Patent Grant
6955907
The present invention relates to an alkaline protease VapK suitable for a laundry detergent, produced by Vibrio metschnikovii KS1, and to the gene vapk coding for said protease.
Enzymes used as laundry detergents have been continually improved since their commercialization in the 1970s. To date, protease, lipase, amylase, and cellulase have been developed for laundry detergents. Among these, protease has been the most widely used as an additive of laundry detergent. Widely used alkaline protease for laundry detergents exhibit their optimal activity at pHs 8 to 12 and at temperature of 40° C. to 60° C. Most proteases used as detergent enzymes are produced by the Bacillus species. They are classified as a serine protease and are widely known as subtilisin.
Subtilisin exhibits stability in high temperature but is not stable under certain conditions. Specifically, its activity is not maintained for a prolonged period upon exposure to urea, a protein denaturizing agent. Subtilisin also quickly and irreversibly loses its proteolytic activity at pH 4 or below. Moreover, the activity of subtilisin is weak at low temperature (15° C. to 25° C.). As such, it cannot be used effectively in Southeast Asia and Central and South America where people usually wash with low temperature water.
U.S. Pat. Nos. 5,741,694 and 5,482,849 describe subtilisin with biochemical properties altered through a site-directed mutagenesis. U.S. Pat. No. 5,401,657 describes bacteria which produce an alkali-resistant enzyme useful as a laundry detergent.
We isolated a vapk gene encoding a protease enzyme VapK, which can be advantageously used in laundry detergent in that it remains active at high temperature and at high alkaline pH, and is resistant to many surfactants and protein denaturants widely used in laundry detergents. The gene vapk has been cloned, sequenced and brought to expression in useful host cell. The gene vapk consists of 1,266 bp coding for 422 amino acids, and the molecular weight of the protease VapK is 27 kda. This enzyme is very resistant to various surfactants such as AOS or LAS and exhibits optimum activity at pH 10.5 and at the temperature of 50° C. Moreover, the protease VapK is more active at low temperatures than are commercially availiable enzymes.
The present invention provides a novel protease VapK having the following amino acid sequence (SEQ ID NO. 1):
The present invention also provides a vapk gene having the following nucleotide sequence (SEQ ID NO. 2):
In addition, the present invention provides a recombinant plasmid pSB1 containing the vapk gene and expressing it.
Further, the present invention provides a recombinant plasmid pSBCm containing the vapk gene and expressing it in a high yield.
Furthermore, the present invention provides a Vibrio metschnikovii KS1 (pSBCm) formed by transformation of V. metschnikovii KS1 with the recombinant plasmid pSBCm.
The present invention also provides a process for producing the protease VapK comprising the steps of culturing the V. metschnikovii KS1 (pSBCm) under conditions which allow the expression of protease VapK, and purifying it from the culture broth.
The characteristics of the recombinant plasmids pSB1 and pSBCm according to the present invention and the processes for producing said plasmids are described below.
Recombinant Plasmid pSB1 (
For cloning the vapk gene, chromosomal DNA of V. metschnikovii KS1 was partially digested and inserted into a vector pT7T3 19U. The resulting recombinant vectors were introduced into a host cell. The transformed microorganisms were cultured on a solid agar medium containing skim milk and the transformant, showing a clear halo, was isolated. The plasmid was isolated and identified from the transformant. The identified plasmid was revealed that it contains a 3.0 kb DNA insert and two Hind III sites. The E. coli cells having the isolated plasmid DNA were cultured. The alkaline protease in the culture solution was analyzed and determined to have a molecular weight of 27 kDa and to exhibit optimum activity at pH 10.5.
Recombinant Plasmid pSBCm (
The recombinant plasmid was transformed into V. metschnikovii KS1 to increase the productivity of the alkaline protease. Therefore, selection factors, such as antibiotic-resistance or colony-discoloration, were used to isolate the clone. The 3.0 kb vapk gene contained in plasmid pSB1 was subcloned into vector pKF3 which includes the cat-5 gene for chloramphenicol resistance (Cm). V. metschnikovii KS1 is sensitive to the antibiotic chloramphenicol. Therefore, it is easy to select the clone formed by transforming V. metschnikovii KS1 with the recombinant vector. The ampicilin-resisting factor is not suitable for the selection of the clone because V. metschnikovii KS1 shows resistance to the antibiotic ampicilin. The plasmid pSB1 was digested with Hind III and the resulting fragment was subcloned into the vector pKF3 digested with the same restriction enzyme Hind III. The recombinant vector obtained therefrom was introduced into E. coli. A strain exhibiting resistance to chloramphenicol and showing halo arround the colony on the skim-milk plate was isolated. The isolated strain contains the recombinant plasmid pSBCm. This pSBCm vector was used in transforming V. metschnikovii KS1.
