KR20110119386A - Gene coding for cellulase from bacillus velezensis a-68 and production method of cellulase by transformed escherichia coli a-68 thereof - Google Patents

Abstract

PURPOSE: A gene of Bacillus velezensis-derived cellulase and a recombinant E.coli A-68 recombinant strain transformed by introducing the same are provided to enhance productivity and efficiency of strains. CONSTITUTION: A Bacillus velezensis A-68(deposit number KACC 91178P)-derived cellulase is encoded by a gene having a sequence of sequence number 1. A recombinant vector for expressing cellulase in E.coli is prepared by linking the gene of sequence number 1 to p-TGEM T-easy vector. The recombinant E.coli A-68(deposit number KACC 91335P) is prepared by transforming through introduction of the recombinant vector pTA-68. A method for producing cellulase comprises a step of culturing the recombinant E.coli A-68 in a medium containing 50g/L of rice bran and 2.5 g/L of yeast extract at pH 7.3 and 35°C.

Description

The fibrinolytic enzyme gene derived from Bacillus bellenchensis A-68 and the transformed Escherichia coli A-68 strain by introducing the same, and the production method of fibrinolytic enzyme using the same {Gene coding for cellulase from Bacillus velezensis A-68 and production method of cellulase by transformed Escherichia coli A-68 thereof}

The present invention relates to a gene of a fibrinolytic enzyme protein derived from Bacillus velezensis A-68, strain accession number KACC 91178P, and a recombinant strain transformed by introducing the same, Escherichia coli A-68 (Escherichia coli A-68, strain Accession No. KACC 91335P), and more specifically, a fibrinolytic enzyme gene was isolated from the Bacillus belenchesis A-68 strain isolated and identified in the ocean, and the gene of the fibrinolytic enzyme was isolated using an expression vector, and E. coli (E. coli). ) To prepare a transformed recombinant strain Escherichia coli A-68, and use this strain to increase the productivity of fibrinolytic enzyme.

Cellulose is a linear polymer of glucose units linked by β-1,4-glucosidic bonds and is the main component of wood biomass. Cellulose is finally decomposed into glucose by fibrinolytic enzyme (cellulase), so bioenergy can be produced by producing ethanol using this.

Therefore, in the United States, the National Renewable Energy Laboratory (NREL) of the Department of Energy (DOE), and Genencor International and Novozymes, which mainly produce enzymes, are mainly trico. Research is being conducted to produce more efficient and inexpensive fibrinolytic enzymes using the enzyme system obtained from Trichoderma reesei. In Japan, the Forest Research Institute uses supercritical water to efficiently produce sugar from wood. Research is in progress for the technology development of the fast saccharification method. In addition, Watanabe and others of Kyoto University are conducting research on the conversion of wood to useful substances such as ethanol after selectively decomposing lignin using white erosion bacteria. In Korea, many studies are being conducted for the practical use of technology for producing bioethanol from lignocellulosic biomass, but no great results have been achieved.

In general, three groups of hydrolytic enzymes, endoglucanase (EG), exoglucanase (CBH), and β-glucosidase (BGL), are involved in the degradation of cellulose by fungi. Is considered. EG randomly hydrolyzes β-1,4 bonds in the inner chain of cellulose, and CBH gradually hydrolyzes the reduced and non-reduced ends of the cellulose polymer produced by hydrolysis to EG, resulting in a cell with two molecules of glucose bound. Produces bios. Since cellobiose acts as a potent inhibitor for EG and CBH, BGL hydrolyzes these cellobiose to eliminate the strong inhibition of EG and CBH, respectively. In general, EG can hydrolyze the amorphous region of a cellulose molecule, and CBH can bind to the crystalline region to produce soluble reducing sugar from the reducing and non-reducing ends of the cellulose chain.

Cellulase enzyme is obtained from bacteria and fungi, as a mold to produce cellulose-decomposing enzyme and the like, Aspergillus (Aspergillus), Penny room Solarium (Penicillium), Fusarium (Fusarium), Spokane roteuri glutamicum (Sporotricum), bacteria in the cell V. (Cellovibrio), Clostridium (Clostridium), acetonitrile Vibrio (Acetovibrio), bacteriophage des (Bacteriodes), cellulite in Pseudomonas (Cellulomonas), Pseudomonas (Pseudomonas) also actinomycetes the thermopile liquid Martino fine test (Thermoactinomycetes) , Thermomonospora (Thermomonospora) and Streptomyces ( Streptomyces ) enzymatic studies have been continuously conducted.

Looking at the previously registered patented technology for fibrinolytic enzyme, yeast that secretes cellulase and feed additive containing the same (Korean Patent No. 10-0377954), Chrysosporium fibrinolytic enzyme and method of use (Korean registered patent) No. 10-0641878), a novel Bacillus 79-23 strain producing a neutral fibrinase ((Korea Patent Registration No. 10-0318554), Aspergillus strain producing fibrinase and xylanase, and thereby Prepared enzymes and solid cultures (Republic of Korea Patent No. 10-0449170), and the like.

In particular, the present inventors identified Bacillus beretensis A-68 having the ability to produce fibrinase from seawater in the Korean Patent Registration No. 10-0738007, "New microorganism Bacillus beretensis A-68 and its use," By culturing in a medium containing one or more selected from the group consisting of rice bran, rice husk, starch and CMC as a carbon source, polysaccharides can be produced at a lower cost compared to the existing method of producing polysaccharides using an expensive carbon source. We have established an eco-friendly polysaccharide production process that reduces environmental pollution by using rice bran and rice husk, which are one of the causes of contamination, to reduce environmental pollution. Korean Patent No. 10-0882021 “Bacillus beretensis in a medium containing rice hull By producing the Bacillus beretensis A-68 strain, which produces fibrinolytic enzyme using a liquid culture method in “Production Method of A-68 Strain”, it increases the growth rate and economically mass-produces fibrinolytic enzyme. It has improved productivity.

