KR100560375B1 - Gene coding mannanase and recombinant mannanase expressed from transformant thereof - Google Patents

Abstract

The present invention relates to a novel mannanase gene encoding a mannanase of Bacillus licheniformis WL-12 strain, a recombinant plasmid comprising the gene, and an E. coli transformant comprising the recombinant plasmid. In addition, it is highly reactive at neutral pH and medium temperature, which is close to the intestinal tract of animals, and has an excellent effect of providing a mannase that decomposes galactomannans into mannose, mannobiose and mannose.

Bacillus licheniformis WL-12 Strain, Mannanase, Mannanase Gene, Galactomannan Hydrolyzate

Description

Gene coding mannanase and recombinant mannanase expressed from transformant

1 is a restriction map of the recombinant plasmid pMWL13 containing a gene encoding a metase of Bacillus licheniformis WL-12 strain.

Figure 2 is a cleavage map of the recombinant plasmid pMWL13 containing a gene encoding a metase of Bacillus licheniformis WL-12 strain of the present invention.

Figure 3 is a graph showing the activity according to the reaction temperature and pH of the met kinase produced by E. coli transformants containing the recombinant plasmid pMWL13 of the present invention.

Figure 4 is a thin layer chromatography photograph of the final degradation product of acacia fruit resin-derived galactomannan by the mannase produced by the E. coli transformant containing the recombinant plasmid pMWL13 of the present invention.

5 is a thin layer chromatography photograph of the final degradation product of mannose, mannose, mannose, mannose, and mannose by mannose produced by an E. coli transformant containing the recombinant plasmid pMWL13 of the present invention. It is also.

The present invention relates to a metase biosynthesis gene and its enzyme production. More specifically, the present invention relates to a nucleotide sequence and an amino acid sequence of a gene encoding a mannanase derived from Bacillus licheniformis WL-12 strain isolated from soybean paste with excellent galactomannan resolution.

Mannanase is an enzyme that decomposes a mannan-containing substance, which is a major component of hemicellulose, and its use is increasing. Hemicellulose is a component of the plant cell wall and is present in combination with cellulose and lignin, the second most abundant polysaccharide in the world after cellulose.

Hemicelluloses include met substances such as galactomannan, arabinogalactan, coniferous glucomannan and galactoglocomannan, which are present in the seeds of legumes. The most important of the availability of the hemicelluloses mannase, along with xylanase and glucanase, is the glycation of hemicellulose, a renewable plant resource on earth, to be converted into a carbon source available to organisms, but as yet not realized. Not in condition.

Hemicellulase is currently in practical use, such as coffee, chocolate, cocoa, tea, cereals and food and pulp.

Grain containing hemicellulose, which is used in food and feed, is processed into hemicellulose to convert it into a form suitable for food and feed, increasing energy yield and reducing viscosity. However, if the available energy content of food and livestock feed is increased, particularly in feed, there is an advantage of lowering the production cost of livestock.

Soy protein is used in livestock feed for dogs, cats, pigs, fish and chickens. Although not a major energy source component, soybean meal is an excellent protein source with essential amino acids. About 10% of the carbohydrates in the soybean meal consist of galactan and pentosan. These carbohydrates are excreted without significant digestion by the unit animal, and the metase breaks down the galactan from carbohydrates, which is the energy source of soybean meal, into small molecules of oligosaccharides or monosaccharides, so that the unit animal can easily metabolize.

Met kinase is produced by microorganisms such as Bacillus subtilis, as well as the bacteria belonging to the genus Pseudomonas and difficulties (Aeromonas), Enterococcus (Enterococcus), Pseudomonas (Pseudomonas), Streptomyces (Streptomyces) fungi and yeasts. And higher plants and animals are also produced to meet metase.

However, impediment actual microorganisms used in the production of the enzyme is mainly encountered in Trichoderma (Trichoderma), Aspergillus (Aspergillus) in fungal strains, fungal-derived kinase met shows a maximum activity in the acidic condition.

On the other hand, the physiological conditions of the digestive system of pigs and chickens vary by site, but the pH condition after the small intestine to which the kinase should act is around 6.5. Therefore, it is required as an enzyme for feed addition, which has a high activity under acidic conditions in neutral vicinity, rather than a fungal-derived mannanase at pH 3.6 to 5.5.

