KR20030085679A - Gene sequence of xylanase biosynthesis enzyme and transformant thereof - Google Patents

Abstract

PURPOSE: A xylanase encoding gene and a recombinant xylanase produced from a transformant thereof are provided. The produced xylanase has improved xylane hydrolyzing activity. CONSTITUTION: A xylanase encoding gene has the nucleotide sequence of SEQ ID NO: 1. A recombinant vector containing the gene of SEQ ID NO: 1 is constructed. A transformant, Escherichia coli transformed with the recombinant vector containing the gene of SEQ ID NO: 1 is produced. A recombinant xylanase has the amino acid sequence of SEQ ID NO: 2. The recombinant xylanase of SEQ ID NO: 2 is produced by culturing the transformed Escherichia coli. The recombinant xylanase may be added into the domestic animal fodder.

Description

Gene sequence of xylanase biosynthesis enzyme and its transformant {Gene sequence of xylanase biosynthesis enzyme and transformant}

The present invention relates to xylanase biosynthesis genes and their enzyme production. More specifically, the E. coli transformant containing the xylanase gene reveals the nucleotide sequence and amino acid sequence of the gene encoding the xylanase derived from the Bacillus AMX-4 strain isolated from the soil. It relates to confirming the produced xylanase activity after producing xylanase by expressing.

Xylanase is one of a variety of enzymes produced by microorganisms, and is an enzyme that is highly available at present and is expected to be developed for various purposes in the future. As the availability of this enzyme, the most important thing is to decompose and sugarify vegetable fiber material, which is the most abundant and renewable energy resource by solar energy, to become energy resource. These include industrial production such as production of high-quality paper and recycling of waste paper, removal of turbidity from fruit drinks and beer, extraction of coffee and vegetable oils and starch, and increased feed use of unit animals and xyloligosaccharide production, which will be mainly mentioned in the present invention. Application is mentioned.

Recently, the usefulness of xylanase as an enzyme for feed addition and a production enzyme of xyloligosaccharide has been increased. Feed grains, which provide carbohydrates and small amounts of protein, which are primarily needed for animal energy metabolism, contain fiber that is not available to most unit animals. The fiber in the grain consists of non-starch polysaccharides and lignin, and the cell wall of the plant is made up of a combination of lignin and non-starch polysaccharides consisting of hemicellulose and beta-glucans covering the entire outer layer of cells and the whole grains inside. This non-starch polysaccharide is not only degraded by the digestive enzymes of livestock, but also surrounds starch or protein in grains, which causes a problem that the livestock uses less nutrients. In the small intestine, non-starch polysaccharides are thickly melted in a thick gel, which prevents digestive enzymes from accessing nutrients and inhibits digestion in the digestive tract. Therefore, the microorganisms are attached to the digestion causing abnormal fermentation, resulting in the loss of nutrients.

The addition of xylanase to grain feed can hydrolyze the hemicellulose layer that surrounds the grains, thereby increasing the availability of nutrients in the grains and improving the condition of the intestinal digest of livestock. In particular, when the wheat products with high hemicellulose content are used as feed, it is easy to cause stool or reddening in livestock. Xylanase can prevent these phenomena, raise the feed value and improve the quality variation of the wheat products. The product can be used as a stable feedstock and its value as a feed additive enzyme. Therefore, xylanase is very important as an enzyme for feed addition of unit animals such as pigs and chickens. In addition, the meat consumption of not only developed countries but also people of many countries with improved living conditions and incomes is increasing, which makes diversifying feed resources and improving feed efficiency for livestock raising.

In addition, xyloligosaccharides are well known as growth factors of useful microorganisms present in the intestinal tract of animals. In particular, xylobioses and xylotriose have excellent effects. Therefore, xylanase acts as a key enzyme to produce xyloligosaccharide from xylan material by enzymatic method.

In order to completely decompose and glycosylate xylan, the main component of hemicellulose, there are generally three types of enzymes: endo-β-xylanase, exo-β-xylanase and xylosidase ( β-xylosidase) is known to work together, these are collectively referred to as xylanase enzymes. On the other hand, the present invention focuses on the first endocyanase among these enzymes, and then assumes that the enzyme is called xylanase for convenience.

