JPH0365150B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an endo-alkaline cellulase produced by an endo-alkaline cellulase-producing bacterium belonging to the genus Streptomyces.

(Prior Art) In recent years, there has been significant progress in washing clothes. That is, washing clothes has become significantly easier due to the development of raw materials suitable for detergents, improvements in water quality, improvements and spread of washing machines, improvements in fibers, etc. In particular, improvements in raw materials for detergents have been remarkable, and it appears that the composition of laundry detergents has almost reached perfection through modifications to surfactants, builders, dispersants, fluorescent dyes, bleaching agents, etc. . However, the idea behind the development of laundry detergents is that (1) surfactants and builders adsorb to the dirt and/or fiber surface, thereby reducing the bond between the dirt and/or fiber and water; (2) Disperse and solubilize dirt with surfactants and inorganic builders; (3) Chemically decompose dirt with enzymes such as protease; (4) Bleaching colored stains with bleach, etc., (5) Adsorbing fluorescent dyes etc. on the fiber surface,
(6) Using a chelating agent to prevent the precipitation of divalent metal ions in ingredients that are effective for cleaning.

That is, the basis of conventional clothing cleaning has been how to effectively incorporate components that directly attack dirt or components that assist in the attacking power of such components as one component of the detergent composition.

(Problems to be Solved by the Invention) However, at present, the cleaning performance of cleaning compositions based on this basic principle has almost reached a saturation point in a sense, and cleaning compositions with even higher cleaning power are currently being developed. development was desired.

From this perspective, the present inventors discovered alkaline cellulase as a substance that provides a novel cleaning mechanism, and identified Streptomyces sp. as a microorganism that produces this enzyme.
KSM-2 (Feikoken Bibori No. 7621), Streptomyces sp. KSM-
9 (Microtechnical Research Institute No. 7622) was found.

However, the alkaline cellulases produced by these microorganisms are a mixture of exo-type and endo-type cellulases, and their productivity is low.

(Means for Solving the Problems) The present inventors have cloned the chromosomal gene site that produces alkaline cellulase of an alkaline cellulase-producing bacterium belonging to the genus Streptomyces using the genetic cloning method of genetic engineering, and The present invention was completed by successfully producing only alkaline cellulase.

That is, the present invention relates to an endo-alkaline cellulase produced by an endo-alkaline cellulase-producing bacterium belonging to the genus Streptomyces.

The endo-alkaline cellulase used in the present invention belongs to the genus Streptomyces and has been transformed with a recombinant vector containing endo-alkaline cellulase-producing chromosomal DNA of an alkaline cellulase-producing bacterium belonging to the genus Streptomyces. It is produced by collecting and collecting endo-type alkaline cellulase-producing bacteria from a culture medium.

The preparation of chromosomal DNA involved in the production of endo-type alkaline cellulase from an alkaline cellulase-producing bacterium belonging to the genus Streptomyces is not particularly limited. For example, after dissolving the cell wall with lysozyme or the like, gentle lysis is performed using a surfactant to obtain a cell-free extract. Next, bacterial cell residue and DNA fraction are separated by centrifugation, and a DNA fraction is further obtained by cesium chloride equilibrium density gradient centrifugation.

Alternatively, after lysis with a surfactant such as SDS, phenol treatment and chloroform treatment are performed.
After purifying and separating the DNA fraction, DNA is obtained by ethanol precipitation.

The prepared donor chromosomal DNA is then cut for ligation with the vector. Cleavage of the donor chromosome is usually carried out by a method using a restriction endonuclease, but is not particularly limited to this method; for example, a method in which the donor chromosome is cleaved by physically applying a shearing force may be used. When using a restriction enzyme to cleave donor DNA, any restriction endonuclease that does not have a cleavage site in the alkaline cellulase gene can be used as long as reaction conditions that cause complete cleavage are used. Also, any restriction endonuclease can be used, provided that reaction conditions that cause only partial cleavage are used. Although various restriction enzymes can be selected depending on the conditions used, it is preferable to use a restriction enzyme that has a unique cleavage site for the vector used for ease of ligation with the vector. For example, Pst, Bam H, Sac, Kpn
, Bgl etc. can be used.