On the other hand, the existing method of producing fibrinolytic enzymes on an industrial scale is producing fibrinolytic enzymes by solid state fermentation of fungi such as Tricoderma species or Aspergillus species. Silver is inefficient compared to liquid culture, and since the growth rate of mold is lower than that of bacteria, the productivity is low, and the cost of fibrinolytic enzyme is high. In order to improve the productivity of fibrinolytic enzyme, the fibrinolytic enzyme gene of fungi is introduced into bacteria such as E. coli , and the mutant strain containing the fibrinolytic enzyme gene is liquid cultured to increase the productivity of fibrinolytic enzyme. Although research is being conducted, there is no industrialization yet, and the production efficiency is low.

Accordingly, as a result of continuing research to increase the productivity of fibrinolytic enzyme, the present inventors have isolated and identified strains with excellent fibrinolytic ability from the soil. After separating the fibrinolytic enzyme gene using the identified strain, E. coli ( E coli ), it was confirmed that the production capacity of fibrinolytic enzyme can be improved through gene recombination, and the present invention was reached.

Therefore, it is an object of the present invention to isolate the fibrinase protein gene from the strain Bacillus velezensis A-68 (Bacillus velezensis A-68, strain accession number KACC 91178P) isolated and identified in seawater, and use the fibrinase gene as a foreign host. It is to increase fibrinolytic enzyme production by culturing this strain after introducing into it to prepare a recombinant strain.

The above object of the present invention is to isolate and identify the fibrinolytic enzyme-producing strain, isolate the fibrinolytic enzyme gene produced by the strain, and confirm the nucleotide sequence to confirm that the fibrinase-producing strain is a novel nucleotide sequence, and the optimal production conditions for the fibrinase protein After confirming, it was achieved by confirming that the fibrinolytic enzyme productivity was increased compared to the existing strain by introducing it into a foreign host.

The present invention is characterized by providing a fibrinolytic enzyme protein derived from Bacillus velezensis A-68 (Bacillus velezensis A-68, strain accession number KACC 91178P), which is encoded by the DNA sequence of SEQ ID NO: 1.

The Bacillus belenchesis A-68 strain was identified through 16s rDNA and gyr A sequencing analysis, and it is characterized by being able to grow using agricultural byproducts such as rice husk, rice bran, and yeast extract as a nutrient source.

The present invention is characterized in that it provides a recombinant vector containing the gene of SEQ ID NO: 1, and a preferred example of the recombinant vector is a recombinant vector pTA- prepared by linking the gene of SEQ ID NO: 1 to a pGEM T-easy vector and then binding. It could be 68.

The present invention is characterized by providing a strain of Escherichia coli A-68 (Escherichia coli A-68, strain accession number KACC 91335p) transformed by the introduction of the recombinant vector pTA-68 to increase fibrinolytic enzyme production capacity. .

The present invention is to prepare a recombinant vector by isolating the fibrinolytic enzyme gene derived from Bacillus velezensis A-68 (Bacillus velezensis A-68, strain accession number KACC 91178P) strain, and introducing it into Escherichia coli strain to transform It relates to the converted recombinant strain Escherichia coli A-68 (Escherichia coli A-68, strain accession number KACC 91335p), and a recombinant produced by introducing a fibrinolytic enzyme gene having excellent fibrinolytic activity into E. coli (E.coli). As strains can increase productivity and efficiency compared to existing strains, mass production of fibrinolytic enzymes used in grain processing, ethanol fermentation from biological resources, liquor production, waste treatment and laundry or dishwashing detergent at an economical production cost There is an outstanding effect that has made this possible.

FIG. 1 shows starch, protein, and fibrinolytic enzyme activity after separating various types of microorganisms collected from seawater.
Figure 2 is a schematic diagram of the phylogenetic tree between Bacillus belenchesis A-68 strain and other strains based on partial determination of the nucleotide sequence of the gyrase A gene.
3 shows a map of the pGEM T-easy vector.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Examples of the present invention include Example 1 for isolating and identifying a fibrinolytic enzyme-producing strain, Example 2 for separating and sequencing a fibrinase gene from the strain, and introducing the fibrinolytic enzyme into E. coli It consists of Example 3 and Experimental Examples 1, 2 and 3 for establishing the optimal conditions for the fibrinolytic enzyme.

Example 1: Isolation, identification and culture of fibrinase-producing strain

Isolation of strains producing fibrinolytic enzymes in seawater

A certain amount of seawater collected from the East and South Seas of Gyeongsang-do was diluted appropriately in sterilized physiological saline (0.85% NaCl), and then cultured at 30°C using marine agar, the microorganisms were separated by plate smearing. . The isolated microorganism was inoculated with one platinum tooth in Marine broth, and the seed culture solution cultured at 25 to 35°C at 150 to 200 rpm for 55 to 85 hours was inoculated with 3 to 5% (v/v) in the electric medium. And, incubated for 3 to 5 days at 150 to 200 rpm at 25 to 35 ℃. The culture medium was centrifuged in the range of 5000 to 9000 × g for 10 to 30 minutes to remove the cells, and the supernatant was used for the fibrin degradation test.

40 μL of the supernatant was dropped on a paper disk of 1.0 cm in diameter, dried, and placed on agar medium to which 2.0% (w/v) of carboxymethyl cellulose (CMC), starch and protein (skim milk) was added. Placed and incubated for 3 days at 37°C, each degrading enzyme was identified through the size of the inhibitory source, and the productivity of the fibrinase was compared with the size of the resulting inhibitory source. As a result of comparing the size of the jersey as shown in FIG. 1, the strain having the best productivity of fibrinolytic enzyme was selected.

Identification of strains producing fibrinolytic enzyme

For the identification of the selected strain, the base sequence of 16S rDNA and gyrase A was partially determined. For 16s rDNA sequencing, 5'-AGG AGG AAA AGA TCA GAT ATG AAA CGG TCA ATC-3' and 5'-TCC AGT ATT TCA TCC ACA ACG ACC TCC-3' were used as primers, and under the following conditions PCR was performed to amplify the 16s rDNA gene. PCR reaction conditions were 94 ℃, 3 minutes → [94 ℃, 30 seconds → 50 ℃, 30 seconds, → 72 ℃, 5 minutes] 30 times → post-elongation: 72 ℃, 10 minutes.

