JP2008048675A - Cellulase and cellulase gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cellulase having a potent decomposing activity to CMC (carboxymethyl cellulose) and chitosan, and to determine an amino acid sequence of the enzyme and a base sequence encoding the enzyme by performing the cloning of a cellulase gene encoding the celulase for providing them. <P>SOLUTION: This cellulase as a protein consisting of amino acids capable of being expressed by a specific sequence, the cellulase gene encoding the amino acids, a recombinant DNA including the gene and a constitution of a recombinant body containing the same are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cellulase that is an enzyme that degrades carboxymethylcellulose (hereinafter referred to as “CMC”) and a cellulase gene that encodes the cellulase.

As disclosed in Patent Documents 1 and 2, the inventors have disclosed peptide bonds (-Lys-X-linkage, X = 20 types of amino acid residues) on the carboxyl group terminal side of lysine residues in proteins and peptides. ) Lysobacter sp. IB-9374, which produces a high yield of lucyl endopeptidase (hereinafter referred to as “LEP”) that specifically cleaves only the bacterium, is isolated from the soil. Announcing.
JP 2000-83652 A JP 2001-231571 A

In addition, Lysobacter sp. IB-9374 is a freeze-dried product in the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba City 1-1-1 Tsukuba City, Ibaraki Prefecture) As of August 3, 1998, it has been entrusted with the deposit number “FERM P-16928”.

  Furthermore, in addition to the LEP, the bacterium secretes cellulase (hereinafter referred to as “Cel8A”), which is an enzyme that degrades CMC. Therefore, Cel8A was purified and its substrate specificity was examined.

As a result, as shown in Non-Patent Document 1, “Cel8A” was purified from the culture medium by ammonium sulfate fractionation, SP-Toyopearl 650M, Superdex 200, etc., and an electrophoretically uniform enzyme preparation was obtained. The purified enzyme had a molecular weight of 41 kDa, the optimum pH for CMC was 5.0, and the optimum temperature was 40 ° C. In addition to CMC activity, this enzyme exhibited chitosan activity and hydrolyzed in the order of CMC> colloidal chitosan>chitosan> glycol chitosan. The N-terminal amino acid sequence was APNYPFGSHRRA-, and the N-terminal amino acid was identical, but otherwise different compared to those from B. subtilis and Myxobacter sp. Showing both known activities. Further, 1/160 amount (w / w) of enzyme was reacted with CMC at pH 5.0 for 24 hours at 37 ° C., and the main degradation product was Cellobiose.
Agricultural Chemistry Society 2005, Annual Meeting Proceedings p29 “Purification and Properties of Lysobacter sp. Cellulase”

  Cellulase consists of a complex enzyme system that catalyzes an enzymatic reaction that degrades cellulose and its similar polysaccharides to glucose, cellobiose, or cellooligosaccharide, and depending on its mechanism of action, C1 enzyme, Cx enzyme and β-glucosidase, or exo-β -It is understood as a general term for enzymes called by names such as glucanase, endo-β-glucanase, cellobiase and the like.

  Among the above cellulases, the one having a particularly high action on CMC, that is, endo-type hydrolytic action is called carboxymethyl cellulase.

As a conventional cellulase, in Patent Document 3, it acts strongly on CMC and lichenan, has a wide working pH range, is stable in the presence of various surfactants, and is particularly excellent in alkali resistance. The carboxymethyl cellulase 5430 to which it belongs is published.
Japanese Unexamined Patent Publication No. 7-87972

  However, the cellulase described in Patent Document 3 is supposed to use “Rikenan” as a substrate in addition to CMC, but does not use “chitosan” as a substrate.

Therefore, the present invention performs cloning of a cellulase having a strong degradation against CMC and chitosan and a cellulase gene encoding the cellulase (hereinafter referred to as “ cel 8A gene”), and the amino acid sequence of the enzyme, the enzyme It is an object to determine a nucleotide sequence that encodes and to provide them.

