KR101591440B1 - Novel strain of bacillus methylotrophicus sci producing protease with superior heat resistance - Google Patents

Abstract

The present invention relates to a novel Bacillus methylotrophicus SCI (Bacillus Methylotrophicus) strain which is isolated from the dark patches of Hacchun County, Gyeongsangnam-do, Gyeongsangnam-do and has excellent proteolytic ability and produces proteolytic enzymes with excellent heat resistance which do not deteriorate even after heat treatment at 100-110 ≪ / RTI > The protease produced by the strain according to the present invention is characterized by exhibiting optimal activity at a temperature of 70 ° C, a pH of 11, and a metal ion absent condition, and is capable of effectively removing a trypsin inhibitor contained in poultry slaughter by- Therefore, the culture solution of the strain or strain can be usefully used for producing feeds having excellent effects on the promotion of the growth of livestock using poultry slaughter by-products.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a Bacillus methylotrophicus SCI strain, which produces a proteinase having excellent heat resistance,

The present invention relates to a novel Bacillus methylotrophicus SCI (Bacillus Methylotrophicus) strain which is isolated from the dark patches of Hacchun County, Gyeongsangnam-do, Gyeongsangnam-do and has excellent proteolytic ability and produces proteolytic enzymes with excellent heat resistance which do not deteriorate even after heat treatment at 100-110 ≪ / RTI >

Proteases are found in many living organisms such as animals, plants and microorganisms, and they are important enzymes that play various physiological roles.

The proteolytic enzyme is classified into serine protease, metalloprotease, aspartic protease, cysteine protease and the like according to functional groups acting on the active site, and acid protease, neutral Proteolytic enzymes, and alkaline proteases.

Industrially useful proteases have been found in various microorganisms such as actinomycetes, bacillus, and fungi. In recent years, microorganisms have been isolated from various habitats in order to industrially utilize proteolytic enzymes that meet new chemical and physical conditions.

Proteolytic enzymes are widely used in various industrial fields such as food industry, leather industry, detergent industry, environment purification industry, and pharmaceutical industry. Recently, due to the necessity of active use of recycled resources and development of eco-friendly process, Use process development is increasing.

Currently, among the more than 30 kinds of industrialized enzymes, the protease is an enzyme that plays an important role in the application to the industrial field as well as the physiological role of the enzymes. Protein enzymes can be used as an additive to surfactants to aid in the washing of protein contaminants or in the food industry such as baking, beverages, pharmaceutical industry such as the dissolution of dead tissue or the dissolution of coagulated blood, leather It is used in industrial, diagnostic industries, waste disposal such as recovery of silver from X-ray and photographic film, and accounts for 60% of the world's sales enzymes.

Proteolytic enzymes catalyze the hydrolysis of peptide bonds of amino acids present in polypeptides that form proteins as enzymes. This enzyme, which plays an essential role in life, is produced by plants, animals and microorganisms, all of which can be sources of proteolytic enzymes.

Since enzymes produced in plants and animals can not meet global demand and are difficult to meet the biotechnological applications, researchers are increasingly using microbial proteases.

A variety of microbial proteases have been reported from a variety of species, and the productivity of the same strain has been extensively studied. In particular, microorganisms that act as decomposers in ecosystems secrete extracellular proteases in order to absorb hydrolysis and decomposition products of proteins present in the extracellular environment, as well as intracellular proteases in cell metabolism.

Among them, Bacillus is known to be a microorganism producing an economical protease, which is excellent in adaptability to the environment, has a short generation and is easy to cultivate.

On the other hand, Korean Patent Laid-Open No. 10-2009-0114905 (published on November 11, 2009) discloses Bacillus licheniformis EDS-2, which promotes the liquefaction of pig manure and has excellent ability to remove various odors, Patent Publication No. 10-2010-0004505 (published on Jan. 13, 2010) discloses a novel Bacillus licheniformis KJ2 having an offensive odor-removing ability. Korean Patent Laid-Open No. 10-2010-0054497 (published on May 25, 2010) discloses microbial preparations for livestock manure liquefaction and wastewater treatment containing Bacillus licheniformis CMS-1 microorganism as an effective ingredient.

