WO2005056787A1 - 界面活性剤に耐性なセルラーゼ及びその変換方法 - Google Patents
界面活性剤に耐性なセルラーゼ及びその変換方法 Download PDFInfo
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- WO2005056787A1 WO2005056787A1 PCT/JP2004/018184 JP2004018184W WO2005056787A1 WO 2005056787 A1 WO2005056787 A1 WO 2005056787A1 JP 2004018184 W JP2004018184 W JP 2004018184W WO 2005056787 A1 WO2005056787 A1 WO 2005056787A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
Definitions
- the present invention is directed to converting a protein having an endalcanase activity (particularly a cellulase belonging to the family 45 and having an endalcanase activity) at the N-terminus to pyroglutamic acid, and in the presence of a surfactant,
- the present invention relates to a method for converting into a cellulase with a small decrease in the activity of an endalcanase, and a cellulase thereof.
- Cellulosic biomass is said to be the most naturally occurring resource. Therefore, efficient use of a cellulase system that degrades this is expected in various fields. During this process, various cellulases have been purified and their properties have been elucidated. Furthermore, various cellulases have been cloned, the homology of their sequences has been analyzed, and families have been classified (see Non-Patent Document 1).
- cellulase is utilized in various industrial fields by virtue of its properties, but its use in the field of textile is important. For example, the texture of cellulose-containing fibers and
- Cellulases have been used to improve Z or appearance, or for "biowash” to provide a "stone wash” -like appearance that provides a localized change in color to the colored cellulose-containing fibers.
- lyocell a regenerated cellulosic fiber derived from wood pulp, which has recently attracted attention due to its strength, water absorbency, and other properties, as well as its production method that is less likely to cause environmental pollution, is used to remove fluff on the fabric surface generated during the production process. Cellulase is used for processing except for the above.
- cellulase has been considered to degrade cellulose by a synergistic effect of a plurality of enzymes, that is, a synergistic effect.
- This complex enzyme cellulase group also includes enzyme systems having unfavorable properties in the field of fiber processing such as reducing fiber strength. Therefore, attempts have been made to separate and produce enzyme components suitable for fiber calories from these cellulases using protein separation technology and genetic engineering techniques.
- components such as CBH I, CBH II, EG II, EG II are isolated
- cellulase preparations containing a large amount of specific cellulase components suitable for each application have been produced.
- cellulases belonging to family 45 such as NCE4 (see Patent Document 2), NCE5 (see Patent Document 3), RCE1 (see Patent Document 4), and STCE1 (see International Patent Application No. PCTZJP2004Z15733), are in the fields mentioned above. It is also clear that it is very useful.
- cellulase when used for a detergent for clothing, it is desired to improve not only the required cellulase component quantitatively but also qualitatively.
- various detergents are mixed in clothing detergents, and when they are dissolved in water, they show alkalinity (pHIO-11). It is required to exhibit resistance to various surfactants under alkaline conditions. So far, Otzen, DE et al. Have introduced a mutation into the internal amino acid sequence of Cel45 derived from Humicola insolens to introduce a linear alkylbenzene at pH 7 as an example of suppressing the activity decrease in the presence of a surfactant.
- Non-Patent Document 2 It is reported that the activity in the presence of sulfonic acid (LAS) is improved about 3.3 times (see Non-Patent Document 2). However, the suppression of activity reduction in the presence of a detergent conferred by this mutation is limited to Cel45 or its homologues, and is limited to the endalcanase belonging to family 45, which has low homology to Cel45. Is not applicable, it is clear!
- Patent Document 1 International Publication No. 91Z17243 pamphlet
- Patent Document 2 International Publication No. 98Z03667 pamphlet
- Patent Document 3 International Publication No. 01Z90375 pamphlet
- Patent Document 4 International Publication No. 00Z24879 pamphlet
- Non-patent literature 1 Henrissat B., Bairoch A. Updating the sequence—based classification of glycosyl hydrolases. Biochem. J. 316: 695—696 (1996)
- Patent Document 2 Daniel E. Otzen, Lars Christiansen, Martin Fischin. A comparative study of the unfolding of the endoglucanase Cel4 5 from Humicola insolens in denaturant and surfactant.Protein Sci. 8: 1878-1887 (1999)
- the present invention relates to a method for converting a protein having an endalcanase activity, particularly a protein having an endalcanase activity belonging to family 45 into a protein having a small decrease in activity in the presence of a surfactant
- An object of the present invention is to provide a vector used in the method, a protein having a small decrease in activity in the presence of a surfactant, having an endalcanase activity, and a polynucleotide encoding the same. Furthermore, by using them, it is intended to provide a microorganism that efficiently produces a protein that can be used as an enzyme for washing clothes.
- pQ pyroglutamic acid
- peptide containing pyroglutamic acid is added to the N-terminal side of a protein belonging to family 45 and having an endalcanase activity, that is, an N-terminal addition type Cellulase did not show a marked decrease in endalcanase activity in the presence of a surfactant, compared to its native form.
- a peptide containing pyroglutamic acid or pyrodaltamic acid in a cellulase that does not want to decrease its activity in the presence of a surfactant and has all N-terminal amino groups protected.
- pyroglutamic acid or a peptide containing pyroglutamic acid The cellulase that can be added is not particularly limited. For example, a cellulase belonging to family 45 is preferable.
- the present invention includes the following inventions.
- An endoprotein in the presence of a surfactant characterized in that a protein having an endodalcanase activity and whose N-terminus is not pyroglutamic acid is modified into a protein having an N-terminus of pyroglutamic acid.
- a method for suppressing a decrease in dalcanase activity characterized in that a protein having an endodalcanase activity and whose N-terminus is not pyroglutamic acid is modified into a protein having an N-terminus of pyroglutamic acid.
- N-terminal amino acid of a protein having an endorcanase activity and whose N-terminus is not pyroglutamic acid, or a region containing the N-terminal amino acid is composed of pyroglutamic acid, pyroglutamine oxidizable amino acid, pyroglutamic acid or pyroglutamic acid.
- (c) contains an amino acid sequence having at least 85% homology with a protein containing the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40, and has reduced activity in the presence of a surfactant A homologous protein having an endalcanase activity.
- (c) hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 37, or SEQ ID NO: 39, and exhibits reduced activity in the presence of a surfactant.
- a protein having endoglucanase activity belonging to family 45 (hereinafter sometimes simply referred to as "cellulase I which is swollen in family 45").
- family 45 refers to those which are classified into family 45 by Henrissat B. et al. By Carbohydrate activating enzyme (Hydrophobic cluster analysis). [Henrissat B., Bairoc A. Updating the sequence—based classification of glycosyl hydrolases. Biochem. J. 316: 695-696 (1996).].
