KR100221204B1 - Gene sequence of cmc-xylanase and expression thereof - Google Patents

Abstract

The present invention is useful for mass production of CMC-xylanase, a kind of fibrinolytic enzyme synthesized and secreted from Fibrobacter succinogenes strain, a fibrinolytic microorganism.

The present invention comprises the steps of separating chromosomal DNA from Fibrobacter succinogenes S85, anaerobic ruminant; Cloning the CMC-xylanase gene; Selecting an E. coli transformant carrying a CMC-xylanase gene and determining a DNA sequence; Comparing the enzyme activity of transformants with Fibrobacter succinogenes S85 and CMC-xylanase gene.

Description

Nucleotide Sequences of the CMC-ｘｙｌａｎａｓ Gene of フ ｉｂｒｏｂａｃｔｅｒ ｕｕｃｃｉｎｏｇｅｎｅｓ S85 and Expression of the Gene

The present invention relates to the nucleotide sequence of the CMC-xylanase gene of Fibrobacter succinogenes S85 and the expression of the gene. More specifically, using pUC19 vector from chromosomal DNA of Fibrobacter succinogenes S85 for mass production of CMC-xylanase, a kind of fibrinolytic enzyme synthesized and secreted from fibrolytic microorganism Fibrobacter succinogenes S85 using genetic engineering techniques. The present invention relates to a CMC-xylanase selected from the DNA library prepared thereafter, and an expression vector containing the gene and a transformed microorganism.

More than 68% of the natural carbohydrates of fiber exist in the world of about 700 billion tons, about 40 billion tons of new biosynthesis each year. Fibrin is a substance in which glucose is continuously connected by β-1 and 4 bonds. As a result, studies are being actively conducted to use as an alternative energy and food resource by efficiently using such fiber.

Recently, research on converting these fibers into glucose or other substances by enzymatic hydrolysis has been the focus of attention. To this end, various studies have been conducted to produce large amounts of fibrinolytic enzymes with high fibrinolytic activity from bacteria or fungi. There are several attempts being made. However, the activity of these degrading enzymes is not caused by a single enzyme, but several enzymes exist in the form of complexes, which cooperatively degrade the fiber (Eveleigh, in cellulase: a perspective. Phil. Traans. Roy. Soc. Lond. A321). , 435 (1987); Lamed and Bayer, Adb. Appl. Microbiology 33, 1 (1988); Lamed et al., J. Bacteriol. 169, 3792 (1987)). In general, fibrinase is somewhat different depending on the type of microorganism produced, but in most cases endoglucanase (endocellulase, EC 3.2.1.4), exoglucanase (exocellulase, EC 3.2.1.91) and β-glucosidase (cellobiase, EC 3.2. All of these three enzymes are required to obtain glucose as an enzyme complex composed of 1.21). In addition, natural cellulose was embedded on the matrix of hemicellulose (xylan) and lignin, so it was necessary to cloning CMC-xylanase, an endoglucanase with xylanase activity, because fibrinase alone could not induce efficient enzymatic hydrolysis.

Therefore, the present invention was made on the basis of the above facts, cloned the CMC-xylanase gene, which is an endoglucanase having xylanase activity, prepared an expression vector containing a homogene, and then provided with E. coli transformed by the vector. The aim is to mass produce CMC-xylanase.

Hereinafter, with reference to the accompanying drawings, the configuration and operation of the present invention will be described.

Figure 1 illustrates the cloning of CMC-xylanase gene into E. coli expression vector.

Figure 2 shows that the recombinant Escherichia coli containing the CMC-xylanase gene of Fibrobacter succinogenes S85 decomposes the substrate (CMC and xylan) present on the solid LB medium to form a clear zone, respectively, expressing the CMC-xylanase enzyme.

Figure 3 shows the DNA base sequence of the cloned CMC-xylanase gene.

The present invention comprises the steps of separating the chromosomal DNA from Fibrobacter succinogenes S85 (ATCC 19169), an anaerobic ruminant; Cloning the CMC-xylanase gene; Selecting an E. coli transformant carrying a CMC-xylanase gene and determining a DNA sequence; Comparing the enzymatic activity of CMC-xylanase derived from transformed E. Coli.

Figure 1 shows the CMC-xylanase gene cloning process into E. coli expression vector. FIG. 2 shows that CMC-xylanase enzyme expressed through recombinant E. coli containing CMC-xylanase gene of Fibrobacter succinogenes S85 decomposes CMC (a) and oat spelt xylan (b), which are substrates present on solid LB medium, respectively. Formation of a clear zone (clear zone) indicates that the CMC-xylanase enzyme is expressed. Figure 3 shows the DNA sequence of the cloned CMC-xylanase gene.

