
= -'(Tcr) to AP (50tip/ln-'
) and -rc (15 ppAm)), and those that do not grow on AP-1-C plates are selected as recombinant plasmid-carrying strains.

■ E. coli that has xylan degrading enzyme gene
Transformed strain: 1 in each well of microplate
my/m, i P-nitrophenyl-β-D-xyloside solution (100μ of 20mM phosphate buffer, pH 7.0)
), and add 1 of each of the above Aps-Tcr 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. This was cultured overnight in 100 ml of LB medium (containing Tc), and the resulting cells were disrupted by ultrasonication to obtain a cell extract. The results of quantifying β-xylosidase and xylanase in this extract.

Both enzyme activities were observed. The reason why the xylanase gene was also included in those screened for β-xylosinase was because the nine genes were close to each other.

■ Identification of the Sakaki plant plasmid pOXN29 containing β-xylosidase 2 and episilanase genes
The plasmid was prepared using the same method as for pBR322. Check this out
The APS1FCr strain introduced with IC exhibits ambidextrous β-gyrosinase activity and xylanase activity. PO this Tamirin body plasmid
Name it XN29. At 21%+, 2 g of pOXN was digested with various ^11 restriction enzymes and after acarose electrophoresis 1)
The NA was stained with ethidium bromide and the restriction enzyme cut point was pine pink. ]) The number of cut WI points for each restriction enzyme can be determined from the equation of IN A node (marked in white). You can understand the molecular weight of each hand position and force.

On the left, Mizuki is λ phage I) NA cut by t(indnl).

Mark with a marker of known molecular weight. As shown in Figure 2, there is only one cut point by +3am H1 and Kpm I, so the molecular weight of N A is 10.4M da l. be. I) st ■ produces fragments of 7.7 and 2.7 Mdal. PBK322
Since the molecular weight of is 2.7mda+, 7.7Mdal is an inserted chromosome fragment. The total was 10.4 Mda+, which corresponds well with the molecular weight of 29 for the excreta plasmid pOX. Therefore, pOXN29 is confirmed to be the recombinant plasmid of interest.

i g, Sal I, Bgl II and Av
aI has 2 locations, EcoR■ has 7 locations (minimum fragment 0.09
Mda+ is kenena(,1o), l' in the 21st gl.
There are 8 locations for vurl and Hindll+. Furthermore, the cleavage position of the restriction enzyme can be determined by combining and decomposing nine different enzymes and analyzing the fragments. FIG. 3 is a restriction enzyme cleavage map of the silk recombinant plasmid pOXN29. pOXN29, PBR322 (D
The Pst I site contains 7.7 mdal of chromosomal DNA. 13. , S and P indicate the positions of cleavage by Bgl, KpnI, Sal I and PstI, respectively. X indicates the position of cleavage by the restriction enzyme XbaI.

Production of enzymes) ■Culture conditions for E. coli transformed strain: 1 volume of medium prepared by adding 15 μp/mjl of tetracycline to LB medium was placed in a 3I sloping flask, and E. coli cultured in advance on slanted agar medium was added to this medium. Three platinum loops of the transgenic strain were planted and cultured overnight at 37°C with continuous shaking (120rP
m) Do. The host strain E, coli C3QQ, was also cultured under the same conditions. B. pumilus I
For PO, put 1 volume of a medium prepared by adding xylose in tLS medium into the same flask and inoculate it with 3 platinum loops of B and pumilus from the slant culture medium.
Culture at 0°C for about 40 hours.

Table 2 shows the β-xylosidase and suprasilanase activities of each strain. As shown in Table 2, the E. coli C600 strain that does not have 29 in the silk recombinant plasmid pOX does not have both the silicase activity and the xylanase activity. E holding P-OXN29,
Coccinoptera C600 (pOXN29) (Accession number: Microtechnical Research Institute No. 6996 (Ff!IM P-6996))
33 shows the β-xylositase activity of vI204 of pum-11us.

