JP2007028924A - Alkaline protease gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene encoding an alkaline protease, to provide a recombinant vector containing the gene, and to provide a transformant containing the recombinant vector. <P>SOLUTION: This gene which comprises a DNA encoding the alkaline protease derived from Bacillus, a protein which comprises an amino acid sequence encoded by the gene DNA, and a DNA which comprises an amino acid sequence given by subjecting one or several amino acids in the above amino acid sequence to deletion, substitution, or addition and encodes a protein having protease activity are provided, respectively. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a gene encoding a novel alkaline protease useful as an industrial enzyme.

  Alkaline proteases blended in various detergents including clothes detergents have played an important role as one component indispensable for improving detergency. Alkaline protease is the most produced enzyme among industrial enzymes because it is used as a mass consumer of detergents. Examples of such alkaline proteases include Savinase (registered trademark), Cannase (registered trademark; Novozymes), Maxacal (registered trademark; Genencor), Blapp (registered trademark; Henkel), KAP (Kao) and the like ( For example, see Patent Documents 1 to 3).

These alkaline proteases are derived from bacteria belonging to the genus Bacillus and form family A in the subtilisin superfamily. This family is further classified into intrinsic subtilisin, highly alkaline protease, intracellular alkaline protease, and the like (see Non-Patent Document 1). In general, detergent proteases belong to highly alkaline proteases, and their properties, molecular weight, and amino acid sequence homology are known to be high. Therefore, it has been desired to find a protease for detergents having a molecular weight and amino acid sequence different from those of known alkaline proteases and to develop them as new gene sources.
JP-A-4-349882 No. 6-292577 Special table hei 8-508643 report Siezen, RJ & Leunissen, JAM, Protein Sci., 6, 501-523, 1997

  The present inventors have succeeded in obtaining a gene encoding a novel alkaline protease having a low amino acid sequence identity with a conventionally known alkaline protease from the genomic DNA of the alkalophilic Bacillus bacterium that has been possessed.

That is, the present invention relates to a gene comprising the following DNA (a) or (b):
(A) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10,
(B) Provided is a DNA encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having protease activity.

The present invention also provides a gene comprising the following DNA (c) or (d):
(C) DNA consisting of the base sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9,
(D) Provided is a DNA that hybridizes with a DNA comprising the base sequence of (c) under a stringent condition and encodes a protein having protease activity.

  The present invention also provides a recombinant vector containing the gene and a transformant having the recombinant vector.

  By using the gene of the present invention, it is possible to produce a single and a large amount of alkaline protease useful as an enzyme for various industries including a detergent for clothing.

  The gene of the present invention comprises (a) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10, and (b) one or several amino acids in the amino acid sequence of (a) A DNA comprising a deleted, substituted or added amino acid sequence and encoding a protein having protease activity, (c) a DNA comprising a base sequence represented by SEQ ID NO: 1, 3, 5, 7 or 9, or (d ) A gene comprising a DNA that hybridizes with a DNA comprising the base sequence of (c) under a stringent condition and encodes a protein having protease activity.

  Here, in the amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10, one or several, preferably 1 to An amino acid sequence in which 10 amino acids have been deleted, substituted or added is included, and the addition includes the addition of one to several amino acids at both ends.

  The DNA encoding the amino acid sequence can be obtained from nature, but can also be prepared using a known technique such as site-directed mutagenesis. For example, it can be prepared by introducing a mutation using a mutagenesis kit [Mutan-super Express Km kit (Takara)] using site-directed mutagenesis.

The amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 of the present invention contains the amino acid sequence of a putative mature protein that functions as a serine protease, and is necessary for the expression of protease activity highly conserved in serine protease. Holds a region or amino acid sequence. When the identity between the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 of the present invention and the amino acid sequence of a conventionally known protein is examined, the amino acid sequence shown in SEQ ID NO: 2 is alkaline serine produced by Bacillus pseudofirmus. The highest identity with protease Vpr (Mol. Gen. Genet. 229 (2), 292-300 (1991)), the identity is 54.7%, and the amino acid sequence shown in SEQ ID NO: 4 is Bacillus sp. -On the database of extracellular minor serine protease (NCBI (HYPERLINK "http://www.ncbi.nlm.nih.gov/" http://www.ncbi.nlm.nih.gov/ )) produced by K16 strain published Bacillus sp. KSM-K16 protein number to; the highest identity with the sequences) represented by BAD63291, its identity is 38.6% and the amino acid sequence shown in SEQ ID NO: 6, Bacillus sp. Extracellular minor alkaline serine protease SprB (Appl. Enviro) produced by LG12 strain n. Microbiol. 61 (12), 4490-4493 (1995)), the identity is 52.8%, and the amino acid sequence shown in SEQ ID NO: 8 is produced by the Bacillus sp. It has the highest identity with alkaline serine protease SprA (Appl. Environ. Microbiol. 61 (12), 4490-4493 (1995)), and its identity is 76.6%. The amino acid sequence shown in SEQ ID NO: 10 is Bacillus The highest identity with the alkaline serine protease SprB (Appl. Environ. Microbiol. 61 (12), 4490-4493 (1995)) produced by sp. LG12 strain was 70.4%. This suggests that the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 is a novel alkaline protease, particularly an alkaline serine protease that does not completely match the conventionally known alkaline serine protease. Is.

