JP4752024B2 - Cell wall degrading enzyme, producing microorganism, and protoplast preparation method using the same - Google Patents

Description

【００１１】
この組成の寒天平板培地に、各地の海洋より採取した海洋性生物体サンプルを滅菌した50％人工海水で適度に希釈して接種し、15℃以下で３〜５日間培養した。出現したコロニーを表１の培地組成から寒天を除いた液体培地に植菌して、15℃以下で静置培養した。低温活性細胞壁分解酵素産生能は後記の方法により検定した。これらのサンプルはいずれもスコットランド沖の北海海域で採取された。ビブリオ sp. SCRC-37964及びシュードアルテロモナス sp.SCRC-38753は次の表２に示す菌学的性質を有する。
【００１２】
【表２】
表２

【００１３】
上記の菌学的性質からSCRC-37964は運動性を有する好気的なグラム陰性桿菌で、カタラーゼ及びオキシダーゼ共に陽性、ブドウ糖からガスを発生することなく発酵的に分解し、0/129に感受性を示すことからVibrioと推定される。オルニチンデカルボキシラーゼ陰性、b−ガラクトシダーゼ陽性などの性質から最も近いVibrioとして、文献イからV. fluvialis、V. augillarumなどが挙げられるが、アラビノース、ソルビトール陰性の点では一致しない。SCRC-38753は運動性を有するグラム陰性桿菌でカタラーゼ及びオキシダーゼ共に陽性を示し、ゼラチン加水分解とDNase陽性であるので文献ロからAlteromonasと推定される。白糖利用、セロビオース非利用の点では文献イからA. haloplanktisが最も近いと考えられるが、マンノース利用能の点で一致しない。
【００１４】
近年、種の同定に用いられている16S rDNA塩基配列を行った結果、SCRC-37964はV. carchariaeに最も近いが相同率は94％であること、又文献ハからウレアーゼ活性が陰性であることから一致しない。 SCRC-38753はPseudoalteromonas undinaに最も高い相同性を示したが、文献イから菌体の形状が一致しない。
以上の結果からSCRC-37964はVibrio属の新種、SCRC-38753はseudoalteromonas属の新種と考えられる。
【００１５】
文献
イ N.R. Krieg et al., Berger's Manual of Systemtic Bacteriology, vol. 1, Williams & Wilkins, (1984)
ロ 門田 元、多賀暢夫、海洋微生物研究法、学会出版センター、1985。
ハ D.J. Grimes et al., System. Appl. Microbiol., 6, 221-226 (1985).
【００１６】
以上、自然界から分離した菌株について詳述したが、これらの菌に変異を生じさせて一層生産性の高い菌株を得ることもできる。本発明の菌株は常法に従って保存することができ、例えば寒天スラント培地上で、又は凍結乾燥法により、又はグリセロール法により保存することができる。寒天スラント培地としては、例えば菌の分離に関して前記した培地を使用することができる。
【００１７】
(2) 細胞壁分解酵素の生産法
前記の微生物を培養して細胞壁分解酵素を製造しようとする場合、基礎栄養培地として本発明の微生物が増殖し得るものであればいずれを使用してもよい。この培地は窒素源として例えば酵母エキス、ペプトン、肉エキスなどの１種類又は複数種類を含む。又、この培地には必要に応じて炭素源として各種の糖類を加えることができる。この培地には天然海水や人工海水を加えることが好ましい。培養は固体培地又は液体培地のいずれを使用してもよいが、目的とする細胞壁分解酵素を多量にかつ容易に利用或いは精製するには液体培地を用い、静置培養若しくは振盪培養、通気・撹拌培養などにより好気的条件下で培養を行うことが好ましい。培養温度は菌が生育し、細胞壁分解酵素が生産される温度範囲であればいずれの温度でもよく、４〜３０℃である。pHは５〜１０、好ましくは６〜８の範囲である。培養時間は利用し得る量の細胞壁分解酵素が生産される時間を選べばよく、通常は１０〜１２０時間である。
【００１８】
次に得られた培養液から細胞壁分解酵素が採取される。その方法は利用目的によって異なり、培養液をそのまま利用することも可能であり、菌体が不要或いは邪魔になるときは菌体を遠心除去し更に滅菌フィルターで濾過することによって菌体を完全に除去した後利用する。又、細胞壁分解酵素の濃度が低いときは常法に従い、硫安沈殿などの塩析後透析、更にアフニティークロマトやゲルろ過などで濃縮が可能である。
【００１９】
(3) プロトプラスト調製法
プロトプラストを調製する場合、従来法と同様に標的となる微生物を培養して集菌し、浸透圧を調節する物質（ショ糖、ソルビトールなど）を含む高張緩衝液に懸濁し、前記の微生物の培養液或いは前記の方法により濃縮した酵素を添加して、２０℃以下で緩やかに振盪することによって細胞壁を除去する。プロトプラスト化は菌体形状の変化、及び蒸留水で希釈し浸透圧を低下させることによる菌体破砕を光学顕微鏡で観察する。
【００２０】
【実施例】
以下実施例により、本発明を詳細に説明する。但し、本発明はこれらに限定されるものではない。
【００２１】
実施例１；.ビブリオ sp. SCRC-37964(FERM P-18219)及びシュードアルテロモナス sp. SCRC-38753(FERM P-18220)からの細胞壁分解酵素の生産
ペプトン1％、酵母エキス0.5％を50％濃度の人工海水に溶解し、pH７に調整した培地100 mlを121℃、20分間加熱滅菌した後、ビブリオ sp. SCRC-37964(FERM P-18219)或いはシュードアルテロモナス sp. SCRC-38753(FERM P-18220)を接種し、15℃で72時間好気的に培養した。培養後、遠心分離機で菌体を除去した培養上清をフィルターでろ過滅菌した。培養上清中の細胞壁分解活性を評価するため、大腸菌から文献（Eur. J. Biochem., 13, 336, (1970)）の方法により細胞壁を調製し、寒天に懸濁したプレートを作製した。そのプレート上に抗菌試験用ディスクを置き、前記のろ過滅菌した培養上清50mlを含浸させて15及び30℃に静置した。約36時間後にディスクの周囲に形成されたハローの面積を測定した。そのハローの面積を表３に示す。コントロールとして、卵白リゾチーム0.1mg/mlの水溶液0.1mlによって形成されたハローの面積を併記する。両株の培養上清の細胞壁分解活性は反応温度15℃の方が30℃よりも高く、卵白リゾチームと逆であった。
【００２２】
【表３】
表３

