JPH08259A - Novel cell wall lysing enzyme producing bacterium, cell wall lysing enzyme and method for producing enzyme preparation containing the same - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a bacterium that produces an enzyme that lyses the cell wall of yeast Phaffia rhodozyma, a cell wall lysing enzyme of the bacterium, and a method for producing an enzyme preparation containing the same. [Structure] Streptomyces Rochey PHA-34
A strain and a cell wall lysing enzyme for lysing the cell wall of yeast Phaffia rhodozyma produced by the strain, and a method for producing the enzyme-containing product. [Effect] Streptomyces Rochey PHA-34
The cell wall lysing enzyme of the strain can very easily lyse the cell wall of the yeast Phaffia rhodozyma.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is directed to the yeast Phaffia
The present invention relates to a novel cell wall lysing enzyme-producing bacterium having a cell wall lysing activity of Rhodozyma, a cell wall lysing enzyme thereof, and a method for producing an enzyme preparation containing the cell wall lysing enzyme.

[0002]

2. Description of the Related Art Cell wall lytic enzymes have been effectively used for elucidating the physiological functions of the structure of the cell wall of microorganisms and for preparing protoplasts necessary for studies such as cell fusion. Also, in terms of practical use, the bactericidal effect in foods and pharmaceuticals,
Alternatively, it is a useful substance which is considered to be applied in a wide range such as extraction of active ingredients existing in microbial cells.

On the other hand, astaxanthin, a natural pigment, has recently been attracting attention as a coloring agent and an antioxidant. Yeast Phaffia rhodozyma is attracting attention as the source. However, since the cell wall of yeast Phaffia rhodozyma is difficult to be dissolved by a commercially available enzyme agent, pigment extraction is mainly performed by physical disruption. However, this method is labor-intensive, and thus there is a demand for a low-cost and simple extraction method.

[0004]

In view of such circumstances, the present invention has found a strain producing a cell wall lysing enzyme that rapidly acts on yeast Phaffia rhodozyma cell wall and can efficiently extract intracellular astaxanthin, and the cell wall lysis The purpose is to establish a method for producing an enzyme-containing enzyme preparation.

[0005]

[Means for Solving the Problems] As a result of intensive studies, the present inventors have surprisingly found that one strain of Streptomyces rochei very strongly lyses the yeast Phaffia rhodozyma cell wall. The present invention has been completed.

That is, the present invention provides (1) a Streptomyces rochey PHA-34 strain that produces an enzyme that dissolves the cell wall of yeast Phaffia rhodozyma, and (2) a Streptomyces rochey PHA-34 described in (1). A strain bred by mutagenesis or gene recombination from the strain,
(3) Streptomyces rochey PH described in (1)
A cell wall lysing enzyme produced by the A-34 strain or the strain described in (2), characterized by the ability to lyse the cell wall of yeast Phaffia rhodozyma, and capable of lysing other bacterial cells, mainly yeast cells, (4) The cell wall lysing enzyme according to (3), which has one or more of β1 → 3 glucanase (laminarinase), xylanase, and neutral protease activity, and (5)
(1) Streptomyces rochey PHA-3
Production of an enzyme preparation containing a cell wall lysing enzyme, which comprises aerobically culturing 4 strains or the strain described in (2) in a liquid medium for enzyme production containing an inducing substrate and recovering the cell wall lysing enzyme from the culture solution. It provides the law.

Hereinafter, the present invention will be described in detail. The Streptomyces rochei strain of the present invention secretes a cell wall lytic enzyme outside the cell. This bacterium has been registered under the contract number FERM on April 21, 1994 at the Institute of Biotechnology, Institute of Biotechnology, AIST.
Deposited under P-14284. Hereinafter, in the present specification, this bacterium is abbreviated as PHA-34 strain.

