JPS5959190A - Preparation of protease - Google Patents

Abstract

PURPOSE:To prepare a large amount of protease with suppressing admixture of coexisting enzymes to a low level, by introducing a protease gene obtained from a bacterium belonging to the genus Bacillus to the same or different kind of bacterium belonging to the genus Bacillus, carrying out transformation, cultivating the transformed strain. CONSTITUTION:Chromosome DNA of a bacterium such as Bacillus amyloliquefaciens, Bacillus natto, etc. belonging to the genus Bacillus, capable of producing neutral protease excreted by the bacterium is cleavaged by a conventional procedure to give a DNA segment, which is introduced to the same or different kind of a bacterium belonging to the genus Bacillus by a method to use a vector, transformation is carried out, a bacterium to which the desired neutral protease gene or a DNA segment of donor chromosome containing the protease gene is introduced is selected from the transformed strain, the bacterium is cultivated under aerobic conditions by a conventional procedure, and neutral protease is collected from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves introducing a protease gene obtained from a Bacillus bacterium into the same or different Bacillus bacterium for transformation using genetic engineering techniques, and transforming the transformed strain. The present invention relates to the production of protease using.

Bacillus bacterium proteases are widely used in industrial applications, and the development of a useful protease production method has great industrial significance. Conventionally, Bacillus amyloliquefacens (B, a4erflL) has been used as its production method.
gw4aCL<ni ) or Bacillus amylosacchulicicus (B, ηb, mCdLahLiLcu, i
A method has been implemented in which bacteria of the genus Bacillus having a high protease-producing ability such as ``Bacillus'' are cultured, and protease is recovered from the culture supernatant. It is widely known that the Bacillus bacteria used in this method, which have a high protease production ability, accumulate other enzymes such as protease, amylase, ribonuclease, and levansucrase in the culture solution in addition to the target protease. ing. Therefore, in the conventional method, many difficulties and complicated steps were required to separate and recover only one enzyme from a culture supernatant in which many types of enzymes were accumulated. Furthermore, in conventional methods, no practically effective method was known for the purpose of improving protease productivity other than mutational manipulation that relies on chance.

The present inventors have deeply considered a method to solve these problems of the conventional method at once, and have found that the protease gene obtained from a high protease producing bacterium can be used with other extracellular enzymes or with a different type of bacterium that does not like to be mixed into the final product. By introducing an extracorporeal enzyme into a Bacillus bacterium that hardly accumulates in the culture solution and transforming it, it is possible to accumulate only a large amount of the target protease in the culture solution, and to efficiently collect and purify the protease. It was found that the transformed strain can accumulate a larger amount of the target protease than the chromosomal DNA donor strain, which has high protease productivity, and was also introduced to improve protease productivity. The present invention was achieved by discovering that the purpose can be achieved by improving the expression or increasing the copy number of the protease gene.

That is, the present invention involves cleaving the chromosomal DNA of an extracellular neutral protease-producing bacterium of the genus Bacillus, and using the resulting DNA.
A microorganism into which a neutral protease gene of interest or a donor chromosomal DNA fragment containing the protease gene has been introduced from the transformed strain by introducing the fragment into a Bacillus bacterium of the same or different species using a vector and performing transformation. The Bacillus bacteria used as the chromosomal DNA donor bacteria used in the Renki invention include extracellular neutral proteases such as Bacillus amyloliquefaciens, Bacillus amylosactucariticus, or Bacillus naratou (B, -μα). High productivity is desirable, but there are no particular restrictions, such as Bacillus subtilis (B, z
JfQJQ, +) or Bacillus stearothermophilus) 730.1la4ff-, ti-e, 侃σ
Any Bacillus bacterium capable of producing extracellular neutral protease such as P/L Note wi) can be used.

The extracellular neutral protease described in the present invention refers to meta-3-taloprotease, which has an optimal pH near neutrality and does not have a serine residue in its active center, among proteases accumulated outside the cell. .

Preparation of chromosomal DNA from extracellular neutral protease producing bacteria is carried out using 5abi, 6-ML x 4L using phenol.
L method (BLLyC/iL#m, PrLtyp~j
, ACC Sumi 72 619 (1963))
Alternatively, the Ma4mw4 method using chloroform-isoamyl alcohol (J, Mσ1. B=J, 3-
208 (1961)) and the like.

The prepared donor chromosomal DNA is then cut for ligation with the vector. The cutting of donor chromosomal DNA is
This is usually carried out by a method using a restriction endonuclease, but it is not limited to this. For example, it can also be carried out by physically applying a shearing force to cut it, as long as it does not cut the neutral protease gene of interest. be. When cutting donor DNA using a restriction endonuclease, if reaction conditions that cause complete cleavage are used,
Any restriction endonuclease that does not have a cutting site in the target neutral protease gene can be used, and various restriction endonucleases can be used depending on the conditions used, such as those that cut only partially. However, from the viewpoint of ease of ligation with a vector, it is desirable to have a unique cleavage site in the vector to be used.