- a protein having endoglucanase activity [0013] A protein having endoglucanase activity
- protein having an endalcanase activity refers to an enzyme exhibiting an endalcanase activity, that is, an endo 1,4
- surfactant refers to a detergent component to be added to a detergent for clothing, and is mainly composed of an anionic surfactant, a cationic surfactant, and a nonionic surfactant. It is used for Preferable examples of the present invention include linear alkylbenzene sulfonic acid (hereinafter sometimes abbreviated simply as “LAS”) as an anionic surfactant.
- LAS linear alkylbenzene sulfonic acid
- EGU endalcanase (hereinafter sometimes abbreviated simply as“ EGU ”) activity” refers to measuring a decrease in the viscosity of a carboxymethylcellulose solution and measuring the enzyme activity. The details of the experiment are as follows.
- carboxymethyl cellulose manufactured by Hercules
- carboxymethyl cellulose was dissolved in 0.1 mol IZL Tris-HCl buffer ( ⁇ . 0) to a final concentration of 3.5%.
- 5 mL of this substrate solution was preliminarily heated at 40 ° C. for 10 minutes, and 0.15 mL of the enzyme solution was added thereto, stirred well, and reacted at 40 ° C. for 30 minutes.
- the viscosity of the reaction solution was measured with an R-type viscometer (Toki Sangyo RE100) set at 40 ° C.
- the enzymatic activity was defined as the amount of the enzyme that reduced the initial viscosity to 1Z2 under each reaction condition as one unit.
- a linear surfactant, a linear alkylbenzene sulfonic acid manufactured by Wako Pure Chemical Industries, Ltd.
- the decrease in endalcanase activity in the presence of a surfactant is small means that the protein of the present invention in which the N-terminus is modified (N-terminal modified protein) and the protein before the modification.
- the unmodified protein As a result of comparing the protein (hereinafter referred to as the unmodified protein) with the index of the endalcanase activity in the presence of the surfactant, the N-terminally modified protein was found to have a higher endulcanase activity than the unmodified protein. Means high.
- the protein before modification to which the method of the present invention can be applied is not particularly limited as long as it has an endalcanase activity and is a protein whose N-terminal is not pyroglutamic acid. Endodalcanase, cellopiohydrase, or j8-dalcosidase), and a cellulase belonging to family 45 is preferable.
- the protein before modification may be a protein having only the endalcanase activity as long as it has at least the endalcanase activity, or may have another enzyme activity instead of the endalcanase activity. It can be a protein. Further, the protein may be a natural protein or a protein modified by genetic engineering.
- Cellulases belonging to this family 45 can be produced by conventional genetic engineering techniques, such as recombinant DNA technology, polypeptide synthesis technology, etc. Naturally, both isolated and isolated bacterial strains can be obtained. Furthermore, mutants of cellulase belonging to family 45, which can also provide natural strength, can be included.
- Cellulases belonging to family 45 can be obtained from microorganisms such as filamentous fungi or zygomycetes.
- filamentous fungi include the genus Humicola (eg, Humicola 'insolens), the genus Trichoderma (eg, Trichoderma viride), the genus Staphylotrichum (eg, Staphylotrichum c cocosporum), A microorganism belonging to the genus Myriococcum [eg, Myriococ cum thermophilum] can be obtained.
- cellulases derived from the genus Humicola include, for example, CBH I, EGV, NCE2, NCE4, and NC E5, and cellulases derived from the genus Trichoderma include, for example, CBH I, CBH II, and EG II.
- Cellulases derived from the genus Staphylotrichum such as EGII include, for example, cellulases derived from the genus Forcemyocockum such as STC El, STCE3, and the like, for example, MTE1 and the like.
- zygomycetes include, for example, Rhizopus (eg, Rhizopus oryzae), Muconore (Mucor) [eg, Muconore'Mucor circinelloides], Phycomyces (Phycomyces). ) [f column, can be obtained from microorganisms belonging to Phycomyces nitens]. Specifically, there are, for example, RCE I, RCE II, RCE I II derived from Rhizopus 'Oryzae, MCE I, MCE II derived from Mucor' Sarcineroides, and PCE I derived from Phycomyces tensis (WO 00Z24879). Pamphlet).
- the term “pyroglutamic acid” means that the N-terminal glutamine or glutamic acid of a mature protein is cyclized to pyroglutamic acid.
- This pyrodaltamic acid is characterized by not presenting an amino group on the N-terminal side, and is capable of oxidizing pyroglutamine either in vivo (in vivo) or in vitro (in vitro).
- a dani protein is obtained.
- Glutamine or glutamic acid at the N-terminus using an acidic solution such as formic acid By treating such a protein, pyroglutamic acid-digested protein can be obtained.
- peptide refers to a group of amino acids polymerized by a peptide bond, which has one or more amino acids. Therefore, in the present specification, “a peptide containing pyrodaltamic acid” means a peptide having pyroglutamic acid as an N-terminal amino acid. Peptides containing pyroglutamic acid also have 2 to more amino acids, for example, 2 to 40, preferably 2 to 30, more preferably 2 to 20, even more preferably 2 to 10, and particularly preferably Preferably, 2 to 5, more preferably 2 to 4 amino acid sequences are crosslinked. The amino acid here is not particularly limited as long as it can be used by those skilled in the art.
- the means for modifying the protein before modification to a protein having a pyroglutamic acid at the N-terminus is not particularly limited as long as the protein modification is possible. Examples include a genetic engineering technique or a chemical technique.
- the protein before modification can be modified by genetic addition and Z or substitution of an appropriate amino acid or amino acid sequence.
- the protein before modification protein having an endalcanase activity and the N-terminus is not pyroglutamic acid
- N-terminal of the protein, pyrodaltamate is added by genetic engineering
- the protein can be modified by genetically adding a peptide containing pyroglutamic acid at the N-terminus.
- the aspect is, for example,
- a polynucleotide encoding an amino acid capable of pyroglutamic acid for example, glutamic acid or glutamine
- an amino acid capable of pyroglutamic acid addition at the N-terminus
- a polynucleotide encoding a peptide comprising;
- the ability to genetically substitute the N-terminal amino acid of the protein before modification or a region containing the same with pyroglutamic acid, or the ability to replace oral glutamic acid at the N-terminal can be modified by genetically substituting the peptide with the peptide.
- the aspect is, for example,
- polynucleotide encoding the amino acid capable of pyroglutamic acid for example, glutamic acid or glutamine
- the region containing the 5 ′ end of the polynucleotide encoding the protein before modification can be used. Replacing the amino acid with a polynucleotide encoding a peptide containing an N-terminus;
- protein modification by a chemical technique includes, for example,
- the N-terminal amino acid is chemically An embodiment in which pyroglutamic acid is applied;
- a more specific modification method will be described using a cellulase belonging to family 45 as an example.