  That is, in the gene of the present invention, when the corresponding sequence in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 is appropriately aligned, the amino acid sequence shown in SEQ ID NO: 2 has a sequence of 60% or more. 40% or more in the case of the amino acid sequence shown in No. 4, 65% or more in the case of the amino acid sequence shown in SEQ ID No. 6, 80% or more in the case of the amino acid sequence shown in SEQ ID No. 8, and the amino acid sequence shown in SEQ ID No. 10 In some cases, a gene that shows 75% or more identity and encodes a protein having protease activity is included. Among these, the identity of each sequence with respect to each amino acid sequence is preferably 85% or higher, more preferably 90% or higher, still more preferably 95% or higher, most preferably 98% or higher, and a protein having protease activity The gene encoding is preferred.

  The amino acid sequence identity described here is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, using the homology analysis (search homology) program of GENETYX-WIN (Ver. 5.1.1; software development), the unit size compare (ktup) can be calculated by performing analysis. it can.

  The gene of the present invention may be any protein as long as it encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 or a protein consisting of an amino acid sequence equivalent to the amino acid sequence, as described above. Of these, a gene comprising a DNA that hybridizes under stringent conditions to a DNA comprising the base sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9 and encodes a protein having protease activity is preferable. The base sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9 encodes a precursor containing the prepro region and mature protein of the serine protease-like protein of the present invention.

  Here, the “stringent conditions” include, for example, conditions described in Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989). For example, a solution containing 6XSSC (composition of 1XSSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / mL herring sperm DNA at 65 ° C. with the probe. Examples include conditions of constant temperature for 8 to 16 hours for hybridization.

  The gene of the present invention consisting of DNA encoding a protein having such protease activity is particularly preferably a gene consisting of DNA encoding alkaline serine protease.

The gene of the present invention is cloned from, for example, a microorganism belonging to the genus Bacillus, preferably Bacillus sp. KSM-LD1 strain (FERM P-18576) described in JP-A-2004-066511, etc., using a shotgun method, a PCR method or the like. In addition, it can be obtained in a large amount by using an appropriate vector and a host bacterium.

Using the gene of the present invention, an alkaline protease can be produced in a single and large amount. Specifically, the alkaline protease gene can be used in any vector suitable for expressing the gene in the target host. And transforming the host using the recombinant vector, culturing the resulting transformant, and collecting alkaline protease from the culture. The host used here is not particularly limited, but a microorganism is preferable. As the microorganism, a Gram-positive bacterium such as Bacillus (Bacillus subtilis), a Gram-negative bacterium such as Escherichia coli (Escherichia coli), a Streptomyces genus (actinomycetes), Saccharomyces Examples include fungi such as genus (yeast) and Aspergillus genus (mold), among which Escherichia coli (E. coli) HB101 is preferred. Examples of the vector used here include pUC18, pBR322, pHY300PLK and the like when Escherichia coli is used as a host, and pUB110, pHSP64 (Sumitomo et al., Biosci. Biotechnol, Biocem., 59, 2172-2175, 1995) or pHY300PLK. The culture may be carried out in accordance with a conventional method by ingesting into a medium containing a carbon source capable of qualitative microorganisms, a nitrogen source and other essential nutrients.

  Collection and purification of alkaline protease from the thus obtained culture can be performed according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized and granulated by a known method.

