KR20000030134A - Nucleotide sequences and amino acid sequences encoding a thermostable protease - Google Patents

Abstract

PURPOSE: A gene sequence of subtilisin-like serine protease isolated from the thermopile bacteria(Thermoanaerobacter yonseii), and an amino acid sequence thereof are identified, and are provided to apply such enzyme property, being able to maintain its activity under condition of high temperature and alkali pH, to the field of detergent industry. CONSTITUTION: A genome DNA of Thermoanaerobacter yonseii isolated from the volcanic region is extracted with solvent and is cleaved with a restriction enzyme, so that its DNA library is prepared. A short oligonucleotide primer being able to hybridize with a template strand, which contains a consensus sequence of serine protease, is prepared by PCR method and is used for screening the genome DNA library. After sequencing the screened DNA library, the serine protease gene sequence consisting of 315 amino acids and the amino acid sequence thereof are identified.

Description

DNA sequence of novel thermophilic protease and protein derived therefrom {Nucleotide sequences and amino acid sequences encoding a thermostable protease}

The present invention relates to DNA sequences of novel thermophilic proteases and amino acid sequences derived therefrom. More specifically, the present invention relates to a novel subtilisin isolated from thermophile thermophile thermoaerobacter Yonsei (Thermoanaerobacter yonseii KB1; patent strain No.-(KFCC 11116: Korean spawn association)). A DNA sequence of a subtilisin-like serine protease and an amino acid sequence translated therefrom.

Thermophilic proteolytic enzymes from high temperature bacteria have attracted much attention due to their high temperature and their resistance to detergents and organic solvents. Moreover, microbial alkaline proteolytic enzymes are one of the commercially important enzymes as the major constituents for detergents in the laundry industry. The best known alkaline proteases are subtilisin BPN 'from Bacillus amyloliquefaciens and subtilisin Carlsberg from Bacillus licheniformis (see M. Jacobs, M. Elisson, M. Uhlen, and J.-I. Flock, Nucl. Acids Res., 13, 8913-8926 (1985)). Alkaline serine-based proteolytic enzymes with high titration pH include alkalophilic Bacillus (K. Horikoshi, Agric. Biol. Chem., 35, 1407-1414 (1971)) and Streptomyces (MHJ Zuidweg, CJK Bos, and H. van Welzen, Biotechnol. Bioeng., 16, 685-714 (1972). Serine-based proteolytic enzymes from alkalophilic microorganisms are generally stable in high alkaline environments while weakly resistant to very high temperatures. On the other hand, proteolytic enzymes derived from high temperature bacteria are stable at high temperatures but unstable at high pH. For this reason, proteases with high activity and stability at high temperatures and high alkali conditions are of interest for their biotechnological applications. Thermophilic neutrophil proteolytic enzymes from thermophilic bacteria, alkaline proteolytic enzymes derived from alkalescent microorganisms stable at high pH and few thermophilic alkaline proteolytic enzymes have been reported (see MJ O'donohue). , BP Roques, and A. Beaumont, Biochem. J., 300, 599-603 (1994)). The previously reported amino acid sequences of alkaline proteolytic enzymes are alkaline serine proteolytic enzymes from Bacillus subtilis, subtilisin Carlsberg and subtilisin BPN '(see Koide, Y., A. Nakamura, T. Uozumi, and T. Beppu, J. Bacteriol., 167, 110-116 (1986)) and thermophilic neutrophil proteolytic enzymes from subtilisin E, subtilisin J, subtilisin DY, Bacillus stearothermophilus (see Takagi, M., T. Imanaka, and S. Aiba, J. Bacteriol., 163, 824-831 (1985)), and keratinase from Bacillus licheniformis, keratin proteases (Lin X, Kelemen DW, Miller ES, Shih JC, Appl. Environ. Microbiol., 61 (4), 1469-1474 (1995). It is known that Asp, His, and Ser, which are active sites of these amino acid sequences, are well preserved.

Accordingly, the inventors of the present invention have found that the DNA of the proteolytic enzyme similar to subtilisin cloned from the genomic DNA library of the thermophilic Thermoaerobacter Yonsei obtained from volcanic regions and the amino acid sequence translated therefrom is novel and the present invention is novel. To complete.

Accordingly, the present invention provides a DNA sequence of a novel subtilisin-like serine protease isolated from a thermophile, Thermophile aerobacter yonseii, and its translation from volcanoes. It is an object to provide an amino acid sequence.

FIG. 1 is a schematic diagram showing a process for preparing genomic DNA library of thermophile thermoaerobacter yonseii taken from a volcanic region.

2A shows the DNA sequence of a PCR product derived from genomic DNA and the amino acid sequence translated therefrom.

Figure 2b shows a comparison of the amino acid sequence of the PCR product and other microbial serine protease.

Figure 3 is a photograph showing the results of screening genomic DNA library.

4A schematically shows the DNA sequence of Thermicin.

4b shows the DNA sequence of Thermicin.

FIG. 4C shows the amino acid sequence translated from the DNA sequence of Thermicin as published in FIG. 4B.