On the other hand, any vector can be used regardless of whether it is a plasmid or a phage, as long as it can be replicated in the Streptomyces microorganism used as a host.

When using a plasmid as a vector, any plasmid can be used as long as it can be replicated in the host, but one that has a unique cleavage site with a specific restriction enzyme and has markers such as antibiotic resistance is the donor chromosome. This is desirable from the viewpoint of binding with and ease of selection of transformed strains. Examples of plasmids include pJCP111, pIJ41, pIJ61, and pIJ61 obtained from known Streptomyces microorganisms.
Examples include pIJ361, pIJ702, pIJ365, and pIJ385.

In order to insert and link the cut donor chromosomal DNA into a vector, cut the donor chromosomal DNA and the vector with the same restriction enzyme, and then
A commonly used method is to use DNA ligase to join the two. However, the method is not limited to this method, and any conventionally known method can be used.

An example of DNA ligase is E. coli DNA.
Examples include ligase, T4 phage and DNA ligase.

The recombinant vector, which is a conjugate of the donor chromosomal DNA fragment and the vector prepared in this manner, is then introduced into a host microorganism of the genus Streptomyces.

Various Streptomyces microorganisms to be used as hosts can be appropriately selected and used depending on the purpose of carrying out the present invention.

The method for introducing the recombinant vector into the selected host microorganism, ie, the microorganism belonging to the genus Streptomyces, is not particularly limited, but a protoplast transformation method is optimal.

The method for selecting and isolating microorganisms into which the desired endo-alkaline cellulase gene or the donor chromosomal DNA fragment containing the endo-alkaline cellulase gene has been introduced from the transformed strain is not particularly limited, but may include markers such as antibiotic resistance possessed by the vector. A preferred method is to perform primary selection using expression and then secondary selection using endo-alkaline cellulase activity of the host as an indicator.

There are no particular limitations on the method for accumulating, recovering, and purifying endo-alkaline cellulase in a culture solution using the transformant finally selected in this manner.

The composition of the medium used for culture includes a suitable carbon source, so that the strain used can grow well and the endo-alkaline cellulase can be produced smoothly.
It consists of nitrogen sources, organic nutrients, inorganic salts, etc. As a carbon source, for example, soluble cellulose derivatives such as carboxymethylcellulose (CMC),
Pulp powder, paper powder, cellulose such as solid cellulose such as Avicel; glucose, fructose,
Any substance that can be assimilated can be used, such as carbohydrates such as sucrose or sorbitol; organic acids such as citric acid and succinic acid; and hydrocarbons such as n-dodecane and n-hexadecane. Among these carbon sources, a medium using cellulose or the like, especially a soluble cellulose derivative, is preferable because it produces a large amount of endo-type alkaline cellulase.

In Example 1 of the present invention, a transformant obtained using plasmid pCAS1 obtained by genetic manipulation, Streptomyces lividans HH21 No.
The physicochemical properties of the endo-type alkaline cellulase produced by FERM P-8276 (pCAS1) and FERM P-8276 are as follows.

1 Action: Carboxymethyl cellulose (CMC)
It decomposes cellulose such as, etc., using an endo type mechanism, resulting in a decrease in viscosity. On the other hand, the production of reducing sugars is hardly observed.

2 Substrate specificity: Acts specifically on cellulose such as CMC.

3 Optimal PH: The effect on CMC is optimal around PH8. As shown in FIG.

4 Stable PH range: When treated at 30℃ for 48 hours, PH
6 to 11, showing residual activity of 90% or more against CMC. As shown in FIG.

5 Optimum temperature: At pH 8, when CMC is used as a substrate, it is around 45°C. As shown in FIG.

6 Molecular weight: 32000 (gel filtration method) 7 Thermal stability: More than 90% activity remains after treatment at 40°C for 1 hour at pH 8. As shown in FIG.

The enzyme activity of the endo-alkaline cellulase of the present invention is determined by the enzyme activity of carboxymethyl cellulose (CMC).
When CMCase is used as a substrate, reducing sugars are hardly purified, so the determination was made according to the method for measuring endo-type CMCase activity described below.