As a vector, any vector can be used regardless of whether it is a plasmid or a phage, as long as it is replicable in the Bacillus bacteria used as the host. The plasmid that can be used as a vector may be any plasmid as long as it can be replicated in the intended host as described above, but it has a unique cleavage site by a specific restriction endonuclease,
Those with markers such as antibiotic resistance are desirable for ease of binding with donor chromosomal DNA and selection of transformed strains. An example of such a plasmid is pC1 with the chloramphenicol resistance marker derived from Staphylococcus (SicLP~1erc, σGCu).
94, pc 221. pc 223 (Pwc, N
a1i, Act, 5CjUSA 741680
(1977) ), pUBllo with kanamycin resistance marker (J, 13tLO mul = 1.13
4.318 (1,978)). Naturally, these plasmids are suitable vectors for the present invention, but plasmids derived from bacteria belonging to the genus Bacillus and whose physiological properties are unknown can also be suitable vectors for the present invention by adding some kind of marker to them.

When using a phage as a vector, any vector may be used as long as the phage DNA can be replicated in the host Bacillus bacterium. Examples of such materials include Toshite z1.963T, 96105, and ρ11.

In order to insert and link the cut donor chromosome DNA fragment into the above vector, the donor chromosome and vector are cut with the same restriction endonuclease, and then D
Although a method of binding using NAIJ gauze is common, the method is not particularly limited to this method; any conventionally known method can be used.

The recombinant DNA molecule thus obtained (conjugate of donor chromosomal DNA fragment and vector) is then introduced into a host bacterium belonging to the genus Bacillus. Various bacteria of the genus Bacillus to be used as a host can be selected depending on the purpose of carrying out the present invention. In other words, if you want to selectively accumulate a large amount of the target neutral protease in the culture solution by suppressing the accumulation of other extracellular enzymes, use Bacillus bacteria that have a low accumulation of extracellular enzymes. (Also, if you want to avoid simultaneous accumulation of other specific extracellular enzymes and the target neutral protease, you can use a Bacillus bacterium that is deficient in that specific enzyme as a host.) On the other hand, if you want to accumulate a specific exoenzyme and the desired neutral protease in the culture solution at the same time, use a Bacillus bacterium that accumulates the specific enzyme in the culture solution as a host. It can be used as

In order to introduce the recombinant DNA molecule into the Bacillus genus bacterium, which is the host bacteria selected in this way, and perform transformation, a transformation method using competent cells (M6.p-,
GJiL, G Buddha a, 167251 (1979)
) Transformation method using water or protoplasts (M
v, I-.

7-G, Gtηzf, 111 (1979), )
However, the method is not limited to these two methods. Primary selection is carried out using the desired neutral protein from the transformant obtained by all conventionally known methods. It is easy to carry out and select using changes in host protease activity as an indicator.

No specific method is required to accumulate the desired neutral protease in the culture medium using the selected transformed strain. That is, as a culture medium, a usual medium containing a carbon source, a nitrogen source, inorganic ions, and further organic micronutrients such as vitamins as necessary can be used. Cultivation is carried out under aerobic conditions while adjusting the pH and temperature of the medium as appropriate until a substantially desired amount of neutral protease is accumulated.

Recovery of neutral protease from the culture solution after culture 8- If only 8- is accumulated, the proportion of neutral protease in the precipitate obtained by adding ordinary organic solvent or inorganic salts to the culture supernatant is extremely large. By drying the precipitate, a neutral:1 protease crude enzyme powder containing less mixed enzymes can be easily obtained.

As detailed above, in the present invention, any bacterium of the genus Bacillus is used as a chromosomal DNA donor, and the extracellular neutral protease produced by the donor bacterium is transferred to a bacterium of the genus Bacillus, which is an arbitrarily selected host bacterium. It has the advantage that it can be manufactured using Therefore, in the present invention, by selecting a bacterium of the genus Bacillus that is highly productive of neutral proteases as a chromosomal DNA donor bacterium, and a bacterium of the genus Bacillus that hardly accumulates extracellular enzymes as a host bacterium, It becomes possible to produce a large amount of neutral protease while suppressing the contamination of contaminant enzymes. As described above, the improvement of the productivity of neutral protease and the simplification of the purification process will greatly contribute to the production of protease.

The present invention will be further explained below with reference to Examples.