  In order to solve the above problems, the present invention provides an amino acid sequence represented by SEQ ID NO: 2 or a part thereof, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence And a protein characterized by having cellulase activity, wherein one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 or a part thereof, or in those amino acid sequences A protein comprising an amino acid sequence and having cellulase activity, wherein the protein comprises the chitosanase activity in addition to the cellulase activity, and the protein comprising the amino acid sequence of claim 1. A protein encoding the amino acid sequence of claim 2 A gene encoding a protein having cellulase activity represented by SEQ ID NO: 1, a gene encoding a protein having cellulase activity represented by SEQ ID NO: 3, and having a chitosanase activity in addition to cellulase activity A gene encoding the protein of claim 4 to claim 7, a gene having a homology of 80% or more with the gene of claim 4 to claim 8, and encoding a protein having cellulase activity and chitosanase activity, A gene that hybridizes with the gene of claims 4 to 8 under stringent conditions, a recombinant DNA obtained by incorporating the gene of claims 2 to 6 into a vector DNA, A recombinant organism comprising the recombinant DNA according to claim 2, The gene was a recombinant organism, characterized by being inserted into the genome, and a recombinant organism arrangement according to claim 11 or 12, characterized in that a microorganism.

Here, hybridization under stringent conditions means that two DNA fragments are Sambrook.
Means hybridizing to each other under standard hybridization conditions as described by J et al. (Expression
of cloned genes in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold
Spring harbor Laboratory Press, New York, USA, 9. 47-9.62 and 11.45-11.61).

  More specifically, this means that hybridization and washing (for example, about 2.0 × SSC, 50 ° C.) are performed based on a Tm value of ± 10 ° C. The homology is, for example, 80% to 90% or more, preferably 95% or more, and more preferably 97.5% or more.

The recombinant DNA of the present invention is a vector DNA into which the cellulase activity of the present invention or a gene encoding the cellulase activity and chitosanase activity ( cel 8A gene) is inserted. The vector DNA is not particularly limited as long as it can be replicated and maintained in various prokaryotic and / or eukaryotic hosts, and includes plasmid vectors and phage vectors.

The recombinant DNA of the present invention can be obtained by adding the cellulase activity of the present invention or the gene encoding the cellulase activity and chitosanase activity ( cel 8A gene) to a vector, preferably a plasmid vector or bacteriophage vector, which can be conveniently obtained in the art. ) Can also be prepared by incorporation by a conventional method.

  Specific examples of such vectors include plasmids derived from E. coli such as pBR322, pBR325, pUC12, pUC13, pUC18 and pUC19, yeast derived plasmids such as pSH19 and pSH15, and Bacillus subtilis derived plasmids such as pUB110, pTP5, and the like. For example, pC194. Examples of the phage include bacteriophages such as λ phage, and animal and insect viruses such as retrovirus, vaccinia virus, and nuclear polyhedrosis virus.

An expression vector is useful for the purpose of expressing the cel 8A gene of the present invention and producing these proteins. The expression vector is not particularly limited as long as it has a function of expressing the cellulase gene of the present invention in various prokaryotic and / or eukaryotic host cells and producing these proteins.

When a bacterium, particularly E. coli, is used as a host cell, the expression vector generally comprises at least a promoter-operator region, an initiation codon, the cellulase gene of the present invention, a termination codon, a terminator region, and a replicable unit.

When yeast or animal cells are used as the host, the expression vector preferably contains at least a promoter, a start codon, the cel 8A gene of the present invention, and a stop codon. In addition, DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5 ′ side and 3 ′ side of the cellulase gene of the present invention, splicing junctions, polyadenylation sites, and the like can be incorporated into the expression vector.

The promoter-operator region for expressing the cel 8A gene of the present invention in bacteria contains a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is an Escherichia bacterium, preferred examples include those containing Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and the like.

  Examples of the promoter for expressing the cellulase of the present invention in yeast include PH05 promoter, PGK promoter, GAP promoter, and ADH promoter. When the host is Bacillus, SL01 promoter, SP02 promoter, penP promoter, etc. It is done.

  When the host is a eukaryotic cell such as an animal cell, a SV40-derived promoter, a retrovirus promoter, a heat shock promoter, a polyhedrin promoter possessed by a nuclear polyhedrosis virus, and the like can be mentioned. However, it is not particularly limited to these. In addition, the use of an enhancer is an effective method for expression.

  A suitable initiation codon is exemplified by methionine codon (ATG). Examples of stop codons include commonly used stop codons (eg, TAG, TGA, etc.). The terminator region includes a natural or synthetic terminator. A replicable unit refers to a DNA having a function capable of replicating its entire DNA sequence in a host cell, and includes a natural plasmid, an artificially modified plasmid (a DNA fragment prepared from a natural plasmid) and Synthetic plasmids and the like are included.