Regarding xylanase (1-; 1 B, pumi I
It is difficult to compare the xylanase activity of both methods because the POXN29-carrying strain of E. and E. coli accumulates inside the cell, but when comparing the xylanase activity of the two methods, E.

coli C600(pOXN29) is B, pu
It appears to have an activity about 6 times that of Milus I l'0.

Table 2 β-xylosinase Xylanase B, pumi Ius I PO16,787,1
(Note) E. coli C60000 ■ Production form of xylan-degrading enzyme Bj@ji (Part 1 Bactodorypton, 05th circulation Bacto yeast extract, 0.
54 NaC1) and 11 types of carbon sources such as glycerol, malate, xylose, and arabinose shown in Table 3.
Add Section 1 and culture for 16 hours.
B. Enzyme activity of pumi IPO xylanase (40 hours) was determined. As shown in Table 3, β-xylosylase and xylanase are lj
pumilus, it is produced inducibly, E, coliC
600 (pOXN29), it is produced constitutively. B
.

In E. pumilus (xylose, xylobiose, and xylan are introduced)
N29), all of them are structural without much difference. Therefore, no inducer is required during culture. This is a regulatory gene that is thought to exist in B. pumilus.

This is probably because it is not on the plasmid created here.

The advantage of genetically manipulating bacteria is that it can be desensitized by isolating the gene.

Table 3 0 0 0
2.3 0 4.8 Glycerol 0 0 2.9
0 5.8 malate 0
0 3.3 0
3.9 xylose 7,4
0 4.0 305 4.
0 arabinose 0 0
2.8 11 8.7 Glucose 0 0 2
．． 9 0 4.2 Fructose
0 0 2.7
1 4.3 xylobiose 1,4
0 2.7 52
6.8 Maltose OO3,405,
3 Cellobiose 0 0
2.7 0 7.4
After culturing for 6 hours, the pelleted cells were collected by continuous centrifugation (Sharpless) (260z wet cells), 1mME1) T
It was washed with 50mM J acid buffer containing A. This was suspended in the same buffer solution of 5QQm, A, and Dyro Mil
It was crushed with l ('I Maru Enterprises). This was centrifuged at 1.000 y:p for 20 minutes to prepare 400 ml of cell extract. Next, the cell extract precipitated at a saturation ammonium sulfate concentration of 0.3 to 0.5 and was collected, dissolved in a buffer solution, and dialyzed. Half of this (39 m) was applied to a Sephadex A-50 column (4 x 50 crn) equilibrated with the same buffer, and O
-I MNaCI 771 degree gradient. The active fraction was eluted around 0.3 M NaCl. Active fraction (
440 mja)) was concentrated and desalted using Amicon CUC450 (64 mja). The same D
Elute with EA'E-Sephadex A-50 from 0-0.
．． A 4M NaCl concentration gradient was used to collect active fractions. This was concentrated and desalted to 46 mn. This was applied to a CM-Sephadex C-50 column (4X 30 cm). Activity iE 1) Transferred to the non-adsorption section of TA-IJ acid buffer elution. This was reduced to 6.7 m using the same method. Sephadex G-200 column (2
, 5 x 100 c1n) and collected the active fraction.

At this stage, β-xylosidase was concentrated to a 2.7η peak and purified to a single protein. The β-xylosinase activity was measured as follows.

lmM Ej) 50mM at pH 7.0 containing TA
Concentration 1 (p- of 1 my/mA in potassium phosphate buffer)
Nitrophenyl-β-1)-xylopyranoside (Ko
chLight Laboratories Ltd.
, Coinbrook Bucks) and 1 m of the enzyme solution were reacted for 10 minutes at 37°C.

The reaction was stopped by the addition of Q, 4M Na2co321n.

The resulting P-nitrophenol was measured by absorbance at 4Q5 nm. 1 unit is 1 μmol per minute
This is the amount of enzyme that produces p-nitrophenol.