Example (1) Preparation of chromosomal DNA from neutral protease high producing strains B, twL, yu, and 几Pt41aC2t-bxi p strains; B, arn; yicdli, Ha=-e-nr
F strain (ATCC23842) was grown in 21 NB medium (0.8
% meat extract; 0.8% polypeptone, 0.2% yeast extract, 0.4% NaCl, pH 7.2) at 37°C.
After culturing with shaking for 4 hours and collecting the bacterial cells at the late stage of logarithmic expansion, M
(Method of 147FLu4 (J, Mef.

B'=61.32081961) ) gives chromosome D
NA was prepared. The DNA obtained was 14.5+++47
Met.

(2) B, amyUtyJILP4aCL-e, n
i Cleavage of F strain DNA with restriction enzymes: The donor DNA obtained in (1) was cleaved using restriction endonuclease ECσR1 according to a conventional method.

(3) Preparation of vector DNA and cleavage with restriction enzymes: Cleavage was performed with restriction endonuclease ECσR1 according to a conventional method.

(4) Binding of donor DNA and vector: 10 μl of E, R1-treated plasmid pUB110 obtained in (1)
75μl of 50mM T4i--HCII (pH 8,
0) Suspend in a buffer solution and add 10 μl of bacterial alkaline phosphatase (BAP) manufactured by WvhnLLnN, vn. To 1 μl of this BAP-treated pUB110, add the ECσR1-treated donor DNA fragment (0.4μ91ttGlttll, 13 μl of distilled water, 110 times concentrated ligase buffer solution (660ml).
M T L]-14Cl pH7,6,66mM M'
JC12, 1'00mM Dithio/(Lytol, 2mM
ATP) 2plj and T4 DNA ligase solution 2μ7
(0,1 unit) was added, incubated at 15°C for 3 hours, and heated at 65°C for 5 minutes to stop the reaction.

(5) Introduction and transformation of recombinant DNA molecules into Bo, bd strain 1A289: B, which is amylase deficient and has low protease productivity.
aTuesday bLノlA289(” 1906 m-a-tB5
, aaCA321 α=yE) strain (strain kept at the Ohio University Bacillus Genetics Stock Center)
Cultured in 0i pen assay medium (manufactured by DLiOr).
z-tLnfI method (M, Mu0. QUL, Q-z
Protoplasts were prepared according to the following method (1979). This protoplast suspension was recombined into protoplasts using a 0.125-gauze reaction mixture (20 μl) according to the method of CL-xg (ibid.).
Introduction of A molecules into actual %112P, 0.5% glucose, 0.02% MgC1120, old% bovine serum albumin, 0.8% agar, m147.0) (.

and incubated at 37°C for 24 hours. Thereafter, all the colonies of the kanamycin-resistant transformants formed on the DM3 medium were transferred using toothpicks to an agar medium containing 008% caseiso (containing ~50 μg of kanamycin) and incubated at 37°C. 1
After 8 hours, 6 strains forming a large halo around the colony were found, and among them, the MT-07is strain (FER, M-BP-1 Ayu) with increased neutral protease activity was obtained. From this strain, the method of QhyCynm et al. (J, 13)
aCmuyLerJl i 34.31s (1978
)) After extracting the plasmid and cutting it with the restriction enzyme ECR1, agarose gel electrophoresis was performed, and as a result, it was confirmed that the donor DNA fragment had been inserted into this plasmid. Furthermore, when strain IA289 was transformed using this plasmid, it was confirmed that all kanamycin-resistant transformants had high productivity of neutral protease.

110: Tease and amylase productivity was determined from chromosome 1111; every DNA donor strain B, am, yJ16.1L1
Table 1 shows the results of comparison with the tt-cfacL<-F strain and the recombinant DNA molecule host strain B, 1JrL muno IA2F+9 strain.

Cultured in BY medium (0.5% meat extract, 0.2% yeast extract, 1% polypeptone, 0.2% NaCl pH 7.2)
The mixture was shaken for 24 hours at 30°C.

The protease activity was measured using the method of Hagiwara et al. (J, BQycLt7rL4' to 185 (19
58)) was followed. Fractionation of neutral protease and alkaline protease 1 = 22mM diimpropylfluorophosphate or 10mM ethylenediaminetetraacetate is added, and when each is added, the remaining Table 1

Claims (1)

[Claims] Cleaving the chromosomal DNA of an extracellular neutral protease-producing bacterium of the Bacillus genus and introducing the resulting DNA fragment into the same or different Bacillus genus bacterium using a vector 1
- performing transformation, selecting a microorganism into which the target neutral protease gene or a donor chromosomal DNA fragment containing the protease gene has been introduced from the transformed strain;
A method for producing protease, which comprises culturing the microorganism and collecting neutral protease accumulated in the culture solution.