Microorganism used in the production of xylene Feed added Rana claim is in the mold, and fungi, among Trichoderma (Trichoderma) sp that is mainly used these enzyme productivity and substantially superior to the enzyme production of the bacteria to produce xylene Rana claim It mainly shows maximum activity under acidic conditions However, the physiological conditions of the digestive system of pigs and chickens vary by region, but the xylanase produced by the strains of Trichoderma is inadequate because the pH condition after the small intestine to which xylanase should act is around 6.5. Therefore, xylanase, which has a high acidity under neutral conditions and produces xyloligosaccharide, a growth factor of microorganisms in useful intestines, as a reaction product, rather than fungal-derived xylanase near pH 5.0, is valuable as a feed enzyme. Is high.

Therefore, an object of the present invention is to determine the nucleotide sequence of the gene encoding the xylanase from the AMX-4 isolate strain of the genus Bacillus, which produces a highly active xylanase in acidity. Another object of the present invention is to produce a xylanase by expressing an E. coli transformant transformed by a recombinant plasmid containing a xylanase gene. Another object of the present invention is to identify the activity of the produced xylanase and its final product.

The object of the present invention is to smear E. coli into agar agar containing oat xylan and introduce a gene library prepared using chromosomal DNA extracted from AMX-4 strain of Bacillus sp. E. coli transformants containing the xylanase gene were obtained, the recombinant plasmids containing these transformants were isolated, the nucleotide sequences of the genes encoding xylanase were identified and restriction enzyme maps were prepared. After purifying the xylanase produced by the transgenic strains, we analyzed the effect of reaction temperature and pH on the activity of xylanase, and then purified olyxyl, including xylobio, xylotriose and xyl Decomposition products of each of Lotetraoz and Xylopentose are analyzed and added to food and feed. Achieved by confirming utility as a solvent.

1 is a restriction map of the recombinant plasmid pXT4 containing a gene encoding xylanase of the AMX-4 strain of Bacillus sp.

Figure 2 is a graph showing the activity according to the reaction temperature and pH of the xylanase produced by the E. coli transformant containing the recombinant plasmid pXT4 of the present invention.

Figure 3 is a thin layer chromatography photograph of the final degradation product of oat xylan by xylanase produced by the E. coli transformant containing the recombinant plasmid pXT4 of the present invention.

Figure 4 is a thin layer chromatography photograph of the final degradation product of xylobiose, xylotriose, xylotetraose, xylopentose by xylanase produced by the E. coli transformant containing the recombinant plasmid pXT4 of the present invention. .

The present invention comprises the steps of isolating and identifying a bacterium of the genus AMX-4 having excellent xylan resolution in nature; Extracting the total chromosomal DNA of the isolated Bacillus genus AMX-4 strain, treating it with a restriction enzyme, linking it with a plasmid, and introducing the same into E. coli to prepare a gene library; Screening transformants containing the xylanase gene by spreading the E. coli transformants of the prepared genetic library onto a solid agar medium to which oat xylan is added to observe the xylan degradation ring; Extracting a recombinant plasmid from an E. coli transformant containing a xylanase gene to reveal a xylanase nucleotide sequence; After expressing the E. coli transformant containing the xylanase gene of the present invention to produce and purify the xylanase, the activity according to the reaction temperature and the reaction pH of the xylanase of the present invention is analyzed and the hydrolytic activity according to the xylan species is analyzed. Comparing; The oat xylan degradation product and xylobioses, xylotriose, xylotetraose and xylopentose of the present invention may be irradiated with thin layer chromatography.

Hereinafter, the present invention will be described in detail.

In this step, oat xylan 5 g / L, yeast extract 5 g / L, tryptone 10 g / L, NaCl 5 g / L, ampicillin 50 LB-Xylan agar medium consisting of mg / L, 15 g / L agar and pH adjusted to 7.0 is used. As culture medium for E. coli transformants, ampicillin (50 mg / L) is added to an LB complex liquid medium consisting of 5 g / L yeast extract, 10 g / L tryptone, and 5 g / L NaCl. .

