JP7162365B2 - Exiguobacterium sibilicum producing cryoproteases and uses thereof - Google Patents

Description

CGMCC 20358

FIELD OF THE INVENTION The present invention relates to the field of microbial screening and use, and in particular to Exiguobacterium sibilicum, which produces cryoproteases, and uses thereof.

Enzymes generally refer to enzymes that can catalyze the hydrolysis of peptide bonds at room temperature and even at low temperatures. A low-temperature protease is a protein hydrolase that has an optimum catalyst temperature of 40°C or less and can maintain high enzyme activity (50% or more) even at 20 to 30°C, and its optimum catalyst temperature is the temperature of the natural environment. Because it is close to , the heating or cooling process can be omitted during use, and compared to high-temperature proteases, it saves energy and time, and is expected to be applied in the food and laundry industries in the future. can. Currently discovered cold protease-producing strains include Bacillus licheniformis and Bacillus pumilus from glacial permafrost, Clostridium and Cyclobacter from Antarctica, Colwellia from sea ice floes, Exygobacterium from which it originates, and Serratia, Vibrio, Xanthomonas from other cold environments, and Penicillium chrysogenum of the Shewanella genus. Most of the cryoproteases produced by these strains have an optimum catalytic temperature between 30 and 40°C, and only a few have an optimum catalytic temperature below 20°C, but are less stable.

Antarctic krill Antarctickrill and Euphausiasuperba, which live in the frigid environment of Antarctica, have a survival temperature of -1.7°C to -3°C and are important species in the Antarctic ecosystem. It has a huge biomass, feeds on bacteria, algae and microzooplankton in the Antarctic biological chain, and is also the prey of apex predators, playing an important role in the Antarctic ecosystem. Cold-adapted organisms require dedicated enzymes to maintain their functional integrity at low temperatures, while enzymes in the body of psychrophilic organisms are more active and catalytic at low temperatures than thermophilic animals. have efficiency; The body of Antarctic krill contains many types of proteolytic enzymes, and previous studies have shown that the proteolytic enzymes isolated from the body of Antarctic krill have a degrading effect on necrotic tissue and reduce fat. It has been proved to be useful for medical use as a lead man agent.

In 1972, Nabou Kato et al. identified the marine bacterium Pseudomoassp. No. 548 producing proteases, many research groups at home and abroad in China have been studying proteases from marine microorganisms, mainly new enzyme-producing microorganisms and novel proteases, such as , cold-adapted proteases, high-temperature proteases, alkaline proteases, neutral proteases, etc. In Chinese patent CN104818225A, a cold protease-producing fungus, Planococcus sp. has been disclosed, which Planococcus originates from deep-sea sediments in the South Indian Ocean. Chinese patent CN110724701A discloses a method for evaluating the enzymatic activity of Antarctic krill trypsin at low temperature, and the low-temperature trypsin has excellent enzymatic activity even at low temperature. However, to date, no strains producing cryoproteases have been isolated or extracted from Antarctic krill, and cryoproteases isolated from other low-temperature environments have poor thermostability and are decomposed and inactivated when heated. It's easy to do.

The present invention provides a cryoprotease-producing Exiguobacterium sibilicum and its use in response to the existing problems in the prior art of poor cold adaptability and thermostability of mesophilic proteases. This cryoprotease-producing Exiguobacterium sibiricum was screened from Antarctic krill, and its optimum growth temperature and optimum enzyme production temperature are both close to those in the natural environment, so it is not necessary to heat or cool in the fermentation process. process can be skipped. The protease produced by this strain has an optimum reaction temperature of about 37°C, and has excellent catalytic activity at 0 to 60°C (60% or more of the maximum catalytic enzyme activity), low temperature adaptability, and excellent Has thermal stability.

The present invention is achieved by the following technical solutions.

Exiguobacterium sibiricum producing low-temperature protease, which is deposited in China Microbial Species Deposit Management Commission Common Microorganism Center, Deposit No. CGMCC No. 20358.

Furthermore, said Exiguobacterium sibilicum is a short, rod-shaped Gram-positive bacterium.

The present invention is the use of said cryopretease-producing Exiguobacterium sibilicum in the production of cryoproteases.