- a method of adding a peptide containing pyroglutamic acid or pyrroglutamic acid to the N-terminal side of a cellulase belonging to family 45 can be obtained by using a genetic engineering technique.
- a general cellulase production method for example, using a filamentous fungus, This can be achieved by properly linking the translation region of the polynucleotide encoding the cellulase of interest with a promoter and a terminator that function in the host, and introducing this expression cassette into the host.
- a polynucleotide encoding a secretory signal sequence that functions in a host cell is added and introduced into the host cell, it is secreted into the medium, so that the target cellulase can be easily recovered.
- the cellulase is added by adding a polynucleotide encoding the amino acid to be added immediately below the secretory signal sequence.
- a desired amino acid can be added to the N-terminal side. Furthermore, modification of the amino group of the N-terminal amino acid can be achieved by using the secretory signal sequence of the host.For example, cbhl and cbh2 derived from Trichoderma 'viride', and NCE2 and NCE5 derived from Humicola ' Therefore, it can be prepared by using these secretory signal sequences and expressing them using Trichoderma viride or Humicola insolens, respectively. According to a preferred embodiment of the present invention, the cellulase belonging to family 45 prepared as described above has such advantageous properties that the decrease in activity is small in the presence of a surfactant.
- the protein of the present invention refers to a protein in which the N-terminus of an unmodified protein having endalcanase activity has been modified to pyroglutamic acid (for example, a cellulase belonging to family 45 is obtained, and a pyrolase is added to the N-terminal amino acid side of the mature protein.
- pyroglutamic acid for example, a cellulase belonging to family 45 is obtained, and a pyrolase is added to the N-terminal amino acid side of the mature protein.
- pQ glutamic acid
- pyrroglutamic acid a protein which can be obtained by the above-mentioned method and has a small decrease in activity in the presence of a surfactant and has an endalcanase activity.
- proteins selected from the group consisting of:
- (c) contains an amino acid sequence having at least 85% homology with a protein containing the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40, and has reduced activity in the presence of a surfactant A homologous protein having an endalcanase activity.
- the amino acid sequence represented by SEQ ID NO: 2 is obtained by adding a 5-amino acid peptide (N-terminal is pyrodaltamic acid) to the N-terminal of endulcanase NCE4 derived from Humicola insolens MN200-1 strain
- the amino acid sequence represented by SEQ ID NO: 4 which is the amino acid sequence of type NCE4 (see Example A2) is obtained by replacing the N-terminal amino acid (Ala) of staphylotricum 'cocosporum IFO 31817 strain-derived endalcanase STCE1 with 4 amino acids.
- 4 is an amino acid sequence of an N-terminal-substituted STCE1 (see Example B2) substituted with an peptide (N-terminal of which is pyroglutamic acid).
- the amino acid sequence represented by SEQ ID NO: 38 is obtained by adding a pyroglutamic acid-added STCE1 (see Example B3) in which pyroglutamic acid is added to the N-terminus of an endorcanase STCE1 derived from staphylotricum 'cocosporum IFO 3181 7 strain. It is an amino acid sequence.
- the amino acid sequence represented by SEQ ID NO: 40 is an N-terminal in which a peptide having 4 amino acids (N-terminal is pyroglutamic acid) is added to the N-terminal of staphylotricum 'Cocosporum IFO 3181 7 strain-derived endalcanase STCE1.
- 4 shows the amino acid sequence of addition type STCE1 (see Example B4).
- the "modified protein” in the present invention refers to one or more (eg, one or several) amino acids in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40.
- the number of amino acids for modification such as “deletion, substitution, insertion, or addition” is preferably 110, more preferably 110, and still more preferably 116.
- the modified protein includes one or more amino acid residue-conserved amino acids in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40. It includes a protein having a sequence and having a small decrease in activity in the presence of a surfactant and having endodalcanase activity.
- ⁇ conservative substitution '' means replacing one or more amino acid residues with another chemically similar amino acid so as not to substantially alter the activity of the protein. I do. For example, a case where a certain hydrophobic residue is substituted with another hydrophobic residue, a case where a certain polar residue is substituted with another polar residue having the same charge, and the like can be mentioned.
- non-polar (hydrophobic) amino acid examples include alanine, palin, isoleucine, leucine, purine, tryptophan, furaralanine, and methionine.
- polar (neutral) amino acids examples include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cystine.
- positively charged (basic) amino acids examples include arginine, histidine, and lysine.
- negatively charged (acidic) amino acids examples include aspartic acid and glutamic acid.
- the "homologous protein” in the present invention is at least 85%, preferably 90%, and most preferably a protein containing the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40.
- the homology values shown here are based on the homology search program FASTA3 [Science, 227, 1435-1441 (1985); Proc. Natl. Acad. Sci. USA, 85, 2444-2448, which is a homology search program known to those skilled in the art. (1988); http: //www.ddbj.nig.ac.jp/Email/homology-j.html], using the default (initial setting) parameters to calculate numerical values (identity; identity).
- the portion excluding the peptide having 5 amino acids added to the N-terminus is: It is derived from the endalcanase NCE4.
- the protein of the present invention may be a protein having the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40.
- the portion excluding the N-terminal amino acid or peptide is derived from the endalcanase STCE1.
- Endoglucanases NCE4 and STCE1 are endoglucanases belonging to family 45, and examples of known endoglucanases belonging to family 45 include NCE5 derived from the genus Humicola (WO 01Z90375 pamphlet). be able to.
- FIGS. 1 and 2 show the amino acid sequence of endoglucanase STCE1 [signal peptide (SEQ ID NO: 43) and mature protein (SEQ ID NO: 44)], and the endoglucanase NCE4 [sidanal peptide (SEQ ID NO: 45) and mature protein].
- SEQ ID NO: 46 amino acid sequences of protein
- NCE5 amino acid sequences of protein
- SEQ ID NO: 47 amino acid sequences of protein
- SEQ ID NO: 48 mature protein
- Figure 1 shows the results for the first half (N-terminal side), and Figure 2 shows the results for the second half (C-terminal side).
- the symbol “*” indicates that the amino acid is common to STCE1.
- the endalcanase belonging to family 45 has a catalytic domain (No. 1 to No. 207) as a common domain, and optionally further comprises a linker. It may have one region (Linker) (No. 208-258) and Z or cellulose binding domain (CBD) (No. 259-295).
- Linker No. 208-258
- CBD cellulose binding domain
- the number shown in parentheses after each domain is the amino acid number in the amino acid sequence (SEQ ID NO: 44) of the endalcanase STCE1.