Suitable plasmids include pBR322 or an artificially modified product thereof (DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) in E. coli , yeast 2μ plasmid, or yeast chromosomal DNA in yeast. Examples of animal cells include plasmid pRSVneoATCC37198, plasmid pSV2dhfrATCC37145, plasmid pdBPV-MMTneoATCC37224, plasmid pSV2neoATCC37149, and the like.

  Examples of the enhancer sequence include SV40 enhancer sequence (72 bp), DNA major viruses such as polyoma, adeno and papilloma, retrovirus LTR (Long Terminal Repeat), immunoglobulin H chain, L chain gene and the like.

An expression vector can be prepared by linking a promoter, a start codon, the cel 8A gene of the present invention, a stop codon, and a terminator region continuously and circularly to appropriate replicable units.

  At this time, if desired, an appropriate DNA fragment (for example, a linker, other restriction sites, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

  The recombinant organism of the present invention includes a transformant and a transductant. A transformant can be prepared by introducing the above-described recombinant DNA into a host cell. Examples of host cells include microorganisms [bacteria (for example, Escherichia, Bacillus), yeast (for example, Saccharomyces), animal cells, insect cells, and the like).

  Specifically, Escherichia bacteria include Escherichia coli K12, DH1, M103, JA221, HB101, C600, XL-1Blue, JM109, etc., and Bacillus bacteria include Bacillus subtilis MI114, 207-21, etc. . Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, and DKD-5D. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells, and human FL cells. Examples of insect cells include BmN4 and Sf9, but are not particularly limited thereto.

  As a host cell, a prokaryotic cell is generally preferred for cloning of DNA sequences and assembly of vectors. The assembled vector is then transformed into a suitable host cell, where prokaryotic and eukaryotic cells can be used.

Introduction (transformation) of an expression vector into a host cell can be performed using a conventionally known method. In the case of bacteria (for example, E. coli and Bacillus subtilis ), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, (1972) 69, 2110) Protoplast method (Mol. Gen. Genet., (1979 168,111) or competent methods (J. Mol. Biol., (1971) 56, 209) etc., Saccharomyces
In the case of cerevisiae, for example, by the method of Hinnnen et al. (Proc. Natl. Acad. Sci. USA, (1978) 75, 1927) or the lithium method (J. Bacteriol., (1983) 153, 163), etc. For example, it can be transformed by Graham's method (Virology, (1973) 52, 456), but is not particularly limited thereto.

  Cel8A of the present invention can be produced by culturing a transformant containing the expression vector prepared as described above in a nutrient medium. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of the host cell (transformant).

  Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include soybean koji and potato extract.

  If desired, other nutrients [for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride], vitamins, and antibiotics may be included. Culturing is performed by methods known in the art.

  The culture conditions such as temperature, medium pH and fermentation time are selected to obtain the highest titer of Cel8A of the present invention. In addition, although the specific culture medium and culture | cultivation conditions used according to a host cell are illustrated below, this invention is not limited to these at all. When the host is a bacterium, actinomycetes, yeast or filamentous fungus, for example, a liquid medium containing the above nutrient source is suitable. In that case, it is desirable that pH is 5-8.

When the host is E. col , a preferred medium is M9 medium (Miller. J. Exp. Mol. Genet., (1972) p. 431, Cold Spring Harbor Laboratory, New York). In such a case, the culture can be performed usually at 14 to 42 ° C., preferably 28 to 39 ° C. for about 3 to 24 hours, with aeration and stirring as necessary. When the host is Bacillus genus, the treatment can be performed usually at 14 to 42 ° C, preferably at 28 to 39 ° C for about 3 to 96 hours with aeration and stirring as necessary.

  When the host is yeast, examples of the medium include Burkholder's minimum medium (Bostian.KLet.al (1980) Proc. Natl. Acad. Sci. USA, 77, 4505), and the pH is 5-8. It is desirable. The culture is usually performed at 14 to 42 ° C., preferably 20 to 35 ° C. for about 12 hours to 10 days, and if necessary, aeration and agitation can be performed.

  When the host is an animal cell, for example, a MEM medium (Science (1952) 122,501) containing about 5 to 20% fetal bovine serum, a DMEM medium (Virology, (1959) 8,396), an RPMI1640 medium [J. Am. Assoc., (1967) 199, 519], 199 medium [proc. SSoc. Exp. Biol. Med., (1950) 73, 1], etc. can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C., preferably 34 to 38 ° C. for about 12 to 72 hours, and aeration and agitation can be performed as necessary. .