For gyrase A sequencing, 5'-CAG TCA GGA AAT GCG TAC GTC CTT-3' and 5'- CAA GGT AAT GCT CCA GGC ATT GCT-3' were used as primers and the following conditions PCR was performed under the following conditions, and a gyrase A gene was amplified. PCR reaction conditions were 94 ℃, 3 minutes → [94 ℃, 30 seconds → 50 ℃, 30 seconds, → 72 ℃, 5 minutes] 30 times → post-elongation: 72 ℃, 10 minutes

As a result of identification by comparing the sequence with the strains reported in the database on Genbank, it was identified as Bacillus velezensis , a type of bacteria, and named as Bacillus velezensis A-68. It was deposited with the Korea Agricultural Microbial Conservation Center under the strain deposit number KACC 91178P.

The 16S rDNA sequence and the Xyrease A sequence of Bacillus velezensis A-68 (KACC 91178P) were amplified through PCR, and genetic associations with other species were investigated through Genbank's database. Tables 1 and 2 show similarities between 16S rDNA and other strains analyzed based on the partial nucleotide sequence of the gyrase A gene.

Similarity of Bacillus belenchesis A-68 strain (16S rDNA) Strain Similarity
(%) Nucleotide differences/
compared Bacillus velezensis LMG 22478T 99.77 2/871 Bacillus amyloliquefaciens ATCC 23350T 99.65 3/865 Bacillus atrophaeus JCM 9070T 99.54 4/874 Bacillus vallismortis DSM1103T 99.43 5/874 Bacillus subtilis subsp .subtilis NCDO 1769T 99.31 6/870 Bacillus majavensis IFO 15718T 99.08 8/874 Bacillus subtilis subsp .subtilis DSM 10T 99.08 8/873 Bacillus licheniformis DSM 13T 97.59 21/873 Bacillus pumilus NCDO 1766T 96.47 30/850 Bacillus carboniphilus JCM 973T 95.06 43/870 Bacillus oleronius DSM 9356T 94.68 46/865 Bacillus sporothermodurans DSM 10599T 94.59 47/868 Bacillus indicus Sd/3T 94.54 47/861 Bacillus firmus IAM 12464 94.23 50/866 Bacillus methanolicus NCIMB 13114T 94.15 51/872 Bacillus azotoformans ATCC 29788T 93.98 50/830

Similarity of Bacillus belenchesis A-68 strain (gyrase A) Strain Similarity
(%) Nucleotide differences/
compared Bacillus vlezensis LMG 22478T 99.77 11/896 Bacillus amyloliquefaciens KCTC 1660T 95.95 38/938 Bacillus mojavensis NRRL B-14698T 84.10 124/780 Bacillus subtilis subsp. spizizenii NRRL 82.84 139/810 Bacillus subtilis subsp. subtilis KCTC3135T 82.48 133/759 Bacillus atrophaeus KCTC 3701T 82.07 156/870 Bacillus vallismortis NRRL B-14890T 81.49 167/902 Bacillus licheniformis KCTC 1918T 78.25 186/855

Fig. 2 shows the similarity with strains of other species analyzed based on the partial nucleotide sequence of the gene. The mycological properties of this strain analyzed through nucleotide sequence were similar to that of conventional Bacillus belenchesis, and identified as Bacillus velezensis A-68, strain accession number KACC 91178P) through 16s rDNA and gyr A nucleotide sequences. I did. It was confirmed that this strain was grown using agricultural by-products such as rice husk and rice bran and yeast extract as a nutrient source.

Example 2: Isolation and nucleotide sequence analysis of fibrinolytic enzyme from Bacillus belenchesis A-68 strain

In order to clone the fibrinolytic enzyme gene of the strain selected in Example 1, a primer prepared based on a Bacillus-derived gene known from GenBank (p1 5'-AGG AGG AAA AGA TCA GAT ATG AAA CGG TCA ATC-3', p2 PCR was performed using 5'-TCC AGT ATT TCA TCC ACA ACG CAA ACC TCC-3'. The initial denaturation step was performed at 94°C for 1 minute, 60°C for 1 minute, 72°C for 2 minutes, and 35 cycles. The stretching step was performed at 72° C. for 10 minutes.

Through PCR, a gene having a size of 1.5 kb could be obtained. In order to analyze the nucleotide sequence of the gene, it was performed by ligation to a pGEM T-easy vector (Promega, USA). The pGEM T-easy vector used as the gene cloning vector is shown in FIG. 3 and is a vector commonly used for DNA sequence analysis. While typical vectors are mainly circular plasmids, the pGEM T-easy vector is straight, and has a T sequence with overhangs at both ends of the straight DNA. This is the principle of recombination in E. coli after making it into a circular shape by connecting the pGEN T-easy vector.

The nucleotide sequence of the fibrinolytic enzyme gene was analyzed using a DNA sequencer. The amino acid sequence of the fibrinolytic enzyme gene of Bacillus belenchesis A-68 is attached to SEQ ID NO: 1. As a result of the fibrinolytic enzyme analysis, it was confirmed that it was composed of 1497 bp nucleotides encoding 499 amino acids, and its molecular weight was 55,059 Da. As a result of examining the homology of the amino acid sequence with other fibrinolytic enzymes of the genus Bacillus registered in GenBank, Bacillus subtilis M28332.1 and 98%, Bacillus WRD-2 AY859492.1 and 98%, and Bacillus amyl It showed 94% homology with Lorequacy FZB AJ576102.1.

Example 3: Bacillus Belenchesis A-68 Introduction of derived fibrinolytic enzyme into foreign host

Introduction to recombinant vectors

The fibrinolytic enzyme gene of Bacillus belenchesis A-68 was ligated to a pGEM T-easy vector and then ligated using a ligase. The recombinant vector was named pTA-68.

Introduction to heterogeneous host (Esherician coli)

An experiment was performed in which the pTA-68 vector was introduced into Escherichia coli JM109. The E. coli JM109 is a widely used strain and was used after being sold in the Biological Resource Center. The transformed strain into which the pTA-68 was introduced was named Escherichia coli A-68 and was deposited with the Agricultural Biotechnology Research Institute as strain accession number KACC 91335p on November 26, 2007.