- non-common Amino acids may be modified to another amino acid (preferably a similar amino acid that can be conservatively substituted) to maintain their enzymatic activity even if they are modified.
- the modified protein or homologous protein maintaining the above can be obtained at a high probability without requiring undue experimentation.
- the modified protein or homologous protein of the present invention includes any region, for example, a protein in which an amino acid contained in a catalytic region, a linker region, or a cellulose binding region is modified, as long as it has an endalcanase activity. It is.
- a protein comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 38, or SEQ ID NO: 40, a modified protein thereof, and a homologous protein (hereinafter, simply referred to as "the protein of the present invention” ")
- the amino acid sequence of a protein the nucleotide sequence encoding it can be easily determined, and thus various nucleotide sequences encoding the protein of the present invention can be selected.
- the term “polynucleotide” includes both DNA and RNA, and DNA is preferable.
- Examples include polynucleotides selected from the group consisting of:
- (c) hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 37, or SEQ ID NO: 39, and exhibits reduced activity in the presence of a surfactant. Coating proteins with low endalcanase activity The polynucleotide to be loaded.
- the number of bases that may be deleted, substituted, inserted, or added is specifically 1 to 90, preferably 1 to 30, more preferably 1 to 90. Eighteen, more preferably one to nineteen.
- the “stringent conditions” in the above (c) means that the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 37, or SEQ ID NO: 39 is used V, According to the attached operation method of the ECL Direct DNAZRNA labeling detection system (manufactured by Amersham), after pre-hybridization at 42 ° C for 1 hour, the above-mentioned polynucleotide was added, and then added at 42 ° C.
- the polynucleotide according to the present invention may be of natural origin, may be wholly synthesized, or may be one which has been synthesized using a part of naturally occurring polynucleotide. Good.
- a typical method for obtaining the polynucleotide according to the present invention a method commonly used in the field of genetic engineering, for example, an appropriate DNA probe prepared based on partial amino acid sequence information from a genomic library of a microorganism is used. And a method for screening.
- the protein of the present invention is a polynucleotide molecule containing a polynucleotide fragment encoding the same in a state where it can be replicated in a host cell and expresses the gene, and is particularly useful for transforming a host cell in the form of an expression vector. In this case, it can be produced by the host cell.
- the polynucleotide fragment encoding the protein of the present invention can be replicated in a host microorganism and the protein encoded by the polynucleotide fragment can be expressed.
- An expression vector is provided.
- the present expression vector is a self-replicating vector, that is, it exists as an extrachromosomal independent entity, and its replication does not depend on chromosomal replication. Can do.
- the present expression vector may be one which, when introduced into a host microorganism, is integrated into the genome of the host microorganism and replicated together with the chromosome in which it has been integrated.
- the procedures and methods for constructing a vector according to the present invention can be those commonly used in the field of genetic engineering.
- the expression vector according to the present invention is, in addition to the above-described polynucleotide fragment according to the present invention, a polynucleotide that controls the expression, in order to actually introduce the vector into a host microorganism and express a protein having a desired activity. It is desirable to include nucleotides and genetic markers for selecting transformants!
- Examples of the polynucleotide for controlling expression used in the present invention include a transcription control signal and a translation control signal such as a promoter, a terminator and a polynucleotide encoding a secretory signal peptide. Ligation of these controlled polynucleotides and insertion into the vector can be performed by a conventional method.
- the promoter used in the present invention is not particularly limited as long as it exhibits transcriptional activity in the host microorganism, and is a polynucleotide that controls the expression of a gene encoding a protein that is either the same or different from the host microorganism.
- promoters include, in Escherichia coli, for example, promoters such as ratatose operon and tryptophan operon, and in yeast, for example, alcohol dehydrogenase gene, acid phosphatase gene, galactose assimilation gene, and glyceraldehyde aldehyde triphosphate dehydrogenase.
- a promoter or a mold of a zeogene for example, a promoter such as an ⁇ -amylase gene, a darcoamylase gene, a cellobion, an idolase gene, and a glyceraldehyde aldehyde triphosphate dehydrogenase gene in a filamentous fungus can be selected.
- the signal peptide is not particularly limited as long as it contributes to the secretion of the protein from the host microorganism. Genetic ability encoding a protein of the same or different type as the host microorganism is also induced. Can be obtained from the polynucleotide.
- the gene marker in the present invention may be appropriately selected depending on the method for selecting a transformant.
- a gene encoding drug resistance and a gene complementing auxotrophy may be used. it can.
- the host used is a bacterium, for example, an ampicillin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, etc.
- the host to be used is yeast, for example, tributophan biosynthesis gene (trpl, trpC), peracil biosynthesis gene (ura3), leucine biosynthesis gene (leu2), histidine biosynthesis gene (his3) are used.
- a filamentous fungus for example, a destomycin resistance gene, a nitrate assimilation gene (niaD), an arginine biosynthesis gene (argB), a peracil biosynthesis gene (pyr4), a hygromycin resistance gene, bialaphos resistance Gene, a bleomycin resistance gene, an aureobasidin resistance gene and the like.
- a filamentous fungus for example, a destomycin resistance gene, a nitrate assimilation gene (niaD), an arginine biosynthesis gene (argB), a peracil biosynthesis gene (pyr4), a hygromycin resistance gene, bialaphos resistance Gene, a bleomycin resistance gene, an aureobasidin resistance gene and the like.
- the host vector system is not particularly limited, and for example, a system using Escherichia coli, actinomycetes, yeast, filamentous fungi, etc., and a fusion protein expression system with other proteins using them can be used.
- yeast is used as the host, microorganisms belonging to the genus Saccharomyces, the genus Hansenula, the genus Pichia, and the like, preferably Saccharomyces cerevisiae, can be mentioned.
- any filamentous fungus may be used, but it is preferably a microorganism belonging to the genus Humicola, the genus Trichoderma, the genus Aspergillus, the genus Acremon-Aum (Acremonium), or the genus Fusarium. And more preferably a genus of Humicola or Trichoderma.
- Humicola insolence Humicola thermoidea, Trichoanorema viride, Trichotenorema reseu (Trichod erma reeseu, Trichoanorema longifunfuntoum, Trichoderma longibrachiatumu, Stafikorotokum Aspernoreginoles 'Niger, Aspernoreginoles' oryzae, Fusarium 'oxysporum, or Acremo-Pam' Acremonium cellulolyticus (Frenchola insolens, or even more preferably) Trichoderma 'Viride.