That is, 3 ml of Wako Pure Chemical's CMC 1.0% solution, 2 ml of glycine buffer 0.5 M solution (PH8), and 3 ml of ion-exchanged water were mixed and kept at 40° C. in an Ubbelohde viscometer. Add 1 ml of the enzyme solution to this, stir quickly, then measure the viscosity, and take this value as V ₀ .
After standing at 40°C for 5 minutes, measure the viscosity again and take the value at this time as _V1 .

End type when the value of V ₀ −V ₁ /5 shows 1 cSt/min
CMCase1 unit.

(Effects of the Invention) Endo-type alkaline cellulase produced by a transformant derived from an alkaline cellulase-producing bacterium belonging to the genus Streptomyces can be used to stain clothing, especially collar fabrics, when added to detergents, especially clothing detergents. It has a remarkable cleaning effect on dirt on cuffs, dirt on shoes, etc.

Next, creation examples and examples of the present invention will be shown.

Creation example 1 (Preparation method of chromosomal DNA) Streptomyces sp. KSM-9
When preparing chromosomal DNA from (FERM P-7620), add 100 ml of MY/Na ₂ CO ₃ medium (meat extract 1.5
%, yeast extract 0.5%, _{KH2PO4 0.1} %,
After collecting the bacteria grown in Na ₂ CO ₃ 0.2%), washing, suspending in 8 ml of 25% sucrose, 50 mM Tris-HCl, 20 mM EDTA (PH 8.0) buffer, and adding 3.2 ml of EDTA (PH 8.0). Add 0) and 5 mg of lysozyme, let stand at 37℃ for 1 hour, and then add 2 ml of 10% SDS.
was added to lyse the bacteria. Add 4ml of 5M NaCl to this,
After standing at 0°C for 3 hours, centrifugation at 48,000g for 1 hour to obtain cleared lysate. PEG6000 was added to a final concentration of 10% to precipitate DNA. This was dissolved in TE buffer, and cesium chloride was added. Chromosomal DNA and plasmid DNA were isolated by density gradient centrifugation.

Creation Example 2 (Construction and Preparation of Recombinant Plasmid) The chromosomal DNA obtained in Creation Example 1 was transformed into Pst.
Perform complete separation at 37°C for 2 hours. Separately plasmid
pIJ385 is also completely disassembled using Pst.

Next, both are mixed, phenol extraction is performed, ethanol precipitation is performed, and ligation is performed.

Ligation was performed using T4 ligase at 12°C for 12 min.
A time reaction was performed.

Complete ligation was confirmed by agarose gel electrophoresis.

Creation Example 3 (Isolation of Recipient Bacteria) Streptomyces lividans strain HH21 is used as the recipient bacterium for transformation, but this bacterium
Since it has CMCase productivity, it cannot be used as a recipient bacterium as it is, so CMCase
A deletion mutant strain was induced.

After the spores of Streptomyces lividans HH21 were well dispersed in 0.05M phosphate buffer (PH7.0), NTG (N-methyl-N′-nitro-N
-Nitrosoguanidine) was added in an amount of 500 g, and the mixture was shaken at 30°C for 1 hour. This is diluted with physiological saline and plated on a BM agar medium containing CMC.

After colony formation, bacteria with reduced halo-forming ability were selected using the Congo Red staining method.

Although we were not able to obtain a complete CMCsae-deficient strain, we isolated a strain with a halo diameter less than 1/5 that of the wild type.
It was named Streptomyces lividans HH21 No. 1 strain.

Creation Example 4 (Protoplast Preparation and Transformation) Protoplast preparation and transformation were performed by the method of Bibb et al. (Bibb, MJ; JMWard & D.A. Hopwood, Nature 274, 398-400 1978).

That is, the bacterial cells grown in S medium are collected,
After washing with 0.3M Sucrose, suspend in 2mg/ml lysozyme (dissolved in P medium shown below) and incubate at 30°C.
Let stand for 1 hour to form protoplasts.

