FI86438B - Plasmids which induce extracellular secretion of xylanase, methods for production thereof, and methods for production of xylanase - Google Patents

Description

1 86438

Plasmids inducing extracellular secretion of xylanase, methods for their preparation and methods for producing xylanase 5 Divided by FI patent application 840843.

This invention relates to novel plasmids, methods for making plasmids, and methods for culturing novel microorganisms carrying the plasmids to produce xylanase. The new plasmids contain DNA encoding the extracellular secretion of xylanase. Microorganisms are transformed with plasmids and cultured to produce xylanase extracellularly.

A plasmid is a non-chromosomal gene present in the cell of a microorganism that is circular DNA.

The plasmid is commonly used for recombination of the microorganism gene and is becoming increasingly important in the fermentation industry.

Recently, studies have been performed on plasmids carrying foreign DNA that has genetic information about metabolic products or is specifically required for the growth of the microorganism, as demonstrated in the production of amino acids and peptides. Some plasmids have been transferred to the host microorganism to obtain transformants. Methods have been devised for the preparation of relatively small molecule compounds such as amino acids and peptides by culturing transformants. However, the degree of proliferation of plasmids carrying the genes required for the production of macromolecular agents depends on the nature of the host microorganisms, and these plasmids have not been efficiently expressed. Moreover, no efficient methods have been provided for culturing such transformants.

35 It has not been possible to selectively obtain 2 86438 specific extracellular, high molecular weight products by culturing a microorganism transformed with a plasmid containing a foreign piece of DNA containing macromolecular substances which are metabolic products of other microorganisms. genetic information on extracellular production. Such extracellular production has not been successfully implemented, although a transformant of Escherichia species commonly used as the host microorganism has been used.

10 U.S. Patent No. 4,411,994 to W. Gilbert et al. Discloses a method of making specific proteins in bacteria and their secretion from a bacterial cell. In this method, DNA representing a desired non-bacterial protein or portion of a protein is recombinantly attached to a plasmid or phage gene to obtain either a periplasmic or extracellular protein, hereinafter referred to as a "carrier protein", by transforming the bacterial host with the recombinant gene and culturing. The method of this invention provides means for producing a selected protein using a gene to produce a carrier protein having a leader sequence formed at its amino terminus by hydrophobic amino acids.

The Escherichia coli cell wall, which has often been used to produce useful physiologically active substances, comprises three types of membranes: inner membrane, peptidoglycan and outer membrane. The space between the inner and outer membranes is called the periplasmic 30 space. The method of U.S. Patent No. 4,411,994 has succeeded in secreting products into the periplasmic space but not into the medium through the outer membrane, i.e. outside the bacterial cell. According to the present invention, by inserting into the plasmid DNA containing 3,864,338 genetic data for the extracellular production of macrophilic substances to obtain a hybrid plasmid, and by culturing the host transformed with the hybrid plasmid, it is possible to secrete and obtain useful physiologically active substances across the outer membrane. back from the medium. Thus, the present invention enables the mass production of useful, physiologically active substances, which has never been possible with previous methods.

Therefore, it is an object of the present invention to provide a recombinant plasmid characterized in that it is obtainable by: a) preparing with a first restriction enzyme, preferably Hind III, Bacillus sp. A chromosomal DNA fragment of strain C 125 (FERM BP-469) encoding extracellular secretion of xylanase; b) digesting the vector plasmid DNA, preferably pBR 322, with another restriction enzyme that does not interfere with the genetic information contained in the chromosomal DNA fragment; C) treating said chromosomal DNA fragment and said digested vector plasmid DNA with DNA ligase to form recombinant plasmid DNA; and d) isolating the recombinant plasmid DNA from the transformed host cell expressing the xylanase-producing phenotype.