- Trichotenorema reseu Trichod erma reeseu
- Trichoanorema longifunfuntoum Trichoderma longibrachiatumu
- Stafikorotokum Aspernoreginoles 'Niger Aspernoreginoles' oryzae
- transformation of a microorganism with the expression vector can be carried out according to a method commonly used in this field.
- a method commonly used in this field Preferable specific examples include, for example, a conventional method such as a calcium ion method, a lithium ion method, an electoral poration method, a PEG method, an agglomerate method, a particle gun method, and the like. It can be selected according to the host.
- the transformant of the present invention is cultured, and the desired The protein of the invention can be obtained. Culturing of the transformant according to the present invention can be performed by appropriately selecting a medium, culture conditions, and the like according to a conventional method.
- Examples of the medium include a carbon source that can be assimilated by the transformant of the present invention, a nitrogen source that can be assimilated, inorganic salts, various vitamins, various amino acids such as glutamic acid or asparagine, micronutrients such as nucleotides, and antibiotics.
- a selection drug such as the above can be added.
- an organic substance, Z or an inorganic substance which promotes the growth of the transformant of the present invention and promotes the production of the protein of the present invention can be appropriately added.
- a synthetic medium or a natural medium containing moderately other nutrients can be used.
- the carbon source and the nitrogen source used in the medium any type can be used as long as the transformant of the present invention can be used.
- carbon sources that can be assimilated include various carbohydrates such as sucrose, starch, glycerin, glucose, fructose, maltose, ratatose, cellulose, cellobiose, dextrin, animal oil, vegetable oil, and hydrolysates thereof. Is available. Usually, the concentration is preferably 0.1% to 5% with respect to the medium.
- Nitrogen sources that can be assimilated include, for example, animal and plant components such as peptone, meat extract, corn 'steep' liquor, defatted soy flour, leached extracts, ammonium succinate salts, and ammonium tartrate salts.
- Nitrogen-containing compounds such as organic acid ammoniums, urea, or other various inorganic acids or organic acids can also be used.
- the inorganic salts for example, sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, or other salts capable of generating ions can be used as appropriate.
- the growth of the transformant of the present invention and the present invention even in a medium containing fine powder of a plant such as a cell of a microorganism such as yeast, an exudate and an exuded extract. Any of them can be appropriately used as long as the production and accumulation of the protein are not prevented.
- a substance capable of satisfying the auxotrophy is added to the medium, but this kind of nutrient is especially added when a medium containing natural products is used. May not be required.
- Culture conditions such as the composition of the medium, the liquid properties of the medium, the culture temperature, the agitation speed, and the aeration rate are appropriately adjusted according to the transformant to be used and external conditions, etc., so as to obtain results. Needless to say, it is selected.
- Antifoaming agents such as silicone oil, vegetable oil, mineral oil, and surfactant can be used as appropriate.
- the protein of the present invention accumulated in the culture thus obtained is contained in the transformant of the present invention and in the culture filtrate, the culture is centrifuged to transform the culture with the culture. It is possible to collect the protein of the present invention separately from the body.
- the means used for collecting the protein of the present invention from the culture can be used alone, in combination in any order, or repeated in a usual manner. Used as That is, for example, extraction filtration, centrifugation, dialysis, concentration, drying, freezing, adsorption, desorption, a method utilizing the difference in solubility in various solutions (e.g., precipitation, salt precipitation, crystallization, recrystallization, Means such as chromatography).
- the protein of the present invention can be obtained from the culture in the transformant of the present invention.
- techniques such as extraction (grinding treatment, pressure crushing, etc.), recovery (filtration, centrifugation, etc.) and purification (salting out method, solvent precipitation method, etc.) from the culture are used.
- the obtained crude substance can be purified by a conventional method, for example, by various column chromatography using a carrier such as dextran, cellulose, agarose, a synthetic polymer, or silica gel.
- a carrier such as dextran, cellulose, agarose, a synthetic polymer, or silica gel.
- the desired pure protein of the present invention can be obtained from the culture of the transformant of the present invention by the methods described above or by appropriately combining them.
- the above-described method for producing a novel protein according to the present invention there is provided the above-described method for producing a novel protein according to the present invention.
- the cultivation of the transformants and their conditions may be essentially equivalent to those for the microorganism used.
- After culturing the transformant a target protein can be recovered by a method commonly used in this field.
- the Escherichia coli JM109 strain (FERM BP-5973) transformed with the expression vector pEGDOl according to the present invention was obtained from the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center [formerly] (Address: 305 305-8566, Tsukuba, Ibaraki, Japan, 1-chome, 1-1-1, Chuo No. 6)] and deposited in Japan on July 12, 1996. (1997) June 13 Power has also been transferred to the international depositary.
- the international accession number (the number in parentheses following the international accession number) is FERM BP-59 73 [FERM P-15729].
- Escherichia coli JM109 strain transformed with the expression vector pMKDOl according to the present invention
- BP-5974 is the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center [formerly, Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (address: 305-8566, Tsukuba, Higashi, Ibaraki, Japan 1-chome, Japan) No. 1 No. 1 Chuo No. 6)], deposited in Japan from July 12, 1996 (1996), and also transferred to international deposit on June 13, 1997 (1997) .
- the international accession number (the number in parentheses following the international accession number) is FERM BP-59
- the Escherichia coli JM109 strain (FERM BP-6045) transformed with the plasmid pCBl-M2XR was obtained from the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (formerly Ministry of International Trade and Industry) (Address: 305305-8566, Tsukuba, Ibaraki, Japan, 1-chome, 1-1-1, Chuo No. 6)] and deposited in Japan on September 9, 1996. August 11 (1997) Power has also been transferred to the international depositary.
- the international accession number (the domestic accession number following [] is FERM BP-6045 [FER M P-15840]).
- the plasmid pCBl-M2 according to the present invention may be obtained, for example, by cloning the plasmid pCBl-M2XR with the restriction enzyme Xbal and self-closing the plasmid as an embodiment other than the method described in Examples described later. You can also get it.
- Humicola's Insolens MN200-1 strain (FERM BP-5977), which can be a host of the expression vector according to the present invention, was obtained from the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, Japan [(formerly Ministry of International Trade and Industry) Deposited at the National Institute of Bioscience and Biotechnology (Title: ⁇ 305-8566, Tsukuba, Higashi, Ibaraki, Japan 1-1, 1-1, Chuo No. 6)] on July 15, 1996 It was transferred to the International Depositary on June 13, 1997.
- the international accession number (the national accession number following the international accession number in []) is FER M BP-5977 [FERM P-15736].