In order to completely decompose and saccharify mannan, the main component of hemicellulose, there are generally three types of enzymes: endo-β-mannanase, exo-β-mannanase and mannosidase ( β-mannosidase should work together. Among them, endomannans catalyze the reaction of random hydrolysis of β-D-1,4-mannopyranosyl bonds of met polysaccharides having a degree of polymerization of mannose tetraose of 4 mannose polymerization, Plays a major role in hydrolysis.

In the present invention, the first endomannanase of these enzymes has been focused on, and for the sake of convenience, this enzyme is referred to as a metnase for convenience.

In view of the above facts, the present inventors have identified Bacillus licheniformis WL-12 strain, which produces a highly active mannase at neutral pH, from a traditional food, Doenjang, and identified it. The gene encoding the aze was cloned and its nucleotide sequence was determined. In addition, the present invention was completed by identifying the reaction characteristics of the mannase produced in the E. coli transformant transformed by the recombinant plasmid containing the mannase gene.

Accordingly, an object of the present invention is to determine the nucleotide sequence of the gene encoding the met kinase from Bacillus licheniformis WL-12 strain that produces a mannase having a high acidity in neutral.

Another object of the present invention is to express the transformed E. coli transformants by recombinant plasmid containing the metase gene to produce a metase.

Another object of the present invention to provide a feed containing the produced met kinase.

The object of the present invention is a solid agar medium in which colonies of Escherichia coli introduced with a gene library prepared using chromosomal DNA extracted from Bacillus licheniformis WL-12 having excellent galactomannan resolution isolated from doenjang. By amplifying agar media containing acacia resin galactomannan and observing galactomannan degradation ring, E. coli transformants containing the mannanase gene were obtained, and recombinant plasmids contained in the transformant were isolated to code the mannase. After identifying the nucleotide sequence of the gene, and preparing a restriction enzyme map, partially purified the met kinase produced by E. coli transformants, the effect of reaction temperature and pH on the activity of the met kinase was analyzed. Mannobiose, Mannotriose, bay including fruit resin galacto The degradation products of Nottetraoz, Mannopentose, and Mantohexose were analyzed and analyzed to confirm their usefulness as food and feed additives.

The present invention is to isolate and identify Bacillus licheniformis WL-12 strain having excellent galactomannan resolution in nature; Extracting the total chromosomal DNA of the isolated Bacillus licheniformis WL-12 strain, treating it with a restriction enzyme, linking it with a plasmid, and introducing it into E. coli to prepare a gene library; The E. coli transformant of the prepared gene library was plated on a solid agar medium, and colonies were formed, and then agar medium containing acacia fruit resin galactomannan was layered to observe the galactomannan degradation ring. Screening the transformants; Extracting a recombinant plasmid from an E. coli transformant containing a mannase gene to reveal a mannase base sequence; After the production and partial purification of the met kinase by expressing the E. coli transformant containing the met kinase gene of the present invention, the activity according to the reaction temperature and pH of the mannase of the present invention is analyzed and the hydrolysis activity according to the type of met step; The acacia fruit resin galactomannan degradation product and the decomposition products of mannobiose, mannose triose, mannose tetraose, mannose pentose and mannose hexose by the mannase of the present invention are prepared by thin layer chromatography.

Hereinafter, the present invention will be described in detail.

In this step, as an intermediate medium for selecting a transformant showing galactomannan degradability among E. coli transformants, 2 g / L of acacia fruit resin galactomannan, 5 g / L of yeast extract, 10 g / L of tryptone, and NaCl 5 LB-galactomannan agar medium consisting of g / L, ampicillin 50 mg / L, agar 8 g / L, and adjusted to pH 7.0 is used.

E. coli transformant culture medium consists of yeast extract 5 g / L, tryptone 10 g / L, NaCl 5 g / L, and ampicillin (50 mg / L) is added to the LB complex liquid medium .

In order to isolate the microorganism producing the mannase, the doenjang sample suspension is incubated in a nutrient plate medium, and the mannase-producing bacteria are separated by observing the galactomannan degradation ring around the colonies formed on the plate medium.

Identification of the isolated strain is carried out by examining the morphological, biochemical properties and nucleotide sequence of the 16S rRNA gene. The characteristics of the isolates were identified to be aerobic, Gram-positive, spore-forming, and short-term bacillus with catalase activity.