【００２３】
実施例２；ビブリオ sp. SCRC-37964(FERM P-18219) からの細胞壁分解酵素の生産
実施例１と同様にビブリオ sp. SCRC-37964(FERM P-18219)を培養し、その培養上清100mlをフィルターでろ過滅菌した。次に硫酸アンモニウムを最終濃度が90％になるように加えて撹拌し、遠心後上清を除いた。残渣に0.1Mリン酸緩衝液(pH6) 5mlを加えて溶解し、同じ組成の緩衝液に対して4℃で２日間透析した。この試料及びこの試料を100℃で5分間加熱処理した場合について、実施例１と同様にして15、30℃及び37℃における細胞壁分解活性を調べた。その結果を表４に示した。
【００２４】
【表４】
表４

【００２５】
実施例3；ビブリオ sp. SCRC-37964(FERM P-18219) からの細胞壁分解酵素の生産
実施例１と同様にビブリオ sp. SCRC-37964(FERM P-18219)を培養し、5℃から30℃までの各温度における細胞壁分解活性を調べた。その結果を表５に示した。
【００２６】
【表５】
表５

【００２７】
実施例４；ビブリオ sp. SCRC-37964(FERM P-18219) から調製した細胞壁分解酵素によるグラム陰性細菌のプロトプラスト調製
実施例２と同様にビブリオ sp. SCRC-37964(FERM P-18219)を培養し、その培養上清を硫安沈殿と透析処理により培養上清中のタンパクを濃縮した。BIORADのタンパク測定キットで測定したところ、タンパク濃度は1.3 mg/mlであった。この試料を同量のプロトプラスト緩衝液（ショ糖 40％、 140mM 塩化カリウム、 30mM EDTA、 60mM Bicine）と混合し、細胞壁分解酵素溶液を調整した。Pseudomonas putida ATCC-11172の対数増殖後期の培養液から遠心で菌体を集め、およそ10⁸cells/ml程度の菌体密度になるように上述の細胞壁分解酵素溶液に懸濁し、15℃で緩やかに振盪した。約２時間後に光学顕微鏡で観察したところ約２０％が桿菌から球形に変化した。この反応溶液の一部を蒸留水或いはプロトプラスト緩衝液で３倍に希釈したところ、前者の菌体数は後者の６０％に減少した。これらの結果からP. putida ATCC-11172の一部はプロトプラストになったと判断した。
【００２８】
【発明の効果】
本発明の微生物を使用することにより、20℃以下の低温で細胞壁分解活性を示し、30℃以上ではその活性が著しく低下する酵素を製造することができる。本酵素はその温度特性を利用して、バクテリアのプロトプラスト融合におけるプロトプラスト調製の際、反応終了後に遠心や透析などプロトプラストの収率を著しく低下させる操作を行うことなく、30℃以上に加熱することによって酵素活性を失活させ、次のステップに進むことができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microorganism producing a cell wall degrading enzyme, a cell wall degrading enzyme produced by the microorganism, a culture solution containing the enzyme, and a protoplast preparation method using the enzyme or the culture solution.
[0002]
[Prior art]
Lysozyme, a typical cell wall degrading enzyme, is present in animal tissues and egg whites and plays a protective role against foreign bacterial infections. In addition to animal-derived lysozyme, cell wall degrading enzymes produced by microorganisms are also known. All of these have an optimum reaction temperature of 30 ° C. or higher, and even an enzyme with low heat stability maintains activity up to about 50 ° C.
[0003]
The purified lysozyme is used for cell wall degradation prior to protoplast preparation for cell fusion and DNA preparation from bacterial cells. After the decomposition reaction, a washing operation such as centrifugation or dialysis is required for removing lysozyme, leading to a decrease in the yield of the target product. In particular, the yield of a physically unstable object such as protoplast is significantly reduced by the washing operation.
[0004]
[Problems to be solved by the invention]
The present invention has found a cell wall degrading enzyme that can remove the activity after completion of the reaction without performing an enzyme removal operation such as centrifugation or dialysis, and by using it for protoplast preparation, the protoplast yield and the overall protoplast fusion strain can be obtained. Aim to improve yield.
[0005]
[Means for Solving the Problems]
