JP7068511B2 - A novel halophilic Bacillus polyfermenticas strain that produces salt-tolerant γ-glutamyl transpeptidase - Google Patents
A novel halophilic Bacillus polyfermenticas strain that produces salt-tolerant γ-glutamyl transpeptidase Download PDFInfo
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- 102000006640 gamma-Glutamyltransferase Human genes 0.000 title claims description 31
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 title claims description 30
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 title claims description 30
- 241000193830 Bacillus <bacterium> Species 0.000 title claims description 28
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- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 6
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- 239000004480 active ingredient Substances 0.000 description 4
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- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
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- 241000251468 Actinopterygii Species 0.000 description 2
- 241001669263 Bacillus amyloliquefaciens DSM 7 Species 0.000 description 2
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Description
本発明は、耐塩性γ－グルタミルトランスペプチダーゼを生産する新規な好塩性バチルス・ポリファーメンチカス（Ｂａｃｉｌｌｕｓ ｐｏｌｙｆｅｒｍｅｎｔｉｃｕｓ）菌株（ＫＣＴＣ１１７５１ＢＰ）、及びそれを用いたγ－グルタミルトランスペプチダーゼの生産方法に関する。 The present invention relates to a novel halophilic Bacillus polyfermenticus strain (KCTC11751BP) that produces a salt-tolerant γ-glutamyl transpeptidase, and a method for producing a γ-glutamyl transpeptidase using the same.
γ－グルタミルトランスペプチダーゼ（ｇａｍｍａ－ｇｌｕｔａｍｙｌ ｔｒａｎｓｐｅｐｔｉｄａｓｅ）は、グルタチオン（Ｇｌｕｔａｔｈｉｏｎｅ）のような物質でグルタミル結合を切って、グルタミルの一部を水に伝達する加水分解作用と他のアミノ酸またはペプチドに伝達するペプチド転移（ｔｒａｎｓｐｅｐｔｉｄａｔｉｏｎ）作用とを行う酵素である。この酵素は、グルタチオン代謝過程に関与し、抗酸化防御、解毒作用、炎症反応で重要な役割を果たす。また、この酵素は、飲食産業に活用が可能である。例えば、アミノ酸の苦味の改善、発酵飲食のうま味の増進などの効果が証明されて、食品産業で多様に活用が可能である。 Gamma-glutamyl transpeptidase is a peptide that breaks glutamil bonds with substances such as glutathione, hydrolyzing parts of glutamil to water and transferring to other amino acids or peptides. It is an enzyme that performs a transeptidation action. This enzyme is involved in glutathione metabolism and plays an important role in antioxidant defense, detoxification and inflammatory response. In addition, this enzyme can be utilized in the food service industry. For example, it has been proven to have effects such as improving the bitterness of amino acids and improving the umami of fermented foods and drinks, and can be used in various ways in the food industry.
γ－グルタミルトランスペプチダーゼを生産する酵素生産菌株として好塩性の菌株も活用可能性が高い。発酵食品であるキムチの初期発酵には、バチルス菌株が重要な役割を果たす。その理由は、バチルス菌株が生産する酵素は、キムチと塩辛内のタンパク質を分解して有用なペプチドを生産し、キムチの風味を増進させる役割を行うためである。キムチは、塩、塩辛を用いて発酵させるために、塩濃度が一般食品に比べて高い側である。したがって、好塩性のバチルス菌株は、塩濃度が高い発酵環境で作用範囲が広い。今まで探索開発されたものは、高活性の酵素であって、好塩性バチルス菌は開発された例が多くない。したがって、好塩性の酵素を生産する好塩性のバチルス菌株は、食品分野で活用価値が広いと考えられる。 Halophile strains are also highly likely to be used as enzyme-producing strains that produce γ-glutamyl transpeptidase. The Bacillus strain plays an important role in the initial fermentation of kimchi, which is a fermented food. The reason is that the enzyme produced by the Bacillus strain decomposes proteins in kimchi and salted fish to produce useful peptides and plays a role in enhancing the flavor of kimchi. Since kimchi is fermented using salt and salted fish, the salt concentration is higher than that of general foods. Therefore, the halophilic Bacillus strain has a wide range of action in a fermentation environment with a high salt concentration. What has been explored and developed so far is a highly active enzyme, and there are not many examples of halophilic Bacillus being developed. Therefore, halophilic Bacillus strains that produce halophilic enzymes are considered to have wide utility value in the food field.