Biochemical characterization of Bacillus spp. With a VITEK reader using a back card (BAC card) showed 97% similarity to Bacillus licheniformis and the base of the 16S rRNA gene. As a result of examining the sequence, 99% homology with the gene sequence of Bacillus licheniformis is considered to be the highest similarity.

The separation and identification of Bacillus piece nipo Miss E. coli transformant that for the met kinase gene cloned (Bacillus licheniformis) WL-12 producing a gene library of Bacillus piece nipo miss (Bacillus licheniformis) WL-12 and containing a met kinase gene Screen sieves.

The Bacillus licheniformis WL-12 strain was grown to logarithmic growth and centrifuged to extract chromosomal DNA from the cells, which were then digested with restriction enzyme Sau3AI and electrophoresed to recover DNA fragments of 1 to 10kb in size. do.

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 and plated on LB agar medium for obtaining a transformant containing a metase gene. As Escherichia coli which introduce | transduces the said recombinant plasmid, any thing can be used as long as it can introduce | transduce, express, and maintain the said recombinant plasmid. For example, XL-1, C600, Y1088, Y1090, NM522, K802, JM105 and the like are used. Subsequently, E. coli transformant colonies were formed, followed by incubation for 5 hours in an LB-galactomannan stratified medium, and a transformant showing galactomannan degradation ring was selected.

The E. coli transformants are cultured in LB liquid medium with ampicillin to determine the nucleotide sequence of the gene encoding the mannase. Subsequently, the plasmids obtained by the culture are separated by alkali decomposition.

The recombinant plasmids contained cloned DNA fragments of about 1.3 kb in size, and the recombinant plasmid was named pMWL13. To determine the nucleotide sequence of the cloned 1.3 kb DNA fragment in plasmid pMWL13, fragments were divided into small fragments and inserted into pUC19 using a variety of restriction enzymes. Determine the sequence.

This base sequence contains a total of 1,083 bp of open reading frame corresponding to the structural gene of the met kinase (SEQ ID NO: 1), and the amino acid sequence of the met kinase enzyme inferred from the nucleotide sequence of the structural gene is sequenced. As shown in number 2, the protein is composed of a total of 361 amino acids.

The amino acid sequence of the met kinase derived from the base sequence of the met kinase gene of Bacillus licheniformis WL-12 was found to have the highest similarity in the amino acid sequence of the met kinase of the Bacillus strain. It is confirmed that high homology shows about 80% homology, so that the met kinase gene of Bacillus licheniformis is a novel gene.

E. coli transformants transformed with pMWL13 in LB liquid medium containing ampicillin to investigate the activity according to temperature and pH of recombinant mannase produced by expressing E. coli transformant containing recombinant plasmid pMWL13 The enzyme is purified partially by crushing and concentrating the cultured cells. The enzyme activity was measured by varying the reaction temperature and pH for this enzyme.

As a result, as shown in Figure 3 shows the highest enzyme activity at 65 ℃ and pH 6.0 and at least 90% activity in the pH 5.5 ~ 6.5 range. In particular, at pH 6.5, the physiological condition of the animal intestine, the activity corresponds to more than 93% of the highest activity. In order to measure the enzymatic activity according to the type of mannan, the hydrolytic relative activities of acacia fruit resin galactomannan, guar resin glucomannan and gongnac were compared using galvantomannan as shown in Table 1. It can be seen that the hydrolysis power for the highest.

In order to analyze the met hydrolyzate of the mannase produced by the expression of the E. coli transformant containing the mannase gene of the present invention, the hydrolysis reaction was carried out under optimum reaction conditions using acacia fruit resin galactomannan as a substrate. The final product is analyzed by thin layer chromatography (TLC). As a result, as shown in FIG. 4, mannose, mannobiose, mannose triose and the like are produced as final products. In addition, the mannose of the present invention was reacted with mannose, mannose, mannose, mannose, mannose, and mannose, respectively, and the hydrolysis products were examined by thin layer chromatography. As shown, it was confirmed that mannose was not decomposed, and mannose, mannose, mannose, and mannose were decomposed into mannose, mannose, and mannose.

Therefore, it can be seen that the nanase produced by the nanase gene of the Bacillus licheniformis WL-12 strain of the present invention decomposes a substance to produce a small sugar.