To isolate the microorganism producing xylanase, the soil sample suspension is incubated in a nutrient plate medium, and the xylanase producing bacteria are separated by observing the xylan degradation ring around the colonies formed on the plate medium. Identification of the isolated strain is carried out by examining the morphological, biochemical properties, fatty acid composition and nucleotide sequence of the 16S rRNA gene. The characteristics of the isolates were identified as aerobic, Gram-positive, spore-forming, and short-lived bacterium with catalase activity. As a result of analysis of cell membrane fatty acid composition of Bacillus sp., Bacillus subtilis showed a similarity value of 0.753 and 0.683 to the fatty acid composition of Bacillus subtilis and Bacillus amyloliquefaciens, respectively. The homology with the nucleotide sequence of the gene appears to be the highest.

For cloning the xylanase gene of the isolated and identified Bacillus genus AMX-4 strain, a gene library of Bacillus AMX-4 is prepared and an E. coli transformant containing the xylanase gene is selected. The AMX-4 strains in Bacillus were grown to logarithmic growth stage and centrifuged to extract the chromosomal DNA from the recovered cells, and then digested with restriction enzyme Sau3AI and electrophoresed to recover 0.5 ~ 10Kb DNA fragments. These fragments are inserted into plasmid pUC19 digested with BamHI to prepare a gene library. The recombinant plasmid prepared above is transformed into Escherichia coli XL-1, and a transformant showing a xylan degradation ring is selected by spreading on LB-xyl agar medium for obtaining a transformant containing a xylase gene.

In order to determine the nucleotide sequence of the gene encoding xylanase, the plasmid is isolated from the cells obtained by culturing the E. coli transformant in LB liquid medium containing ampicillin by alkaline decomposition. Treatment with various restriction enzymes revealed that the recombinant plasmid contained a cloned DNA fragment of about 0.8 kb in size, which was named pXT4. In order to determine the nucleotide sequence of the cloned 0.8 kb DNA fragment in plasmid pXT4, fragments were divided into small fragments and inserted into pUC19 using various restriction enzymes, and a total of 748 bp was determined by the dideoxynucleotide sequencing method. Determine the base sequence. The base sequence contains a total of 642 bp open reading frame corresponding to the structural gene of xylanase (SEQ ID NO: 1), and the amino acid sequence of the xylanase enzyme inferred from the base sequence of the structural gene. Is a protein consisting of 213 amino acids shown in SEQ ID NO: 2. The amino acid sequence of xylanase inferred from the base sequence of the xylanase gene of Bacillus AMX-4 was found to have high similarity to the amino acid sequence of xylanase of the Bacillus strain. 10-9 of 213 amino acids with homology of about 95.3% to xylanase (Jeong, KJ, PC Lee, IY Park, MS Kim, and SC Kim, Enzyme Microb.Technol. 22, 599-605 (1996)) There is a difference in amino acid sequence.

E. coli transformation transformed with pXT4 in LB liquid medium containing ampicillin to investigate the activity according to temperature and pH of recombinant xylase produced by expressing E. coli transformant containing recombinant plasmid pXT4 The enzyme is purified by culturing the sieve, crushing and concentrating the cultured cells. For this enzyme, xylanase activity is measured by varying the reaction temperature and pH. As a result, as shown in Figure 2 shows the highest enzyme activity at 50 ~ 55 ℃ and pH 7.0, 90% or more activity in the pH 5.0 ~ 7.0 range. In particular, at pH 6.5, the physiological condition of the animal intestine, the activity corresponds to more than 98% of the highest activity. On the other hand, it is important not to be inactivated during the passage of the stomach when used as a feed additive, so to analyze the stability of xylanase according to pH, the purified enzyme was left in different buffers in the range of 3.0 to 9.0 for 1 hour. The remaining activity of the enzyme is then measured. As a result, as shown in Table 2, the residual activity is 90% or more in the pH 4.0 to 8.0 range, and the residual activity is about 88% in the pH 3.0. Therefore, as a result of investigating the remaining activity at different conditions of pH 3.0, which is similar to the above, the residual activity was shown to be about 85% or more until 4 hours. In addition, in order to measure the enzyme activity according to the type of xylan using purified xylanase, the relative hydrolytic activities of arabino xylan, oat xylan and birch xylan are compared. As a result, as shown in Table 1, it can be seen that the hydrolytic power of arabino xylan, which is a wheat-derived xylan, which is widely used as a feedstock, is higher than that of oat xylan. Looking at the above results it can be seen that the xylanase produced by the xylanase gene of the AMX-4 strain of the Bacillus genus of the present invention is suitable for feed addition purposes.