The present invention provides fibrobacter succinogenes S85 (ATCC 19169), which is a ruminant microorganism, each 50 ml of Sweet E. broth (Holdman et al., In Anaerobic laboratory manual (4th ed.), Virginia Polytech. Inst. And State Univ. Blackburg, Virginia (1977). )) Was used as a subculture under anaerobic conditions. The obtained subculture was inoculated again in 1 liter of PY medium (Holdman et al., In Anaerobic laboratory manual (4th ed.), Virginia Polytech. Inst. And State Univ. Blackburg, Virginia (1977)), and then incubated for 36 hours at 37 ° C. Political culture in an anaerobic state. To isolate the chromosomal DNA, the culture was centrifuged (4 ° C, 5500 rpm, 10 min) to collect the cells. Chromosomal DNA was isolated from the collected cells (Mamur, J. Mol. Biol. 3, 208 (1961): Thomas, J. Mol. Biol. 11, 476 (1965): Saito and Miura, Biochem. Biophys. Acta 72 619 (1963)), and finally isolated chromosomal DNA was dissolved in 3 mL of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.4). To prepare a gene library from chromosomal DNA, it was partially cut with restriction enzyme Sau3AI. DNA fragments of 2-8 kb were collected by centrifugation (20 ° C, 25,000 rpm, 24 hours) at 10-40% sucrose gradient to obtain the appropriate size fragment from the gene library generated by cleavage with restriction enzyme Sau3AI. .

Meanwhile, pUC19 was used as a cloning vector, which was digested with restriction enzyme BamHI and treated with calf intestine phosphate (CIP). E. coli DH5 _α was used as a microbial strain for transformation, and CaCl ₂ was used to make competent cells.

Cohesive termini conjugation was used to clone the CMC-xylanase gene. First, CIP-treated vector (0.1㎍) and external DNA (0.3㎍) were cut into BamHI, mixed in a microfuge tube, and the total volume was made to 7.5μl with secondary distilled water, and then 10x Bacteriophage T ₄ DNA ligase buffer solution. 1 μl (200 mM Tris-Cl, pH 7.6, 50 mM dithiothreitol, 500 mg / mL bovine serum albumin), 0.1 unit of Bacteriophage T ₄ DNA ligase and 1 μl of 5 mM ATP were added to the mixture, followed by reaction at 16 ° C. for at least 4 hours. . After the reaction was completed, 2 μl was taken and used to transform competent E. coli.

In order to confirm the insertion of external DNA in transformed E. coli, a screening method using α-complementation was used. First, 40 μl of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) stock solution (20 mg / mL in dimethylformamide) was added to an LB agar plate containing 50 μg of ampicilin per mL. 4 μl of IPTG (200 mg / mL) solution was spread. 150 μl of transformant was sprayed onto the plate thus prepared and incubated at 37 ° C. for 12-16 hours. After the incubation was completed, the plate was stored at 4 ° C. for several hours to further change colony color. Colonies with only the vector are blue, but colonies with the recombined DNA show the original white color of E. coli. E. Coli transformed in the present invention was deposited on September 4, 1997, the deposit number KTCC 8830P to the International Institute of Biotechnology.

White colonies selected using the α-complementation method were used in the second screening process to confirm the presence of the CMC-xylanase gene. In order to select E. coli transformants with CMC-xylanase gene, first, the transformants were picked on LB agar plates containing 50 μg of ampicilin per mL and 0.5% CMC (carboxymethylcellulose) or oat spelt xylan. Incubated at 37 ° C. for 16-24 hours. 0.5% congo-red solution was added to the colony-formed plates and allowed to stand for 30 minutes. Congo-red that was not bound to the polymer CMC or oat spelt xylan was removed by adding 1M NaCl. Enzyme activity was confirmed with or without the formed clear zone. In order to confirm whether the selected microorganisms express CMC-xylanase enzyme, inoculate the liquid LB medium again and incubate with shaking at 37 ° C for 12 hours to examine CMC-xylanase enzyme activity and clone the CMC-xylanase gene. Check the presence or absence. Sanger method was used to determine the DNA sequence of the cloned CMC-xylanase gene. In order to identify the substrate used by the CMC-xylanase enzyme expressed by the CMC-xylanase gene, all substrates were used as a result of using cellobiose, PNPG, p-nitrophenol cellobioside (PNPC), and carboxymethylcellulose as substrates. . In addition, the localization of intracellular CMC-xylanase enzymes revealed that 67.9% of the cells secreted extracellularly.