These plasmids can be prepared by a method characterized by the following steps: a) prepared with the first restriction enzyme Bacillus sp. A chromosomal DNA fragment of strain C 125 (FERM BP-469) encoding extracellular isolation of xylanase; b) digesting the vector plasmid DNA with another restriction enzyme that does not interfere with the genetic information contained in the chromosomal DNA fragment 4 86438; c) treating said chromosomal DNA fragment and said digested vector plasmid DNA with DNA ligase to form recombinant plasmid DNA; and 5 d) isolating the recombinant plasmid DNA from a transformed host cell expressing a xylanase-producing phenotype.

The present invention also provides a method for producing an extracellular xylanase comprising culturing E. coli transformed with the recombinant plasmid described above. The method is characterized in that a) a medium containing a selected carbon source is inoculated together with an inorganic salt which is a sodium salt or a potassium salt with a transformant; and b) continuing culturing the transformant after the cell concentration has peaked and until the xylanase production and accumulation in the medium has peaked.

Figures 1 and 2 show bacterial growth and xylanase production by Eschrichia coli HB101 (pCX311), Figure 3 shows xylanase production by Bacillus sp. With C125, Figure 4 shows a restriction enzyme cleavage map of plasmid pCX311.

dry 5 shows the pH similarity of the xylanase produced by Eschrichia coli HB101 (pCX311) in Bacillus sp. With the pH activity of the enzyme produced by C125.

The present invention is explained in detail below.

(a) The construction of the plasmid as well as the novel plasmids of the invention is such that they are formed by inserting foreign DNA into a plasmid (extra-86438 chromosomal DNA or vector DNA), such as col E, which present in Escherichia cells.

Vector DNA that can be used in the present invention includes a vector isolated from natural sources or a vector from which a DNA fragment unnecessary for propagation has been removed, such as ColE1, pMB9, pBR322, pSCIOI, R6K, and lambda phage.

Foreign DNA fragments, or exogenots, that can be inserted into vector DNA are genes with information about high molecular weight substances such as enzyme proteins, e.g., xylanase, penicillinase, alkaline phosphatase, β-galactosidase, and the like. Exogenous or DNA fragments that can be used in the present invention include genes that have genetic information on the extracellular production and secretion of macromolecular substances and are obtained from a microorganism belonging to, e.g., the genus Bacillus.

The microorganism containing DNA having genetic information on extracellular xylanase production that can be used in the present invention is Bacillus sp. C125.

Bacillus sp. C125 isolated from a soil sample collected in Tsurugashima, Saitama, Japan, has the following characteristic microbiological properties. Examination and classification of this strain was performed according to "Aerobic Spore-forming Bacteria" (United States Department of Agriculture, November 1952, by N.R. Smith, R.E. Gordon & F.E. Clark) and "Bergey's Manual of Deter-30 minative Bacteriology (1957)".

6 86438

Characteristic microbiological properties: (a) Morphology (1) Rods, 0.5-0.7 μ x 3.0-4.0 μ (2) Spore formation oval, 0.6-0.8 μ x 1.0- 1.2 5 μ and the spore colony is swollen (3) Gram-positive.

(b) Growth in medium

Medium Growth pH7 pH 10 10 1) Broth growth good 2) Broth agar '· "3) Glucose-broth low growth" 4) Glucose-broth agar' 5 "agar 15 5) Tyrosine agar" "6) Medium I" "7) Glucose-nitrate growth" 8) Glucose-asparagine- none "ni-agar 20 9) Medium-I + 5% growth"

NaCl (c) Physiological properties 1) Nitrate reduction almost no 25 2) In favor of propionate - no use 3) VP test negative negative 4) Urease test neutral 5) Starch hydro-positive positive lysis 6) Casein hydrolysis M "7) Growth conditions growth below pH 11.0 and below 55 ° C 8) Growth in oxygen-free with little growth under 35 conditions (medium I) 7 86438

In summary of the above characteristics, it is clear that the microorganism belongs to the genus Bacillus because it is an aerobic, spore-forming bacterium. The microorganism Bacillus sp. C125 was concluded to be a new strain of alkalo-5 bacterial bacteria because it clearly differs from known bacteria in that it grows below pH 11.0 and below 55 ° C.