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 Bacillus ( Bacillus Separation and Identification of Genus WL-12

To isolate microorganisms producing mannanase, 1 g of soybean paste was suspended in 10 mL of 0.85% NaCl solution, and an appropriate amount of suspension was added to the nutrient plate with 0.5% acacia locust bean gum. It was plated in a medium (beef extract, 3 g; bacto-peptone, 5 g; water, 1 liter) and incubated at 37 ° C. The galactomannan degradation ring around the colonies formed on the plate medium was observed to select the mannanase producing bacteria, and finally the bacteria growing at 60 ° C were finally separated for 1 week. Name the isolate WL-12 and use the formula milk (1%), starch (0.2%), tributyllin (1%), and to analyze if it can decompose other polymers other than galactomannan. The degradation ring was examined by culturing in a plate medium to which carboxymethyl cellulose (0.5%) was added, respectively. As a result, all of them were decomposed.

Identification of the isolated strain was carried out by examining the morphological, biochemical properties and nucleotide sequence of the 16S rRNA gene. Gram staining and spore staining were performed for morphological observation of the isolates, and VITEK (bioMerieux Inc. France) was used to investigate biochemical properties. In order to identify the isolate WL-12, it was found to be aerobic and Gram-positive bacteria. It was identified as a simple bacillus with spore formation and catalase activity.

In addition, the carbohydrate availability of WL-12 was investigated using BAC CARD. Sucrose, glucose, inositol, arabinose, mannose, salicin, Amygdalin, maltose, trehalose, and palatinose were used, but no other carbohydrates were found.

In view of the morphological and biochemical properties of WL-12, Bacillus Among the genus strains, Bacillus licheniformis had the highest similarity and showed similarity value of 97%.

Additionally, in order to compare the 16S rRNA gene sequence of the WL-12 strain with other strains, it was amplified by PCR using the conserved sequence of the bacterial 16S rRNA gene sequence as a primer and the total chromosomal DNA of the isolate as a template. The base sequences of the DNA fragments were compared with those of other strains. As a result, Bacillus licheniformis The gene showed the highest homology with the nucleotide sequence. In view of the above characteristics, the isolate was determined as Bacillus licheniformis .

Example 2 Bacillus Richenformis ( Bacillus licheniformis ) Screening of the metase gene of WL-12 strain

(One)Step 1: Bacillus licheniformis ( Bacillus licheniformis ) Genetic Library Preparation of WL-12

Bacillus licheniformis WL-12 strain isolated and identified in Example 1 was inoculated into 200 mL of LB liquid medium, shaken and cultured at 37 ° C. to a logarithmic growth period, and the cells recovered by centrifugation of cell culture medium were TEN. SET buffer (20% sucrose, 50 mM Tris-HCl, pH 7.6, 50 mM EDTA) after washing with buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 10 mM NaCl) and adding lysozyme 20 It was suspended in mL and reacted at 37 ° C for 30 minutes. 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 1 to 10 kb. A gene library of Bacillus WL-12 was prepared by ligated plasmid pUC19 completely digested with restriction enzyme BamHI and chromosomal cleavage. Then, T4 DNA ligase was added and reacted for 15 hours at 14 ° C.

(2) Step 2: selection of E. coli transformants having a metase gene

E. coli XL-1 was transformed using a gene library of Bacillus licheniformis WL-12 obtained in step 1 and plated on LB agar medium to obtain about 10,000 transformants. In this case, E. coli may be selected from C600, Y1088, Y1090, NM522, K802 and JM105 instead of E. coli XL-1. LB-galactomannan medium was layered on these transformants to select a transformant showing galactomannan degradation ring around the colony for 1 week to obtain an E. coli transformant containing a mannanase gene.

Example 3 Determination of Gene Sequencing Encoding the Mannase of the Invention

Alkali-decomposition method of the transformants obtained in step 2 of Example 2 from the cells obtained in LB medium added with ampicillin (50 mg / L) (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 1.3 kb, which was named pMWL13. A restriction map of pMWL13 is shown in FIG. 1.

In Figure 1, Man is a DNA fragment containing the metase gene of the present invention.

To determine the nucleotide sequence of the cloned 1.3 kb DNA fragment in plasmid pMWL13, the fragment was divided into small fragments and inserted into pUC19 using various restriction enzymes, resulting in a total of 1,300 bp by means of the dedeoxynucleotide sequencing method. The base sequence was determined. In this base sequence, a total of 1,083 bp open reading frames corresponding to the structural genes of the met kinase existed (SEQ ID NO: 1). The amino acid sequence of the mannase enzyme deduced from the nucleotide sequence of the structural gene is shown in SEQ ID NO: 2 and is a protein consisting of a total of 361 amino acids.