Hereinafter will be described in detail step by step the specific configuration and operation of the present invention.

Step 1: cleavage of DNA with restriction enzymes

Restriction enzyme and reaction buffer used in the present invention was purchased from Kosko Biotech Co., Korea, the reaction volume in a sterile 1.5mL microfuge tube was usually from 10μl to 100μl and the reaction temperature was 37 The reaction was carried out at 1 ° C. for 2 hours, and after the reaction was completed, the result was heat treated at 65 ° C. for 15 minutes. The 10-fold buffer composition is as follows.

10-fold buffer A: 6 mM Tris-HCl, 6 mM NaCl, 6 mM MgCl ₂ , 1 mM DTT, pH 7.5

10-fold buffer B: 6 mM Tris-HCl, 50 mM NaCl, 6 mM MgCl ₂ , 1 mM DTT, pH 7.5

10-fold buffer C: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT, pH 7.9

10-fold buffer D: 6 mM Tris-HCl, 150 mM NaCl, 6 mM MgCl ₂ , 1 mM DTT, pH 7.9

10-fold buffer for EcoR1: 100 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl ₂ , pH 7.5

10-fold buffer for Smal: 10 mM Tris-HCl, 50 mM KCl, 7 mM MgCl ₂ , pH 7.5

Step 2: Phenol Extraction and Ethanol Precipitation

After the enzymatic reaction was completed, phenol was used to deactivate the enzyme or extract the nucleic acid. The phenol was saturated with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA solution. The phenol extraction method is to mix the sample and phenol in a ratio of 1: 1 (v: v), shake vigorously, and then centrifuge (15,000 g, 5 min) to transfer the supernatant to a new tube, and then to the same volume of phenol-chloroform solution. After extraction by adding (a solution of phenol-chloroform ratio of 1: 1 (v: v)), the supernatant was transferred to a new tube, and 0.1 volume of 3M sodium acetate and 2 volumes of ethanol (-20 ° C.) were added. ) Was added and allowed to stand at -20 ° C for about 12 hours, followed by centrifugation (15,000g, 20min, 4 ° C) to precipitate nucleic acid.

Step 3: conjugation reaction

Bonding enzyme was used as the enzyme T ₄ DNA junction _{(T 4 DNA ligase, KOSCO Biotech} Co.) and 10 times the bonding buffer (0.5M Tris _{-HCl (pH 7.8) 0.1M MgCl 2} , 0.2M DTT, 10mM ATP) Usually, 100 units of conjugate enzyme was used in a volume of 10 μl, and the DNA conjugate having a blunt end was used for 1,000 units of conjugate enzyme. Reaction conditions were made to react at 15 degreeC for 12 hours or more.

Step 4: E. coli transformation

DH5α was used as E. Coli host cell. Host cells were inoculated in 50 mL of liquid LB medium (1% bactotrypton, 0.5% bacterium yeast extract, 1% NaCl), and then cultured at 37 ° C. until the optical density at 650 nm reached 0.25 to 0.5. After standing on ice for 10 minutes, centrifuge (4 ℃, 10min, 3,000g) to separate the cells, and add 10mL of 0.1M CaCl ₂ solution for 10 minutes on ice, then centrifuge (4 ℃, 10min, 3,000g). Cells were isolated, and 2 mL of 0.1M CaCl ₂ was added and uniformly dispersed, stored at 4 ° C., and used after 24 hours. In the transformation, 10 µl of the conjugated reaction solution was added to 200 µl cells obtained above, and left to stand on ice for 1 hour, followed by heat treatment at 42 ° C. for 90 seconds, incubation at 0.8 ° C. in liquid LB medium for 45 minutes, and 50ug. It was applied to a petri dish with solid LB medium containing / mL of empicillin and incubated overnight at 37 ° C.

Hereinafter, the specific configuration and operation of the present invention will be described by the embodiment of the process. Of course, the scope of the present invention is not limited only to the examples described below.