  When the host is an insect cell, examples of the medium include Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA (1985) 82, 8404), and the pH is 5-8. Is desirable. The culture is usually performed at 20 to 40 ° C., preferably 25 to 30 ° C., for about 12 hours to 10 days, and if necessary, aeration and agitation can be performed.

  Cel8A of the present invention can be obtained from the culture obtained by the above culture as follows. That is, when the Cel8A of the present invention is present in the culture medium of the culture, the culture culture or supernatant is obtained by a method such as filtration or centrifugation, and the culture filtrate or culture supernatant is obtained. Then, Cel8A of the present invention is purified and isolated according to a conventional method generally used for purifying and isolating natural or synthetic proteins.

  As an isolation and purification method, for example, a method using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, a method using molecular weight difference such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods using charge such as ion exchange chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing hydrophobic differences such as reverse phase high performance liquid chromatography, isoelectric focusing etc. For example, a method using the difference in isoelectric point.

  On the other hand, when the Cel8A of the present invention is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to a conventional method such as filtration or centrifugation to collect the cells or cells, and an appropriate buffer solution. The cell wall and / or cell membrane of cells and the like is disrupted by a method such as ultrasonic wave, lysozyme and freeze-thawing, and then a crude extract containing the cellulase of the present invention is obtained by a method such as centrifugation or filtration. The crude extract can be isolated and purified by using a conventional method as exemplified above. The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

  Cellulase digests the cell walls of cells such as plants and microorganisms, is used to adjust protoplasts, and to decompose and solubilize plant fibers, and is used to maintain and enhance human health. On the other hand, chitosanase is used for the adjustment of mold protoplasts and as an enzyme for the adjustment of low molecular weight chitosan oligosaccharides having activity of inhibiting the growth of harmful microorganisms, antitumor activity and immunopotentiating activity.

  Accordingly, Cel8A having both cellulase activity and chitosanase activity can be efficiently and inexpensively produced by the gene encoding cellulase of the present invention, recombinant DNA incorporating the cellulase gene, and recombinant organisms containing them. The expenses in the conventional usage form can be kept low.

  The object of providing cellulase and its gene was realized by using a protein consisting of the amino acid sequence of SEQ ID NOs: 2 and 4 and a gene encoding the protein represented by SEQ ID NOs: 1 and 3.

[ Lysobacter
Determination of the nucleotide sequence of the cellulase (Cel8A) gene ( cel 8A gene) produced from sp. IB-9374]
1. Extraction of genomic DNA
Lysobacter
sp. IB-9374 is a medium composed of 0.5% milk casein, 1% sucrose, 1% meat extract, 0.01% monopotassium phosphate, 0.01% dipotassium phosphate and 0.01% magnesium sulfate (pH 7.2). 100 ml), and cultured with shaking at 30 ° C. for 16 hours, and then the cells were collected by centrifugation.

  To the obtained cells, 10 mM 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM EDTA was added and suspended uniformly. After adding 250 mM Tris-HCl buffer (pH 8.0) containing 1 ml of 10 mg / ml lysozyme to the suspension and placing on ice for 45 minutes with occasional mixing, 0.5% SDS, 0.4 MEDTA, 1 mg / ml proteinase 2 ml of STEP buffer composed of K and 50 mM Tris-HCl buffer (pH 8.0) was added and incubated at 50 ° C. for 16 hours.

  11 ml of 10 mM Tris-hydrochloric acid buffer (pH 8.0, TE buffer) containing 1 mM EDTA was added to the solution completely lysed by this operation and mixed gently. After adding 24 ml of PCI to the aqueous solution and mixing gently, the denatured protein was removed by centrifugation. This operation was repeated until no denatured protein was formed, and then the supernatant was mixed with an equal amount of chloroform: isoamyl alcohol (24: 1) to completely remove PCI.

  The DNA solution was layered with 2.5 times the amount of ethanol, and the interface was slowly swirled with a glass rod, and the DNA precipitated in the form of a filament was wound up with the glass rod. The wound DNA was rinsed with 70% ethanol, air-dried, and dissolved in 5 ml of TE buffer. Through these operations, 6.5 mg of genomic DNA of Rhizobacter genus IB-9374 was obtained.

2. Bam HI treatment of genomic DNA 10 units of Bam HI (Nippon Gene) was added to 25 μg of the genomic DNA obtained in Step 3, and incubated at 37 ° C. for 3 minutes. The genomic DNA solution partially digested with Bam HI was treated with PCI, chloroform, and ethanol precipitated, and then dissolved in 20 μl of TE.