Experimental Example 1: Escherichia coli A-68 according to a carbon source and a nitrogen source ( Escherichia coli A-68)-derived fibrinolytic enzyme activity measurement

Recombinant strain Escherichia coli A-68 was cultured at 35°C for 3 days using various carbon sources (glucose, fructose, malt sugar, sugar, rice bran, rice husk) and nitrogen sources (malt extract, yeast extract, peptone, tryptone, etc.) as a medium. I did it. The growth of the cells and the activity of the fibrin degrading enzyme produced in the culture medium were measured to confirm the types of the optimal carbon source and nitrogen source.After preparing a medium by combining the optimal carbon source and the optimal nitrogen source of different concentrations, the fibrin degrading enzyme was produced. The strain was inoculated, and the growth of the cells and the activity of the fibrinolytic enzyme produced in the culture medium were measured to optimize the concentration of the optimal carbon source and the optimal nitrogen source, respectively.

Optimal carbon source and nitrogen source measurement

20.0 g/L carbon source (glucose, fructose, malt sugar, sugar, rice bran, rice husk), 2.5 g/L nitrogen source (malt extract, yeast extract, peptone, tryptone) in order to obtain the optimal carbon source and nitrogen source of the fibrinolytic enzyme of Example 3 Etc.), 0.1% sodium chloride, 0.5% potassium hydrogen phosphate (K ₂ HPO ₄ ), 0.02% magnesium sulfate (MgSO ₄ 7H ₂ O), and 0.06% ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) in 500 mL The flask was cultured at 30° C. for 3 days using 200 mL as the total amount. After collecting the culture solution for each time period, the cells were removed using a centrifuge at 6000 rpm for 10 minutes. Using the removed supernatant, take 0.5 mL of the fibrinolytic enzyme supernatant dissolved in a buffer solution (10 mM sodium phosphate buffer pH 6.5), add 0.5 mL of 1% (w/v) carboxymethyl cellulose solution, and then at 50°C for 20 minutes. After reacting, the reducing sugar present in the reaction solution was measured by the DNS method to examine the activity of fibrinolytic enzyme.

465.8 in a medium using rice husk as a carbon source and yeast extract as a nitrogen source, as shown in Table 3 below. The highest fibrinolytic enzyme activity of U/mL was confirmed.

Effects of Optimal Carbon and Nitrogen Sources on Fibrinolytic Enzyme Activity Produced by Escherichia coli A-68 Strains CMCase (U/mL)
Nitrogen sources Carbon sources Glucose Fructose Maltose Starch Rice bran Rice hulls Malt extract 91.1 ± 9.4 47.4 ± 6.4 88.1 ± 8.6 84.5 ± 7.4 256.1 ± 23.2 329.6 ± 27.3 Peptone 72.2 ± 6.8 55.9 ± 6.3 67.6 ± 7.7 56.7 ± 6.3 222.1 ± 24.4 245.8 ± 22.6 Tryptone 83.2 ± 8.5 43.4 ± 5.2 87.4 ± 6.7 114.4± 7.6 249.8 ± 23.1 326.0 ± 28.2 Yeast extract 106.4± 9.3 72.3 ± 9.1 135.1 ± 12.9 68.5 ± 7.8 343.2 ± 27.7 465.8 ± 32.3 Ammonium chloride 93.1 ± 8.5 51.3 ± 6.2 67.2 ± 5.4 77.6 ± 8.1 167.8 ± 14.2 273.3 ± 26.4 Ammonium nitrate 78.5 ± 6.8 66.8 ± 7.4 84.1 ± 6.3 245.2 ± 22.1 254.1 ± 18.7 224.3 ± 18.6

Optimal carbon source and nitrogen source concentration

This is an experiment to confirm the optimal concentration of rice husk and yeast extract determined in the previous experiment. 0-100 g/L rice husk and 0-100 g/L yeast extract, 0.1% sodium chloride, 0.5% potassium hydrogen phosphate (K ₂ HPO ₄ ), 0.02 Combine% magnesium sulfate (MgSO ₄ ·7H ₂ O) and 0.06% ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and incubate at 30°C for 3 days using 200 mL as a total amount in a 500 mL flask. After collecting the culture solution for each time period, the cells are removed using a centrifuge at 6000 rpm for 10 minutes. Using the removed supernatant, take 0.5 mL of the fibrinolytic enzyme supernatant dissolved in a buffer solution (10 mM sodium phosphate buffer pH 6.5), add 0.5 mL of 1% (w/v) carboxymethyl cellulose solution, and then at 50°C for 20 minutes. After reacting, the reducing sugar present in the reaction solution was measured by the DNS method to examine the activity of fibrinolytic enzyme.

As shown in Table 4 below, the fibrinolytic enzyme produced by the recombinant strain Escherichia coli A-68 has the highest activity of 531.7 U/mL in the combination of 50.0 g/L of rice husk and 2.5 g/L of yeast extract. Indicated.

Effect of Optimal Carbon and Nitrogen Source Concentrations on Fibrinolytic Enzyme Activity Produced by Escherichia coli A-68 Strains CMCase (U/mL)
YE
(g/L) Rice hulls (g/L) 0.0 10.0 20.0 30.0 50.0 75.0 100.0 0.0 23.5 ± 5.4 114.8 ± 9.6 182.4 ± 16.2 263.6 ± 23.4 271.5 ± 24.7 240.5 ± 22.1 215.8 ± 17.7 0.5 35.6 ± 4.8 167.4 ± 15.7 287.2 ± 26.2 325.4 ± 27.5 365.7 ± 31.3 317.2 ±27.8 264.5 ± 21.2 1.0 56.2 ± 5.2 186.3 ± 17.4 315.6 ± 27.7 421.5 ± 34.8 485.6 ± 36.0 387.2 ± 31.4 357.9 ± 29.3 2.5 76.8 ± 8.8 214.2 ± 23.4 421.6 ± 32.1 492.1 ± 34.2 531.7 ± 32.6 445.8 ± 37.8 394.4 ± 38.6 5.0 89.3 ± 9.2 223.1 ± 19.3 384.5 ± 32.1 446.1 ± 34.6 485.6 ± 35.6 416.7 ± 34.6 352.1 ± 32.1 7.5 72.4 ± 6.4 185.4 ± 17.3 322.4 ± 28.9 405.3 ± 31.4 442.1 ± 38.4 375.2 ± 29.0 315.7 ± 28.9 10.0 63.1 ± 6.3 154.6 ± 15.3 289.7 ± 24.3 372.5 ± 27.4 398.2 ± 35.1 321.8 ± 31.4 256.9 ± 27.1