Step 1: Isolation of Chromosomal DNA

Extraction of DNA Fibrobacter succinogenes S85 (ATCC 19169), an anaerobic microorganism, was inoculated into 100 mL Swelet E broth and used as a subculture under anaerobic conditions. Subcultures were inoculated again in 1 liter of Peptone Yeast Extract (PY) medium and then incubated in anaerobic conditions at 37 ° C. for 36 hours. The cells were collected by centrifugation (4 ° C, 5,500 rpm, 10 min), washed twice with 300 mL of saline-EDTA solution (0.15 M NaCl, 0.1 M EDTA, pH8.0), and 40 mL of saline-Tris solution (0.1 M NaCl, 10 mM EDTA, 0.1M Tris-HCl, pH9.0). After dissolution, lysozyme was added at a concentration of 1.8 mg / mL and gently shaken at 37 ° C for 10 minutes. 12 mL of Tris-SDS solution (5% (W / V) SDS, 0.14 M NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH7.5) was slowly added dropwise and gently shaken at 60 ° C. for 20 minutes. Proteinase K was added at a concentration of 1mg / mL, and then left at 3 ° C for 3 hours, followed by phenol extraction and ethanol precipitation to precipitate chromosomal DNA, and then the DNA was washed with a glass rod to wash in 70% (V / V) ethanol solution. Then dissolved in 30ml of SSC solution (0.1M NaCl, 0.015M Na ₃ citrate). 30 μl of RNAase solution (50mg / mL) was added to the SSC solution in which DNA was dissolved, followed by incubation at 37 ° C. for 1 hour to remove RNA, followed by phenol extraction and ethanol precipitation. The DNA precipitated with the glass rod was collected, washed in 70% (V / V) ethanol solution, dissolved in 3 mL of TE buffer solution (10 mM Tris-HCl (pH8.0), 1 mM EDTA), and then dialyzed with 0.1 times TE buffer solution. Pure chromosomal DNA was obtained.

Step 2: Cloning the CMC-xylanase Gene

Preparation of Gene Library

70 μl of the chromosomal DNA solution obtained in step 1, 10 μl of 10-fold buffer B, 19 μl of secondary distilled water, and 3 units of restriction enzyme Sau3AI were reacted at 37 ° C. for 8 minutes, followed by heat treatment at 75 ° C. for 10 minutes. The restriction enzyme was inactivated. To obtain a DNA fragment of 2-8kb from the gene library generated by cleavage with the restriction enzyme Sau3AI, 10-40% sucrose gradient centrifugation (20 ° C, 25,000rpm, 24hours) was received in a microcentrifuge tube at 400µL and then 5M NaCl. 30 μl of the solution was added, followed by addition of 2 volumes of ethanol, followed by standing at -20 ° C. for at least 12 hours. After centrifugation at 14,000 rpm for 20 minutes at 4 ℃ washed with 70% ethanol and dissolved in 30 μl of TE buffer. 1 μl of each fraction was electrophoresed to select fractions 3 and 4 containing 2-8 kb DNA fragments.

Preparation of the vector for cloning

For cloning, the vector used pUC19, which was cut with BamHI identical to the cleavage site of the restriction enzyme Sau3AI. 30 μl (5 μg) of pUC19 vector digested with restriction enzyme BamHI, 103 μl of secondary distilled water, 15 μl of 1M Tris-HCl (pH 8.0), and 2 units of calf intestine phosphatase (KOSCO Biotech Co) were mixed and mixed at 37 ° C. After incubation for 30 minutes, phenol was extracted, and then extracted twice with the same volume of ether (ether saturated with distilled water), precipitated with ethanol, and dissolved in 20 μl TE buffer.

Gene cloning

1 μl of the selected fractions 3 and 4 for cloning into the pUC19 vector, 1 μl of the pUC19 vector digested with BamHI, 1 μl of 10-fold conjugation buffer, 5 μl of secondary distilled water, and 1 μl of T ₄ DNA conjugated enzyme. The mixture was added and mixed to react at 15 DEG C for at least 12 hours. After completion of the reaction, E. coli was prepared as described in the fourth step, 10 μl of the conjugated reaction solution and 200 μl of the E. coli dispersion solution were mixed, left on ice for 1 hour, heat treated at 42 ° C. for 90 seconds, and then 0.8 mL of liquid LB medium was added and incubated at 37 ° C. for 45 minutes. In order to confirm the insertion of external DNA in the transformed E. coli was used a screening method using α-complementation. First, 40 µl of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto pyranoside) stock solution (20 mg / mL in dimethylformide) was added to a solid LB medium containing 50 µg / ml of ampicillin. ) And 4 μl of IPTG (20 mg / mL) solution were spread. 150 μl of transformed E. coli was plated on the solid LB medium thus prepared and incubated at 37 ° C. for 12-16 hours. Colonies with only the pUC19 vector are blue, but colonies are white when the vector and the external DNA are recombined. White colonies selected using the α-complementation method were used in the second screening process to confirm the inclusion of the CMC-xylanase gene.

