JP2643264B2 - New thermostable alkaline protease and its manufacturing method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a thermostable alkaline protease and a method for producing the same. For more details,
Bacillus stearothermophil
The present invention relates to a thermostable alkaline protease having high thermostability obtained by extracting from a culture supernatant of a strain belonging to us) and a method for producing the same.

[Prior art] Conventionally, alkaline protease has been used as Bacillus subtilis (Baci
subtilisin produced by llus subtilis)
It is also widely used industrially. Subtilisins are also known as Carlsberg, Novo, BPN ', etc., and have a molecular weight of 26,000 and active site residues Asp32, His64 and Ser221. Examples of alkaline proteases other than subtilisin include Aspergillus oryzae Aspergillopeptidase B, C, Sorangium α-lytic proatase, and Streptomyces griseus pronase.

These alkaline proteases are widely used not only as research reagents but also for detergent addition and various food processing.

[Problems to be Solved by the Invention] As described above, alkaline protease is a proteolytic enzyme widely used industrially. However, if the heat resistance to heat is further increased, it can withstand use at high temperatures and the range of use can be expanded. Also, the stability of the enzyme is increased even at room temperature.
The present invention focuses on these points, and an object of the present invention is to provide an alkaline protease having higher heat resistance than conventional alkaline proteases.

[Means for Solving the Problems] As a result of intensive studies on the above problems, the present inventors have found that
Alkaline protease (hereinafter referred to as “protease”) with significantly improved heat resistance compared to conventional alkaline protease
NCA1503), which led to the present invention.

That is, the present invention is a thermostable alkaline protease having the following enzymatic chemical properties.

a) Molecular weight: about 28000 (gel filtration method, SDS-PAGE) b) pH stability: After treatment at 37 ° C. for 30 minutes, 90 at pH 6-12.
%) The activity is stable. C) Optimum pH: 10.0 to 10.5 d) Temperature stability: Residual activity after heat treatment at 60 ° C for 30 minutes: 100% Residual activity after heat treatment at 65 ° C for 30 minutes Activity: 50% e) Inhibitor: phenylmethylsulfonyl fluoride (PMSF) diisopropyl fluorophosphate (DF
P) f) Ultraviolet absorption spectrum: around 280 nm g) Substrate specificity: Gln-His, Ser-His, Leu-Tyr, Tyr-Le in proteins or peptides such as casein, azocasein, albumin, insulin, polylysine, etc.
specifically degrades the u, Phe-Tyr portion. h) Amino acid sequence and composition: Ala 32, Arg 12, Asp 12, Asn 4, Cys 2, Gln 13, Glu
23, Gly 17, His 11, Ile 14, Leu 22, Lys 19, Met
6, Phe 9, Pro 13, Ser 6, Thr 12, Trp 1, Tyr 15, Val
18 i) Nucleotide sequence of structural gene Conditions for inactivation by pH and temperature consisting of 783 base pairs: pH 4.0, 37 ° C, about 50% inactivation in 30 minutes. Further pH10,70
Deactivates almost completely at ℃ for 30 minutes.

The present invention also relates to a method for producing a thermostable alkaline protease, comprising culturing a bacterium belonging to the genus Bacillus stearothermophilus, which produces the protease NCA1503, and collecting the protease NCA1503 from the culture supernatant. And a method for producing the protease NCA1503.

The protease NCA1503 producing microorganism used in the present invention is a Bacillus stearothermophilus (Bacillus stearofils) capable of producing the protease NCA1503.
thermophilus), including all strains, mutants and variants belonging to the genus thermophilus. A particularly preferred strain is Bacillus stearothermophilus NCA1503 (Bacillus s.
tearothermophilus NCA1503)
It is.

To produce the protease NCA1503 of the present invention, for example, the above Bacillus stearothermophilus NCA1
503 (Bacillus stearothermophilus NCA1503) is ingested into a medium containing a carbon source, a nitrogen source, salts, and other substances, and at an appropriate temperature, for example, about 50 to 60 ° C., for an appropriate time until the enzyme titer reaches its maximum, Under aerobic conditions, for example, there is a method of producing by shaking or aeration and stirring culture.

The culture cultured as described above is clarified by a method such as centrifugation or filtration, and then the target enzyme can be precipitated by adding, for example, ammonium sulfate to the clarified liquid and collected. Further, the precipitate can be further purified by using an ion exchange method or a gel filtration method to obtain an extremely high protease NCA1503.