Experimental Example 2: Escherichia coli A-68 according to the initial pH and culture temperature of the medium ( Escherichia coli A-68)-derived fibrinolytic enzyme activity measurement

A medium was prepared using an optimized carbon source and nitrogen source, and the growth of the cells and the activity of the fibrinolytic enzyme produced in the culture medium were measured while culturing at the initial pH and culture temperature of each different medium. Optimize the pH and culture temperature of the initial medium for fibrinolytic enzyme production.

Initial pH of optimal medium, culture temperature

50 g/L of rice husk, 2.5 g/L of yeast extract, 0.1% sodium chloride, 0.5% potassium hydrogen phosphate (K ₂ HPO ₄ ), 0.02% magnesium sulfate (MgSO ₄ 7H ₂ O) and 0.06% ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) in combination, the initial pH is 5.8, 6.3, 6.8, 7.3, 7.8 and 8.3 and the temperature is 25, 30 , 35 and 40°C, respectively, and incubate for 3 days using 200 mL as a total amount in a 500 mL flask. After collecting the culture solution for each time period, the cells are removed using a centrifuge at 6000 rpm for 10 minutes. Using the removed supernatant, take 0.5 mL of the fibrinolytic enzyme supernatant dissolved in a buffer solution (10 mM sodium phosphate buffer pH 6.5), add 0.5 mL of 1% (w/v) carboxymethyl cellulose solution, and then at 50°C for 20 minutes. After reacting, the reducing sugar present in the reaction solution was measured by the DNS method to examine the activity of fibrinolytic enzyme.

As shown in Table 5 below, the fibrinolytic enzyme produced by the recombinant strain Escherichia coli A-68 exhibited the highest activity of 651.4 U/mL at an initial pH of 7.3 and a culture temperature of 35° C. of the medium.

Effect of Optimal Medium Initial pH and Optimal Culture Temperature on Fibrinolytic Enzyme Activity Produced by Escherichia coli A-68 Strain CMCase (U/mL) Temp.
(℃) pH 5.8 6.3 6.8 7.3 7.8 8.3 25 354.7 ± 25.6 443.2 ± 32.3 462.2 ± 32.1 502.9 ± 40.8 423.8 ± 36.7 276.2 ± 22.4 30 386.8 ± 27.4 468.1 ± 35.7 528.7 ± 42.8 574.6 ± 42.5 517.5 ± 46.3 382.5 ± 29.1 35 436.7 ± 35.7 553.1 ± 41.6 614.6 ± 48.5 651.4 ± 48.3 581.4 ± 48.1 432.6 ± 33.5 40 311.5 ± 28.4 385.6 ± 27.2 483.2 ± 41.4 513.6 ± 46.2 453.4 ± 37.9 223.6 ± 22.6

Experimental Example 3: Measurement of fibrinolytic enzyme derived from Bacillus belenchesis A-68 strain and recombinant strain Escherichia coli A-68

50 g/L of rice husk, 2.5 g/L of yeast extract, 0.1% sodium chloride, 0.5% potassium hydrogen phosphate (K ₂ HPO ₄ ), 0.02% magnesium sulfate (MgSO ₄ 7H ₂ O) and 0.06% ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) in combination, the initial pH is 5.8, 6.3, 6.8, 7.3, 7.8 and 8.3 and the temperature is 25, 30 , 35 and 40°C, respectively, and incubate for 3 days using 200 mL as a total amount in a 500 mL flask. After collecting the culture solution for each time period, the cells are removed using a centrifuge at 6000 rpm for 10 minutes. Using the removed supernatant, take 0.5 mL of the fibrinolytic enzyme supernatant dissolved in a buffer solution (10 mM sodium phosphate buffer pH 6.5), add 0.5 mL of 1% (w/v) carboxymethyl cellulose solution, and then at 50°C for 20 minutes. After reacting, the reducing sugar present in the reaction solution was measured by the DNS method to examine the activity of fibrinolytic enzyme.

As shown in Table 6 below, the fibrinolytic enzyme productivity of the recombinant strain Escherichia coli A-68 transformed by introducing the fibrinolytic enzyme gene of Bacillus belenchesis A-68 strain was 50.0 as a carbon source: glucose, rice bran and rice husk. When used at the concentration of g/L, the productivity was 144.3 U/mL, 465.2 U/mL, and 668.3 U/mL, respectively, and 3.35 times and 5.89 compared to the fibrinolytic enzyme productivity of Bacillus belenchesis A-68 strain, respectively. And 3.44 times higher.