Furthermore, the cryoproteases are obtained by fermenting cryoproteases-producing Exiguobacterium sibiricum.

Furthermore, the fermentation temperature used for said fermentation is 10-20°C.

1. In the present invention, the fermentation conditions of the cryoprotease-producing Exiguobacterium sibilicum are easy to control, and both the optimum growth temperature and the optimum enzyme production temperature of this strain are close to the temperature of the natural environment, so that heating or The cooling process can be omitted.
2. In the present invention, the cryoprotase produced by Exiguobacterium sibilicum, which produces cryoproteases, has an optimum catalytic temperature of about 37°C, which is close to the normal temperature of the human body, and exhibits high catalytic activity between 30 and 40°C. can be maintained. The low-temperature protease maintains high enzymatic activity even at 0 to 60° C., has a maximum catalytic enzymatic activity of 60% or more, and is adaptable to a low-temperature environment and has excellent thermostability, so that the range of application is widened.
Bacterial species deposit information Deposit time: July 14, 2020 Depositary institution: China Microbial Species Deposit Management Committee Ordinary Microorganism Center (International depositary authority based on the Budapest Treaty) ;
Deposit number: CGMCC No. 20358;
Depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, postal code: 100101;
Category nomenclature: Exiguobacterium sibiricum.

Photographs of the screened strains, a: Transparent circles produced by plate hydrolysis, b: Purification streak. 1 is a Gram-stained photomicrograph of Exiguobacterium sibiricum. It is an agarose gel electrophoresis image. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a phylogenetic tree of Exiguobacterium sibiricum of the present invention. Tyrosine standard curve. Relative enzymatic activity of cryogenic protease at each temperature.

Examples of the present invention will be described in detail below. Although the present examples are implemented on the basis of the technical solutions of the present invention and describe detailed embodiments and specific operating processes, the patent scope of the present invention is not limited to the following examples.

４）再スクリーニング
成分が表１と同じで、ｐＨが７．２～７．４である液体発酵培地を調製し、三角フラスコに分注し、２０ｍｉｎ滅菌して使用に備えた。
容量２５０ｍＬの三角フラスコに発酵培地１００ｍＬを入れて、１２１℃で高圧蒸気により３０ｍｉｎ滅菌し、一次スクリーニングにより得られた菌株を接種した後、１５℃の恒温培養床に置いて、１８０ｒ／ｍｉｎで５日間培養し、再スクリーニングした菌株を得た。
再スクリーニングした菌株の平板における加水分解円の直径とコロニーの直径との直径比は２．２であった。
５）低温プロテアーゼ産生菌株の同定
ａ）グラム染色
グラム染色方法のステップは、塗抹、固定、一次染色、媒染染色、脱色、再染色であり、最後に、顕微鏡下で観察した。図２の顕微鏡像に示すように、菌体は青紫色であり、単一の細胞は短い棒状のものであり、不規則に配列されており、グラム陽性菌であると同定された。
ｂ）細菌ゲノムＤＮＡの抽出
細菌ゲノムＤＮＡ抽出キッドの原理ステップに従ってゲノムＤＮＡを抽出し、高品質の細菌ゲノムＤＮＡを得た。
ｃ）ＰＣＲ増幅
ＤＮＡ増幅反応系（５０μＬ）：
Ｍｉｘ ２５μＬ
ｄｄＨ_２Ｏ ２０μＬ
上流プライマー（Ｆ） ２μＬ
下流プライマー（Ｒ） ２μＬ
テンプレート１μＬ；
ＰＣＲ反応条件：
９４予備変性４ｍｉｎ
９４変性１ｍｉｎ
５５アニーリング１ｍｉｎ
７２伸長２ｍｉｎ
３０サイクル
７２ １０ｍｉｎまで補充；
ｄ）ＰＣＲ産物の電気電気泳動
アガロースゲル電気泳動後、紫外線ランプでバンドを観察した（図４参照）。この菌株は、１６ｓｒＤＮＡ断片の長さが３５００ｂｐ程度である。同社でシーケンシングして、１６ｓｒＤＮＡ配列をデータベースと類似性を比較して、系統樹を作成した。本特許のエグジゴバクテリウムの系統樹を図４に示す。データベースと比較した結果、配列類似性は９９．９％であり、配列相同性は９９．９％である。それにより、スクリーニングした菌株はエキシグオバクテリウム・シビリカム（Ｅｘｉｇｕｏｂａｃｔｅｒｉｕｍ ｓｉｂｉｒｉｃｕｍ）であると同定された。 Example 1
The cryoprotease-producing Exiguobacterium sibiricum of the present invention is derived from Antarctic krill, has been screened multiple times, and is categorized as Exiguobacterium sibiricum. This strain was deposited in the General Microbiology Center of the China Microbial Species Deposit Management Committee on July 14, 2020. The deposit address is the Institute of Microbiology, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. is CGMCC No. 20358.
Strain Screening: Sample Collection, Sample Processing, Primary Screening, Re-Screening, Enzyme Activity Measurement, High Yield Strain Identification, Strain Deposit.
1) Sample collection: Antarctic krill was purchased from a seafood market in Shandong Province.
2) Sample processing: Grind Antarctic krill with a mortar at a low temperature, weigh 3 g, add sterilized water to a constant volume of 30 mL, make a stock solution, dispense into test tubes, and place the test tubes in a constant temperature culture bed. It was put in and fixed, set to 180 r/min, and measured the time for 30 minutes.
3) Primary screening (plating): The stock solution was diluted with sterile water in a concentration gradient of 10 ⁻¹ to 10 ⁻⁵ g/ml, dispensed into tubes, inoculated into the prepared separate culture plates, and the temperature was adjusted to It was placed in a constant temperature incubator set at 15° C. and cultured for 2 weeks. A single colony with a large colony shape and a large transparent circle around the colony was selected from the grown colonies, and the strain with the largest hydrolysis circle was screened by plate separation. The ratio of hydrolysis circle diameter to colony diameter was 2.1, and further selected single colonies were streaked and purified on a clean bench, which was repeated three times to obtain a purer strain. FIG. 1 shows transparent circles and streak purification diagrams produced by tabular hydrolysis of screened strains.
The solid medium used during the plate separation consists of the components shown in Table 1 and agar. ready for use.