However, the above technologies are aimed at liquefying livestock manure at room temperature, purifying wastewater, and the like, and therefore, they are difficult to apply to protein source decomposition such as poultry slaughter by-products based on high temperature decomposition. In addition, studies have been carried out in Korea to search for strains producing high efficiency of proteolytic enzymes and feed the poultry slaughter by-products, but the results are still insufficient.

Therefore, the present inventors have recognized the above problems and have searched for various strains in the field of dandruff in Hacchin County, Gyeongsangnam-do, and have developed a strain producing a protease capable of decomposing protein sources such as poultry slaughter by-products based on high- The present invention has been completed by identifying and isolating.

The present invention relates to a novel protein degrading enzyme capable of degrading poultry slaughter byproducts such as chicken embryos and the like under high temperature conditions to produce an animal feed having an excellent effect for promoting the growth of livestock, It is intended to provide a microorganism.

In order to accomplish the above object, the present invention provides a method for producing a proteinase having excellent heat resistance, which is isolated from the stomach, comprising the steps of: Bacillus methylotrophicus methylotrophicus ) SCI strain (Accession No. KFCC11557P).

Further, the present invention provides a Bacillus methylotrophicus SCI strain having 16S rDNA comprising the nucleotide sequence of SEQ ID NO: 1.

Also, the present invention provides a Bacillus methylotrophicus SCI strain characterized in that the protease produced by the strain has a temperature range of 50 ° C to 80 ° C, which indicates optimal activity at pH 11.

In addition, the present invention provides a Bacillus methylotrophicus SCI strain, wherein the protease produced by the strain has a pH ranging from pH 9 to pH 12, the optimum range of which exhibits the optimum activity at a temperature of 70 ° C.

In addition, the present invention provides a Bacillus methylotrophicus SCI strain characterized in that the protease produced by the strain exhibits optimal activity at a temperature of 70 ° C, a pH of 11, and a metal ion absence condition.

The present invention also provides a composition for the hydrolysis of poultry slaughter by-products, which comprises a Bacillus methylotrophicus SCI strain or a culture thereof.

The present invention provides a novel strain of Bacillus methylotrophicus SCI which is isolated from the dark spot of Hacchun County, Gyeongsangnam-do, Gyeongsangnam-do, and produces proteinase having excellent proteolytic ability and high heat resistance even under high temperature conditions.

The protease produced by this strain not only has a high activity of decomposing poultry slaughter byproducts such as chicken embryos at high temperatures but also efficiently cleaves protein nutrients by removing trypsin inhibitors.

Therefore, when the strain according to the present invention is used, it is possible to produce an animal feed having an excellent effect for accelerating the growth of livestock by efficiently decomposing a protein source such as poultry slaughter by-products, and thus can be usefully used for industrial purposes.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing a bacterial count according to the culture temperature of Bacillus methylotrophicus SCI strain,
2 is a graph showing the results of a proteolytic enzyme production and bacterium counting graph while culturing the strain of Bacillus methylotrophicus SCI in a sterilized soybean meal at 40 ° C,
FIG. 3 is a graph showing the effect of the protease produced from Bacillus methylotrophicus SCI strain on the temperature at a pH of 11,
FIG. 4 is a graph showing the influence of the proteolytic enzyme produced from Bacillus methylotrophicus SCI strain on the pH at a fixed temperature of 70 ° C.,
FIG. 5 is a graph showing the results of measurement of protease activity at different metal ion concentrations,
Fig. 6 shows the result of analysis of the flexible relationship with the similar species by the 16S rDNA sequence of Bacillus methylotrophicus SCI strain.

Hereinafter, the novel strain of the present invention will be described as follows.