The amino acid sequence of the met kinase derived from the base sequence of the met kinase of Bacillus licheniformis WL-12 was found to have the highest similarity between the amino acid sequence of the met kinase of the Bacillus strain. The homology was confirmed to show about 80% homology, so the met kinase gene of Bacillus licheniformis was a novel gene.

Example 4 Investigation of Mannanase 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 met kinase produced by expressing the E. coli transformant containing the recombinant plasmid pMWL13 prepared in Example 3. E. coli transformants transformed with pMWL13 were inoculated into one LB liquid medium, shaken and cultured at 37 ° C. for 16 hours, and the cultured cells were ultrasonically crushed, and the lysate was fractionated by ammonium sulfate fractionation and ion chromatography. After purification, the partially purified enzyme was reacted with acacia fruit resin galactomannan at different reaction temperatures and pHs to determine the activity of the metnanase.

As a result, as shown in Figure 3 showed the highest enzyme activity at 65 ℃ and pH 6.5, and showed a 90% or more activity in the pH 5.5 ~ 6.5 range. In particular, at pH 6.5, the physiological condition of the animal intestine, it showed activity corresponding to more than 93% of the highest activity.

In addition, the hydrolysis relative activity of acacia fruit resin galactomannan, guar resin glucomannan and gongnac was compared to measure enzyme activity according to the type of mannan using partially purified mannanase. At this time, the reaction conditions were performed at pH 6.0 and 50 ℃. As a result, as shown in Table 1, it was found that the high hydrolytic ability to galactomannan, gongnac, glucomannan in order.

Looking at these results, it can be seen that the met kinase produced by the E. coli transformant containing the met kinase gene of the Bacillus licheniformis WL-12 strain of the present invention is suitable for feed addition.

Comparison of activities according to the met type of met kinase produced by the expression of transformed Escherichia coli Type of substrate Relative Activity (%) Acacia Fruit Resin Galactomannan 100.0 Gongnac 91.6 Guar resin glucomannan 12.2

Example 5 Investigation of Met Hydrolysates of Mannanase Produced by Expression of Transfected Escherichia Coli

In order to analyze the final reaction product by the partially purified mannanase obtained in Example 4, the hydrolysis reaction was carried out under optimum reaction conditions using acacia fruit resin galactomannan as a substrate, and the final hydrolysis product was subjected to thin layer chromatography (TLC). ).

As a result, as shown in FIG. 4, mannose, mannobiose, mannose, and the like were produced as final products.

In addition, after the reaction of the mannanase of the present invention using mannobiose, mannose triose, mannose tetraose, mannose, and mannohexose as substrates, the hydrolysis products were examined by thin layer chromatography, and FIG. 5. Mannobiodes were not decomposed as shown in the following, and it was confirmed that the mannotriose, the mannotetraose, the mannose, and the mannohexose were decomposed into mannose, mannose, and mannose.

Therefore, the metase of the present invention has been shown to break down the met material to produce met sugars.

As described above, the antagonist produced by the expression of E. coli transformed by introducing the antagonist gene of the Bacillus licheniformis WL-12 strain of the present invention may be derived from acacia fruit resin, gongnac, and guar resin-based nannan. Not only does it have an excellent effect on hydrolysis, but the hydrolysis product is very useful for the food and beverage industry because it is a small amount of mannose, including mannose, mannose bios and mannose, which act as growth factors for the intestinal useful bacteria of animals. It is an invention.