4) Re-screening A liquid fermentation medium having the same ingredients as in Table 1 and a pH of 7.2 to 7.4 was prepared, dispensed into Erlenmeyer flasks, sterilized for 20 minutes, and ready for use.
Put 100 mL of fermentation medium in a 250 mL conical flask, sterilize with high pressure steam at 121 ° C. for 30 min, inoculate the strain obtained by primary screening, place on a constant temperature culture bed at 15 ° C., and 5 at 180 r / min. A rescreened strain was obtained after culturing for days.
The diameter ratio between the hydrolysis circle diameter and the colony diameter in the rescreened strain plates was 2.2.
5) Identification of cold protease-producing strains a) Gram staining The steps of the Gram staining method were smearing, fixing, primary staining, mordant staining, destaining, restaining, and finally observed under a microscope. As shown in the microscopic image of FIG. 2, the cells were bluish-purple and the single cells were short rod-shaped and irregularly arranged, which was identified as a Gram-positive bacterium.
b) Bacterial Genomic DNA Extraction Genomic DNA was extracted according to the principle steps of the Bacterial Genomic DNA Extraction Kit to obtain high quality bacterial genomic DNA.
c) PCR amplification DNA amplification reaction system (50 μL):
Mix 25 μL
20 μL of _ddH2O
Upstream primer (F) 2 μL
Downstream primer (R) 2 μL
1 μL template;
PCR reaction conditions:
94 pre-denaturation 4 min
94 denaturation 1 min
55 Annealing 1 min
72 extension 2min
Refill up to 30 cycles 72 10 min;
d) Electrophoresis of PCR products After agarose gel electrophoresis, bands were observed with an ultraviolet lamp (see FIG. 4). This strain has a 16s rDNA fragment with a length of about 3500 bp. Sequencing in-house, the 16s rDNA sequences were compared for similarity with databases to generate a phylogenetic tree. FIG. 4 shows the phylogenetic tree of the Exygobacterium of this patent. The sequence similarity is 99.9% and the sequence homology is 99.9% as compared with the database. The screened strain was thereby identified as Exiguobacterium sibiricum.