Comparison of Fibrinolytic Enzyme Productivity of Bacillus Bellensis A-68 and Escherichia coli A-68 Strain Carbon source Cell weight
(g/L) Fibrinolytic enzyme
(U/mL) Bacillus Belenchesis A-68 glucose 1.75 ± 0.13 43.0 ± 8.5 Rice bran 1.12 ± 0.15 113.4 ± 7.2 chaff 1.87 ± 0.18 136.4 ± 12.1 Escherichia coli A-68 glucose 1.02 ± 0.08 144.3 ± 18.6 Rice bran 1.40 ± 0.14 465.2 ± 32.1 chaff 1.84 ± 0.19 668.3 ± 24.3

Institute of Agricultural Biotechnology KACC91335 20071107

<110> Dong-A University Research Foundation for Industry-Academy Cooperation Jinsang Company <120> Gene coding for cellulase from Bacillus valezensis A-68 and production method of cellulase by transformed Escherichia coli A-68 thereof <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 2000 <212> PRT <213> Bacillus velezensis A-68 <400> 1 Ala Thr Gly Ala Ala Ala Cys Gly Gly Thr Cys Ala Ala Thr Thr Thr 1 5 10 15 Cys Thr Ala Thr Thr Thr Thr Thr Ala Thr Thr Ala Cys Gly Thr Gly 20 25 30 Thr Thr Thr Ala Thr Thr Gly Ala Thr Thr Gly Cys Gly Gly Thr Ala 35 40 45 Thr Thr Gly Ala Cys Ala Ala Thr Gly Gly Gly Cys Met Lys Arg Ser 50 55 60 Ile Ser Ile Phe Ile Thr Cys Leu Leu Ile Ala Val Leu Thr Met Gly 65 70 75 80 Gly Gly Cys Thr Thr Gly Cys Thr Gly Cys Cys Thr Thr Cys Gly Cys 85 90 95 Cys Gly Gly Gly Ala Thr Cys Ala Gly Cys Ala Gly Cys Ala Gly Gly 100 105 110 Gly Ala Cys Ala Ala Ala Ala Ala Cys Gly Cys Cys Ala Gly Cys Ala 115 120 125 Gly Cys Cys Ala Ala Gly Ala Ala Thr Gly Gly Gly Gly Leu Leu Pro 130 135 140 Ser Pro Gly Ser Ala Ala Gly Thr Lys Thr Pro Ala Ala Lys Asn Gly 145 150 155 160 Cys Ala Gly Cys Thr Thr Ala Gly Cys Ala Thr Ala Ala Ala Ala Gly 165 170 175 Gly Ala Ala Cys Ala Cys Ala Gly Cys Thr Cys Gly Thr Ala Ala Ala 180 185 190 Cys Cys Gly Gly Gly Ala Cys Gly Gly Cys Ala Ala Ala Gly Cys Gly 195 200 205 Gly Thr Ala Cys Ala Ala Thr Thr Gly Ala Ala Ala Gln Leu Ser Ile 210 215 220 Lys Gly Thr Gln Leu Val Asn Arg Asp Gly Lys Ala Val Gln Leu Lys 225 230 235 240 Gly Gly Gly Ala Thr Cys Ala Gly Thr Thr Cys Ala Cys Ala Thr Gly 245 250 255 Gly Ala Thr Thr Gly Cys Ala Gly Thr Gly Gly Thr Ala Thr Gly Gly 260 265 270 Cys Gly Ala Thr Thr Thr Thr Gly Thr Cys Ala Ala Thr Ala Ala Ala 275 280 285 Gly Ala Cys Ala Gly Cys Thr Thr Ala Ala Ala Ala Gly Ile Ser Ser 290 295 300 His Gly Leu Gln Trp Tyr Gly Asp Phe Val Asn Lys Asp Ser Leu Lys 305 310 315 320 Thr Gly Gly Cys Thr Gly Ala Gly Ala Gly Ala Cys Gly Ala Thr Thr 325 330 335 Gly Gly Gly Gly Cys Ala Thr Ala Ala Cys Cys Gly Thr Thr Thr Thr 340 345 350 Cys Cys Gly Cys Gly Cys Gly Gly Cys Gly Ala Thr Gly Thr Ala Thr 355 360 365 Ala Cys Gly Gly Cys Ala Gly Ala Thr Gly Gly Cys Trp Leu Arg Asp 370 375 380 Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr Thr Ala Asp Gly 385 390 395 400 Gly Gly Thr Thr Ala Thr Ala Thr Thr Gly Ala Thr Ala Ala Thr Cys 405 410 415 Cys Gly Thr Cys Cys Gly Thr Gly Ala Ala Ala Ala Ala Thr Ala Ala 420 425 430 Ala Gly Thr Ala Ala Ala Ala Gly Ala Ala Gly Cys Gly Gly Thr Thr 435 440 445 Gly Ala Ala Gly Cys Gly Gly Cys Ala Ala Ala Ala Gly Tyr Ile Asp 450 455 460 Asn Pro Ser Val Lys Asn Lys Val Lys Glu Ala Val Glu Ala Ala Lys 465 470 475 480 Gly Ala Ala Cys Thr Cys Gly Gly Gly Ala Thr Ala Thr Ala Thr Gly 485 490 495 Thr Cys Ala Thr Cys Ala Thr Thr Gly Ala Cys Thr Gly Gly Cys Ala 500 505 510 Thr Ala Thr Cys Thr Thr Ala Ala Ala Thr Gly Ala Cys Gly Gly Cys 515 520 525 Ala Ala Cys Cys Cys Ala Ala Ala Cys Cys Ala Ala Glu Leu Gly Ile 530 535 540 Tyr Val Ile Ile Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln 545 550 555 560 Cys Ala Thr Ala Ala Ala Gly Ala Gly Ala Ala Gly Gly Cys Ala Ala 565 570 575 Ala Ala Gly Ala Ala Thr Thr Thr Thr Thr Thr Ala Ala Gly Gly Ala 580 585 590 Ala Ala Thr Gly Thr Cys Ala Ala Gly Thr Cys Thr Thr Thr Ala Cys 595 600 605 Gly Gly Ala Ala Ala Cys Ala Cys Gly Cys Cys Ala His Lys Glu Lys 610 