<110> CTC BIO, INC. <120> Gene coding mannanase and recombinant mannanase expressed from          transformant <130> 2959 <150> KR1020030042182 <151> 2003-06-26 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1083 <212> DNA <213> Bacillus licheniformis WL-12 <220> <221> CDS (222) (1) .. (1083) <400> 1 tta gtg aaa aaa aac atc gtt tgt tca atc ttc gcg ctg ctc ctt gct 48 Leu Val Lys Lys Asn Ile Val Cys Ser Ile Phe Ala Leu Leu Leu Ala   1 5 10 15 ttt gcc gtt tcg cag ccg agc tat gca cac acc gtt tct ccg gtg aac 96 Phe Ala Val Ser Gln Pro Ser Tyr Ala His Thr Val Ser Pro Val Asn              20 25 30 ccg aat gcc cag ccg acg acg aaa gcg gtg atg aac tgg ctc gcc cac 144 Pro Asn Ala Gln Pro Thr Thr Lys Ala Val Met Asn Trp Leu Ala His          35 40 45 ctg ccc aat cgg acg gaa agc cgg gtg atg tcc ggg gcg ttc gga gga 192 Leu Pro Asn Arg Thr Glu Ser Arg Val Met Ser Gly Ala Phe Gly Gly      50 55 60 tac agc ctc gac aca ttt tca acg gct gaa gcc gac cgg atc aaa cag 240 Tyr Ser Leu Asp Thr Phe Ser Thr Ala Glu Ala Asp Arg Ile Lys Gln  65 70 75 80 gca aca gga cag ctg ccg gcc ata tac ggc tgc gat tat gca aga gga 288 Ala Thr Gly Gln Leu Pro Ala Ile Tyr Gly Cys Asp Tyr Ala Arg Gly                  85 90 95 tgg ctg gag ccg gaa aag atc gcc gat acg att gac tac agc tgc aac 336 Trp Leu Glu Pro Glu Lys Ile Ala Asp Thr Ile Asp Tyr Ser Cys Asn             100 105 110 cgt gat ttg atc gca tac tgg aaa agc gga ggc att ccg caa atc agc 384 Arg Asp Leu Ile Ala Tyr Trp Lys Ser Gly Gly Ile Pro Gln Ile Ser         115 120 125 atg cac ctc gca aac ccc gcg ttt act tcg ggt cat tat aaa act cag 432 Met His Leu Ala Asn Pro Ala Phe Thr Ser Gly His Tyr Lys Thr Gln     130 135 140 att tca aac agc cag tat gag aga att tta gat tct tcc act cct gaa 480 Ile Ser Asn Ser Gln Tyr Glu Arg Ile Leu Asp Ser Ser Thr Pro Glu 145 150 155 160 gga aag cgg ctt gag gcg atg ctg agc aaa atc gcg gac ggc ctc cag 528 Gly Lys Arg Leu Glu Ala Met Leu Ser Lys Ile Ala Asp Gly Leu Gln                 165 170 175 gag ctt gaa aat gaa ggc gtg ccc gtt cta ttc aga ccc ctt cac gaa 576 Glu Leu Glu Asn Glu Gly Val Pro Val Leu Phe Arg Pro Leu His Glu             180 185 190 atg aac ggc gaa tgg ttc tgg tgg ggg ctg acg caa tat aat caa aaa 624 Met Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Gln Tyr Asn Gln Lys         195 200 205 gac agc gaa aga atc tcc ttg tac aaa cag ctc tat gtg aaa atc tat 672 Asp Ser Glu Arg Ile Ser Leu Tyr Lys Gln Leu Tyr Val Lys Ile Tyr     210 215 220 gac tat atg aca aaa aca aga ggc ctg gat cat ctc ttg tgg gtg tat 720 Asp Tyr Met Thr Lys Thr Arg Gly Leu Asp His Leu Leu Trp Val Tyr 225 230 235 240 gcg ccg gac gcc aac aga gac ttt aaa acg gac ttt tat ccg ggc gca 768 Ala Pro Asp Ala Asn Arg Asp Phe Lys Thr Asp Phe Tyr Pro Gly Ala                 245 250 255 tca tat gtg gac att gtc ggg ctt gac gct tat ttt gat gac ccg tac 816 Ser Tyr Val Asp Ile Val Gly Leu Asp Ala Tyr Phe Asp Asp Pro Tyr             260 265 270 gcc att gat ggc tac gaa gag ctc aca tcg ctg aac aag ccg ttt gcc 864 Ala Ile Asp Gly Tyr Glu Glu Leu Thr Ser Leu Asn Lys Pro Phe Ala         275 280 285 ttt aca gaa gtc gga ccg cag acg aca aac ggc ggg ctg gat tac gcg 912 Phe Thr Glu Val Gly Pro Gln Thr Thr Asn