The novel protease NCA150 thus obtained
The physicochemical properties of 3 are shown below.

(1) Action The preferred pH for proteolytic action at 37 ° C. using casein as a substrate is pH 9 to 11, as shown in FIG. 1, and is an alkaline protease having an optimum pH of 10 to 10.5.

(2) Substrate specificity Decomposition of B-chain as a substrate at 37 ° C and pH 8.5 Figure 2
As shown in Gln-His, Ser-His, Leu-Tyr, Tyr-Leu, Ph
Decompose the e-Tyr part.

(3) Optimum pH FIG. 1 shows the effect of pH on the proteolytic action at 37 ° C. using casein as a substrate. The optimum pH is 10-0.5.

(4) pH stability FIG. 3 shows the residual activity after treatment at 37 ° C. for 30 minutes at various pHs using casein as a substrate. It showed a residual activity of 90% or more in the pH range of 6 to 12. At pH 4.0, about 50% was inactivated.

(5) Method for measuring titer 1 ml of a 2% casein solution (50 mM Tris-HCl, pH 8.5)
1 ml of each sample, and after a certain period of time, stop reaction solution (0.33 M
Acetic acid, 0.22M sodium acetate, 0.1M trichloroacetic acid) 2ml
In addition, it was left at room temperature for 30 minutes. This was filtered through Whatman No. 1 filter paper. Alternatively, put stop solution to casein solution, the sample was added, A ₂₇₅ as a reference which was filtered
nm was measured and tyrosine solution to create a calibration curve of A ₂₇₅ relative to the measured protease activity. One unit was defined as the amount of enzyme that releases 1 μg of a tyrosine-equivalent substance to a trichloroacetic acid-soluble fraction per minute at 37 ° C.

(6) Optimum temperature FIG. 4 shows the temperature dependence of the proteolytic action at pH 8.5 using casein as a substrate. The optimal temperature of action of this enzyme is about 55 ° C.

(7) Temperature stability FIG. 5 shows the temperature stability at pH 8.5 using casein as a substrate. This enzyme has a residual activity of 100% after heat treatment at 60 ° C for 30 minutes, which is very stable as an alkaline protease. 50% residual activity after heat treatment at 65 ° C for 30 minutes
It is. Deactivated almost completely by heat treatment at 70 ℃ for 30 minutes.

(8) Effect of inhibitor Table 1 shows the effect of the inhibitor.

(9) Protease Purification Method A crude enzyme sample obtained by ammonium sulfate precipitation, filtration, and the like from a culture supernatant can be separated and purified by the following method, for example.
That is, about 1 g of the crude enzyme sample was added to 10 ml of 0.1 M phosphate buffer.
It was dissolved in pH 8.0 and equilibrated with the same buffer. Toyopearl-HW
50 (trade name, manufactured by Tosoh Corporation)
Pour gently into the upper part and elute with the same buffer.
The active fraction obtained here is further applied to TSKgel-G2000SW (trade name, manufactured by Tosoh Corporation) to collect a peak fraction. Active fraction is ammonium sulfate salting out (70% saturation)
Or ultrafiltration membrane (UF-10PS, trade name, Tosoh Corporation)
), Dialysis, and freeze-dried.

(10) Molecular weight The molecular weight measured by gel filtration and SDS-polyacrylamide gel electrophoresis is about 28000, and the molecular weight from the amino acid sequence is 29,460.

(11) Ultraviolet absorption spectrum As shown in FIG. 6, the absorption maximum is around 280 nm.
The absorption minimum is at about 245-255 nm.

(12) Amino acid composition As shown in column h) Amino acid sequence and composition of [Problems to be Solved by the Invention] Consist of 261 amino acids.

(13) Nucleotide Sequence As shown in the column i) of the structural gene in [Problems to be Solved by the Invention], the sequence consists of 783 base pairs.

[Effects of the Invention] The protease NCA1503 according to the present invention has higher heat resistance than conventional alkaline protease, withstands use at high temperatures, has a wide range of use, and has increased enzyme stability even at room temperature. Protease NCA1503 is
It can be obtained by the method of the present invention.

Example Next, an example will be described. However, the present invention is not limited to only these examples.