The present invention relates to a novel Bacillus methylotrophicus SCI (Bacillus Methylotrophicus) strain which is isolated from the dark patches of Hacchun County, Gyeongsangnam-do, Gyeongsangnam-do and has excellent proteolytic ability and produces proteolytic enzymes with excellent heat resistance which do not deteriorate even after heat treatment at 100-110 ≪ / RTI >

The strain according to the present invention was identified as Bacillus bacterium through Gram stain, catalase test and spore staining. It was identified as Bacillus methylotrophicus through 16S rDNA sequencing analysis and found to be Bacillus methylotrophicus SCI And deposited by the applicant on August 29, 2013 at the Korean Society for Microbiological Protection (accession number KFCC11557P).

The Bacillus methylotrophicus SCI strain has the 16S rDNA gene sequence of SEQ ID NO: 1.

The strain is most active in the temperature range of 35 to 40 캜, and the protease production is maximum at about 44 hours after the cultivation.

The proteolytic enzyme produced by the strain exhibits high activity at a temperature range of 50 to 80 ° C. and exhibits an activity value of 90% or more at a pH of 9 to 12 at 70 ° C. showing the highest activity, Lt; / RTI >

In addition, the protease has a characteristic that the enzymatic activity does not deteriorate even when heat treatment is performed at a temperature of 100 to 110 ° C. for 30 minutes, and the ability to remove a trypsin inhibitor is also excellent. Therefore, a protein such as a poultry slaughter by- It is possible to produce an animal feed having an excellent effect for accelerating the growth of livestock.

Therefore, an excellent animal feed can be produced by hydrolyzing poultry slaughter by-products such as chicken embryos and the like using a culture medium of Bacillus methylotrophicus SCI strain or a strain thereof.

Hereinafter, the present invention will be described in detail in the following examples, but the scope of protection of the present invention is not limited to the following examples.

Example

Example  1: Isolation and identification of proteolytic enzyme producing microorganisms

The strains used in the present invention were isolated from the dark patches around the Hacchin group in Gyeongnam, and were collected in a disposable sterilized bag using a sterilized spoon, and then collected in an ice box and transported to a laboratory. 1 g of the collected dwellings was put into 9 ml of sterilized water and mixed thoroughly. This sample was diluted 10 times with the stock solution, and inoculated on each sterilized medium and cultured at 30 ° C for 2 days. YM agar (Difco) medium was used for culture of yeast fungi or acid tolerant bacteria, and TSA agar (Difco) medium was used for culturing general bacteria.

For the purpose of isolating the species producing the protease, 500 isolates were used for the measurement of proteolytic activity. Each isolate was streaked on a Skim milk agar medium and cultured at 25 ° C for 48 hours to isolate strains showing a transparent ring formed by proteolytic enzymes. The strain was stored frozen in sterile Glycerol stock solution. The experiment was carried out in subculture on Skim milk medium to maintain the activity of Extra cellular protease.

Isolated strains were identified as Bacillus spp. By Gram stain, catalase test and spore staining.

16S ribosomal DNA sequencing of isolated strains was performed as follows. (5'-AGAGTTTGATCATGGCTCAG-3 ') and 1492R (5'-GGATACCTTGTTACGACTT-3') primers used for 16S rRNA sequencing were isolated using the Wizard genomic DNA purification kit (Promega, USA) PCR amplification was performed using MyCycler Thermal Cycler system (Bio-Rad). The amplified PCR products were purified using Wizard SV Gel and PCR clean-up system (Promega, USA). The purified PCR products were sequenced using ABI PRISM BigDye ™ Terminator Cycle Sequencing kits (Applied Biosystems Co.) using ABI PRISM 3730XL Analyzer (96 capillary type). The results were compared with GENEBANK ribosomal DNA sequence using BLASTN program. Sequence homology was compared using Clustal X and Mega 2 program. As a result of identification, Bacillus methylotrophicus was identified as Bacillus methylotrophicus SCI and deposited by the applicant on August 29, 2013 at the Korean Society for Microbiological Protection (Accession No. KFCC11557P). The 16S rDNA sequence of the Bacillus methylotrophicus SCI strain is shown in SEQ ID NO: 1, and Fig. 6 is a result of analysis of the flexible relationship with the similar species by the 16S rDNA sequence of the strain.