615 620 Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly Asn Thr Pro 625 630 635 640 Ala Ala Cys Gly Thr Cys Ala Thr Thr Thr Ala Thr Gly Ala Ala Ala 645 650 655 Thr Thr Gly Cys Ala Ala Ala Cys Gly Ala Ala Cys Cys Ala Ala Ala 660 665 670 Cys Gly Gly Thr Gly Ala Thr Gly Thr Gly Ala Ala Cys Thr Gly Gly 675 680 685 Ala Ala Gly Cys Gly Thr Gly Ala Thr Ala Thr Thr Asn Val Ile Tyr 690 695 700 Glu Ile Ala Asn Glu Pro Asn Gly Asp Val Asn Trp Lys Arg Asp Ile 705 710 715 720 Ala Ala Ala Cys Cys Gly Thr Ala Thr Gly Cys Gly Gly Ala Ala Gly 725 730 735 Ala Ala Gly Thr Gly Ala Thr Thr Thr Cys Cys Gly Thr Thr Ala Thr 740 745 750 Cys Cys Gly Cys Ala Ala Ala Ala Ala Thr Gly Ala Thr Cys Cys Ala 755 760 765 Gly Ala Cys Ala Ala Cys Ala Thr Cys Ala Thr Cys Lys Pro Tyr Ala 770 775 780 Glu Glu Val Ile Ser Val Ile Arg Lys Asn Asp Pro Asp Asn Ile Ile 785 790 795 800 Ala Thr Thr Gly Thr Cys Gly Gly Ala Ala Cys Cys Gly Gly Thr Ala 805 810 815 Cys Ala Thr Gly Gly Ala Gly Cys Cys Ala Ala Gly Ala Thr Gly Thr 820 825 830 Gly Ala Ala Thr Gly Ala Thr Gly Cys Ala Gly Cys Cys Gly Ala Thr 835 840 845 Gly Ala Thr Cys Ala Gly Cys Thr Ala Ala Ala Ala Ile Val Gly Thr 850 855 860 Gly Thr Trp Ser Gln Asp Val Asn Asp Ala Ala Asp Asp Gln Leu Lys 865 870 875 880 Gly Ala Thr Gly Cys Ala Ala Ala Cys Gly Thr Cys Ala Thr Gly Thr 885 890 895 Ala Cys Gly Cys Gly Cys Thr Thr Cys Ala Thr Thr Thr Thr Thr Ala 900 905 910 Thr Gly Cys Cys Gly Gly Cys Ala Cys Ala Cys Ala Cys Gly Gly Cys 915 920 925 Cys Ala Ala Thr Cys Thr Thr Thr Ala Cys Gly Gly Asp Ala Asn Val 930 935 940 Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Ser Leu Arg 945 950 955 960 Gly Ala Thr Ala Ala Ala Gly Cys Ala Ala Ala Cys Thr Ala Thr Gly 965 970 975 Cys Ala Cys Thr Cys Ala Gly Thr Ala Ala Ala Gly Gly Ala Gly Cys 980 985 990 Gly Cys Cys Thr Ala Thr Thr Thr Thr Cys Gly Thr Gly Ala Cys Gly 995 1000 1005 Gly Ala Ala Thr Gly Gly Gly Gly Ala Ala Cys Ala Asp Lys Ala Asn 1010 1015 1020 Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr Glu Trp Gly Thr 1025 1030 1035 1040 Ala Gly Cys Gly Ala Cys Gly Cys Gly Thr Cys Thr Gly Gly Ala Ala 1045 1050 1055 Ala Thr Gly Gly Cys Gly Gly Thr Gly Thr Ala Thr Thr Cys Cys Thr 1060 1065 1070 Thr Gly Ala Cys Cys Ala Gly Thr Cys Gly Cys Gly Gly Gly Ala Ala 1075 1080 1085 Thr Gly Gly Cys Thr Gly Ala Ala Thr Thr Ala Thr Ser Asp Ala Ser 1090 1095 1100 Gly Asn Gly Gly Val Phe Leu Asp Gln Ser Arg Glu Trp Leu Asn Tyr 1105 1110 1115 1120 Cys Thr Cys Gly Ala Cys Ala Gly Cys Ala Ala Gly Ala Ala Cys Ala 1125 1130 1135 Thr Cys Ala Gly Cys Thr Gly Gly Gly Ala Gly Ala Ala Cys Thr Gly 1140 1145 1150 Gly Ala Ala Thr Cys Thr Thr Thr Cys Thr Gly Ala Thr Ala Ala Gly 1155 1160 1165 Cys Ala Gly Gly Ala Ala Thr Cys Ala Thr Cys Cys Leu Asp Ser Lys 1170 1175 1180 Asn Ile Ser Trp Glu Asn Trp Asn Leu Ser Asp Lys Gln Glu Ser Ser 1185 1190 1195 1200 Thr Cys Ala Gly Cys Gly Thr Thr Ala Ala Ala Gly Cys Cys Gly Gly 1205 1210 1215 Gly Ala Gly Cys Ala Thr Cys Thr Ala Ala Ala Ala Cys Ala Gly Gly 1220 1225 1230 Cys Gly Gly Cys Thr Gly Gly Cys Cys Gly Cys Thr Thr Ala Cys Ala 1235 1240 1245 Gly Ala Thr Thr Thr Ala Ala Cys Thr Gly Cys Thr Ser Ala Leu Lys 1250 1255 1260 Pro Gly Ala Ser Lys Thr Gly Gly Trp Pro Leu Thr Asp Leu Thr Ala 1265 1270 1275 1280 Thr Cys Ala Gly Gly Ala Ala Cys Ala Thr Thr Cys Gly Thr Ala Ala 1285 1290 1295 Gly Ala Gly Ala Ala Ala Ala Cys Ala Thr Thr Cys Thr Cys Gly Gly 1300 1305 1310 Cys Ala Ala Cys Ala Ala Ala Gly Ala Thr Thr Cys Ala Ala Cys Gly 1315 1320 1325 Ala Ala Ala Gly Ala Ala Cys Gly Cys Cys Cys Thr Ser Gly Thr Phe 1330 1335 1340 Val Arg Glu Asn Ile Leu Gly Asn Lys Asp Ser Thr Lys Glu Arg Pro 1345 1350 1355 1360 Gly Ala Ala Ala Cys Gly Cys Cys Ala Gly Cys Ala Cys Ala Ala Gly 1365 1370 1375 Ala Thr Ala Ala Cys Cys Cys Cys Gly Cys Ala Cys Ala Gly Gly Ala 1380 1385 1390 Ala