Figure 5 shows a comparison of the amino acid sequence of Thermicin and known microbial serine proteolytic enzymes.

In order to achieve the above object, the present invention provides oligonucleotide primers for amino acid sites well preserved in serine-based proteolytic enzymes, and PCR products from the genomic DNA library of Thermoaerobacter Yonsei. DNA encoding a protease similar to the subtilisin of was cloned. The cloned DNA has an open reading frame of 1239 nucleotides encoding 413 amino acids. The estimated amino acid sequence is a signal peptide consisting of 13 amino acids, a prop peptide consisting of 85 amino acids, and a mature enzyme consisting of 315 amino acids. It was confirmed that it is separated into a serine proteolytic enzyme (hereinafter referred to as 'Thermicin') of the mature enzyme. Thermicin has an estimated molecular weight of 44.4 kD from the amino acid sequence and is expected to have a molecular weight of 32.9 kD when it becomes a mature enzyme, and the isoelectric point (pI) is estimated to be 8.51 from the amino acid component.

On the other hand, from the information search results of the protein, it was confirmed that this serine protease has similarity in the amino acid sequence of 61% to 51% with the subtilisin-like proteases of other known microorganisms. In addition, the novel protein of the present invention was confirmed that the amino acid sequence of the Asp, His, Ser and its constituents constituting the active site of the serine protease is well preserved as known enzymes, the present invention It is expected that the novel protein of has similar proteolytic activity to subtilisin.

The DNA sequence of the novel subtilisin-like serine protease isolated from the thermophile thermoaerobacter Yonsei of the present invention can be expressed by an expression system established in recombinant E. coli, yeast, Subtilisin-like proteases produced in large quantities in the same manner may be useful for the production of detergents and essential amino acids.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Example 1 Genomic DNA Extraction and Library Preparation of Thermophile Thermoaerobacter

The thermophilic thermoaerobacter Yonsei was cultured at 2 L at 80 ° C., and genomic DNA was extracted therefrom. The hot bacteria harvested by centrifugation at 5,000 × g for 20 minutes were reacted with Lysozyme and proteolytic enzyme K. Then, the same amount of chloroform / isoamyl alcohol (24: 1) was added to extract protein and carbohydrate complexes. After centrifugation, transfer the supernatant to a new tube, add the same amount of phenol / chloroform / isoamyl alcohol (25: 24: 1), extract it again, centrifuge for 20 minutes, add 0.7 times the volume of isopropanol and 3M sodium acetate. Sterile Pasteur pipettes were used to rescue genomic DNA from solution. The remaining salt was removed by washing with 70% ethanol, dried in a cleanbench and the DNA pellet was suspended in 1 ml of TE buffer to prepare genomic DNA. The prepared genomic DNA was partially digested with Sau3AI restriction enzymes, inserted into lambda ZAP Express (Stratagene, USA), and subjected to laboratory packaging to complete the library. Genomic DNA libraries were assayed to contain 10 ⁶ independent plaques. A process for preparing a genomic DNA library from thermophilic thermoaerobacter Yonsei is shown schematically in FIG. 1.

Example 2: Preparation of Probes

To select probes for screening subtilisin-like proteases using the genomic DNA prepared in Example 1, oligonucleotide primers were synthesized for amino acid sites well conserved in serine-based proteases of microorganisms. .: In other words,

As a 5'-primer, a degenerate oligonucleotide having a 5'- AAY GGN CAY GGN CAN CAY-3 'sequence based on the amino acid sequence NGHGTH around histidine, one of the active sites of the serine protease, is used. It was done; The 3'-primer is a degenerate oligonucleotide primer having a 5'-YGG YGT YGC CAT NGA NGT NCC -3 'sequence based on the amino acid sequence GTSMATP around serine, one of the active sites of the serine protease. (Wherein Y represents nucleotides in which C and T are mixed; N represents each nucleotide of A, G, C, and T).

Using the primers and Taq DNA polymerase as a template using the genomic DNA prepared in Example 1, 25 polymerase chain reactions were performed at 95 ° C. for 2 minutes, 45 ° C. for 30 seconds, and 72 ° C. for 30 seconds. Polymerase chain reaction (PCR) was performed to give a 585 bp PCR product. The obtained PCR product was subcloned into plasmid pGEM T easy (Promega, USA) and the DNA sequence was determined (FIG. 2A).

The amino acid sequence derived from the DNA sequence (PCR 585) is a known serine protease, that is, an alkaline serine protease isolated from Bacillus subtilis and a subtilisin like protease isolated from Streptomyces albogriseolus, respectively. It was confirmed to have a similarity of% and 56% (FIG. 2B). 2A and 2B, the underlined portion is a DNA sequence derived from a PCR primer and the box portion is a well conserved portion with other proteolytic enzymes.

Therefore, 585bp PCR product was used for the screening of the actual DNA library.