In addition to V. metschnikovii KS1, E. coli HB101, E. coli JM101, E. coli Top10F′, V. metschnikovii RH530 N4-8, etc. can be used as a host.
To produce the desired alkaline protease, the transformed V. metschnikovii KS 1 was cultured in an LSC medium consisting of 1% Bacto-tryptone, 0.5% yeast extract, 1% sodium chloride, and 100 mM sodium carbonate buffer solution, pH 10.5.
The activity of the alkaline protease was determined by the method of Yanagida et al with minor modifications. The reaction mixture was prepared by mixing 100 mM sodium carbonate solution with the supernatant formed after centrifuging the culture broth at 6,000 rpm for 15 minutes. 0.5 ml of the fermentation broth was mixed with 2.5 ml of 1% prewarmed casein solution in 100 mM sodium carbonate buffer (pH 10.5). The resulting mixture was incubated at 37° C. for 10 minutes. The reaction was terminated by adding a solution consisting 0.22 M trichloroacetic acid, 0.22 M sodium acetate and 0.22 M acetic acid. The reaction mixture was placed on ice for 10 minutes followed by centrifugation. 1 ml of the supernatant was mixed with 9 ml of distilled water. The optical density (O.D.) was measured at 280 nm. The result is indicated as unit. One unit of the enzyme is defined as the amount of protein which produces an increase of 0.1 absorbance unit under the assay conditions.
The gene sequencing was conducted by a dideoxy chain termination method (Sanger et al., 1997, Proc. Natl. Acad. Sci. USA. 74: 5463-5467).
The invention will now be described with reference to the following illustrative Examples.
The recombinant plasmid pSB1 was constructed by a shot-gun cloning method. After growth of V. metschnikovii KS1, an alkaline overproducing-mutant, its chromosomal DNA was separated by the Mamur method (Mamur, et al., J. Mol. Biol. 3:208-218, 1961). The chromosomal DNA was partially digested with Hind III, and the fragment was precipitated with ethanol and recovered. The plasmid pT7T3 19U was prepared by the Birnboim and Doly method (Nucleic acids Res. 7:1513-1523, 1979), and digested with Hind III. Both DNAs were ligated. The resulting recombinat plasmid was introduced into E. coli HB101. The transformant was plated on an LB-Ap-skim milk medium containing 1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl, 40 μg/ml of ampicilin, and 1% skim milk. Several colonies, each forming a clear halo around the colony, were selected as the clones possessing the alkaline protease gene. One of them was confirmed as harboring the vapk gene encoding the alkaline protease VapK. The recombinant plasmid with the vapk gene was designated as pSB1. The nucleotide sequence of the vapk gene of pSB1 and the amino acid sequence of the expressed alkaline protease VapK were determined. In addition, the properties of the alkaline protease VapK were studied.
The 3.0 kb vapk gene was digested with various restriction enzymes. Each of the resulting fragments was subcloned into plasmid pUC19. The overlapping sites were drawn. The nucleotide sequence was determined based on such overlapping sites (SEQ ID Nos 1 and 2, and FIG. 5). A single open reading frame (ORF) of 1,266 bp exists between base 331 and base 1,596. The real molecular weight of the protease VapK is 27 kDa as mentioned above. However, the MW of the enzyme deduced from the ORF is 45 kDa. The difference results from the processing of the precursor region during the extracellular secretion of the protease VapK.
The precursor region is the signal sequence inducing the extracellular secretion of the expressed protein and consists of 25 amino acids. The site between Ala 25 and Ser 26 is expected to be processed. The amino acid positions 3 and 4 which are highly basic amino acids are lysine. The amino acid positions 5 through 25 include strongly hydrophobic amino acids. The −35 region (5′TTGACA3′), −10 region (5′TATAAAT3′) and Shine-Dalgarno (SD) sequence (5′CAAGGA3′) located 8 bp upstream of the initiation codon (ATG) are similar to those previously reported (FIG. 5). The dyad symmetrical sequence, located 18 bp downstream of the stop codon TAA, forms a stem-loop structure and functions as a rho (ρ) independent terminator. The amino acid sequence of the protease VapK deduced from the nucleotide sequence of the vapk gene was compared to a variety of known proteases (FIGS. 6 and 7).