3. Cosmid vector Lorist6 treatment with Bam HI and binding to genomic DNA fragments
After mixing Bam HI digested and dephosphorylated with alkaline phosphatase cosmid vector Lorist6 (Nippon Gene) 60ng and Bam HI partially digested genomic DNA solution (11μg-DNA) obtained in 2, DNAligation kit Ver.2 (Takara Bio) ) Ligation I solution was added in an equal amount and ligated at 16 ° C. for 6 hours. This solution was subjected to ethanol precipitation, and then dissolved in 20 μl of TE buffer.

4). In vitro packaging into bacteriophage λ Promega's Packagene Lambda DNA Packaging System (stored at −80 ° C.) was used for this operation. After melting 50 μl of Packagene Extract on ice, 5 μl of the DNA solution obtained in 3 (6 μg-DNA) was added and incubated at 22 ° C. for 3 hours. To this solution, 445 μl of Phage buffer and 25 μl of chloroform were added, mixed gently, and stored at 4 ° C.

5. Infection with E.coli DH5αMCR
E. coli DH5αMCR was cultured with shaking in LB medium (20 ml) containing 0.2% maltose and 10 mM magnesium sulfate at 37 ° C. for 16 hours. 10 ml of the culture solution was transferred to a sterilized Kolben, and 0.2 ml of the packaging solution prepared in 4 was added. After standing at 37 ° C. for 30 minutes, 25 ml of LB medium previously warmed to 37 ° C. was added, and cultured with shaking for 1 hour. The culture solution was centrifuged at 500 × g for 15 minutes at room temperature, the supernatant was discarded, and the cells were suspended in 1.2 ml of LB medium.

6). Selection of neomycin resistant transformants
50 μl each of the bacterial cell solution obtained in 5 was spread on an LB agar medium containing 25 μl / ml neomycin and cultured at 37 ° C. for 16 hours. Add 1 ml of LB medium containing 15% glycerol to an LB agar plate with neomycin-resistant transformants, scrape the transformants, transfer the cell solution to a sterile 1.5 ml plastic tube, − Stored at 80 ° C.

7). Preparation of specific primer Analyzing the amino terminus and internal amino acid sequence of Cel8A purified uniformly by electrophoresis, primerC12 ^{* 1} predicted from the sequence in the amino terminus sequence -APNYPFG- and sequence in the internal amino acid sequence -FFTAPFA -Primer C20 ^{* 2} predicted from-was synthesized respectively.

2μM
primerC12, 2μM primerC20, 0.3μg of Rhizobacter genomic DNA prepared in 1, 10μl of 10xGC
BufferI, 0.4 mM dNTP mix and 1 unit LA
PCR was performed on a 20 μl total volume of reaction solution consisting of Taq DNApolymerase (Takara Bio) using the following program ^{* 3} .

The amplified fragment of about 1,000 bp was ligated to pGEMT-vector (Promega) using DNA ligation kit Ver.2 (Takara Bio), and E. coli JM109 was transformed. As a result of purifying the plasmid from the transformant and determining the base sequence of the inserted fragment, the N-terminal and internal amino acid sequence of purified Cel8A was included in the amino acid sequence predicted from the base sequence of the fragment of about 1,000 bp. It was.

Therefore, this DNA fragment was confirmed to be a part of the gene encoding Cel8A. When determining the base sequence, BigDyeTerminator using the Sanger method
Ver. 3 (ABI) and ABI PRISM 3100 DNA sequencer (ABI) were used. Accordingly, the deduced amino acid sequence of this DNA fragment was synthesized cel 8A gene-specific primers primerF ^{* 4} and primerR ^{* 5.}

^{* 1} primerC12
5'-GC (A / C / G / T) CC (A / C / G / T) AA (C / T) TA (C / T) CC (A / C / G / T) TT (C / T ) GG-3 '(SEQ ID NO: 7)
^{* 2} primerC20
5'-GC (A / G) AA (A / C / G / T) GG (A / C / G / T) GC (A / C / G / T) GT (A / G) AA (A / G ) AA-3 '(SEQ ID NO: 8)

^{* 3} PCR Program Step 1 94 ° C 4 minutes 1 cycle Step 2 94 ° C 1 minute 30 cycles
45 ° C for 1 minute
72 ℃ 1 minute step 3 72 ℃ 5 minutes 1 cycle

^{* 4} primerF
5′-AGCCATCGCCAGGCCTACGTCAG-3 ′ (SEQ ID NO: 9)
^{* 5} primerR
5′-GCGTGACCAGCTTGGTGTTGGCG-3 ′ (SEQ ID NO: 10)

8). 5. Screening of cel 8A gene using PCR The transformant obtained in (1) was thawed on ice, diluted with sterilized physiological saline, plated on an LB agar medium containing 25 μl / ml neomycin, and cultured at 37 ° C. for 24 hours. About 300 single colonies were inoculated on an LB agar medium containing 25 μl / ml neomycin to prepare a PCR screening plate.