The cell wall lysing enzyme of the PHA-34 strain can be obtained as follows. First, the PHA-34 strain was treated with an enzyme-producing medium (for example, glucose 0.1%, Phaffia rhodozyma 0.3%, yeast nitrogen base 0.67%, and potassium monophosphate 0.68).
% Liquid culture medium having a pH of 6.8), and shaking culture or aeration-agitation culture is performed. Here, Phaffia rhodozyma cells are used as an inducing substrate, and the form thereof may be alkali-treated dry powder or wet cells, or untreated dry powder or wet cells. Also,
The activity can be obtained by adding yeast cells other than Rhodozyma cells to the medium. Further, the activity can be obtained even in a medium supplemented with mannose, xylose and the like which are monosaccharides constituting the cell wall of Phaffia rhodozyma. The PHA-34 strain can secrete a cell wall lysing enzyme without adding such an inducing substrate to the medium, but it is preferable to culture in a medium to which the inducing substrate is added from the viewpoint of increasing the enzyme activity, and Phaffia rhodozyma. The body is particularly preferred as the inducing substrate.

Further, for example, the PHA-34 strain may be subjected to mutation treatment or gene recombination so as to increase the secretory productivity of cell wall lysing enzyme.

From the culture broth obtained as described above, a supernatant is separated by a known method such as centrifugation or filtration, and the supernatant is separated into, for example, a molecular weight exclusion of 5,000 or 10,000.
The dry powder can be obtained by using a known drying method such as freeze-drying without concentration or by ultrafiltration using the filtration membrane described above or concentration with ammonium sulfate. The concentrated solution may be dialyzed before drying.

The enzyme preparation thus obtained withstands long-term storage at low temperature in a dry powder state. Moreover, even if it is not made into a dry powder, there is no problem in using the enzyme. Therefore, the enzyme preparation containing the cell wall lysing enzyme of the present invention may be in the form of either a dry powder or a concentrated liquid. Since the cell wall lysing enzyme of the present invention contains any one or more of laminarinase, xylanase, and neutral protease activity, not only Phaffia rhodozyma but also other cells, mainly basidiomycete yeast, It also shows a good action on the cell wall of complete yeast. Therefore, the cell wall lysing enzyme of the present invention can be used not only for lysing the yeast Phaffia rhodozyma cell wall and efficiently extracting the red pigment, but also for preparing various yeast protoplasts, which is useful for industrial and research purposes. Extremely high

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Isolation and characterization of PHA-34 strain Screening for Phaffia rhodozyma cell wall lysing enzyme-producing bacteria from soil and mulch, the highest activity among them is a strept with a wide lytic spectrum that lyses basidiomycete yeast and imperfect yeast. One Myces rochei strain was selected and named PHA-34 strain. Below is PHA-3
The properties of the four strains are described. The PHA-34 strain secreted a cell wall lytic enzyme extracellularly. The growth temperature of the PHA-34 strain is 10
~ 45 ° C, and about 25-40 ° C was suitable for culture. The growth pH was near neutral (6.8), and the growth was good. On Emerson agar and enzyme-producing agar, the strain initially appeared white and gradually matured into beige colonies. Bennet
t) Colorless and transparent basal hyphae were observed on the agar medium, and the diameter was 0.8 to 1.5 μm, and some were rarely cut, but many were branched in many directions without being cut. .
The aerial mycelium was spiral, and when matured, its tip was cut to form spores. Smooth spore surface, but wrinkles at high magnification
Was observed and many were oval. The size was 0.8 to 1.5 μm in length and 0.5 to 1.0 μm in diameter. No sporangia, motile spores or segmented spores were observed in this bacterium that grew on the above medium, and melanin production was negative. Further, the cell wall of the bacterium obtained by liquid culture had L-type diaminopimelic acid and glycine.

Example 1 Production of Crude Enzyme Powder and Enzymatic Properties FIG. 1 shows a method for producing a cell wall lysing enzyme-containing product of the present invention from PHA-34 strain. A liquid medium having the composition shown in Table 1 was used for culturing the PHA-34 strain.