Example  2: Analysis of mycorrhizal growth by incubation temperature

To determine the optimal culture temperature for the growth of Bacillus methylotrophicus SCI strain and to investigate the changes of proteolytic enzyme activity according to the growth temperature, soybean meal was used as a basic medium and incubated at 25 ° C to 45 ° C The samples were collected at 3, 6, 12, 48, 72, and 96 hours after the incubation at temperature, diluted by decidual dilution method, and cultured in TSA medium at 37 ° C for 24 hours.

FIG. 1 is a graph showing the number of bacteria according to the culture temperature of Bacillus methylotrophicus SCI strain. It was confirmed that the temperature range of 35 ° C. to 40 ° C. is most suitable for the growth of Bacillus methylotrophicus SCI strain.

FIG. 2 is data on the production and number of proteolytic enzymes of Bacillus methylotrophicus SCI strain inoculated on sterilized soybean meal and cultured at 40 ° C. From 0 hrs to 4 hrs, we had a preparation machine and from 4 hrs to 16 hrs we went through algebra and from 16 hrs later we were in a stagnant period. The proteolytic enzyme reached the maximum value after 44 hours, and this strain was found to produce protease in the stasis.

Example  3: Proteolysis Crude enzyme solution  Manufacturing and Enzyme Activity Measurement

To measure the proteolytic activity, each culture was incubated at different temperatures, and the culture broth was collected at each time point. The supernatant was used as a crude enzyme solution by centrifugation at 12,000 × g for 3 minutes. In order to measure protease activity of the isolates, Azocasein was added to the crude enzyme solution at 50 ° C for 10 minutes and 0.44M TCA was added to stop the reaction. After centrifugation, commercially available Folin reagent reagent was diluted 3-fold and then added to develop color, and absorbance was measured at 660 nm. The activity of the enzyme was defined as 1 Unit of the amount by which 1 ml of the crude enzyme solution produced 1 μg of tyrosine per minute. Protein quantification was performed according to the Lowy method using bovine serum albumin as a standard protein.

Enzyme activity was measured at 5 ℃ intervals from 10 ℃ to 105 ℃ in order to investigate the optimum temperature of protease. Other conditions except the reaction temperature were measured in the same manner as described above.

As a result, the comparative activity was investigated using the highest activity at 70 ° C as a control. And more than 90% in the temperature range of 50 ° C to 80 ° C. When the temperature exceeds 90 ° C or falls below 40 ° C, the reaction activity is markedly lowered.

The optimum pH was determined by adjusting the pH of the buffer suspension containing Azocasein to 5.0 ~ 11.0 at intervals of 1.0. The buffer used was 100 mM citrate buffer for pH 5.0 ~ 6.0, 100 mM sodium phosphate buffer for pH 6.0 ~ 8.0, 100 mM tris-HCl buffer for pH 8.0 ~ 9.0 and 100 mM glycine-NaOH buffer for pH 9.0 ~ 11.0 .

The optimum temperature for the enzyme activity was 70 ℃ and the effect of each enzyme on the pH was investigated. The pH of 9 ~ 12 was more than 90% and the activity was significantly lowered when the pH dropped below 8.

FIG. 3 is a graph showing the effect of the protease produced from Bacillus methylotrophicus SCI strain on the temperature under a fixed pH of 11, FIG. 4 is a graph showing the effect of FIG. 4 is a graph showing the results of examining the effect of the protease produced from Bacillus methylotrophicus SCI strain on pH. FIG.

Example  4: Proteinase trypsin inhibitor Removability  Measure

Trypsin inhibitor is a major anti-nutritional factor that lowers protein digestion and absorption by inhibiting the activity of trypsin, which is involved in digesting proteinaceous nutrients. Are also reported to exist.