本発明の目的は、耐塩性γ－グルタミルトランスペプチダーゼを生産する好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株（ＫＣＴＣ１１７５１ＢＰ）、それを用いた耐塩性γ－グルタミルトランスペプチダーゼの生産方法、及び該菌株を培養して得られた菌体、培養物またはこれにより分離精製された耐塩性γ－グルタミルトランスペプチダーゼを有効成分として含む食品組成物を提供するところにある。 An object of the present invention is to use a halophilic Bacillus polyfermenticas KMU01 strain (KCTC11751BP) for producing a salt-tolerant γ-glutamyl transpeptidase, a method for producing a salt-tolerant γ-glutamyl transpeptidase using the strain, and the strain. It is an object of the present invention to provide a food composition containing a cell, a culture obtained by culturing, or a salt-tolerant γ-glutamyl transpeptidase separated and purified by the same as an active ingredient.
前記目的を果たすために、本発明は、耐塩性γ－グルタミルトランスペプチダーゼを生産する好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株（ＫＣＴＣ１１７５１ＢＰ）を提供する。 To achieve the above object, the present invention provides a halophilic Bacillus polyfermenticas KMU01 strain (KCTC11751BP) that produces a salt-tolerant γ-glutamyl transpeptidase.
また、本発明は、前記好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株を培養する段階を含む耐塩性γ－グルタミルトランスペプチダーゼの生産方法を提供する。 The present invention also provides a method for producing a salt-tolerant γ-glutamyl transpeptidase, which comprises the step of culturing the halophilic Bacillus polyfermenticas KMU01 strain.
また、本発明は、前記好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株を培養して得られた菌体、培養物またはこれにより分離精製された耐塩性γ－グルタミルトランスペプチダーゼを有効成分として含む食品組成物を提供する。 Further, the present invention is a food containing as an active ingredient a cell, a culture obtained by culturing the halophilic Bacillus polyfermenticas KMU01 strain, or a salt-tolerant γ-glutamyl transpeptidase separated and purified thereby. The composition is provided.
本発明は、耐塩性γ－グルタミルトランスペプチダーゼを生産する新規な好塩性バチルス・ポリファーメンチカス菌株に関するものであって、本発明による好塩性のバチルス・ポリファーメンチカスＫＭＵ０１菌株（ＫＣＴＣ１１７５１ＢＰ）は、好塩性のγ－グルタミルトランスフェラーゼを生産する。本発明の菌株を用いて食品を発酵させる場合、塩濃度が高い食品にも活用が可能であり、タンパク質の結合を分解する高い酵素活性によって風味に優れ、多様なペプチドを含有した発酵食品を製造することができる。 The present invention relates to a novel halophilic Bacillus polyfermenticas strain that produces a salt-tolerant γ-glutamyl transpeptidase, the halophilic Bacillus polyfermenticas KMU01 strain (KCTC11751BP) according to the present invention. Produces a halophilic γ-glutamyltransferase. When fermenting foods using the strain of the present invention, it can also be used for foods with high salt concentration, and it produces fermented foods containing various peptides with excellent flavor due to high enzymatic activity that decomposes protein bonds. can do.
本発明は、耐塩性γ－グルタミルトランスペプチダーゼを生産する好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株を提供する。 The present invention provides a halophilic Bacillus polyfermenticas KMU01 strain that produces a salt-tolerant γ-glutamyl transpeptidase.
望ましくは、前記菌株は、韓国生命工学研究院にＫＣＴＣ１１７５１ＢＰで受託されたものであるが、これに限定されるものではない。 Desirably, the strain is consigned to the Korea Research Institute of Bioscience and Biotechnology under KCTC11751BP, but is not limited thereto.
望ましくは、前記菌株は、０．５～１５％ ＮａＣｌ濃度及び３０～４０℃の温度で成長し、より望ましくは、５％ ＮａＣｌ濃度及び４０℃の温度で最適成長することができるが、これに限定されるものではない。 Desirably, the strain can grow at 0.5-15% NaCl concentration and a temperature of 30-40 ° C, and more preferably optimally grow at a 5% NaCl concentration and a temperature of 40 ° C. Not limited.