According to the present method, any of the conventional methods can be used to insert the foreign DNA into the vector DNA. For example, chromosomal DNA is digested with a suitable restriction enzyme or endonuclease to obtain a foreign DNA fragment, i.e., an exogenous, which is then mixed with vector DNA treated with a similar restriction enzyme, and the mixture is ligated with a suitable ligase. Thus, as described above, the novel plasmids of the present invention can be obtained by producing a DNA fragment encoding the extracellular production of xylanase and the like by a restriction enzyme by extracting plasmid-20 vector DNA with a non-interfering restriction enzyme. To the genetic information contained in the DNA fragment, by treating the DNA fragment and the extracted vector plasmid with DNA ligase to construct a recombinant plasmid, and isolating the recombinant plasmid by conventional methods (see Bolivar et al. Gene, 2, 95 (1977)).

The new plasmid pCX311 can be constructed from Bacillus sp. From a chromosomal DNA fragment of C125 and a plasmid from pBR322. Figure 4 shows a restriction enzyme map for plasmid pCX311. As shown in Figure 4, plasmid pCX311 is constructed from plasmid pBR322, in which Bacillus sp. Chromosomal DNA of C125 encoding extracellular xylanase production is inserted between the Hind® restriction site of plasmid pBR322. Thus, plasmid pCX311 is a cyclic DNA molecule of about 9.01 to 35 kb in size containing pBR322 DNA and Bacillus sp. C125 β 86438 DNA with a size of about 4.0 kb.

(b) Manufacture of micro-organisms

Recombinant plasmids constructed from chromosomal DNA fragments as well as vector DNA plasmids are introduced into the Escherichia coli host microorganism using conventional transformation techniques. The culture is continued until a stabilized genotype is obtained to obtain a transformant carrying both genotypes with the selected chromosomal DNA and vector DNA. For this purpose, the usual, so-called "shot gun" method.

A typical host microorganism that can be used in the present invention is Escherichia coli HB101, which is a hybrid strain of Escherichia coli K-12 and Escherichia coli B.

Transformant 11a Escherichia coli HB101 (pCX311), prepared by introducing plasmid pCX311 into Escherichia coli HB 101, has the same microbiological properties as host Escherichia coli HB101 except for penicillin-20 resistance and xylanase production. Molecular Cloning, A Laboratory Manual, p. 504 (1982), genotype: F “, hsd S20 (rB, mB), rec A13, ara-14, proA2, lacYl, galK2, rpsL20, (Sm '), xyl- 5 mtl-1, supE44X ". Escherichia coli HB101 (pCX311) is further characterized by the property of plasmid pCX311, i.e. the ability to produce extracellular xylanase.

The extracellular production of xylanase by Escherichia coli HB101 (pCX311), as shown in the examples described later, reaches more than 80% of the total production, including intracellular production, and remains unchanged after long cultures. In contrast, Bacillus sp. With C125, as shown in Figure 3, xylanase activity as well as extracellular production reaches a maximum slowly and then drops rapidly. Thus, 35 Bacillus sp. The extracellular production of C125 cannot be compared to the extracellular production of the disclosed Escherichia coli HB101 (pCX311).

The present invention, which provides Escherichia coli HB101 (pCX311), which has the ability to produce extracellular enzyme proteins such as xylanase, is not only a novel but also an ingenious invention. E. coli HB100 (pCX311) can produce and secrete large amounts of xylanase from cells into the medium, large amounts of other enzyme proteins that can also be assembled. More specifically, it has been found that Escherichia coli HB (pCX311) produces and secretes from cells into the medium, as shown in the examples later, large amounts of penicillinase, an alkaline phosphatase in addition to xylanase, previously observed only in the periplasmic state.

Such extracellular production by Escherichia HB101 (pCX311) indicates that the DNA carried by plasmid pCX311, which is approximately 4000 nucleotide base pairs, provides a host capable of producing metabolic products extracellularly.