In order to analyze the xylan hydrolyzate of xylase produced by the expression of an E. coli transformant containing the xylase gene of the present invention, a hydrolysis reaction is performed under optimum reaction conditions using oat xylan as a substrate and the final The product uses thin layer chromatography (TLC). As a result, as shown in FIG. 3, xylose is not produced as a final product, but xylobioses, xylotriose, xylotetraose and the like are produced. This is due to xylan hydrolysis products by xylanase of the present invention and xylan hydrolysis by xylanase from Bacillus sp. Strains analyzed as having high similarity between amino acid sequence and xylanase of the present invention as shown in Example 3. Comparison of the products (Jeong, KJ, IY Park, MS Kim, and SC Kim, Appl. Microbiol. Biotechnol. 50, 113-118 (1998)) produced xylose as a hydrolysis product by xylanase of the present invention. The biggest difference is that it is not. In addition, after the reaction of the xylanase of the present invention with the substrates of xylobiose, xylotriose, xylotetraose, and xylopentose as a substrate, the hydrolysis products were examined by thin layer chromatography. Similarly, it is confirmed that the xylobiose and the xylotriose are not decomposed, the xylotetraose and the xylopentose are decomposed into the xylobio and the xylotriose, and the reaction product does not generate xylose. do. Accordingly, it can be seen that the xylanase of the present invention decomposes the xylan material to produce xyloligosaccharides with excellent functionality.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Isolation and Identification of a Novel Strain Bacillus AMX-4

To isolate the microorganisms producing xylanase, 1 g of soil samples were suspended in 10 mL of 0.85% NaCl solution, and an appropriate amount of suspension was added to the nutrient plate medium added with 0.5% oat spelt xylan. (beef extract, 3 g; bacto-peptone, 5 g; water, 1 liter) and incubated at 37 ℃. Xylanase-producing bacteria were selected by observing the xylan degradation ring around the colonies formed on the plate medium, and finally, bacteria having high xylanase productivity were finally separated for 1 week. Name the isolate AMX-4 and use the formula milk (1%), starch (0.2%), tributylin (1%) and carboxymethyl to analyze if you can decompose other polymers other than xylan. The degradation ring was examined by culturing in a plate medium containing cellulose methyl cellulose (0.5%), respectively, but the milk, starch and tributylin were decomposed, but the carboxymethyl cellulose was hardly decomposed. Therefore, the isolated bacterium AMX-4 does not produce cellulase under this medium condition, and it is assumed that the xylanase produced by the isolated bacterium does not have cellulase activity.

Identification of the isolated strain was carried out by examining the morphological, biochemical properties, fatty acid composition and the nucleotide sequence of the 16S rRNA gene. Gram staining and spore staining were performed for morphological observation of isolated bacteria, API 50 CHB kit was used for biochemical characterization, and Microbial Identification System (MIDI Inc., USA) was used for fatty acid composition. . In order to identify the isolate AMX-4, it was found to be aerobic and Gram-positive bacteria. It was identified as a simple bacillus that forms spores and has catalase activity. In addition, the carbohydrate availability of AMX-4 was investigated using the API 50 CHB kit. Mannose, mannitol, alpha-methyl-D-glucoside, N-acethyl-glucosamine, arbutin, esculin esculin, maltose, sucrose and trehalose were used, but other carbohydrates were not found. Based on the morphological and biochemical properties of AMX-4, Bacillus Among the genus strains, Bacillus subtilis (B.subtilis), Bacillus amyloliquefaciens (B.amyloliquefaciens) And Bacillus pumilus (B. pumilus), But the agreement is not high.

Therefore, in order to accurately identify isolates, in order to compare 16S rRNA gene sequences of AMX-4 strains with other strains, the conserved sequences of 16S rRNA gene sequences of bacteria were used as primers, and the total chromosomal DNA of the isolates was used as a template. Bacillus subtilis as a result of comparing the nucleotide sequence of the amplified DNA fragments with those of other strains by PCR The homology with the nucleotide sequence of the gene was found to be the highest. In view of the above characteristics, the isolate was determined to be close to Bacillus subtilis.