望ましくは、前記γ－グルタミルトランスペプチダーゼは、０．５～１５％ ＮａＣｌ濃度及び３０～４０℃の温度で活性を示し、より望ましくは、５％ ＮａＣｌ濃度及び４０℃の温度で最高活性を示すことができるが、これに限定されるものではない。 Desirably, the γ-glutamyl transpeptidase exhibits activity at a temperature of 0.5-15% NaCl concentration and 30-40 ° C, and more preferably exhibits maximum activity at a temperature of 5% NaCl concentration and 40 ° C. However, it is not limited to this.
本発明に使われたバチルス・ポリファーメンチカスＫＭＵ０１菌株は、韓国生命工学研究院にＫＣＴＣ１１７５１ＢＰ（受託日：２０１０．８．２５）「バチルス・アミロリケファシエンス（Ｂａｃｉｌｌｕｓ ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ）Ｋｉｍｃｈｉ」で受託されたが、以後、明確な菌株同定を通じて「バチルス・ポリファーメンチカスＫＭＵ０１」に名称が訂正された。 The Bacillus polyfermenticas KMU01 strain used in the present invention was commissioned by the Korea Research Institute of Bioscience and Biotechnology at KTTC11751BP (contract date: 2010.8.25) "Bacillus amyloliquefaciens Kimchi". However, after that, the name was corrected to "Bacillus polyfermenticas KMU01" through clear strain identification.
また、本発明は、前記好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株を培養する段階を含む耐塩性γ－グルタミルトランスペプチダーゼの生産方法を提供する。 The present invention also provides a method for producing a salt-tolerant γ-glutamyl transpeptidase, which comprises the step of culturing the halophilic Bacillus polyfermenticas KMU01 strain.
望ましくは、前記菌株培養は、０．５～１５％ ＮａＣｌ濃度及び３０～４０℃の温度で培養することができるが、これに限定されるものではない。 Desirably, the strain culture can be cultured at a concentration of 0.5 to 15% NaCl and a temperature of 30 to 40 ° C., but is not limited to this.
また、本発明は、前記好塩性バチルス・ポリファーメンチカスＫＭＵ０１菌株を培養して得られた菌体、培養物またはこれにより分離精製された耐塩性γ－グルタミルトランスペプチダーゼを有効成分として含む食品組成物を提供する。 Further, the present invention is a food containing as an active ingredient a cell, a culture obtained by culturing the halophilic Bacillus polyfermenticas KMU01 strain, or a salt-tolerant γ-glutamyl transpeptidase separated and purified thereby. The composition is provided.
本発明の食品組成物である場合、前記食品の種類には、特別な制限はない。前記有効成分を添加することができる食品の例としては、肉類、ソーセージ、パン、チョコレート、キャンデー類、スナック類、お菓子類、ピザ、ラーメン、その他の麺類、ガム類、アイスクリーム類を含んだ酪農製品、キムチ、塩辛を含んだ発酵製品、各種スープ、飲料水、お茶、ドリンク剤、アルコール飲料及びビタミン複合剤などがある。 In the case of the food composition of the present invention, there are no particular restrictions on the type of the food. Examples of foods to which the active ingredient can be added include meats, sausages, breads, chocolates, candies, snacks, sweets, pizzas, ramen, other noodles, gums and ice creams. There are dairy products, kimchi, fermented products containing salt, various soups, drinking water, tea, drinks, alcoholic beverages and vitamin complex.
以下、本発明の理解を助けるために、実施例を挙げて詳細に説明する。但し、下記の実施例は、本発明の内容を例示するものであり、本発明の範囲が、下記の実施例に限定されるものではない。本発明の実施例は、当業者に本発明をより完全に説明するために提供されるものである。 Hereinafter, in order to help the understanding of the present invention, examples will be given and described in detail. However, the following examples exemplify the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to those skilled in the art to more fully explain the present invention.