(Example 1) 500 ml of a medium having the composition shown in Table 2 is placed in a two-volume Sakaguchi flask and sterilized at 120 ° C. for 15 minutes. 5 ml of a preculture of Bacillus stearothermophilus NCA1503 cultured in a medium having the same composition was added, and the cells were cultured with shaking at 50 ° C., 150 rpm for 24 hours. Ammonium sulfate was added to the culture supernatant to 70% saturation to precipitate an enzyme active fraction, and then centrifuged (1100
(00 rpm, 10 minutes). The collected enzyme was turbidized to a minimum amount of 0.1 M phosphate buffer pH 8.0, packed in a dialysis tube and dialyzed for 24 hours at 4 ° C. After the dialysis, the insoluble matter was removed by centrifugation and freeze-dried, and the obtained powder was used as a crude enzyme sample. About 1 g of the crude enzyme sample is added to 10 ml of 0.1 M phosphate buffer pH
It was dissolved in 8.0 and equilibrated with the same buffer. Toyopearl-HW50
(Trade name, manufactured by Tosoh Corporation)
0 cm), gently poured into the upper part, and eluted with the same buffer. The active fraction obtained here was further subjected to TSKgel-G2000SW (trade name,
Tosoh Corporation) and fractionated the peak fraction. The active fraction was concentrated using an ultrafiltration membrane (UF-10PS, trade name, manufactured by Tosoh Corporation), dialyzed, and lyophilized.
0.05 g of protease NCA1503 was obtained from one culture.

The purity of the purified enzyme was determined by polyacrylamide gel electrophoresis (12.5%) and high-performance liquid chromatography (column
As a result of analysis using TSKgel G2000SW), a single protein band was detected in each case.

(Example 2) The half-life of the enzyme activity at each temperature was measured in an alkaline solution (substrate of boric acid-sodium hydroxide, pH 9.0) of subtilisin Calsberg and protease NCA1503 used in detergents and the like. . Table 3 shows the results.

Thus, subtilin /
Compared with Carlsberg, protease NCA1503 hardly deactivates up to 60 ° C in alkaline solution.

[Brief description of the drawings]

FIG. 1 shows the optimum pH of protease NCA1503. FIG. 2 shows the degradation of oxidized insulin B chain of protease NCA1503, and shows the substrate specificity of this enzyme. FIG. 3 is a diagram showing the pH stability of protease NCA1503. FIG. 4 is a diagram showing the optimal temperature of protease NCA1503. FIG. 5 is a diagram showing the temperature stability of protease NCA1503. FIG. 6 shows an ultraviolet absorption spectrum of protease NCA1503 in an aqueous solution.

Claims (3)

(57) [Claims] 1. A thermostable alkaline protease having the following enzymatic chemical properties. Molecular weight: about 28000 (gel filtration method, SDS-PAGE) pH stability: After treatment at 37 ° C for 30 minutes, it has 90% or more activity in the range of pH 6 to 12, and is stable. 10.5 Temperature stability: Residual activity after heat treatment at 60 ° C for 30 minutes: 100% Residual activity after heat treatment at 65 ° C for 30 minutes: 50% Inhibitor: phenylmethylsulfonyl fluoride (PMS
F) Diisopropylfluorophosphate (DFP) Ultraviolet absorption spectrum: around 280 nm Substrate specificity: Gln-His, Ser-His, Leu-Tyr, Tyr-Leu in proteins or peptides such as casein, azocasein, albumin, insulin, polylysine, etc. , Phe
Amino acid sequence and composition that specifically degrades the Tyr moiety: Ala 32, Arg 12, Asp 12, Asn 4, Cys 2, Gln 13, Glu
23, Gly 17, His 11, Ile 14, Leu 22, Lys 19, Met
6, Phe 9, Pro 13, Ser 6, Thr 12, Trp 1, Tyr 15, Val
18 Structural gene base sequence Conditions for inactivation by pH and temperature consisting of 783 base pairs: pH 4.0, 37 ° C, about 50% inactivation in 30 minutes. Further pH10,70
Deactivates almost completely at ℃ for 30 minutes. 2. Bacillus stearothermophilus (Baci)
llus stearothermophilus)
A method for producing a protease, comprising collecting the protease according to claim 1 from the culture solution. 3. The method according to claim 1, wherein the strain is Bacillus stearothermophilus.
NCA1503 (Bacillus stearothermophilus NCA1503)
3. The method according to claim 2, wherein the method is (Pierced Laboratories No. 8978).