As a means for solving the above problems, the present inventors have sought to search for a low-temperature active cell wall degrading enzyme that loses its activity by heating to a temperature that shows activity at low temperatures and does not reduce the yield of the target product. As a result of repeated studies, microorganisms belonging to the genus Vibrio or Pseudoalteromonas, producing cell wall degrading enzymes that are active at 20 ° C. or lower and whose activity is remarkably reduced at 30 ° C. or higher are secreted outside the cells. To complete the present invention.
[0006]
That is, the present invention relates to a method for producing a cell wall degrading enzyme by culturing a microorganism belonging to the genus Vibrio or Pseudoalteromonas, producing a cell wall degrading enzyme having an activity at low temperature and secreting it outside the cell, and culture As an application example of the cell wall degrading enzyme obtained by concentrating the solution as it is or from the culture solution, it is to provide a protoplast preparation method for protoplast fusion.
DETAILED DESCRIPTION OF THE INVENTION
[0007]
(1) Microorganism The microorganism of the present invention belongs to the genus Vibrio or Pseudoalteromonas and produces a cell wall degrading enzyme that exhibits activity at a low temperature, for example, 20 ° C. or less, and whose activity is significantly reduced at a temperature of normal temperature or more, for example, 30 ° C. or more. Any microorganism can be used as long as it can be secreted outside the cells, and such a microorganism can be newly isolated from the natural world or a mutant thereof.
[0008]
Examples of the microorganism belonging to the genus Vibrio of the present invention include Vibrio sp. SCRC-37964, which is a new strain, and examples of the microorganism belonging to the genus Pseudoalteromonas include Pseudoarteromonas sp. SCRC-38753. These strains were deposited with the National Institute of Advanced Industrial Science and Technology (AIST) as FERM P-18219 and FERM P-18220.
[0009]
The new strain was isolated as follows. First, a medium having the composition shown in Table 1 was prepared.
[0010]
[Table 1]
Table 1

[0011]
An agar plate medium of this composition was inoculated with a marine organism sample collected from various oceans in an appropriate dilution with sterilized 50% artificial seawater and cultured at 15 ° C. or lower for 3 to 5 days. The appearing colonies were inoculated into a liquid medium obtained by removing agar from the medium composition shown in Table 1, and statically cultured at 15 ° C. or lower. The ability to produce a cold-active cell wall degrading enzyme was assayed by the method described below. All of these samples were collected in the North Sea area off Scotland. Vibrio sp. SCRC-37964 and Pseudoalteromonas sp. SCRC-38753 have the mycological properties shown in Table 2 below.
[0012]
[Table 2]
Table 2