＜実施例１＞
バチルス・ポリファーメンチカス菌株の同定
<Example 1>
Identification of Bacillus polyfermenticas strain
１．発酵食品の微生物の分離 1. 1. Isolation of microorganisms in fermented foods
発酵食品であるタチウオキムチを購入して、バチルスの分離を行った。発酵食品を粉砕して、発酵食品１ｇを生理食塩水で懸濁し、希釈した懸濁液の上澄み液０．１ｍｌを塩濃度０．５％、５％、１０％ ＴＳＡ（Ｔｒｙｐｔｏｎｅ １．７％、Ｓｏｙｔｏｎｅ ０．３％、Ｄｅｘｔｒｏｓｅ ０．２５％、Ｓｏｄｉｕｍ Ｃｈｌｏｒｉｄｅ ０．５％、Ｄｉｐｏｔａｓｓｉｕｍ Ｐｈｏｓｐｈａｔｅ ０．２５％、Ａｇａｒ １．５％）培地に塗抹して、３７℃で２４時間培養して分離した。培養された微生物のうち、１０％塩濃度の培地で成長が最も良い菌を選別した。 We purchased fermented hairtail kimchi and separated Bacillus. Fermented food is crushed, 1 g of fermented food is suspended in physiological saline, and 0.1 ml of the supernatant of the diluted suspension is added with a salt concentration of 0.5%, 5%, 10% TSA (Tryptone 1.7%,). Soytone 0.3%, Extractrose 0.25%, Sodium Chloride 0.5%, Dipotassium Phosphate 0.25%, Agar 1.5%) Smeared on medium, cultured at 37 ° C. for 24 hours and separated. Among the cultured microorganisms, the microorganisms having the best growth in a medium having a 10% salt concentration were selected.
２．分離菌株の同定 2. 2. Identification of isolated strains
ＲＮＡを増幅させ、分析した配列情報をＮＣＢＩ（ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ）のＢＬＡＳＴを用いて同定して、系統樹を描くことができた。その結果、本発明のバチルス・ポリファーメンチカスＫＭＵ０１菌株を同定することができた（図１）。 RNA was amplified and the analyzed sequence information was identified using BLAST of NCBI (www.ncbi.nlm.nih.gov), and a phylogenetic tree could be drawn. As a result, the Bacillus polyfermenticas KMU01 strain of the present invention could be identified (Fig. 1).
＜実施例２＞
他の菌株との好塩性の比較
<Example 2>
Comparison of halophilicity with other strains
本発明による菌株及びバチルス・サブチリス１６８（Ｂ．ｓｕｂｔｉｌｉｓ １６８）菌株を０．５％、５％、１０％ ＮａＣｌが添加されたＴＳＢ培地で２４時間培養した後、６００ｎｍで吸光度を測定して、各菌株の成長程度を測定した（図２）。図２から分かるように、本発明による菌株は、バチルス・サブチリス１６８に比べて、１０％以上塩化ナトリウムに対する耐性があることが分かる。
The strain according to the present invention and the Bacillus subtilis 168 (B. subtilis 168) strain were cultured in TSB medium supplemented with 0.5%, 5%, and 10% NaCl for 24 hours, and then the absorbance was measured at 600 nm for each. The degree of growth of the strain was measured (Fig. 2). As can be seen from FIG. 2, the strain according to the present invention is 10% or more resistant to sodium chloride as compared with
＜実施例３＞
他の菌株とのγ－グルタミルトランスペプチダーゼ活性の比較
<Example 3>
Comparison of γ-glutamyl transpeptidase activity with other strains
分離菌株をＴＳＢ培地５０ｍｌに接種して、３７℃で２４時間培養した培養液の上澄み液を取って、８，０００ｒｐｍで２０分間遠心分離した。ｐＮＡ生成量を４１０ｎｍで測定して、酵素活性を分析した。標準菌株であるバチルス・アミロリケファシエンスＤＳＭ７菌株とバチルス・サブチリス１６８菌株でのγ－グルタミルトランスペプチダーゼ活性を測定した。バチルス・アミロリケファシエンスＤＳＭ７菌株とバチルス・サブチリス１６８菌株は、韓国微生物資源センターから得た。
The isolated strain was inoculated into 50 ml of TSB medium, and the supernatant of the culture broth cultured at 37 ° C. for 24 hours was taken and centrifuged at 8,000 rpm for 20 minutes. The amount of pNA produced was measured at 410 nm, and the enzyme activity was analyzed. The γ-glutamyltranspeptidase activity in the standard strains Bacillus amyloliquefaciens DSM7 strain and
その結果、本発明のバチルス・ポリファーメンチカス菌株は、約３５００ｍＵ／ｍｌ以上の酵素活性を示して、他の菌株よりも酵素活性に優れていることが分かった（表１、図３）。 As a result, it was found that the Bacillus polyfermenticas strain of the present invention showed an enzyme activity of about 3500 mU / ml or more and was superior to other strains (Table 1, FIG. 3).