Example 1 Cloning of Alkaline Protease (SF) Gene Derived from Bacillus sp. KSM-LD1 and Determination of Base Sequence Around 64th His and 221 which are highly conserved regions in serine protease family 1 produced by Bacillus subtilis Between these regions for the amino acid sequences His-Gly-Thr-His-Val-Ala-Gly and Gly-Thr-Ser-Met-Ala-Ser-Pro-His-Val-Ala-Gly around the Ser Primer 1 (SEQ ID NO: 11: 5'-CAYGGNANCCAYGTNGCNNGGG-3 '), Primer 2 (SEQ ID NO: 12: 5'-CCNGCNNACGTGNGNGNGCATCATNGANGTNCTNGTCNGTCNGTCNGTCNGTCNGTCGNTGTNTGTGTNCG) -3 ′) was synthesized. Here, amino acid numbers are shown based on subtilisin BPN ′.

Bacillus sp. KSM-LD1 strain was aerobically shaken in a medium consisting of 1% tryptone (Difco), 0.5% yeast extract (Difco), 1% sodium chloride (Wako Pure Chemical Industries) at 30 ° C for 24 hours. Culture was performed, and the cells were collected by centrifugation. Genomic DNA is prepared from the obtained cells in accordance with the method of Saito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963), and PCR is performed using a combination of primers 1, 2, 1 and 3. went. PCR conditions were 10 to 100 ng of genomic DNA, 20 nmol of each primer, Pyrobest (Takara) reaction solution (50 μL), after heat denaturation at 94 ° C. for 1 minute using a Biometra: T-Gradient Thermocycler (Whatman). 30 cycles were performed with 1 cycle at 40 ° C for 1 minute, 40-60 ° C for 1 minute, and 72 ° C for 3 minutes. The obtained PCR fragment was purified using a High Pure PCR Product Purification kit (Roche), ligated to pUC18 digested with SmaI, and introduced into E. coli strain HB101. Plasmids were prepared from the transformants using High Pure Plasmid Isolation Kit (Roche), digested with EcoRI and HindIII, and the presence and size of the inserted fragment were confirmed by 1.2% agarose gel electrophoresis. An insert having a size of 200 bp to 1500 bp was selected, and the nucleotide sequence was determined using a BigDye Terminator Cycle Sequencing kit (Perkin Elmer) and a DNA sequencer (type 377: Applied Biosystems). BcaBEST Sequencing Primer RV-M (Takara) or BcaBEST Sequencing Primer M13-47 (Takara) was used as a primer. By comparing base sequences and selecting different ones, primers suitable for determining the base sequences in the upstream and downstream directions (same species, two types in the same direction: outside and inside) were synthesized. The genomic DNA of Bacillus sp. KSM-LD1 strain is digested with EcoRI or HindIII, and the digested fragments are ligated to the EcoRI or HindIII cassette in the LA PCR in vitro cloning kit (Takara), respectively. PCR was performed with primer C1. The reaction conditions for PCR were 0.1 to 1.0 ng of DNA fragments linked to the cassette, 10 to 20 pmol of each primer and Pyrobest (50 μL), and after heat denaturation at 94 ° C. for 1 minute, 94 ° C. For 1 minute, 55 ° C. for 1 minute and 72 ° C. for 4 minutes for 30 cycles. Thereafter, the amplified DNA fragment was amplified by PCR with each inner primer and attached primer C2. The PCR conditions at this time were as follows: 1 μL of the previously obtained amplified DNA fragment diluted 10000 times, using 10-20 pmol primer as a template (50 μL), heat denaturation at 94 ° C. for 1 minute, and then at 94 ° C. for 1 minute. 1 cycle at 55 ° C. and 2 minutes at 72 ° C. for 30 cycles. The base sequence of the amplified fragment was determined in the same manner as described above. Based on the newly obtained nucleotide sequence information, a primer for determining the nucleotide sequence of the upstream region and the downstream region was synthesized, and the region considered to encode alkaline protease (SF) and the upstream and downstream regions by the same technique. The entire base sequence (SEQ ID NO: 1) of the region was determined.

Example 2 Cloning of Bacillus sp. KSM-LD1 Strain Alkaline Protease (SG, SH, SI, SJ) Gene and Determination of Base Sequence According to the method of Example 1, Bacillus sp. KSM-LD1 strain alkaline protease ( SG, SH, SI, SJ) all base sequences (SEQ ID NO: 3, 5, 7, 9) were determined.

Claims (5)

A gene comprising the following DNA (a) or (b):
(A) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10,
(B) A DNA encoding a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having protease activity. A gene comprising the following DNA (c) or (d):
(C) DNA consisting of the base sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9,
(D) DNA that hybridizes with a DNA comprising the base sequence of (c) under a stringent condition and encodes a protein having protease activity.   A recombinant vector containing the gene according to claim 1 or 2.   A transformant comprising the recombinant vector according to claim 3.   The transformant according to claim 4, wherein the host is a microorganism.