Colonies were inoculated from the plate into LB liquid medium containing 25 μl / ml neomycin and cultured at 37 ° C. for 16 hours, and then the cosmid vector was purified. Using this as a template, PCR was performed under the following conditions ^{* 6} using primerF and primerR prepared in 7. By this operation, an amplified fragment of about 500 bp was defined as a positive clone.

^{* 6} PCR program Step 1: 94 ° C, 4 minutes (1 cycle)
Step 2: 94 ° C, 1 minute → 60 ° C, 1 minute → 72 ° C, 1 minute (30 cycles)
Step 3: 72 ° C, 5 minutes (1 cycle)

9. Sequence 8. 8 positive clones, pGH4-1-24, pGH4-1-41, pGH4-1-44, pGH4-1-54, pGH4-1-61, pGH4-1-65, pGH4-1-75, and pGH4 -1-93 was obtained. A cosmid vector, pGH4-1-24, was obtained from this transformant by the alkali miniprep method, and this was sequenced using BigDyeTerminator Ver.3 (ABI) and ABI PRISM 3100 DNA Sequencer (ABI). The nucleotide sequence of the cel 8A gene was determined.

As a result, Lysobacter
The cel 8A gene derived from sp. IB-9374 strain had a total length of 1,242 bases (SEQ ID NO: 1) and encoded a tampac quality consisting of 414 amino acid residues. By computer analysis using the method of von Heijne (program, PSORT; http://psort.ims.u-tokyo.ac.jp/), regions 1 to 33 are presequences (hereinafter referred to as “prepeptide regions”). ).

  Further, the N-terminal amino acid sequence of purified Cel8A is 1-APNYPFGSHRQAYVS-15, which corresponds to the region of Nos. 34 to 48 of the deduced amino acid sequence, and further the estimated molecular weight of the region of Nos. 34 to 414 (41,241 Da ) Almost coincided with the molecular weight (41 kDa) of purified Cel8A.

  Accordingly, Cel8A was synthesized as a precursor composed of a pre-peptide region of 33 amino acid residues (hereinafter referred to as “signal peptide”) and a mature Cel8A region of 381 amino acid residues, and then subjected to post-translational modification. It was speculated that mature Cel8A with enzymatic activity was produced.

Reference to the accompanying drawings, cel 8A gene is present invention is described in detail Cel8A coding.

FIG. 1 shows the results of SDS-PAGE of Cel8A purified from Lysobacter sp. IB-9374. Lane 1 is a molecular size marker (unit: KDa). Lane 2 is purified Cel8A (2.5 μg / μl).

"Pel8A purification method"
Cel8A is cultured in the culture medium as described above for 30 hours at 30 ° C., and purified by ammonium sulfate fractionation from the culture liquid, followed by SP-Toyopearl 650M, Superdex 200, etc. Obtained an enzyme preparation.

  As a result of electrophoresis, Cel8A was estimated to be 41 kDa (triangle in the figure). The optimum pH for the degradation activity of CMC was 5.0, and the optimum temperature was 40 ° C. Moreover, this enzyme showed chitosan activity in addition to CMC activity, and showed hydrolysis activity in the order of CMC> colloidal chitosan> chitosan> glycol chitosan. The result is shown in FIG.

"Culture method of Lysobacter sp. IB-9374 strain"
In addition, as described in Patent Document 1, the culture conditions of this bacterium may be any of a natural medium and a synthetic medium as long as this microorganism can grow. Any liquid medium may be used.

  These media include, for example, sugars such as glucose, maltose and mannose as carbon sources, organic acids such as citric acid and malic acid, alcohols such as ethanol and glycerol, peptone as a nitrogen source, meat extract, In addition to general natural nitrogen sources such as yeast extract, protein hydrolyzate, and amino acids, various inorganic and organic ammonium salts are included. In addition, inorganic salts such as potassium, magnesium, iron, and calcium salts are required. Depending on the case, it is added appropriately.