- Trichoderma viride MC300-1 strain (FE RM BP-6047), which can serve as a host for the expression vector according to the present invention, is available from the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary [ Institute of Biotechnology, Industrial Technology (Address: ⁇ 305-8566 days) It has been deposited domestically since September 9, 1996 in Tsukuba East 1-chome, 1-Chome 6), Ibaraki Prefecture, Japan, on August 9, 1997, and August 11, 1997. Has been transferred to the International Depositary.
- the international accession number (the national accession number following the international accession number within []) is FERM BP-6047 [FERM P-15842].
- Escherichia coli DH5a (FERM BP-10 127) transformed with plasmid pUC118-STCEex was obtained from the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (address: ⁇ 305-5566 Tsukuba, Ibaraki, Japan) 1 Higashi 1-chome No. 1 1 Central No. 6) Deposited domestically on December 1, 2003 (2003), transferred to international deposit on September 15, 2004 (2004) ing.
- the international accession number (the domestic accession number following the international accession number in []) is FERM BP-10127 [FERM P-19602].
- the plasmid pUC118-STCEex is obtained by inserting the STCE gene into the BamHI site of the plasmid pUC118.
- the endoglucanase STCE1 gene contained in this BamHI fragment contains an intron and has the sequence shown in SEQ ID NO: 5.
- Example A1 Construction of N-terminal addition type cellulase expression vector and natural type cellulase expression vector for Humicola 'insolens >>
- Plasmid pUCl18 (Takara Bio Inc.) was digested with BamHI, and the resulting fragment was blunt-ended using a DNA blanching kit (Takara Bio Inc.). This was self-closed using a DNA ligation kit (manufactured by Takara Bio Inc.) to obtain pUC118-BN. This PUC118BN was digested with Sphl, blunt-ended and self-closed to obtain pUC118BSN. Next, a 3.4 kb Pstl-Xbal fragment containing the full length of the cellulase NCE2 gene was obtained according to the method described in JP-A-8-126492 by Humicola 'Insolens MN200-1 strain (FERM BP-5977).
- the plasmid pM21 was digested with BamHI, and a PCR fragment of the cellobiohydrolase gene (NCE3) derived from Humicola 'insolens MN200-1 strain (NCE3) was ligated with BamHI to obtain a plasmid pKM04.
- the PCR fragment of NCE3 was amplified to the ⁇ type from genomic DNA derived from Humicola's insolence strain MN200-1 (FERM BP-5977) (WO98Z03640) using the following primers MKA-05 and MKA-06. did.
- a promoter and terminator of the trpC gene derived from Aspergillus nidulans were added to the plasmid pKM04.
- a gene was prepared in which the destomycin-resistant gene described in Japanese Patent Application Laid-Open No. 59-175889 was made expressible with Humicola's insolence. This was ligated with a plasmid pKM04 digested with Xbal to construct an N-terminally digested cellulase expression vector pMKDOl (FERM BP-5974).
- This pMKDOl has a BamHI recognition sequence mutated 10 bp downstream from the N-terminus of the mature protein of the NCE2 gene and 3 bp downstream from the stop codon, so that an additional 5 amino acids are added to the N-terminus of the cellulase belonging to the desired family-45.
- the structure is such that residues are added.
- a site-specific mutation was introduced into the plasmid pM21 obtained in the above (1) using the oligonucleotide pMN-Bam to obtain a mutation-introduced plasmid pM21-m-Al.
- This pM21-m-A1 was digested with restriction enzymes Hindlll and BamHI, and a fragment of about lkb obtained was recovered.
- pMKDOl obtained in Example Al (1) was digested with Hindlll and BamHI to recover a fragment of about 7 kb. These fragments were ligated to obtain plasmid pJDOl.
- the BamHI recognition sequence is mutated 15 bp upstream of the translation start point of the NCE2 gene and 3 bp downstream of the stop codon, so that the transcription start point force is such that the cellulase belonging to the desired family 45 is expressed. Become.
- Example A2 Construction of Natural Type NCE4 Expression Vector and N-terminal Added NCE4 Expression Vector, and Transformation into Humicola 'Insolens and Evaluation of Activity >>
- the entire translation region of the NCE4 gene was ligated to pJDOl obtained in Example A1 (2) to construct pN2EX-N4.
- the full-length translation region of the NCE4 gene was subjected to PCR using the combination of the primers NCE4-Ne and NCE4-Ce with the genomic DNA derived from the aforementioned Humicola 'insolens MN200-1 strain, and the resulting PCR product of about 0.9 kbp was purified using BamHI.
- This was digested with the same restriction enzymes and ligated with pJDOl to construct pN2EX-N4.
- TC 3 (SEQ ID NO: 15)
- NCE4-Ce 5 'GGGGGGATCCCTGCGTTTACAGGCACTGATGG-3' (SEQ ID NO: 16)
- the mature protein translation region of the NCE4 gene was subjected to PCR using the combination of the primers NCE4-Ns and NCE4-Cs with the genomic DNA derived from the aforementioned Humicola's insolens MN200-1 strain, and the obtained PCR was performed at about 0.8 kbp.
- PCR was performed at about 0.8 kbp.
- NCE4 Ns: 5, CCGGTGTTGGCCGGATCCGCTGATGGCAAG—3 '(SEQ ID NO: 17)
- NCE4-Cs 5, -TAAGGCCCTCAAGGATCCCTGCGTCTACAG-3 '(IS column number 18)
- Humicola insolens MN200-1 was added to an S medium (3.0% glucose, 2.0% yeast extract, 0.1% popultone, 0.03% Shioridani canoleicum, 0.03% magnesium sulfate). , PH 6.8) at 37 ° C for 24 hours, and the mycelium was recovered by centrifugation and washed with 0.5 mol ZL sucrose. This is suspended in a 0.5 ml olZL sucrose solution containing 3 mg ZmL ⁇ -glucurodase (Sigma), lmg / mL chitinase and lmgZmL Zymoryase 20T (Seikagaku Corporation), and gently suspended at 30 ° C for 60 to 90 minutes.
- the protoplast suspension treated above is Wash with C buffer, and add YMG medium containing 200 ⁇ g ZmL of Hygromycin ((1% darcose, 0.4% yeast extract, 0.2% malt extract, 1% agar, pH 6.8) The cells were overlaid with agar and cultured at 37 ° C for 5 days, and the grown colonies were used as transformants.
- Hygromycin ((1% darcose, 0.4% yeast extract, 0.2% malt extract, 1% agar, pH 6.8)
- the cells were overlaid with agar and cultured at 37 ° C for 5 days, and the grown colonies were used as transformants.
- the obtained transformant was cultured according to International Publication No. 98Z03667, and the culture supernatant was obtained. This culture supernatant was subjected to SDS-PAGE, and a culture supernatant in which a band of about 43 kDa NCE4 was detected favorably was selected.