■ Purification of xylanase: B. pumilus 36
0.2~
Precipitate with 0.6 cm of ammonium sulfate. This precipitate fraction was collected by centrifugation, and 20% of 5QmM phosphate buffer, pH 6.5, was added.
Dissolve to 0ml. This is dialyzed against the same buffer. The resulting precipitate is removed by centrifugation. When the dialyzed enzyme solution (210yn) is applied to a 11EAF,-Sephadex C-50 column (3x50crn) equilibrated with the same buffer and eluted with the same buffer, the active fraction is eluted in the non-adsorbed section. Active fraction (260ys)) was equilibrated with the same buffer in CM
The activity was applied to a Sephadex C-50 column (3 x 50 Crn) and eluted with a gradient of O-0,3M NaCl in 7 acid buffer, eluting with 0.22 M NaCl. This was concentrated into a single protein. Its purity was confirmed by polyacrylamide electrophoresis, 51) S-polyacrylamide electrophoresis, and equilibrium density ultracentrifugation. Measurement of xylanase activity is performed as follows. 1rn
ll (7) 14hoshin (manufactured by Sigma) solution (p
H6,5 (5QmM phosphate buffer) and 0.5 mal enzyme solution are reacted at 40°C for 10 minutes, and reducing sugars forming a living film are quantified by the Somogyi method. The 1st car (unit) is
This is the amount of enzyme that produces reducing sugar equivalent to 17 zmol xylose per minute.

■ Beta-xylosinase gene bovine xylanase gene and promoter jr needle fi'l ii'r on plasmid POXN29: As shown in the 41st line I, the 4th wheel plasmid, pOXl\29 ; P.O.
XN 29 K (chromosome 1.3 N of pOXl\29
Between A and C (~); POXN29
1 (POXN29's Is g ! II @ (l narrowed at S position) excluding I\A): pOXN291K
(A reversed to 1) of pUXN291);
pL) xl'J 391 (Rg of PUXIN 29
Cut out the ln fragment (3,0-67Mdal) and convert it into p
lsj (thread 1 inserted into the tsam H] site on the -1-c gene of 322) 1. ．． 4-Sitep OX N3
91R (pUXN39] 1.) with the NA reversed) was prepared. An example of Tsuguhoho is one example.

POXN29111c+J manufactured by KU, if,
POXN29 Lami-bearing E, coli C600
(pOXN29) Kara MT6e hector plasmid p
The plasmid pOXN29f'g is generated by the same method as the hexagonal connection of Is K322. This is the same as the restriction enzyme PstlT.
Join with ligase and transform E, col+C600 again. This allows pR of chromosome 1) NA
I have a blasmid that has the opposite insertion direction to k322? , E. coli C600 (29k to pOX) strain will be obtained with a probability of 50%. Form @ transferred to Italy E,
Several strains of E. coli were collected and the plasmids were extracted again. This was cut with the restriction enzyme EcoRI (E c
100mM Tris-HCl as oRI buffer, 50
mM NaCl, 100 mM MgCl, pi-1
Using 7,5, all illusions are P of PBi'-322
As a result of measuring the total size of the NA fragment (same as stI cleavage) and agarose gel electrophoresis, a double size pattern was observed. The insertion direction was determined by this, and the plasmid in which the chromosome 1') NA of POXN29 was inserted in the opposite direction was designated as pOXN29.

The enzymatic activities of E. coli containing each of the above plasmids are also shown in FIG. This activity value display (ha.

The activity value per bacterial protein of the POXN29-carrying strain was 1.0.
Relative activity of the Gods plasmid carrying strain (if
It is f. The arrow in column 1 indicates the reading direction of mRNA.

POXN291 (to POXN291) β-xy 0
Cy-I-ase activity activation source (D pOXN29 top
OXN291), and the xylanase activity of POXN29 is up to 50 times that of the original POXN29. By inverting the chromosomal DNA fragment, the β-lactamase load of PBR322-4 is transferred to the progenitor promoter (p3゜pl) so that mKINA synthesis is
One syrositase gene and one doxylanase gene. It is estimated that

Beta-xyloshidase activity with 291 techniques held on pOX (
1゜i strain B, pu+I'1iius I PU It corresponds to 7 times.