(P medium) Seuculose 103g K ₂ SO ₄ 0.25g MgCl ₂ 6H ₂ O 2.03g KH ₂ PO ₄ 0.05g CaCl ₂・2H ₂ O 3.68g TES (0.25M PH7.2) 100ml Water 1 This remains after cotton filtration. The mycelia were separated and protoplasts were obtained by centrifugation. After further washing well with P medium, suspend in a small amount of P medium, add the DNA prepared in Example 2, and add polyethylene glycol #1000 to a final concentration of 20%.
Incorporated DNA. After diluting this with P medium and centrifuging, it was spread on the R2YE agar medium shown in the pair and regenerated.

(R2YE agar medium) Shurose 103g K ₂ SO ₄ 0.25g MgCl ₂・6H ₂ O 10.12g CaCl ₂・2H ₂ O 2.95g KH ₂ PO ₄ 0.05g Glucose 10g Casamino acids 0.1g Yeast extract 5g L-asparagine 3g TES 100ml Agar 22g Water 1 After sufficient spores have settled on the R2YE agar medium, the transformed strain contains 30g of thiostrepton.
Selection was made by replicating on the BM agar medium shown below.

(BM agar medium) Yeast extract 1g Meat extract 1g NZ amine (type A) 2g Maltose 10g CMC 10g Agar 20g Water 1 Strains containing the desired CMCase gene were stained with a transparent halo on a CMC-containing agar plate using the Congo Red staining method. Forms around colonies. Among the halos produced, the strain that produced the largest halo was isolated, and this was used as Streptomyces lividans.
It was named HH21 No.1 (pCAS1). This strain is
It was deposited with the Institute of Fine Technology as FERM P-8276.

Creation example 5 (Confirmation of plasmid pCAS1) Streptomyces lividans HH21 No.1
(pCAS1), FERM P-8276 in MY medium at 30℃
After culturing for 3 days, the cultured cells were lysed, and after ultracentrifugation, the cleared lysate was subjected to cesium chloride density gradient centrifugation to obtain plasmids.
The existence of pCAS1 was confirmed. Plasmid pCAS1
The cleavage map of is shown in Figure 1. Analysis using restriction enzymes revealed that plasmid pCAS1 was derived from Streptomyces sp. KSM-9. It was confirmed that it had a 3.4Kb inserted fragment.

Example 1 (Production of endo-type alkaline cellulase) Meat extract, 1.5% (W/V), yeast extract 0.5%
(W/V), potassium dihydrogen phosphate 0.1% (W/V)
V), sodium carbonate 0.2% (W/V) (separately sterilized)
Streptococcus lividans HH21 containing pCAS1 was added to a 500 ml Sakaguchi flask and sterilized.
No. 1 (pCAS1), inoculated with FERM P-8276, 30
After shaking culture at ℃ for 4 days, the bacterial cells were removed from the culture solution by centrifugation.
When the CMCase activity was measured by a viscosity reduction method, an endo-alkaline cellulase was obtained with an endo-type CMCase activity of 0.106 cSt/min.

[Brief explanation of drawings]

FIG. 1 shows a cleavage map of plasmid pCAS1. The number inside the bracket represents Kb. Figure 2 shows the action of endo-type alkaline cellulase, the optimal
3 is a diagram showing its PH stability, FIG. 4 is a diagram showing its optimum temperature for action, and FIG. 5 is a diagram showing its temperature stability.

Claims (1)

[Scope of Claims] 1. Streptomyces transformed with a recombinant vector containing endo-alkaline cellulase-producing chromosomal DNA of an alkaline cellulase-producing bacterium belonging to the genus Streptomyces in a vector derived from Streptomyces Endo-type alkaline cellulase is produced by endo-type alkaline cellulase-producing bacteria belonging to the genus and exhibits the following physical and chemical properties. 1 Action: Carboxymethyl cellulose (CMC)
It decomposes cellulose such as, etc., using an endo type mechanism, resulting in a decrease in viscosity. On the other hand, the production of reducing sugars is hardly observed. 2 Substrate specificity: Acts specifically on cellulose such as CMC. 3 Optimal PH: The effect on CMC is optimal around PH8. 4 Stable PH range: When treated at 30℃ for 48 hours,
Shows over 90% residual activity against CMC at pH 6-11. 5 Optimum temperature: At pH 8, when CMC is used as a substrate, it is around 45°C. 6 Molecular weight: 32000 (gel filtration method).