(c) Culture of microorganisms

Any medium suitable for the production of specific substances encoded by the specific genetic information and suitable for culturing microorganisms of the genus Escherichia can be used for culturing the transformants prepared in step (b). In the method of the present invention, it is necessary to cultivate the microorganism in a medium containing an inorganic salt necessary for the growth of the microorganism or a selected carbon source together with the inorganic salt and to continue culturing the microorganism after the concentration of cells has reached a maximum and until the production and accumulation of high molecular weight substances in the medium reaches a maximum of 35.

10 86438

Examples of the inorganic salt that can be used in the present invention include sodium and potassium salts such as sodium chloride, sodium sulfate, potassium chloride, and the like, among which sodium chloride is preferable. The medium containing an inorganic salt or a selected carbon source together with an inorganic salt may contain a carbon source such as glucose, sucrose, lactose, maltose, glycerol, bran, xylene and the like, as ammonium water, ammonium salts and the like as a nitrogen source and the like. in the cultivation of a nutrient such as amino acids, vitamins, etc., it is important and necessary to add a carbon source suitable for the selected product to the culture medium for the cultivation of Escherichia coli HB101 (pCX311). The media that can be used in the present invention are those with LB broth (containing tryptone, yeast extract and NaCl) as the basic medium, BPB broth (Difco Laboratories) containing polypeptone, yeast extract and K2HPO4, nutrient broth (Difco 0001) , tryptone-NaCl broth or some other such basic medium.

Since more than 80% of the total products remain in the intracellular fraction in the medium containing no inorganic salt, it is necessary to use a medium containing the inorganic salt in order for most of the products to be secreted into the cellular medium. The amount of inorganic salt used is in the range of 0.5 to 3.0% by weight based on the medium.

Excellent results have been obtained in the culture of Escherichia coli HB101 (pCX311) using LB broth supplemented with bran and / or xylan as carbon sources, preferably in an amount of 0.5% by weight of each based on the medium.

Although culture conditions such as pH, temperature, oxygen conduction, etc. can be changed, for example, 86438

In order to obtain maximum growth of a microorganism of the genus Escherichia, it is necessary in the present invention to continue culturing the microorganism after the concentration of the microorganism cells has reached a maximum, i.e. after a later logarithmic step and until the production and accumulation of macromolecular substances in the medium reaches a maximum.

After inoculation of a microorganism of the genus Escherichia into the medium, the concentration of cells 10 reaches a maximum after 5-20 hours and the production and accumulation of high molecular substances reaches a maximum within 12-48 hours. Although the pH of the medium is not critical, it is preferred in the pH range of 5-8, especially at pH 7. Thus, without adding more inorganic salt, carbon sources, etc. necessary for the growth of the microorganism, during cultivation, the microorganism produces large amounts of extracellular, high molecular weight substances that can be conveniently collected.

Examples of microorganisms that can be used in this invention include (i) Bacillus sp C125 and (ii) Escherichia coli HB101 (pCX311) deposited in the collection of the Fermentation Research Institute, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry , Japan, the International 25 Depository Authority (hereinafter referred to as "FERM") under the following deposit numbers, FERM BP-469 and FERM BP-470, respectively, and are deposited with FERM, from which they are available without restriction.

The deposit and receipt numbers of the above-mentioned microorganisms are as follows:

Micro-organism_Deposit institution Deposit number (s) Bacillus sp. C125 FERM FERM BP-469 (ii) Escherichia coli FERM FERM BP-470 HB101 (pCX311) 35 The applicant will keep the deposits of FERM BP-469 and i2 86 438 FERM BP-470 unlimited until the end of the patent term, if this application is granted a patent, and thus said microorganism strains are available to the third group at any time, until the end of the grant period.

The following are examples of methods for constructing the plasmids of the present invention, methods for preparing transformant Escherichia coli HB101 (pCX311), and extracellular production of xylanase, etc. by means of transformants.