Ｆｏｌｉｎ発色法によりエグジゴバクテリウムの酵素活性を測定した結果、エキシグオバクテリウム・シビリカムの吸光値は０．５３１であり、相対酵素活性は８．６５であった。 Example 2
Measurement of Low-Temperature Protease Enzyme Activity (1) Measurement of Enzyme Activity As a method for measuring the enzyme activity of alkaline protease, the Folin-phenol chromogenic method is generally used. In this experiment, one enzymatic activity unit U is the amount of enzyme required to hydrolyze casein per minute at a constant temperature of 37° C. to produce 1 μmol of tyrosine. was calculated.
U=A*B*C/D
U: enzyme activity value of protease;
A: difference between sample absorbance value and blank absorbance value, tyrosine release (μg/mL) after comparison to standard curve;
B: reaction magnification factor;
C: enzyme dilution ratio;
D: reaction time.
A liquid medium shown in Table 1 of Example 1 was prepared, 100 mL of fermentation medium was placed in an Erlenmeyer flask with a capacity of 250 mL, sterilized with high pressure steam at 121 ° C. for 30 minutes, and Exiguobacterium sibiricum screened in Example 1 ( After being inoculated with Exiguobacterium sibiricum), it was placed on a 15° C. constant temperature culture bed and cultured at 180 r/min for 5 days to obtain a fermented liquid.
The fermented liquid was treated and centrifuged at 4° C. and 6000 r/min for 15 minutes to obtain an enzyme liquid as a supernatant. After filtering the enzyme solution, 800 μL was diluted 10-fold (diluent: phosphate buffer), 1 ml was dispensed per tube, and left at 37° C. for 10 minutes. Add 1 ml of 2% casein buffer per tube (the one to which TCA was added first is used as a blank control), react at 37° C. for 10 minutes, add 2 ml of TCA, centrifuge for 1 minute, and mix with sodium carbonate solution and Folin. A phenol reagent was added to develop the color for 10 min and the value was measured at 660 nm.
Table 2 shows the method for preparing tyrosine standard solutions. Four test tubes were prepared according to Table 2, the following solutions were gradually added to the test tubes, treated in a water bath at 37°C, and left for 30 minutes. Next, absorbance values (660 nm) are measured, a standard curve is plotted for the obtained data, and a linear regression equation is created as shown in FIG.

As a result of measuring the enzymatic activity of Exigobacterium by the Folin chromogenic method, the absorbance value of Exigobacterium sibilicum was 0.531, and the relative enzymatic activity was 8.65.

Example 3
Measurement of optimal enzyme activity Set a temperature gradient (0 to 60°C, one reaction every 5°C) and react for 10 minutes, then add 2 ml of TCA, centrifuge for 1 minute, remove sodium carbonate solution and folinphenol reagent. In addition, the steps of fermentation of Exiguobacterium sibiricum and enzyme solution treatment were the same as in Example 2, except that the color was developed for 10 minutes and the value was measured at 660 nm. Relative enzymatic activity is shown in Figure 6 below.
As a result of measurement, this strain has an optimum enzyme activity temperature of 37°C and has high activity even at 0 to 60°C. This indicates that this cryoprotease can withstand both high and low temperatures and has excellent thermostability.

Claims (5)

It was deposited at the General Microorganism Center of the Chinese Microbial Species Deposit Management Committee, and the deposit number is CGMCC No. 20358 of Exiguobacterium sibiricum, said Exiguobacterium sibiricum producing a cryoprotease characterized in that the single cell is a short rod-shaped Gram-positive bacterium (Exiguobacterium sibiricum). 2. The cryoprotease-producing Exiguobacterium sibiricum according to claim 1 , wherein the length of the 16s rDNA fragment of the Exiguobacterium sibiricum is about 3500 bp. Use of the cryoprotease-producing Exiguobacterium sibiricum according to claim 1 or 2 in the production of cryoproteases. 4. Use according to claim 3, characterized in that the cryoprotease is obtained by fermenting Exiguobacterium sibilicum. Use according to claim 3, characterized in that the fermentation temperature used for the fermentation is 10-20°C.

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