Example 3: Screening of Genomic DNA Libraries

The genomic DNA library prepared in Example 1 was introduced into E. coli XL-1 Blue and plated to obtain a total of 1.2 × 10 ⁵ plaques. The plaques were transferred to a nitro cellulose membrane, and then the membrane was treated with denatured solution (0.5N NaOH, 1.5M NaCl) for 2 minutes, neutralized solution (0.5M Tris HCl (pH7.4), 1.5M NaCl) for 5 minutes, and 2 × Wash for 30 seconds in SSC. The membranes were treated for 2 minutes in a UV crosslinker and then 65 ° C., 3 hours prehybridization in a solution containing 6 × SSC, 5 × Denhardt solution, 0.5% SDS, 100 μg / ml single strand salmon sperm DNA ) The probe selected in Example 2 was randomly labeled with [α- ³² P] dCTP and added to the prehybridization solution at a concentration of 1 × 10 ⁶ cpm / ml, followed by hybridization at 65 ° C. for 4 hours. It was. The membrane is then washed twice at room temperature for 15 minutes in a solution containing 2 × SSC, 0.1% SDS, twice at 15 ° C. for 15 minutes in a solution containing 1 × SSC, 0.1% SDS, and then at 0.1 × SSC. , In a solution containing 0.1% SDS was washed twice at 15 ° C. for 15 minutes.

The membrane was exposed to -70 [deg.] C. for 14 hours on an X-ray film under an intensive screen. As a result, in the duplicate on the X-ray film, 14 positive clones in which spots were formed at the same position were found (FIG. 3).

The photograph of FIG. 3 shows the result of hybridizing phage plaques transferred from the same plate to the nitro cellulose membrane and then exposing them to an X-ray film. Plated at low dilution multiples to repeat secondary and tertiary screening in the same manner as above, resulting in independent plaques, respectively.

Each clone was subcloned into the pBK-CMV vector (Stratagene, USA), and the restriction enzyme treatment resulted in an insert size of 1 kb to 2.6 kb. The partial sequence of each clone was determined to confirm that 14 clones contained a portion of the same gene, which determined the sequence of the entire longest 2.6 kb clone.

Example 4: Determination of DNA Sequences

Determination of the sequence of the 2.6 kb clone was carried out with emphasis on the site including the entire open reading frame in the 2.6 kb total insert (FIG. 4A), resulting in a sequence of 2605 bp. The dark GTG near 5 'indicates the initiation codon, and the dark TAA near 3' indicates the stop codon (Figure 4b).

Example 5: Analysis of DNA Sequences

The cloned DNA has an open reading frame of 1239 nucleotides encoding 413 amino acids. The estimated amino acid sequence is a signal peptide consisting of 13 amino acids, a prop peptide consisting of 85 amino acids, and a mature enzyme consisting of 315 amino acids. It was confirmed that it is separated into a serine proteolytic enzyme (hereinafter referred to as 'Thermicin') of the mature enzyme. Thermicin has an estimated molecular weight of 44.4 kD from the amino acid sequence and is expected to have a molecular weight of 32.9 kD when it becomes a mature enzyme, and the isoelectric point (pI) is estimated to be 8.51 from the amino acid component.

The DNA sequence (PCR 585) and amino acid sequence of this clone (Thermicin) and the 585 bp PCR product obtained in Example 2 were found to be identical in amino acids and nucleotides other than well conserved active sites.

On the other hand, from the results of the information search of the protein using Blast, this serine protease has an identity on the amino acid sequence of 47% to 32% with subtilisin-like proteolytic enzymes of other known microorganisms, and 51% It was confirmed that there is similarity in the amino acid sequence of from 61% (Fig. 5). In addition, the novel protein of the present invention, Asp, His, Ser (indicated by * in FIG. 5) and its surrounding amino acid sequences constituting the active site of the serine-based protease are well preserved as well known enzymes Was confirmed. From these results, it could be expected that there will be very similar serine proteolytic enzymes with different amino acid sequences in Thermoaerobacter Yonsei. In FIG. 5, the box markings indicate conserved amino acids; The asterisk indicates an active site among them. Thus, the novel protein of the present invention was also expected to have similar protein activity as subtilisin, so it was decided to call it 'Thermicin'.

As described above, the present invention provides a DNA sequence of a novel thermophilic subtilisin-like serine protease isolated from thermophile thermophile aerobacter yonseii collected from a volcanic region. Providing amino acid sequences translated therefrom. The DNA sequence of the novel subtilisin-like serine protease isolated from the thermophile thermoaerobacter Yonsei of the present invention can be expressed by an expression system established in recombinant E. coli, yeast, Subtilisin-like proteases produced in large quantities in the same manner may be useful for the production of detergents and essential amino acids.

Claims (4)

Of a novel subtilisin-like serine protease having all or part of the following sequence isolated from the thermophile Thermophile aerobacter yonseii DNA sequence: PCR primers and probes having the following sequences for use in library screening of DNA as claimed in claim 1: Amino acid sequence having all or part of the following sequence derived from DNA as claimed in claim 1 A subtilisin-like proteinase isolated from the Thermoanaerobacter yonseii having the amino acid sequence of claim 3.