Step 3: screening and DNA sequencing of E. coli transformants carrying CMC-xylanase gene

To screen for E. coli transformants with the CMC-xylanase gene, first picking the transformants on an LB agar plate containing 50 μg of ampicillin per mL and 0.5% CMC (carboxymethylcellulose) or oat spelt xylan, respectively. Incubated at 37 ° C. for 16-24 hours. 0.5% congo-red solution was added to the colony-formed plates and allowed to stand for 30 minutes. Congo-red that was not bound to the polymer CMC or oat spelt xylan was removed by adding 1M NaCl. Enzyme activity was confirmed with or without the clear zone formed. In order to confirm whether the selected microorganisms express CMC-xylanase enzyme, inoculate the liquid LB medium again and incubate with shaking at 37 ° C for 12 hours to examine CMC-xylanase enzyme activity and clone the CMC-xylanase gene. Check the presence or absence. The culture solution incubated in liquid LB medium was disrupted by sonication (4 ℃, 4 min, duty cycle 50%) and then centrifuged at 5500 rpm for 30 minutes to use the supernatant as an enzyme solution. The substrate is dissolved in 50 mM potassium phosphate buffer as CMC. After mixing the enzyme solution 1.0mL and substrate 1.0mL shaking reaction for 30 minutes at 37 ℃ and soaked in boiling water for 10 minutes to inactivate the enzyme. In order to measure the reducing sugar, the reaction was performed using DNS (3,5-dinitrosalicylic acid) and then absorbance was measured at 550 nm. One unit of enzyme corresponds to 1 μole of reducing sugar dissociated for 1 minute at 37 ° C.

Fourth Step: Comparison of Enzyme Activity of Transformants with Fibrobacter succinogenes S85 and CMC-xylanase Gene

Fibrobacter succinogenes S85 was inoculated in 1 liter of Peptone Yeast Extract (PY) medium and then incubated in an anaerobic state at 37 ° C. for 36 hours. The cultured culture solution was centrifuged at 5500 rpm for 10 minutes to collect the cells, and then dispersed in 20 mL of 50 mM potassium phosphate buffer, followed by sonication (4 ℃, 4 min, duty cycle 50%) to destroy microorganisms, followed by centrifugation at 5500 rpm for 30 minutes. The supernatant was separated and used as the enzyme solution. E. coli transformants, which were introduced with the CMC-xylanase gene, were inoculated in 1 liter of liquid LB medium (ampicilin 50㎍ / mL) and incubated with shaking at 37 ° C for 12 hours. The cultured culture solution was centrifuged at 5500rpm for 10 minutes to collect the cells, and then dispersed in 50mL of 50mM potassium phosphate buffer, followed by sonication (4 ℃, 4min, duty cycle 50%) to destroy microorganisms and then centrifuged at 5500rpm for 30 minutes. The supernatant was separated and used as the enzyme solution. The substrate is dissolved in 50 mM potassium phosphate buffer with CMC. After mixing 1.0 mL of the enzyme solution and 1.0 mL of the substrate, the reaction is shaken at 37 ° C. for 30 minutes, and then immersed in boiling water for 10 minutes to inactivate the enzyme. Reducing sugar was reacted using DNS (3,5-dinitrosalicylic acid) for the measurement and then the absorbance was measured at 550nm. The CMC-xylanase enzyme activity expressed was 0.013 units / mg protein for Fibrobacter succinogenes S85, and 0.244 units / mg protein for E. coli transformant with CMC-xylanase gene to express CMC-xylanase. In this case, the enzyme activity was 18.77 times higher than the enzyme activity.

The present invention provides a nucleotide sequence of a novel CMC-Xylanase gene and a vector containing the gene, E. Coli transformed by the expression vector as described through the steps and process-specific examples described above In addition, since it has the effect of mass biosynthesis of CMC-Xylanase, which is easy to separate and use from a transformant, it is a very useful invention in the biological industry and the enzyme industry.

Claims (4)

CMC-xylanase gene of Fibrobacter succinogenes S85 having the following DNA base sequence

Expression vector containing CMC-xylanase gene of Fibrobacter succinogenes S85 E. coli transformed by the expression vector of claim 2 (KTCC 8830P) Recombinant CMC-Xylanase enzyme prepared by the transforming microorganism according to claim 3

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