P(JXN29R-T, ?? Schiff -j--seactivated pOXN29 (7) Although the increase is only 4 times at most, POXN29K') In this second, the kerchief is the left 4411 of β-epoxycytase. It's probably Katana-Ra, which is located far away from the promoter.

pOXN291 and 291.1'- to pOX have no moxilanase activity, and pOXN
391 and p(JXN391 K have this cylanase activity, and if this activity is present, it will be transferred to pOXN29s and pUX291(
From being higher than.

POXN 29's 3.0-6.7 M dal (7)
L3g! It is clear that there is a nixylanase gene (rE gene) on the It fragment.Furthermore, we analyzed the pUXN391 1(indlll VR fragment deletion plasmid).
:1-'yra3--ce gene IB PUXN29 top]
It is within 4.0 to 4.8Mdal. P
OXN391Id shows 3 times more xylanase activity than pOXN29.

h jr, E, co due to the miniaturization of Nifrasmid.
This is thought to be due to an increase in the number of copies of ' in li. In this way, by extracting the minimum required gene and placing it under the control of a strong promoter, the activity can be greatly increased.''Y
,・Thick vine. 7 as a strong promoter.

For example, the promoter of the lactose operon can also be used.

■ Immunological proof of plasmid-carrying strain E, coli β silosidase 2 and episilanase: 13, pu
The β single syrosigase and xylanase proenzymes of M. milus I PO were purified and injected into rabbits to prepare 8 volumes of each antiserum. For its preparation, first, purification 1! ＃Round 2 my 1" Complete
e Adjuvant t and 3 times a total of 3 mj & after mixing into an emulsion.

311X! Inject subcutaneously into rabbits every week.

At 4 weeks m1, whole blood was collected and incubated at 37°C for 1 hour. 'Come and give this 10. Centrifuge for 10 minutes. The supernatant was used as a crude supernatant.

Then, the gel was diluted with 1QmM potassium phosphate at pH 7.0.
ii) -50mM NaC1f6 and 1-group agarose (Sigma d type FT) were prepared, and wells were provided therein as shown in Figure 5(a). Into these wells, from the bottom left, 50 μm of purified xylanase (XN), plasmid-carrying strains I, 4, and 50 μm each were added. col
iC600 (pOXN29), extracted with 1,000 ml of purified β-silicase (41')).

In the same manner, put 7r (Anti-XN) of xylanase from -H stage Seri to To1ζ, and coffin plate f* (A old 1-XI) of β~honshishishi-se. When left overnight at 4°C, the Anci-XN, XN and 1':, coli extracts precipitate, and glands are formed and the Anci-XN and Ecoli extracts settle.
The precipitate M, % fi W with the extract is fused. An
Also for t 1-XJ).

E. coli extract and XIJ combine to create sedimentation.

fi:, between coli extract and Anti-X N and E, between coli extract and Anti-XI')! Sections 11 and 11 intersect near the center of Figure 1. LJ-
1-'s l′ yi force): , col i C600(
It is concluded that the pOXN29) strain produces the same enzymes .beta.-1 silochase and supercilanase as Kojidoko R, pumuchusl)'0.

Figure 5(b) ld, shape! Disintegrated convertible strain ic, col
i C600 (pOXN29R) (accession number: FERM P-6997) and F
,, coli C600 (pOXN291K) β-
The results of immunoassay of xylosinase are shown. to pOX29
These E, co containing R and pOXN291R
It was found that the E-coli cells produced the same β-xylosidase as E-coli carrying pOXN29 and pOXN 29 ]. Although not shown here, we conducted a similar experiment with xylanase, and found that E.
co1ic600 (POXN29K) strain is F,,co
It was confirmed that the same xylanase as liC600(pOXN29) was produced.

In addition, th Yoi conversion strain E, coli C600 (pOX
N29) Nokkuru β-xio'i 4--ase is B,
It was electrophoretically identical to that of P. pumilus I PO.