Ala Ala Cys Gly Gly Cys Ala Thr Thr Thr Cys Thr Gly Thr Ala 1395 1400 1405 Cys Ala Ala Thr Ala Cys Ala Ala Ala Gly Cys Ala Glu Thr Pro Ala 1410 1415 1420 Gln Asp Asn Pro Ala Gln Glu Asn Gly Ile Ser Val Gln Tyr Lys Ala 1425 1430 1435 1440 Gly Gly Gly Gly Ala Thr Gly Gly Gly Gly Gly Thr Gly Thr Thr Ala 1445 1450 1455 Ala Cys Ala Gly Cys Ala Ala Cys Cys Ala Ala Ala Thr Cys Cys Gly 1460 1465 1470 Cys Cys Cys Gly Cys Ala Gly Cys Thr Thr Cys Ala Cys Ala Thr Ala 1475 1480 1485 Ala Ala Ala Ala Ala Thr Ala Ala Cys Gly Gly Cys Gly Asp Gly Gly 1490 1495 1500 Val Asn Ser Asn Gln Ile Arg Pro Gln Leu His Ile Lys Asn Asn Gly 1505 1510 1515 1520 Ala Ala Thr Gly Cys Gly Ala Cys Gly Gly Thr Thr Gly Ala Thr Thr 1525 1530 1535 Thr Ala Ala Ala Ala Gly Ala Thr Gly Thr Cys Ala Cys Thr Gly Cys 1540 1545 1550 Cys Cys Gly Thr Thr Ala Cys Thr Gly Gly Thr Ala Thr Ala Ala Cys 1555 1560 1565 Gly Cys Gly Ala Ala Ala Ala Ala Cys Ala Ala Gly Asn Ala Thr Val 1570 1575 1580 Asp Leu Lys Asp Val Thr Ala Arg Tyr Trp Tyr Asn Ala Lys Asn Lys 1585 1590 1595 1600 Gly Gly Cys Cys Ala Ala Ala Ala Cys Thr Thr Thr Gly Ala Cys Thr 1605 1610 1615 Gly Thr Gly Ala Cys Thr Ala Cys Gly Cys Gly Cys Ala Gly Ala Thr 1620 1625 1630 Thr Gly Gly Ala Thr Gly Cys Gly Gly Cys Ala Ala Thr Cys Thr Gly 1635 1640 1645 Ala Cys Cys Cys Ala Cys Ala Ala Ala Thr Thr Thr Gly Gln Asn Phe 1650 1655 1660 Asp Cys Asp Tyr Ala Gln Ile Gly Cys Gly Asn Leu Thr His Lys Phe 1665 1670 1675 1680 Gly Thr Gly Ala Cys Gly Cys Thr Gly Cys Ala Thr Ala Ala Ala Cys 1685 1690 1695 Cys Cys Ala Ala Gly Cys Ala Ala Gly Gly Thr Gly Cys Ala Gly Ala 1700 1705 1710 Thr Ala Cys Cys Thr Ala Thr Cys Thr Gly Gly Ala Ala Cys Thr Gly 1715 1720 1725 Gly Gly Thr Thr Thr Thr Ala Ala Ala Ala Cys Ala Val Thr Leu His 1730 1735 1740 Lys Pro Lys Gln Gly Ala Asp Thr Tyr Leu Glu Leu Gly Phe Lys Thr 1745 1750 1755 1760 Gly Gly Ala Ala Cys Gly Cys Thr Gly Thr Cys Ala Cys Cys Gly Gly 1765 1770 1775 Gly Ala Gly Cys Ala Ala Gly Cys Ala Cys Ala Gly Gly Gly Ala Ala 1780 1785 1790 Thr Ala Thr Thr Cys Ala Gly Cys Thr Thr Cys Gly Thr Cys Thr Thr 1795 1800 1805 Cys Ala Cys Ala Ala Thr Gly Ala Thr Gly Ala Cys Gly Thr Leu Ser 1810 1815 1820 Pro Gly Ala Ser Thr Gly Asn Ile Gln Leu Arg Leu His Asn Asp Asp 1825 1830 1835 1840 Thr Gly Gly Ala Gly Thr Ala Gly Thr Thr Ala Thr Gly Cys Ala Cys 1845 1850 1855 Ala Ala Ala Gly Cys Gly Gly Cys Gly Ala Thr Thr Ala Thr Thr Cys 1860 1865 1870 Cys Thr Thr Thr Thr Thr Thr Cys Ala Ala Thr Cys Ala Ala Ala Thr 1875 1880 1885 Ala Cys Gly Thr Thr Thr Ala Ala Ala Ala Cys Ala Trp Ser Ser Tyr 1890 1895 1900 Ala Gln Ser Gly Asp Tyr Ser Phe Phe Gln Ser Asn Thr Phe Lys Thr 1905 1910 1915 1920 Ala Cys Gly Ala Ala Ala Ala Ala Ala Ala Thr Thr Ala Cys Ala Thr 1925 1930 1935 Thr Ala Thr Ala Thr Cys Ala Thr Cys Ala Ala Gly Gly Ala Ala Ala 1940 1945 1950 Ala Cys Thr Gly Ala Thr Thr Thr Gly Gly Gly Gly Ala Ala Cys Ala 1955 1960 1965 Gly Ala Ala Cys Cys Cys Cys Ala Thr Thr Ala Gly Thr Lys Lys Ile 1970 1975 1980 Thr Leu Tyr His Gln Gly Lys Leu Ile Trp Gly Thr Glu Pro His *** 1985 1990 1995 2000

Claims (4)

Fibrinase protein derived from strain Bacillus velezensis A-68, strain Accession No. KACC 91178P, encoded by the gene sequence of SEQ ID NO: 1.
Recombinant vector pTA-68 for fibrinase expression of Escherichia coli ( E. coli ) prepared by linking the gene of SEQ ID NO: 1 to the p-TGEM T-Easy vector.
Recombinant strain Escherichia coli A-68 transformed by introducing recombinant vector pTA-68 (Esherichia coli A-68, strain Accession No. KACC 91335P).
Production of fibrinase, characterized in that the recombinant strain Escherichia coli A-68 is incubated in a medium consisting of 50 g / L rice hull and 2.5 g / L yeast extract at the conditions of the initial pH of 7.3 and 35 ℃ Way.

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