- Table 3 shows the results obtained by measuring the EGU activity of the culture supernatant at pH 10 and the kasu section with LAS at pH 10 and comparing the degree of the decrease.
- Table 3 shows the EGU activity of pHIO as 100, and the percentage is shown.
- the culture supernatant obtained from the NCE4 added NCE4 transformant showed a small decrease in the activity in the LAS-added group. Comparing the EGU activity of the LAS-added caro group, it was found that N-terminal-caro-type NCE4 had 2.1 times the activity of native NCE4. At this time, since the EGU activity of Humicola's Insolens MN200-1 strain was about 4% of the transformants, most of these EGU activities were due to the recombinant enzyme expressed by the expression vector into which the gene was introduced. It was found to be derived from
- the culture supernatant of the transformant was subjected to SDS-PAGE, and the electrophoresed protein was electrically transferred to a PVDF membrane (Immobilon-PSQ manufactured by Millipore). An approximately 43 kDa NCE4 band was cut out from this plot. This was subjected to Protein Sequencer Model 49 2 (manufactured by Applied Biosystems) to decode the amino acid sequence.
- Table 4 summarizes the N-terminal amino acids and their sequences of native NCE4 and N-terminal-added NCE4. This clearly showed that NCE4 has the N-terminal amino acid sequence of the constructed expression vector.
- Example B1 Construction of N-terminal Substituted Cellulase Expression Vector and Natural Cellulase Expression Vector for Trichoderma viride
- Trichoderma 'viride MC300-1 obtained according to the pamphlet of International Publication No. 98Z11239, including a full-length cbhl gene derived from a 7 kb Pstl fragment containing the full-length cbhl gene.
- the 7 kb Sail fragment was cloned into plasmid PUC119 (plasmid pCBl-H4 and plasmid pCBl-Sl, respectively).
- Escherichia coli JM109 strain carrying this plasmid was infected with helper phage M13K07 to obtain single-stranded DNA.
- the phosphorylated oligonucleotides CBn-Stu and CBc-Xho were annealed to plasmid pCBl-H4 and plasmid pCBl-Sl, respectively, and a sculptor in vitro mutagenesis system (Amersham Biosciences) was used.
- the plasmids PCB1H4-19 and pCBlSl-17 were constructed from each.
- the plasmid pCBlH4-19 is digested with Xbal and Xhol, and the obtained fragment of about 6 kb and the plasmid pCBlSl-17 are digested with XbaI and then partially digested with Xhol, and the obtained fragment of about 1.2 kb is obtained.
- pCBl-M was digested with Sail, and a fragment of about 2.7 kb was cloned into plasmid pUC119 to construct plasmid pCBl-SalM. This was further converted into single-stranded DNA, and the phosphorylated oligonucleotides CB1-SmSph, CB1-Bam and CB1-Pst were annealed, and a mutation was introduced using a sculptor in vitro mutagenesis system.
- CBl-Bam 5, GCCGGGAGAGGATCCAGTGGAGG-3 '(SEQ ID NO: 24)
- CBl-Pst 5' -GCTCGAGTACCTTACTGCAGGCACTGAGAG-3 '(SEQ ID NO: 25)
- plasmid pUC118 was digested with Xbal and EcoRI, blunt-ended using a DNA branching kit, and self-cyclized to construct plasmid pUC118-SBN.
- This pUC118-SBN was digested with Hindlll and Sail, and an approximately 1.4 kb cb hi promoter fragment cut out with the same restriction enzymes was cloned.
- the introduced mutations are as shown below (BamHI mutations are not shown).
- Example B2 Construction of N-terminally Substituted STCE1 Expression Vector and Native STCE1 Expression Vector, and Transformation into Trichoderma viride and Evaluation of Activity
- the plasmid pCBl—M2 obtained in Example B1 was used as a native STCE1 expression vector. Then, the translation region of the STCE1 gene was ligated to construct pCB-Ste.
- the translation region of the STCE1 gene is amplified using a combination of the primers STCE1-TNERV and STCE1-TCET22I with the plasmid pUC118-STCEex (FERM BP-10127) as a type II using PCR, and the resulting PCR product of about lkbp is obtained.
- the resulting PCR product of about lkbp is obtained.
- STCE1-TNERV GGGGATATCGCGCATCATGCGTTCCTCCCCCGT CCTC (SEQ ID NO: 30)
- the plasmid pCB1M2 obtained in Example B1 was ligated with a mature protein translation region obtained by removing the secretory signal sequence from the translation region of the STCE1 gene. — Built Sts.
- the mature protein translation region of the STCE1 gene was amplified using a combination of the primers STCE1-TmNSph and STCE1-TCET22I, and the plasmid pUCl18-STCEex (FER MBP-10127) was amplified by PCR using type III PCR.
- a lkbp PCR product was prepared by digestion with Sphi and EcoT22I. This was digested with Sphl and Pstl! And ligated with pCBl-M2 to construct pCB-Sts.
- STCE1 TmNSph: GGGGCATGCGATGGCAAGTCGACCCGCTAC (SEQ ID NO: 32)
- Trichoderma Trichoderma.
- This selective medium is a minimal medium [0.2% 1 potassium dihydrogen phosphate, 0.4% ammonium sulfate, 0.03% urea, 0.03% magnesium sulfate heptahydrate, 0.03% Shiojiri calcium, 0.5% glucose, 2.5% agar, 0.01% trace element (5 mg iron sulfate heptahydrate, 1.5 6 mg manganese sulfate heptahydrate, 1.4 mg zinc sulfate heptahydrate, 2 mg cobalt chloride dissolved in 1 L of water)] and 10 g / mL pyridine and lmg / mL 5-fluorofluorotinic acid It is made from rice cake.
- Transformation of pCB-Ste and pCB-Sts obtained from Example B2 (1) into Trichoderma viride strain was carried out by adding Trichoderma viride strain 2 obtained by Example B2 (2) to -Eurospora crassa (Neurospora crassa). ) was performed by a co-transformation method with a marker plasmid pPYR4 containing the pyr4 gene of interest, and a strain growing on a minimal medium was used as a transformant.
- Trichoderma viride s train2 was added to 50 mL of a cell formation medium (1% yeast extract, 1% malt extract, 2% polypeptone, 2.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% Magnesium sulfate 7 water, 0.0001% codidine (pH 7.0)) Inoculated in a 200 mL Erlenmeyer flask dispensed with S, cultivated at 28 ° C for 2 days, and cultivated the obtained cells. It was collected by centrifugation and washed with 0.5 mol ZL sucrose.