  The culture conditions of the present bacterium can be performed by a conventional method, but the culture temperature is usually 20 to 35 ° C., preferably 25 to 35 ° C., and the pH of the medium is usually 6 to 8, preferably pH 6. 5 to 7.5. Moreover, it is desirable to carry out by aerobic conditions by means, such as shaking or aeration stirring, and culture | cultivation time is 150-250 hours normally.

  FIG. 2 is a table showing the substrate specificity of Cel8A. The hydrolysis reaction was performed under the following conditions using Cel8A purified as described above. The table left shows various substrates, the table center shows activity (U / mg), and the table right shows relative activity (%). The activity (U / mg) is determined from the standard substance (β-D-glucose for cellulose substrate, β-D-glucosamine for chitosan substrate, N-acetylglucosamine for chitin substrate, and p-nitrophenol for pNP substrate). The activity per 1 ml (U / ml) is calculated from the standard curve prepared by using the obtained value, and the value is obtained by dividing by the Cel8A concentration used. The relative activity (%) is a degradation activity value for each substrate when the degradation activity per unit time of Cel8A is 100%.

Substrate b: 85% deacetylated chitosan hydrate c: 99% deacetylated chitosan d: -40% deacetylated chitosan e: 40-50% soluble deacetylated chitosan Avicel: cellulose powder, Asahi Kasei ), PNP-Glc for standard biochemistry: p-Nitrophenyl-β-D-glucoside, Seikagaku Corporation

-Reaction conditions The reaction composition (0.75 ml) consists of 0.43% (w / w) of each substrate and 0.14 U of Cel8A dissolved in 50 mM acetate buffer (pH 5.0). The reaction temperature was 37 ° C. Here, 1U of Cel8A indicates the amount of enzyme that liberates 1 μmol of reducing sugar per minute. The reaction conditions for the substrate pNP-Glc consisted of 0.1% substrate, 4.5 U Cel8A and 50 mM acetate buffer (pH 5.0), and the reaction temperature was 37 ° C.

  From the results of FIG. 2, it was confirmed that Cel8A has chitosanase activity with respect to chitosan derivatives of about 40% to 15% of cellulase activity. From this, unlike the known bifunctional enzyme having a cellulase activity of about 40% or less of the chitosanase activity, it can be said to be a novel bifunctional enzyme exhibiting the reverse substrate specificity. There has been no report on an enzyme having such cellulase activity and chitosanase activity and having cellulase as a main activity.

FIG. 3 is a thin-layer chromatogram of the hydrolysis reaction product of CMC with Cel8A. Lane 0 is an unreacted product, lane 24 is a spot of a reaction solution using CMC as a substrate, and lane S is a standard product. Standard products, Glc; glucose, _{(Glc) 2;} cellobiose, _{(Glc) 3;} cellotriose, _{(Glc) 4;} cellotetraose, _{(Glc) 5;} using cellohexaose; Cerro maltopentaose, _{(Glc) 6} did.

・ Reaction conditions and results
When the enzyme of 1/160 volume (w / w) was reacted at 37 ° C for 24 hours at pH 5.0 and developed by a conventional method, the main degradation product when CMC was decomposed by Cel8A was cellobiose. Met.

  FIG. 4 is a thin layer chromatogram of the hydrolysis reaction product of colloidal chitosan by Cel8A. Lane 0 is an unreacted substance, Lane 0.5 is a colloidal chitosan substrate, reaction solution spot when reacted for 0.5 hours under the following conditions, Lane 4 is a 4-hour reaction solution spot, Lane Similarly, 24 is a spot of the reaction solution, and lane S is a standard product.

Standard products, GlcN; glucosamine, _{(GlcN) 2;} chitobiose, _{(GlcN) 3;} chitotriose, _{(GlcN) 4;} chitotetraose, _{(GlcN) 5;} Quito maltopentaose, _{(GlcN) 6;} using Quito maltohexaose did.

・ Reaction conditions and results
When the enzyme of 1/160 amount (w / w) was reacted at 37 ° C. for 24 hours with colloidal chitosan at pH 5.0 and developed by a conventional method, the degradation product when colloidal chitosan was decomposed by Cel8A was Chitobiose was the main degradation product.

FIG. 5 shows the nucleotide sequence encoding Cel8A derived from Lysobacter sp. IB-9374, the amino acid sequence of Cel8A deduced therefrom, and the gene sequence adjacent to that gene.