[0014]

[Table 1]

The optimum pH of the obtained enzyme was 5.5 to 6.0 (in MacIlvaine buffer) at 30 ° C. The optimum temperature was 30-40 ° C (pH
The cell wall of Phaffia rhodozyma was used as a substrate in 5.5 McIlvain buffer). The enzyme has a pH of 3 to 1.
It was stable in the 0 range and the temperature range of 10 to 40 ° C. The various enzyme activities contained in the enzyme were examined and the results shown in Table 2 were obtained.

[0016]

[Table 2]

Example 2 Dissolution of Phaffia rhodozyma cells The lysis of the cell wall was regarded as a reduction in the turbidity of the Phaffia rhodozyma cell suspension, and the rate of turbidity reduction was measured by the following method. Phaffia rhodozyma cell suspension (cells in stationary phase treated with alkali) has a turbidity of 500 ppm (CORONA U
T-11, about 1.0 × 10 ⁷ cells / 1 ml) was finally prepared, and the enzyme concentration was adjusted to 5 mg / ml with McIlbein buffer (pH 5.5) and the reaction solution was turbid. The degree was measured over time. As a control, the turbidity was measured in the same manner with the addition of McIlbein buffer instead of the enzyme solution. In addition, other commercially available enzymes Yatalas (Yatalas
The dissolution rate was similarly measured for e) and Zymolyase. The dissolution rate was calculated by the following formula. The results are shown in Table 3.
~ 5.

[0018]

[Equation 1]

[0019]

[Table 3]

[0020]

[Table 4]

[0021]

[Table 5]

Example 3 Measurement of bacteriolytic spectrum Using the novel cell wall lysing enzyme produced in Example 1, the logarithmic phase (log) was measured for 12 typical yeast strains other than Phaffia.
Phase), stationary phase early period (before sta), and late period (after sta), the rate of lysis with this enzyme was measured. As a result, 3 strains of incomplete yeast and 4 strains of basidiomycete were dissolved. Each dissolution rate after time is shown. The dissolution rate was obtained by the formula of Example 2.
Each cell was suspended in a McIlbein buffer solution having a pH of 5.5, and the decrease in turbidity was examined under the optimum conditions of this enzyme.

[0023]

[Table 6]

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, the cell wall of Phaffia rhodozyma, which was difficult to be lysed by conventional commercial enzymes, was added
It can be easily lysed using the cell wall lysing enzyme of the −34 strain, and astaxanthin, the active ingredient present in the cells, can be efficiently extracted. Furthermore, a method for producing the enzyme preparation containing the cell wall lysing enzyme is also provided.

[Brief description of drawings]

FIG. 1 is a diagram showing a method for producing a crude enzyme powder from PHA-34 strain.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location (C12S 3/00 C12R 1: 465) (72) Inventor Yataro Nunokawa 2 Hirata-cho, Ashiya-shi, Hyogo No. 7 of 105

Claims (5)

[Claims] 1. The yeast Phaffia rhodozyma (Phaffia rh
Streptomyces rochei PHA- that produces an enzyme that dissolves the cell wall of odozyma)
34 shares. 2. A strain cultivated by mutagenesis or gene recombination from the Streptomyces rochey PHA-34 strain according to claim 1. 3. Produced by the Streptomyces roshei PHA-34 strain of claim 1 or the strain of claim 2, characterized by the ability to lyse the cell wall of the yeast Phaffia rhodozyma, other bacterial cells, A cell wall lytic enzyme that can mainly lyse yeast cells. 4. One or more of β1 → 3 glucanase (laminarinase), xylanase, and neutral protease activity.
The described cell wall lytic enzyme. 5. The Streptomyces rochey PHA-34 strain according to claim 1 or the strain according to claim 2 is aerobically cultured in a liquid medium for enzyme production containing an inducing substrate,
A method for producing an enzyme preparation containing a cell wall lysing enzyme, which comprises recovering the cell wall lysing enzyme from a culture solution.