[0013]
SCRC-37964 is an aerobic Gram-negative bacilli with motility due to the above bacteriological properties, positive for both catalase and oxidase, fermentatively decomposed without generating gas from glucose, and sensitive to 0/129 It is presumed that Vibrio is shown. Examples of Vibrio that are closest to the properties such as ornithine decarboxylase negative and b-galactosidase positive include V. fluvialis and V. augillarum from literature A, but they do not match in terms of arabinose and sorbitol negative. SCRC-38753 is a gram-negative gonococci with motility and positive for both catalase and oxidase. It is presumed to be Alteromonas from literature B because it is gelatin hydrolyzed and DNase positive. Although A. haloplanktis is considered to be the closest in terms of sucrose use and non-use of cellobiose, it does not match in terms of mannose availability.
[0014]
As a result of 16S rDNA base sequence used for species identification in recent years, SCRC-37964 is closest to V. carchariae but has a homology of 94%, and urease activity is negative from literature c Does not match. SCRC-38753 showed the highest homology to Pseudoalteromonas undina.
Based on the above results, SCRC-37964 is considered a new species of the genus Vibrio, and SCRC-38753 is considered a new species of the genus seudoalteromonas.
[0015]
Literature NR Krieg et al., Berger's Manual of Systemtic Bacteriology, vol. 1, Williams & Wilkins, (1984)
(B) Gen Kadota, Ikuo Taga, Marine Microbial Research Methods, Society Publishing Center, 1985.
DJ Grimes et al., System. Appl.Microbiol., 6, 221-226 (1985).
[0016]
As mentioned above, although the strain isolate | separated from the natural world was explained in full detail, a strain with higher productivity can also be obtained by producing a mutation in these bacteria. The strain of the present invention can be stored according to a conventional method, for example, can be stored on an agar slant medium, by a freeze-drying method, or by a glycerol method. As the agar slant medium, for example, the medium described above for the isolation of bacteria can be used.
[0017]
(2) Production Method of Cell Wall Degrading Enzyme When culturing the aforementioned microorganism to produce a cell wall degrading enzyme, any basal nutrient medium may be used as long as the microorganism of the present invention can grow. This medium contains one or a plurality of types such as yeast extract, peptone, and meat extract as nitrogen sources. Moreover, various saccharides can be added to this culture medium as a carbon source as needed. It is preferable to add natural seawater or artificial seawater to this medium. For the culture, either a solid medium or a liquid medium may be used. However, in order to use or purify the target cell wall degrading enzyme in a large amount and easily, a liquid medium is used, stationary culture or shaking culture, aeration and agitation. Culturing is preferably performed under aerobic conditions such as by culturing. The culture temperature may be any temperature as long as bacteria are grown and a cell wall degrading enzyme is produced, and is 4 to 30 ° C. The pH is in the range of 5-10, preferably 6-8. What is necessary is just to select the time when the cell wall decomposing enzyme of the quantity which can be utilized is produced for culture | cultivation time, and it is 10 to 120 hours normally.
[0018]
Next, cell wall degrading enzyme is collected from the obtained culture broth. The method varies depending on the purpose of use, and the culture solution can be used as it is. If the cells are unnecessary or obstructive, the cells are removed by centrifugation and then filtered through a sterilization filter. After use. When the concentration of the cell wall degrading enzyme is low, it can be concentrated by dialysis after salting out such as ammonium sulfate precipitation, further by affinity chromatography, gel filtration or the like according to a conventional method.
[0019]
(3) Protoplast preparation method When preparing protoplasts, the target microorganism is cultured and collected in the same manner as in the conventional method, and suspended in a hypertonic buffer solution containing substances that regulate osmotic pressure (sucrose, sorbitol, etc.). The cell wall is removed by adding the culture solution of the microorganism or the enzyme concentrated by the method and gently shaking at 20 ° C. or lower. In protoplast formation, changes in cell shape and cell disruption by diluting with distilled water to lower osmotic pressure are observed with an optical microscope.
[0020]
【Example】
Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these.
[0021]
Example 1: Production of cell wall degrading enzymes from Vibrio sp. SCRC-37964 (FERM P-18219) and Pseudoalteromonas sp. SCRC-38753 (FERM P-18220) 50% peptone 1%, yeast extract 0.5% 100 ml of medium dissolved in artificial seawater with a concentration of 7 and adjusted to pH 7 was sterilized by heating at 121 ° C for 20 minutes, and then Vibrio sp. SCRC-37964 (FERM P-18219) or Pseudoalteromonas sp. SCRC-38753 ( FERM P-18220) and aerobically cultured at 15 ° C. for 72 hours. After culturing, the culture supernatant from which the cells were removed with a centrifuge was sterilized by filtration through a filter. In order to evaluate the cell wall degrading activity in the culture supernatant, cell walls were prepared from Escherichia coli by the method of the literature (Eur. J. Biochem., 13, 336, (1970)), and a plate suspended in agar was prepared. An antibacterial test disk was placed on the plate, impregnated with 50 ml of the filter sterilized culture supernatant, and allowed to stand at 15 and 30 ° C. The area of the halo formed around the disc after about 36 hours was measured. The area of the halo is shown in Table 3. As a control, the area of a halo formed by 0.1 ml of an aqueous solution of egg white lysozyme 0.1 mg / ml is also shown. The cell wall degradation activity of the culture supernatants of both strains was higher at the reaction temperature of 15 ° C. than at 30 ° C., which was opposite to that of egg white lysozyme.
[0022]
[Table 3]
Table 3