As shown in
E. coil HB101 harboring the plasmid pSB1 was cultured in an LB medium. The culture broth was recovered and subjected to electrophoresis. As a result (FIG. 8), the expressed protease VapK was detected at the 27 kDa band. In addition, the proteolytic activity of the enzyme was measured. The results are shown in Table 1 below.
It can be seen from the above results that the vapk gene contained in the recombinant plasmid pSB1 was expressed in E. coli HB 101 and the expressed protease VapK was secreted extracellularly. The protease VapK exhibited optimum activity at pH 10.5 and at 50° C. (FIG. 9). This enzyme showed stability in the entire pH range. However, the stability of the enzyme was influenced slightly by temperature. The protease VapK exerted high resistance to surfactants such as LAS (linear alkylbenzen sulfonate), SDS (sodium dodecyl sulfonate), and AOS (Sodium-α-olefin sulfonate), making it suitable as a laundry detergent. The additional characteristics of the expressed recombinant VapK are shown in Table 2 below.
The results indicated that the enzymatic activity of the protease VapK was inhibited only by PMSF, the serine protease inhibitor. Thus, the protease VapK is a serine protease. In addition, the enzymatic activity of the protease VapK at the temperature 4° C. was nearly identical with that at 20° C. That is, the enzyme VapK showed high activity at low temperature. Accordingly, the protease VapK is an advance over proteases used to date which are active only at high temperature.
In addition, the resistance of the protease VapK to various surfactants was tested. The enzyme sample was appropriately diluted in distilled water. The diluted solution was mixed with each surfactant in the same ratio. The mixture was reacted at 25° C. for 25 minutes. A substrate solution of 5 mM N-succinyl-ala-ala-pro-phe-p-nitroanilide in 1 M tris-HCl, pH 9.0 was added to the reaction mixture in the ratio of 1 to 10. The reaction mixture was reacted at 25° C. for 30 minutes. The optical density of the reaction mixture was measured at 410 nm to determine the enzymatic activity of the protease VapK. The results are shown in Table 3 below.
The above results demonstrate that the protease VapK is very stable to anionic surfactants such as SDS and AOS and to nonionic surfactants such as polyoxyethylene alkylether. Under general washing conditions, the amount of surfactant is no more than 1,000 ppm. Therefore, it is believed that the protease VapK remains stable to surfactants during washing.
The V. metschnikovii KS1 was transformed with the recombinant plasmid pSBCm (FIG. 2). Vibrio strains secrete large amount of DNase extracellularly or within the periplasmic space. Furthermore,when the strains are shocked by high temperature, they easily die, even though the DNase is inhibited. Thus, the transformation could not be easily achieved by a conventional heat-shock method. Instead, the transformation was performed by electroporation under the following conditions:
(1) Effect of the Capacitance
The transformation efficiency was tested by varifying the capacitance in the range of 1 μF to 50 μF. The survival rate was 96% at 1 μF and 9% at 50 μF. The survival rate decreased as the capacitance increased. The transformation efficiency was highest at 10 μF.
(2) Effect of the Electric Field Strength
The transformation efficiency was tested by varifying the electric field strength in the range of 5 kV/cm to 10 kV/cm while fixing the capacitance at 10 μF. As a result, the highest transformation efficiency was obtained at 7.5 kV/cm.
(3) Effect of the DNA Concentration
The transformation efficiency was tested by varifying the DNA concentration in the range of 10 ng to 5 μg while fixing 10 μF of capacitance and 10 kV/cm of electric field strength. As a result, the transformation efficiency increased in proportion to the DNA concentration. In addition, the enzyme activity of the transformed V. metschnikovii KS1 (pSBCm) was tested. As shown in Table 4 below, the activity of the transformed V. metschnikovii KS1 (pSBCm) was twice as high as that of the host strain V. metschnikovii KS 1.
The strain V. metschnikovii KS1 of the present invention was deposited with the Korean Culture Center of Microorganisms, Seoul, Korea, on Dec. 15, 1998, as Accession No. KCCM-10141.
Also, the stain E. coli Top10F′ containing the recombinant vector pSBCm of the present invention was deposited with the Korean Culture Center of Microorganisms on Dec. 15, 1998, as Accession No. KCCM-10142.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1999-12588 | Apr 1999 | KR | national |
This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT/KR00/00022 which has an International filing date of Jan. 14, 2000, which designated the United States of America.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR00/00022 | 1/14/2000 | WO | 00 | 10/9/2001 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO00/61769 | 10/19/2000 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5336611 | van Eekelen et al. | Aug 1994 | A |
| 5858748 | Jones et al. | Jan 1999 | A |