＜実施例４＞
培養温度と塩濃度とによる菌の生長
<Example 4>
Bacterial growth depending on culture temperature and salt concentration
菌株の最適培養条件を確立するために、多様な温度で２４時間、２００ｒｐｍで培養した。温度条件は、３０℃、３７℃、４０℃で進行し、塩濃度は、０．５％、５％で培養した。２４時間培養後、６００ｎｍで菌の生長を測定した。 In order to establish the optimum culture conditions for the strains, the strains were cultured at various temperatures for 24 hours at 200 rpm. The temperature conditions proceeded at 30 ° C., 37 ° C., and 40 ° C., and the culture was carried out at a salt concentration of 0.5% and 5%. After culturing for 24 hours, the growth of the fungus was measured at 600 nm.
図４Ａは、０．５％塩化ナトリウム環境で温度別の菌の生長であり、図４Ｂは、５％塩化ナトリウム環境で温度別の菌の生長である。最大ＯＤを示す条件は、４０℃、塩５％である。これを見て、当該菌株は、４０℃、塩５％で最適生長を示し、他の菌株よりも高い塩濃度で最適生長を示すと見て好塩性菌であると言える。 FIG. 4A shows the growth of bacteria by temperature in a 0.5% sodium chloride environment, and FIG. 4B shows the growth of bacteria by temperature in a 5% sodium chloride environment. The conditions showing the maximum OD are 40 ° C. and 5% salt. In view of this, it can be said that the strain is a halophilic bacterium because it shows optimum growth at 40 ° C. and 5% salt, and shows optimum growth at a higher salt concentration than other strains.
＜実施例５＞
培養温度と塩濃度とによるγ－グルタミルトランスペプチダーゼ活性
<Example 5>
Gamma-glutamyl transpeptidase activity depending on culture temperature and salt concentration
γ－グルタミルトランスペプチダーゼの最高活性を示す条件を確立するために、多様な温度で２４時間、２００ｒｐｍで培養した。温度条件は、３０℃、３７℃、４０℃で進行し、塩濃度は、０．５％、５％で培養した。２４時間培養後、８，０００ｒｐｍ、２０分 間遠心分離して、上澄み液を回収して、酵素活性を測定した。 In order to establish the conditions showing the highest activity of γ-glutamyl transpeptidase, the cells were cultured at various temperatures for 24 hours at 200 rpm. The temperature conditions proceeded at 30 ° C., 37 ° C., and 40 ° C., and the culture was carried out at a salt concentration of 0.5% and 5%. After culturing for 24 hours, the mixture was centrifuged at 8,000 rpm for 20 minutes, and the supernatant was collected and the enzyme activity was measured.
図５Ａは、０．５％塩化ナトリウム環境で温度別の酵素活性であり、図５Ｂは、５％塩化ナトリウム環境で温度別の酵素活性である。最大γ－グルタミルトランスペプチダーゼ酵素活性を示す条件は、４０℃、塩５％である。これを見て、当該菌株が生産するγ－グルタミルトランスペプチダーゼは、４０℃、塩５％で最高活性を示し、したがって、好塩性酵素であると言える。 FIG. 5A shows the enzyme activity by temperature in a 0.5% sodium chloride environment, and FIG. 5B shows the enzyme activity by temperature in a 5% sodium chloride environment. The condition showing the maximum γ-glutamyl transpeptidase enzyme activity is 40 ° C. and 5% salt. In view of this, the γ-glutamyltranspeptidase produced by the strain shows the highest activity at 40 ° C. and 5% salt, and therefore can be said to be a halophilic enzyme.
以上、本発明の特定の部分を詳しく記述したところ、当業者において、このような具体的な記述は、単に望ましい実施例であり、これにより、本発明の範囲が制限されるものではないという点は明白である。したがって、本発明の実質的な範囲は、下記の特許請求の範囲とそれらの等価物とによって定義される。 As described above, when a specific part of the present invention is described in detail, those skilled in the art will find that such a specific description is merely a desirable embodiment and does not limit the scope of the present invention. Is obvious. Therefore, the substantial scope of the present invention is defined by the following claims and their equivalents.
Claims (7)
The method for producing a salt-tolerant γ-glutamyl transpeptidase according to claim 6 , wherein the strain culture is performed at a concentration of 0.5 to 15% NaCl and a temperature of 30 to 40 ° C.
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