Example 2 Screening of Xylanase Gene of Bacillus AMX-4 Strain

Step 1: Prepare Gene Library of Bacillus AMX-4

Bacillus AMX-4 strain isolated and identified in Example 1 was inoculated into 200 mL of LB liquid medium and shaken and cultured at 37 ° C. to a logarithmic growth phase. -HCl, pH 7.6, 1 mM EDTA, 10 mM NaCl) and then suspended in 20 mL of SET buffer (20% sucrose, 50 mM Tris-HCl, pH 7.6, 50 mM EDTA) with lysozyme added at 37 ° C. The reaction was carried out for 30 minutes at. 2 mL of 10% sodium dodecyl sulfate (SDS) was added thereto to dissolve the cells, and the protease K was treated with an appropriate amount and left at 37 ° C. for 1 hour. After adding phenol and centrifuging, collecting the supernatant, adding twice the volume of cold ethanol, and then squeezing the chromosomal DNA into glass rods and washing them sequentially in 70%, 80%, and 90% ethanol, Dissolved in solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

50 μg of the isolated chromosomal DNA was partially digested with restriction enzyme Sau3AI, followed by agarose gel electrophoresis to recover a DNA fragment having a size of 0.5 to 10 kb. A plasmid pUC19 completely cut with restriction enzyme BamHI and a chromosome cleavage were mixed in the same amount, and T4 DNA ligase was added to react for 15 hours at 14 ° C. to carry out ligation to prepare a gene library of Bacillus AMX-4.

Step 2: Selection of Escherichia Coli Transformant with Xylanase Gene

E. coli XL-1 was transformed using the gene library of Bacillus AMX-4 obtained in step 1 and plated on LB-Xylan agar medium to obtain about 10,000 transformants. E. coli transformants containing the xylanase gene were obtained by selecting one strain of transformants showing a xylan degradation ring around the colonies among these transformants.

Example 3 Determination of Gene Sequencing Encoding the Invention Xylanase

Alkali digestion method of the transformants obtained in step 2 of Example 2 from the cells obtained in LB liquid medium to which ampicillin (50 mg / L) was added (Birnboim, HC and J. Doly, Nucleic Acid Res. 7, 1513 -1523 (1979)), the plasmid was isolated and examined by treatment with various restriction enzymes. The recombinant plasmid contained a cloned DNA fragment of about 0.8 kb, and the recombinant plasmid was named pXT4. A restriction map of pXT4 is shown in FIG. 1. In Figure 1 Xyl is a DNA fragment containing the xylanase gene of the present invention.

In order to determine the nucleotide sequence of the cloned 0.8 kb DNA fragment in plasmid pXT4, fragments were divided into small fragments and inserted into pUC19 using various restriction enzymes, and a total of 748 bp was determined by the dideoxynucleotide sequencing method. The base sequence was determined. In this base sequence, there was a total open frame of 642 bp corresponding to the structural gene of xylanase (SEQ ID NO: 1). The amino acid sequence of the xylanase enzyme deduced from the nucleotide sequence of this structural gene is shown in SEQ ID NO: 2 and is a protein consisting of a total of 213 amino acids. The amino acid sequence of xylanase inferred from the base sequence of the xylanase gene of Bacillus AMX-4 was found to have high similarity to the amino acid sequence of xylanase of the Bacillus strain. 10-9 of 213 amino acids with homology of about 95.3% to xylanase (Jeong, KJ, PC Lee, IY Park, MS Kim, and SC Kim, Enzyme Microb.Technol. 22, 599-605 (1996)) There was a difference in amino acid sequence.