Comparative Example

Preparation and Purification of Plasmid pEAP2 1. Bacterial cell culture using Escherichia coli HB101 (pEAP2) (FERM BP-468) 15 Preculture in LB Overnight at 37 ° C

Culture in M-9 Chloramphenicol 130 is added

After the Klett unit for 16 hours at 37 ° C 20 /

Centrifugation 5000 rpm, 20 min.

4 ° C

Cell pellet Stored at -80 ° C 2. Plasmid DNA purification Cell pellet 110 ml, 20% sucrose, 50 mM Tris, ImM EDTA pH 8. Slurry, kept on ice.

30 50 ml polypropylene centrifuge tube 12 ml, 0.25 M EDTA. 1 ml, lysozyme (5 mg / ml) 0.025 M Tris, pH 8) 0.1 ml, RNase (10 mg / ml) Cell lysis. Gentle mixing. Let stand for 15-30 min. on ice.

35 5 ml, 3 x Triton. Gentle mixing. Let stand for 15-45 min. on the ice i3 80438

Centrifugation.17000 rpm, 40 min at 4 ° C

Supernatant solution in crumb cylinder 5 250 ml glass bottle 2/3 volume DD H 2 O 2/3 volume cold saturated phenol ψ Gently mix Centrifugation 10 ^ 6500 rpm, 15 min. 4 ° C

Upper verse j, Equal volume of phenol; chloroform Centrifugation ^ 6500 rpm. 15 min. At 4eC 15 Upper fraction in a 250 ml flask 1/25 vol. 5 M NaCl 2 volumes of EtOH at -20 ° C y Overnight at -20 ° C Centrifugation 20 ^ 6500 rpm, 60 min. -20 ° C DNA precipitate. Drying of excess liquid 5 ml A-50 buffer, resuspended in 1 ml sterile 80% glycerol, gentle v mixing 25 A-50 column (2x35 cm, 1 fraction = 4 ml) DNA fraction (A260 peak) 2 vol. ETOH at -20 ° C. Overnight at -20 ° C Centrifugation 30 J, 6500 rpm, 60 min. At -20 ° C DNA precipitate in 2.1 ml TEN buffer in a 5 ml cellulose nitrate tube: 2.2 g CsCl, mixing (in the dark) 35,150 μΐ Pdl (2 mg / ml). Good mix y 2 ml of mineral oil at the top of the tube i4 86 438

CsCl-gradient centrifugation. 36,000 rpm, 40 h at 20 ° C. Upper zone visible under US light; chromosomal truncated DNA Y Lower zone; covalently closed p-DNA 5 Assembly of the lower DNA band dropwise ir

Dowex 50W-X8 column. UV check ^ (light)

Dialysis against 2-4 liters of 10 mM Tris, 1 mM EDTA buffer. pH 8. Overnight at 4 ° C in a 30 ml keratin centrifuge tube, | vol. 1/25 spacious. 5 M NaCl. 2 tilav. EtOAc at -20 ° C. Overnight at -20 ° C

Centrifugation at 15 6500 rpm, 60 min. At -20 ° C, DNA precipitates in 1-2 ml of TEN buffer

Purified plasmid DNA (1 mg / l broth). Storage at -20-70 ° C Example 1 (1) Preparation of chromosomal DNA containing genetic information of xylanase production capacity

Alkalophilic Bacillus sp C125 (FERM BP-469) capable of producing extracellular xylanase was cultured with shaking at 30 ° C for 19 hours in broth containing 10.0 g of bran, 5.0 g of yeast extract, 5.0 g of polypeptide, 0.2 g of MgSO 4 · 7H 2 O and 10 g of Na 2 CO 3 per liter of water and the pH adjusted to 9.0). The cells were harvested at a later logarithmic stage, chromosomal DNA was extracted by phenol extraction, and purified to obtain 5 mg of chromosomal DNA.

(2) Insertion of a chromosomal DNA fragment into vector DNA.