In order to measure the ability of the Bacillus methylotrophicus SCI strain to remove the trypsin inhibitor from the proteolytic enzyme, a 5-fold amount of n-hexane was added to the sample to finely crush the sample, and the mixture was incubated at room temperature for 1 hour And stirring was repeated twice. The resulting powder was mixed with 30 mM Tris-HCl (pH 8.0) and 1 M NaCl at a ratio of 1:10 (W / V), and the protein components were extracted at room temperature for 3 hours. This solution was centrifuged at 4 ° C and 12,000 rpm for 10 minutes, and then the supernatant was used. Trypsin standard curves were prepared by dissolving Trypsin 30 mg, 90 mg, 150 mg, 210 mg, and 270 mg in 0.0025 N HCl, respectively, to 100 mL and taking 1 mL of the solution. The standard protein maker was purchased from Intron, Inc. and analyzed using SDS-PAGE.

The activity of trypsin inhibitor was 5.83 mg / g, 4.75 mg / g, and 0.0031 mg / g, respectively, in chicken intestine hydrolyzed with protease produced by low temperature dried fish meal, chicken meat meal and Bacillus methylotrophicus SCI strain The proteolytic enzyme hydrolyzed chicken intestines showed that many trypsin inhibitors were removed.

From the above results, the protease produced by the strain according to the present invention can effectively remove the trypsin inhibitor contained in the poultry slaughter by-product such as chicken embryo. Therefore, using the culture medium of an enzyme or a strain produced by the strain or strain, Hydrolysis of by - products could be useful for the production of diets with excellent effect on the growth of livestock.

Example  5: Effect of metal ions on the activity of proteolytic enzymes

The final concentrations of various metal ions (Ca, Zn, Mn, Mg) were adjusted to 1 mM or 5 mM during the enzyme reaction in order to investigate the metal ions (minerals) affecting the activity of the extracellular protease. The chelate compounds bound to the metal ion were measured to be the final inhibitory concentration of 1 mM of the enzyme.

The effect of each metal ion on the enzyme reaction was investigated by adding 1 mM and 5 mM of each metal ion with the temperature and pH fixed at 70 ° C and 11, respectively. As a result, when the metal ion was added, Respectively. FIG. 5 is a graph showing the results of measurement of protease activity at different metal ion concentrations. FIG.

Example  6: Thermal resistance of proteolytic enzyme

The enzymes were added to the artificially controlled grains so that the moisture content was 15%, and the enzyme was added at 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes And the heat resistance of the proteolytic enzyme was examined.

Enzyme activity did not decrease even after heat treatment for 30 minutes at 100 ° C to 110 ° C, but slightly decreased at 120 ° C or higher.

[Table 1] shows the result of investigating the proteolytic activity of the protease after treating the protease according to the temperature and time.

[Table 1]

Korea Microorganism Conservation Center (Domestic) KFCC11557P 20130829

<110> SCI Inc. <120> NOVEL STRAIN OF BACILLUS METHYLOTROPHICUS SCI PRODUCING PROTEASE          WITH SUPERIOR HEAT RESISTANCE <130> HP-2013-0113 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1427 <212> DNA <213> Bacillus methylotrophicus SCI <400> 1 gcggcgtgcc tatacatgca gtcgagcgga cagatgggag cttgctccct gatgttagcg 60 gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac 120 cggggctaat accggatggt tgtttgaacc gcatggttca gacataaaag gtggcttcgg 180 ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa 240 ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc 300 cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag 360 caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca 420 agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 540 agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt 600 cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg 660 aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga 720 cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780 aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta 840 agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960 gacatcctct gacaatccta gagataggac gtccccttcg ggggagagt gacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttgatctt agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg 1260 ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttct acacaccgcc cgtcacacca 1380 cgagagtttg taacacccga agtcggtgag gtaacctttt aggagcc 1427

Claims (6)

Bacillus methylotrophicus SCI strain (Accession No. KFCC11557P), which has 16S rDNA comprising the nucleotide sequence of SEQ. delete delete delete delete A composition for the hydrolysis of poultry slaughter by-products, which comprises the strain of claim 1 or a culture of the strain.

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