Example 4 Investigation of Xylanase Activity Produced by Expression of Transfected Escherichia Coli

Ampicillin (50 mg / L) was added to investigate the hydrolysis activity according to the reaction temperature and pH of the xylanase produced by expressing the E. coli transformant containing the recombinant plasmid pXT4 prepared in Example 3. E. coli transformants transformed with pXT4 were inoculated into one LB liquid medium, shaken and cultured at 37 ° C. for 16 hours, and the cultured cells were crushed by ultrasonication. The crushed solution was subjected to ammonia sulfate fractionation, ion chromatography, and concentration. After purifying the enzyme, xylanase activity was measured by reacting the purified enzyme with oat xylan at different reaction temperatures and pHs. As a result, as shown in Figure 2 showed the highest enzyme activity at 50 ~ 55 ℃ and pH 7.0, and showed a 90% or more activity in the pH 5.0 ~ 7.0 range. In particular, at pH 6.5, the physiological condition of the animal intestine, the activity was more than 98% of the highest activity. On the other hand, it is important not to be inactivated during the passage of the stomach when used as a feed additive, so to analyze the stability of xylanase according to pH, the purified enzyme was left in different buffers in the range of 3.0 to 9.0 for 1 hour. The remaining activity of the enzyme was then measured. As shown in Table 2, the residual activity was 90% or more in the pH range of 4.0 to 8.0, and the residual activity was about 88% in the pH 3.0. Therefore, as a result of investigating the residual activity at different pHs of 3.0, the remaining conditions showed that the residual activity was about 85% or more until 4 hours.

In addition, in order to measure the enzyme activity according to the type of xylan using the purified xylanase, the relative hydrolytic activities of arabinoxylan, oat xylan and birch xylan were compared. At this time, the reaction conditions were performed at pH 7.0 and 50 ℃. As a result, as shown in Table 1, the hydrolytic power of arabinoxylan, a wheat-derived xylan used as a feed material, was higher than that of oat xylan.

Looking at these results, it was found that the xylanase produced by the E. coli transformant containing the xylanase gene of the Bacillus AMX-4 strain of the present invention is suitable for feed addition.

Comparison of Activities According to Xylan Types of Xylanase Produced by Expression of Transfected Escherichia Coli Type of substrate Relative Activity (%) Arabino Xylan 100 Birch xylan 83 Oat Xylan 70

PH-dependent Activity of Xylanase Produced by Expression of Transfected Escherichia Coli pH 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Relative activity level of residual activity (%) 88.0 90.3 93.2 100 100 97.3 84.8

Example 5 Investigation of Xylan Hydrolyzate of Xylanase Produced by Expression of Transfected Escherichia Coli

In order to analyze the xylan final reaction product by the purified xylanase obtained in Example 4, the hydrolysis reaction was performed under optimal reaction conditions using oat xylan as a substrate, and the final hydrolysis product was subjected to thin layer chromatography (TLC). As a result, as shown in FIG. 3, xylose was not produced as a final product, but xylobioses, xylotriose, xylotetraose, and the like were produced. This is due to the high degree of similarity between the xylan hydrolysis product of the present invention xylanase and the present invention xylanase and amino acid sequence as shown in Example 3. Comparison of the products (Jeong, KJ, IY Park, MS Kim, and SC Kim, Appl. Microbiol. Biotechnol. 50, 113-118 (1998)) produced xylose as a hydrolysis product by xylanase of the present invention. The biggest difference was confirmed. In addition, after the reaction of the xylanase of the present invention with the substrates of xylobiose, xylotriose, xylotetraose, and xylopentose as a substrate, the hydrolysis products were examined by thin layer chromatography. Similarly, it did not decompose xylobios and xylotriose, and it was confirmed to decompose xylotetraose and xylopentose into xylobis and xylotriose, and the reaction product was also found to be free of xylose. . Accordingly, the xylanase of the present invention has been shown to produce xyloligosaccharides with excellent functionality by decomposing xylan materials.

As described above, xylanase produced by the expression of E. coli transformed by introducing the xylanase gene of the Bacillus genus AMX-4 strain of the present invention has an excellent effect on hydrolysis of xylan derived from wheat, oats, and birch In addition, the hydrolyzate is a very useful invention for the food and beverage industry because it is a xylo-oligosaccharide including xylobioses and xylotrioses, which act as growth factors for the enteric useful bacteria of animals.

Claims (6)

A gene comprising the nucleic acid sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 1. E. coli transformed with the recombinant vector according to claim 3. Recombinant xylanase comprising the amino acid sequence of SEQ ID NO: 2. A method for producing the recombinant xylanase of claim 4, comprising culturing the transformed Escherichia coli according to claim 3. A method for adding a recombinant xylanase according to claim 4 to a feed for unit livestock.