Chromosomal DNA (10 Mg) obtained in step (35) was extracted with the restriction enzyme Hind III

is 86,438 at 37 ° C for 5, 10, 20, 30 and 60 minutes to obtain DNA fragments.

Plasmid pBR322 (Bethesda Research Laboratories, USA, resistant to tetracycline (Tetr) 5 and ampicillin (Ampr) used in the vector was digested with Hind III, then heated at 65 ° C for 5 minutes and then mixed with DNA fragments. The mixture was treated with T4 phage DNA ligase at 10 ° C for 24 hours and then heated at 65 ° C for 10 minutes.

Three volumes of ethanol were added to the mixture. Plasmids carrying chromosomal DNA fragments were mixed and assembled.

(3) Transformation of microorganisms with plasmids containing the algal gene that regulates the extracellular production of xylanase.

Escherichia coli HB101, a hybrid strain of Escherichia coli K-12 and Escherichia coli B, was inoculated into 10 ml of LB broth [containing 10 g of trypsin (Difco Laboratories, Detroit, Mich. ), 5 g of yeast extract, 1 g of glucose and 10 g of NaCl per liter of deionized water; The pH was adjusted to 7.0] and cultured at 37 ° C with shaking until a later logarithmic step. Cells were harvested and slurried in ice-cold 0.03 M CaCl 2 solution (final concentration) to obtain competent cells. The cell suspension and the plasmid solution obtained in step (2) were combined and kept on ice for 60 minutes. The mixture was heated to 42 ° C for 1-2 minutes to obtain plasmid DNA inside the cells. This cell suspension was inoculated into fresh LB broth and cultured at 37 ° C for 3-5 hours with shaking. Cells were harvested and washed to obtain transformants from which to isolate. . Escherichia coli HB101 (pCX311) (FERM BP-470), which has the ability to produce extracellular xylanase as well as penicillin 86438 linase.

Example 2

Preparation and purification of plasmid pCX311

The procedure of Comparative Example 5 was repeated except that Escherichia coli HB101 (pCX311) (FERM BP-470) was used in place of Escherichia coli HB101 (pEAP2) (FERM BP-468).

Purified plasmid pCX311 1 mg / l broth was obtained.

10 Example 3

Escherichia coli HB101 (pCX311) (FERM BP-470) obtained in step (3) of Example 1 was inoculated into 500 ml flasks containing 100 ml of LB broth (containing 10 g tryptone, 5 g yeast extract, 15 l g of glucose, 2 g of glycerol, 10 g of NaCl and 10 mg of penicillin per liter of water) containing 0.5% xylan and cultured at 37 ° C with shaking. Cell growth was measured by optical density at 660 nm. Enzyme activity was determined as follows: 0.05 ml of culture medium, 0.1 ml of xylan solution (Seikagaku Kogyo, Japan) and 0.1 ml of 0.2 M Tris-malate buffer pH 8.0 were mixed and heated to 40 ° C. for 10 minutes. One ml of DNA (3,5-dinitrosalicylic acid) was added to the mixture, and then the mixture was heated at 100 ° C for 5 minutes. Four milliliters of water were added to the mixture. The absorbance was measured at 510 nm. One unit of xylanase reduced one mg of xylose in one minute.

Figure 1 shows the extracellular production and secretion of xylanase by Escherichia coli HB101 (pCX311) cultured in LB broth containing 0.5% xylan.

As shown in Figure 1, cell growth of the transformant peaked after 9 to 12 hours of culture, and extracellular xylanase activity began after 17 8 6 4 3 8, and extracellular xylanase activity began to increase after 6 hours and reached a maximum (about 0.35 units). / ml) after 13 hours of culture.

The extracellular xylanase produced was very sta-5 bile and, as shown in Figure 1, the yield remained high even after 48 hours of culture and reached more than 80% of the total enzyme yield. In contrast, intracellular xylanase production was found to be low in the initial phase (after 9 h of culture).