Gly Gly Leu Asp Tyr Ala     290 295 300 cgg ttt atc cat gcc atc aaa gaa aaa tac ccg aaa acg acg tac ttc 960 Arg Phe Ile His Ala Ile Lys Glu Lys Tyr Pro Lys Thr Thr Tyr Phe 305 310 315 320 ctg gcg tgg aac gat gag tgg agc ccg gct gtg aat aag gga gcg gac 1008 Leu Ala Trp Asn Asp Glu Trp Ser Pro Ala Val Asn Lys Gly Ala Asp                 325 330 335 acc ctc tat ctt cat cca tgg acg ctg aat aaa gga gag ata tgg gac 1056 Thr Leu Tyr Leu His Pro Trp Thr Leu Asn Lys Gly Glu Ile Trp Asp             340 345 350 ggc gat tct ttg acg cct gtc gtg gaa 1083 Gly Asp Ser Leu Thr Pro Val Val Glu         355 360 <210> 2 <211> 361 <212> PRT <213> Bacillus licheniformis WL-12 <400> 2 Leu Val Lys Lys Asn Ile Val Cys Ser Ile Phe Ala Leu Leu Leu Ala   1 5 10 15 Phe Ala Val Ser Gln Pro Ser Tyr Ala His Thr Val Ser Pro Val Asn              20 25 30 Pro Asn Ala Gln Pro Thr Thr Lys Ala Val Met Asn Trp Leu Ala His          35 40 45 Leu Pro Asn Arg Thr Glu Ser Arg Val Met Ser Gly Ala Phe Gly Gly      50 55 60 Tyr Ser Leu Asp Thr Phe Ser Thr Ala Glu Ala Asp Arg Ile Lys Gln  65 70 75 80 Ala Thr Gly Gln Leu Pro Ala Ile Tyr Gly Cys Asp Tyr Ala Arg Gly                  85 90 95 Trp Leu Glu Pro Glu Lys Ile Ala Asp Thr Ile Asp Tyr Ser Cys Asn             100 105 110 Arg Asp Leu Ile Ala Tyr Trp Lys Ser Gly Gly Ile Pro Gln Ile Ser         115 120 125 Met His Leu Ala Asn Pro Ala Phe Thr Ser Gly His Tyr Lys Thr Gln     130 135 140 Ile Ser Asn Ser Gln Tyr Glu Arg Ile Leu Asp Ser Ser Thr Pro Glu 145 150 155 160 Gly Lys Arg Leu Glu Ala Met Leu Ser Lys Ile Ala Asp Gly Leu Gln                 165 170 175 Glu Leu Glu Asn Glu Gly Val Pro Val Leu Phe Arg Pro Leu His Glu             180 185 190 Met Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Gln Tyr Asn Gln Lys         195 200 205 Asp Ser Glu Arg Ile Ser Leu Tyr Lys Gln Leu Tyr Val Lys Ile Tyr     210 215 220 Asp Tyr Met Thr Lys Thr Arg Gly Leu Asp His Leu Leu Trp Val Tyr 225 230 235 240 Ala Pro Asp Ala Asn Arg Asp Phe Lys Thr Asp Phe Tyr Pro Gly Ala                 245 250 255 Ser Tyr Val Asp Ile Val Gly Leu Asp Ala Tyr Phe Asp Asp Pro Tyr             260 265 270 Ala Ile Asp Gly Tyr Glu Glu Leu Thr Ser Leu Asn Lys Pro Phe Ala         275 280 285 Phe Thr Glu Val Gly Pro Gln Thr Thr Asn Gly Gly Leu Asp Tyr Ala     290 295 300 Arg Phe Ile His Ala Ile Lys Glu Lys Tyr Pro Lys Thr Thr Tyr Phe 305 310 315 320 Leu Ala Trp Asn Asp Glu Trp Ser Pro Ala Val Asn Lys Gly Ala Asp                 325 330 335 Thr Leu Tyr Leu His Pro Trp Thr Leu Asn Lys Gly Glu Ile Trp Asp             340 345 350 Gly Asp Ser Leu Thr Pro Val Val Glu         355 360

Claims (6)

A nucleotide encoding a mannase represented by the nucleotide sequence of SEQ ID NO: 1. Cleavage map comprising the nucleotide sequence of claim 1 recombinant vector described in FIG. E. coli transformed with the recombinant vector according to claim 2. Mannanase protein represented by the amino acid sequence of SEQ ID NO: 2. A method for producing a mannase, which is obtained from the culture of Escherichia coli according to claim 3. Livestock feed containing the recombinant mannanase according to claim 4.

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