  The amino acid deduced from the determined base sequence was expressed in one-letter code below the gene sequence. The amino acid sequence is numbered from the first methionine residue. The estimated SD sequence is underlined. The N-terminal of purified Cel8A and the portions that matched the amino acid sequence deduced from the internal amino acid sequence and base sequence were displayed in black and white reversal and gray, respectively. An asterisk (*) is written under the stop codon. In the figure, solid lines “C12” and “C20” indicate positions corresponding to primerC12 and primerC20, respectively, and dotted lines “F” and “R” indicate positions corresponding to primerF and primerR, respectively.

  FIG. 6 shows the results of comparing the homology between a structural protein deduced from the nucleotide sequence of SEQ ID NO: 1 according to the present invention and a known protein. Known proteins are the following (1) and (2).

(1) β-1,3-1,4-glucanase from Bacillus
circulans WL-12 (B.WL12 in the figure)
(2) endo-1,4-β-glucanase from Bacillus
circulans KSM-N257 (B.N257 in the figure)

The structural gene of SEQ ID NO: 6 is translated from the methionine indicated by the arrow (A) and encodes a protein consisting of 414 amino acid residues (hereinafter referred to as “CelA8 precursor”). The signal peptide from arrow (A) to arrow (C) undergoes post-translational modification to form a region consisting of a total length of 381 residues after arrow (C) (hereinafter referred to as “mature cellulase region”). That is, the mature cellulase region is Cel8A produced by Lysobacter sp. IB-9374 strain having cellulase activity and chitosanase activity.

  As a result of comparison between the protein encoded by the gene of the present invention and the known proteins (1) and (2), (1) is 46% identical, homology is estimated to be 63%, and (2) is 45. % Concordance and homology was estimated at 63%. Of the three sequences to be compared, the portion where the amino acid residues matched in the two sequences was surrounded by a line.

As a result of homology search of amino acid sequence with known cellulase, Cel8A has family 8 motif region (ATDGDLDIAYALLLADLQW). Compared to those from B. subtilis and Myxobacter sp., Which exhibit both other known activities, the three N-terminal amino acids are identical, but otherwise. The whole amino acid sequence of the Cel8A precursor showed 46%, 45%, and 42% identity with Bacillus circulans WL-12-derived glucanase, Bacillus circulans KSM-N257-derived endoglucanase N257, and Bacillus sp.-derived chitosanase, respectively.

It is the result of SDS-PAGE of Cel8A purified from Lysobacter sp. IB-9374. It is a table | surface which shows the substrate specificity of Cel8A. The thin-layer chromatogram of the hydrolysis reaction product of CMC by Cel8A. Thin layer chromatogram of colloidal chitosan hydrolysis reaction with Cel8A. A base sequence encoding Cel8A derived from Lysobacter sp. IB-9374, an amino acid sequence of Cel8A deduced therefrom, and a gene sequence near the gene. It is the result of having compared the homology with the structural protein estimated from the base sequence of sequence number 1 which is this invention, and a known protein.

SEQ ID NO: 7 Basic sequence of forward primer for PCR, n is a, c, g, or t
SEQ ID NO: 8 Basic sequence of reverse primer for PCR, n is a, c, g, or t

Claims (14)

  A protein comprising an amino acid sequence represented by SEQ ID NO: 2 or a part thereof, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence, and having cellulase activity .   A protein comprising an amino acid sequence represented by SEQ ID NO: 4 or a part thereof, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence, and having cellulase activity .   The protein according to claim 1 or 2, which has chitosanase activity in addition to cellulase activity.   A gene encoding a protein comprising the amino acid sequence of claim 1.   A gene encoding a protein comprising the amino acid sequence of claim 2.   A gene represented by SEQ ID NO: 1 and encoding a protein having cellulase activity.   A gene represented by SEQ ID NO: 3 and encoding a protein having cellulase activity. The gene which codes the protein of Claims 4-7 which has a chitosanase activity other than a cellulase activity.   A gene encoding a protein having cellulase activity and chitosanase activity having 80% or more homology with the gene of claims 4-8.   A gene that hybridizes with the gene of claim 4 to 8 under stringent conditions.   Recombinant DNA, wherein the gene of claims 2 to 6 is incorporated into vector DNA.   A recombinant organism comprising the recombinant DNA according to claim 7.   A recombinant organism, wherein the gene of claim 2 to 6 is inserted into the genome. The recombinant organism according to claim 11 or 12, which is a microorganism.

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