10 But the maximum was only 0.13 units / ml or about 10% of the total enzyme production and besides, the intracellular production gradually decreased. Figure 2 shows xylanase production by Escherichia coli HB101 (pCX311) cultured in LB broth containing 0.5% bran in place of xylan-15. Figure 2 shows a similar trend to that shown in Figure 1. For comparison, Bacillus sp C125 (FERM BP-469), a DNA donor, was cultured and xylanase activity was determined.

Bacillus sp. C125 was inoculated into 500 ml flasks containing 100 ml of medium containing 10.0 g of xylan. 5.0 g of yeast extract, 5.0 g of polypeptide, 1.0 g of K 2 HPO 4, 0.2 g of MgSO 4 * 7H 2 O and 10 g Na 2 CO 3 in one liter of water and the pH was adjusted to 6.0), and cultured at 37 ° C with shaking. The extracellular xylanase activity of the medium was checked every eight hours. It started to rise after eight hours of culture and reached a maximum (about 0.5 units / ml) after 48 hours, but decreased rapidly (Fig. 3).

(Xylanase identification) Ammonium sulfate was added to the culture medium of the transformant obtained in Example 2 step (3). The precipitate formed was dissolved in water, and the solution was dialyzed overnight against running water. The dialysate was adsorbed onto a CM cellulose column equilibrated with 20 mM 35 phosphoric acid monosodium phosphate buffer, pH 4.5.

ie 8 ό 4 3 8 using a linear gradient. The xylanase was eluted with 0.4 M sodium chloride. Fractions with xylanase activity were pooled and subjected to Sephade® G-100 gel filtration to obtain purified xylanase-5.

In a similar manner, the culture medium of Bacillus sp C125 (FERM BP-469) was obtained to obtain purified xylanase.

To determine iden-10 of Escherichia coli HB101 (pCX311) xylanase with Bacillus sp C125 xylanase, the effect of pH on activity was examined, ultracentrifugal analysis, electrophoresis, and the molecular weight of each xylanase were determined. As a result, both enzymes were found to be identical to each other, as shown below.

(a) Acetate (pH 4-5), Tris malate (pH

5-8), Tris-HCl (pH 7-9) and glycine-NaOH (pH 9-11). These buffer solutions were used to study the effect of pH on activity. The results are shown in Figure 5, which shows the similarity of both enzymes in terms of pH activity.

(b) Ultracentrifugal analysis of both enzymes showed that both enzymes had one peak with a sedimentation constant of 3.5S.

(C) Disk electrophoresis at pH 8.3 showed that both enzymes had one band. Electrofocusing with ampholine revealed one peak. The isoelectric point of both enzymes was pH 6.3.

(d) Determination of the molecular weight of the enzymes was performed by the SDS-30 polyacrylamide method. The molecular weight of both enzymes was about 40,000.

Example 4

Escherichia coli HB101, Escherichia coli HB101 (pBR322) and Escherichia coli HB101 pCX311 (FERM BP-35470) were cultured in the same xylan-containing LB broth used in Example 3 at 37 ° C with shaking for about 20 hours. (penicillinase) or 15 hours (alkaline phosphatase and β-galactosidase). The enzyme activity of alkaline phosphatase and β-galactosidase was measured at 420 nm optical density. The results are shown in Table 1.

Table 1 ^ Microorganism Products _ Activity (U / ml) ___.

Cell- Cell- Total ____external internal E. coli (2 *) 1.31 (98%) KB101 i cis-galactose-0.03 (2%) 1 20 (98%) dase (100 ^)

Alkaline n 4 a E.coli phosphatase · °, 01 <2 *) ° / 47 <«%) HB101 (pBR322) 0 80 20/8-galactos-°, 00 (0¾) 0.80 (100%) dase ( ίου *;

Penicillin 9.72 return 0.20 (3%) 9.5 2 (97%) (100%) * '·, 0.50

Alkaline 0.30 (60%) 0 ^ 20 (40%). E. coli phosphatase (100%) HB101 (pCX311), 8.16

Penicillin 6 ^ 25 (77%) 1 * 91 (23%) (100%) returned 20,86438

Example 5

The effect of inorganic salts in the medium on xylanase production in Escherichia coli HB101 (pCX311) (FERM BP470) was studied. As the basic medium 5, the same medium as used in Example 3 was used. The microorganism was cultured for 14 or 20 hours in the basic medium to which various inorganic salts had been added. The results are shown in Table 2.