Effects of the invention Japanese recombinant plasmid pOXN2 granted by this defense
9b YoU' pOXN291 (,b F coli C600 (p(J
XN29) and E, coliC600(1)OX~
2gtt> contains dual-speed needles of xylanase and β-single syrositase necessary for the decomposition of xylan. Gene to be cloned into vector plasmid p+nt32
Enzyme activity can be increased by inserting 2-lactamase enzymes into the 1' promoter of the β-lactamase enzyme. Furthermore, by extracting only the IV and btiL genes and linking them to a strong promoter, it is possible to increase the copy number and increase productivity, thereby increasing the activities of both enzymes. It is also possible to further increase enzyme production by connecting potent promoters in series. Since the transformed strain constitutively produces both enzymes, culturing is extremely easy and inexpensive.

First (1) is a schematic diagram illustrating the preparation of the silk target plasmid of the present invention and the form f0 transfection carrying the same.
Figure 2 shows the mapping of restriction enzyme cleavage points by agarose pneumophoresis analysis of the silk recombinant plasmid POXN29 treated with control SI enzymes, and Figure 3 shows the mapping of restriction enzyme cleavage points of pOXN29. Figure 4 shows plasmid pOXN29,
POXN29R, pOXN291, pOXN29
1 R, pOXN3 g 1 and pOXI'N 39
1 K restriction enzyme cleavage field diagram, Figure 5 (a) is 凰col
A diagram showing the immunological identity of the xylan degrading enzyme produced by i C5Q Q (POX, N29) and the xylan degrading enzyme produced by parent strain B, pumilus I PO.
51st N1(b)i E, coli Cf'ioo
(pOXN29) b, 1: UE, coli
β-xylosidase and E produced by C3QQ (POXN291).

coli C600 (pOXN29JJ and E, co
β- produced by l i C600 (POX”291K)
FIG.

Figure 1 ↓ し]=i逗】Continuation of the first page of Kuo Shiko ■Int, C1, 3 identification symbol Internal reference number (C
12N 9/42 CI2 R1/185) 0 Inventor Atsuhiko Niina 7-1-61 Satsukidai Nakayama, Takarazuka City No. 1 484-

Claims (1)

[Claims] 1. A silk recombinant plasmid containing the present silane decomposition system gene DNA. 2. A to the xylan degrading enzyme gene IJ is Bac
llus pumilus IPO to white rice in the above-mentioned patent claim? Silk-like body plasmid. 3. Previous @i: BacilXus pumilus
The scope of the above claims is that the IPO rice silane degrading enzyme system gene DNA is linked to the vector plasmid pBR322! 51! Section 2 contains Kikugi's glossy body plasmid. 4. The above-mentioned silane component 4f @ details contains the present shiranase gene and the β-to silanase gene. body plasmid. 5 The front copy of the silanase package is the vector plasmid pR.
4. A Sakaki body plasmid as defined in claim 4, which is located on the promoter side of the β-lactamase gene on No. 322. 6. A recombinant plasmid in which the β-lactamase gene is located on the promoter side of the β-lactamase gene (7”) on the vector plasmid pBR322. 7. -1-silane decomposition enzyme ligation system gene 1) Escb-transformed with a recombinant plasmid containing NA
Microorganisms that rot in Erichia 11. 8. Previous, -it' xylan degrading enzyme system gene 1) N
A is Bacillus pumilus I I'O
The microorganism according to claim 6 [J-encircle 7] for preparing polished rice. 9. The silk recombinant plasmid is Bacillus sp.
umilus IPO Yumai white rice silanase system gene DNA is linked to the vector plasmid p131 (322). -xylosinase gene.11. The microorganism containing the xylanase gene is
The microorganism according to claim 10, which is located on the promoter side of the β-lactamase gene of RK322J. 12. The microorganism according to claim 10, wherein the β-xylosinase gene is located on the promoter side of the β-lactamase gene on vector plasmid pBR322. 13. The microorganism according to claim 7, which constitutively produces the xylan-degrading enzyme, is transformed with a silk recombinant plasmid containing the xylan-degrading enzyme gene DNA and produces the xylan-degrading enzyme. A method for producing a xylan-degrading enzyme, which comprises culturing a competent microorganism belonging to the genus Escheriehia in a medium, producing and accumulating the enzyme within the microorganism or in the medium, and collecting the enzyme from the microorganism or the culture solution.