[0023]
Example 2: Production of cell wall degrading enzyme from Vibrio sp. SCRC-37964 (FERM P-18219) Vibrio sp. SCRC-37964 (FERM P-18219) was cultured in the same manner as in Example 1, and 100 ml of the culture supernatant was obtained. The solution was sterilized by filtration through a filter. Next, ammonium sulfate was added to a final concentration of 90% and stirred, and the supernatant was removed after centrifugation. The residue was dissolved by adding 5 ml of 0.1 M phosphate buffer (pH 6), and dialyzed against a buffer having the same composition at 4 ° C. for 2 days. About this sample and the case where this sample was heat-treated at 100 ° C. for 5 minutes, the cell wall decomposing activity at 15, 30 ° C. and 37 ° C. was examined in the same manner as in Example 1. The results are shown in Table 4.
[0024]
[Table 4]
Table 4

[0025]
Example 3; Production of cell wall degrading enzyme from Vibrio sp. SCRC-37964 (FERM P-18219) Vibrio sp. SCRC-37964 (FERM P-18219) was cultured in the same manner as in Example 1 and cultured at 5 to 30 ° C. The cell wall decomposing activity at each temperature up to was investigated. The results are shown in Table 5.
[0026]
[Table 5]
Table 5

[0027]
Example 4 Preparation of Protoplast of Gram-Negative Bacteria with Cell Wall Degrading Enzyme Prepared from Vibrio sp. SCRC-37964 (FERM P-18219) Vibrio sp. SCRC-37964 (FERM P-18219) was cultured in the same manner as in Example 2. Then, the protein in the culture supernatant was concentrated by ammonium sulfate precipitation and dialysis treatment. The protein concentration was 1.3 mg / ml as measured with the BIORAD protein assay kit. This sample was mixed with the same amount of protoplast buffer (sucrose 40%, 140 mM potassium chloride, 30 mM EDTA, 60 mM Bicine) to prepare a cell wall degrading enzyme solution. Pseudomonas putida ATCC-11172 cells collected from the logarithmic late growth medium by centrifugation, suspended in the above-mentioned cell wall degrading enzyme solution to a density of about 10 ⁸ cells / ml, and gently at 15 ° C. Shake. When observed with an optical microscope after about 2 hours, about 20% changed from gonococci to spheres. When a part of this reaction solution was diluted 3 times with distilled water or protoplast buffer, the number of cells in the former decreased to 60% of the latter. From these results, it was judged that a part of P. putida ATCC-11172 became a protoplast.
[0028]
【The invention's effect】
By using the microorganism of the present invention, it is possible to produce an enzyme that exhibits cell wall decomposing activity at a low temperature of 20 ° C. or lower and whose activity is remarkably reduced at 30 ° C. or higher. This enzyme utilizes its temperature characteristics, and when preparing protoplasts for bacterial protoplast fusion, after the reaction is completed, the enzyme is heated to 30 ° C or higher without any significant reduction in the protoplast yield, such as centrifugation or dialysis. Enzyme activity can be deactivated and proceed to the next step.

Claims (5)

Vibrio sp. SCRC-37964 (FERM-P18219) strain. Pseudoalteromonas sp. SCRC-38753 (FERM-P18220) strain. A method for preparing a protoplast for cell fusion of Escherichia coli using the microorganism culture solution according to claim 1 or 2. A cell wall degrading enzyme of Escherichia coli produced by the microorganism according to claim 1 or 2. A method for preparing a protoplast for cell fusion of Escherichia coli using the cell wall degrading enzyme produced by the microorganism according to claim 1 or 2.