Table 2

Time (h) Inorganic- Inorganic- Xylanase activity (U / fal_ ninen nisen suo- ςπι, 1η <;, salt lan con- Soiunul_ Soiunsi- Total suoxa age κοη koinen saunen concentration _ (M) __ '15 14 None o 0 0

NaCl 0.08 0.07 0.11 0.18 "0.161 0.21 0.10 0.31" 0.32 0.38 0.10 0.48 20 0.48 0.15 0.10 0.25 KCl .16 0.25 0.12 0.37 • 20 None - 0 0 0 25 NaCl 0.03 0.06 0.07 0.13 "0.16 0.28 0.04 0.32" 0.32 0.40 0.13 0.53 0.48 0.25 0.10 0.35 KCl 0.16 0., 28 0.02 0.30 0.16 M [IaCl corresponds to 1% by weight.

Claims (12)

21 86 438 A recombinant plasmid capable of inducing the extracellular secretion of xylanase in Escherichia coli transformed with said plasroid, characterized in that it is obtainable by: a) preparation with a first restriction enzyme, preferably Hind III, of Bacillus sp. A chromosomal DNA fragment of strain C 125 (FERM BP-469) encoding extracellular secretion of xylanase; b) digesting the vector plasmid DNA, preferably pBR 322, with another restriction enzyme that does not interfere with the genetic information contained in the chromosomal DNA fragment; C) treating said chromosomal DNA fragment and said digested vector plasmid DNA with DNA ligase to form recombinant plasmid DNA; and d) isolating the recombinant plasmid DNA from the transformed host cell expressing the xylanase-producing phenotype. Recombinant plasmid according to Claim 1, characterized in that the vector plasmid is plasmid pBR 322. Recombinant plasmid according to Claim 1, characterized in that it is plasmid PCX 311 (FERM BP-470). A method for constructing a recombinant plasmid according to claim 1, characterized in that it comprises the steps of: a) preparing with the first restriction enzyme Bacillus sp. A chromosomal DNA fragment of strain C 125 (FERM BP-469) encoding extracellular secretion of xylanase; B) digesting the vector plasmid DNA with another restriction enzyme that does not interfere with the genetic information contained in the chromosomal DNA fragment; c) treating said chromosomal DNA fragment and said digested vector plasmid DNA with DNA ligase to form recombinant plasmid DNA; and d) isolating the recombinant plasmid DNA from the transformed host cell expressing the xylanase-producing phenotype. The method of claim 4, characterized in that the first and second restriction enzymes are the restriction enzyme Hind III. Method according to claim 4, characterized in that the vector plasmid is pBR 322. Method according to claim 4, characterized in that the recombinant plasmid is plasmid pCX 311 (FERM BP-470). A method for producing an extracellular xylanase, which comprises culturing a transformant obtained by transforming an Escherichia coli host microorganism with a recombinant plasmid according to any one of claims 1 to 3, characterized in that a) a medium containing a selected carbon source is inoculated together with an inorganic salt which is a sodium salt or a potassium salt of a transformant; and b) continuing culturing the transformant after the cell concentration has peaked and until xylanase production and accumulation in the medium has peaked. Process according to Claim 8, characterized in that the inorganic salt is sodium chloride or potassium chloride. Process according to Claim 8 or 9, characterized in that the inorganic salt is used in an amount of 0.5 to 3.0% by weight of the medium. Method according to one of Claims 8 to 10, characterized in that the transformant is cultured for 12 to 48 hours. Process according to one of Claims 8 to 11, characterized in that the carbon source is bran or xylan. 23 86438