- a cell formation medium 1% yeast extract, 1% malt extract, 2% polypeptone, 2.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% Magnesium sulfate 7 water, 0.0001% codidine (pH 7.0)
- the culture supernatant of the transformant was made into 0.05 mol ZL Tris-HCl (pH 8.0) and Imol ZL sodium sulfate solution, and adsorbed to Toyopearl butyl 650C (manufactured by Tosoh Corporation) having an equivalent bed volume (BV).
- the non-adsorbed components were washed with 0.05 mol ZL of Tris-HCl (pH 8.0) and Imol ZL of sodium sulfate.
- This fraction was concentrated, desalted with PD-10 (Amersham Bioscience), made into a 0.05 mol / L Tris-HCl (pH 7.5) solution, and supplied to Resource Q (6 mL, Amersham Bioscience). The non-adsorbed fraction was collected. After desalting, this fraction was made into a 0.05 mol / L sodium acetate (pH 5.0) solution, supplied to Resource S (6 mL, manufactured by Amersham Bioscience), and the non-adsorbed fraction was collected. After concentrating this fraction, it was subjected to gel filtration of Superdettas 75 pg (16 ⁇ 60 mm, manufactured by Amersham Bioscience) to collect a fraction having a molecular weight cut off of about 45,000.
- This fraction contained a single 45 kDa band.
- the purified fraction was subjected to SDS-PAGE, and the electrophoresed protein was electrically transferred to a PVDF membrane. From this plot, a band of STCE1 of about 45 kDa was cut out. This was subjected to a protein sequencer Model 492 (manufactured by Applied Biosystems) to decode the amino acid sequence.
- the native type STCE1 obtained from the pCB- Ste transformant was identical to the DNA sequence of STCE1 derived from Staphylotrichum cocosporum IFO (currently NBRC) strain 31817 (SEQ ID NO: 5). .
- Table 7 summarizes the N-terminal amino acids of native STCE1 and N-terminally substituted STCE1 and their sequences based on these results. This clearly showed that STCE1 has the N-terminal amino acid sequence of the constructed expression vector.
- Example B2 The EGU activity of the purified native STCE1 and N-terminally substituted STCE1 obtained in (5) at pHIO or at pHIO in the LAS-added section was measured at pHIO, and the results of comparison of the degree of decrease were tabulated. See Figure 8. In Table 8, the EGU activity of pHIO was defined as 100 and expressed as a percentage.
- Example B3 Change of N-terminal sequence added to STCE1 and construction of vector, and transformation into Trichoderma viride and evaluation of activity
- the STCE1 expression vector to which only pyroglutamic acid was added was constructed as pCB-Stml by ligating the plasmid pCBl-M2 obtained in Example B1 with the mature protein translation region excluding the secretory signal sequence of the STCE1 gene. .
- the mature protein translation region of the STCE1 gene is amplified using a combination of the primers STCE-TmNSma and STCEl-TCET22I with the plasmid pUCl18-STCEex (FERM BP-10127) as a type III using PCR, and the resulting about lkbp was digested with Smal and EcoT22I. This was digested with Smal and Pstl! And ligated with pCB1-M2 to construct pCB-Stml.
- the Trichoderma viride transformed with the vector pCB-Stml1 constructed as described above has an N-terminal amino acid sequence of pQADGKSTR (SEQ ID NO: 41) (i.e., one amino acid sequence at the amino terminus of native STCE1). Which has a sequence to which pyrroglutamic acid (pQ) is added].
- STCE-TmNSma GGCCCGGGCTCAGGCCGATGGCAAGTCGACCCG (SEQ ID NO: 35)
- Transformation of Trichoderma viride strain 2 with pCB-Stml was performed according to Example B2 (3).
- the obtained transformant was cultured according to International Publication No. 98Z11239, and a culture supernatant was obtained.
- the culture supernatant was subjected to SDS-PAGE, and a culture supernatant from which a band of STC E1 of about 45 kDa was favorably detected was selected.
- Table 9 shows the results obtained by measuring the EGU activity of the culture supernatant with LAS-added kaju at pH 10 and pH 10 and comparing the degree of reduction with the natural STCE1 of Example B2 (4).
- Table 9 shows the EGU activity of pHIO as 100 and the percentage.
- Example B4 Modification of N-terminal sequence added to STCE1 and construction of vector, and transformation into Trichoderma viride and evaluation of activity
- the mature protein translation region of the STCE1 gene is obtained by amplifying the plasmid pUCl18-STCEex (FER M BP-10127) into a type III PCR using a combination of the primers STCE-TmNSph-2 and STCE1-TCET22I.
- a PCR product of about lkbp was digested with Sphi and EcoT22I to prepare a PCR product. This was digested with Sphl and Pstl! And ligated with pCBl-M2 to construct pCB-Stml2.
- the Trichoderma viride transformed using the vector pCB-Stml2 constructed as described above has the N-terminal amino acid sequence of pQSACADGKSTR (SEQ ID NO: 42) [that is, 4 amino acids at the amino terminal of native STCE1. A sequence with an acid peptide (N-terminal pyroglutamic acid added)].
- STCE— TmNSph— 2 GGGCATGCGCCGATGGCAAGTCGACCCGC (SEQ ID NO: 36)
- Transformation of Trichoderma viride strain 2 with pCB- 3 ⁇ 112 was performed according to Example 82 (3).
- the obtained transformant was cultured according to International Publication No. 98Z11239, and a culture supernatant was obtained.
- the culture supernatant was subjected to SDS-PAGE, and a culture supernatant from which a band of STC E1 of about 45 kDa was favorably detected was selected.
- PH 10 and p of this culture supernatant Table 10 shows the results of measuring the EGU activity of the LAS-supplemented kamitsu section in H10 and comparing the degree of the decrease with the natural STCE 1 of Example B2 (4).
- Table 10 shows the EGU activity at pH 10 as 100, and the percentage is shown.
- the protein of the present invention can be used in the presence of a surfactant in the presence of a surfactant, as compared with natural cellulase.
- FIG. 1 The amino acid sequence of endoglucanase STCE1 [signal peptide (SEQ ID NO: 43) and mature protein (SEQ ID NO: 44)] and NCE4 [signal peptide (SEQ ID NO: 45) and the amino acid sequence of mature protein (SEQ ID NO: 46)] and NCE5 [signal peptide (SEQ ID NO: 47)] and mature protein (SEQ ID NO: 48)].
- FIG. 1 The amino acid sequence of endoglucanase STCE1 [signal peptide (SEQ ID NO: 43) and mature protein (SEQ ID NO: 44)] and NCE4 [signal peptide (SEQ ID NO: 45) and the amino acid sequence of mature protein (SEQ ID NO: 46)] and NCE5 [signal peptide (SEQ ID NO: 47)] and mature protein (SEQ ID NO: 48)].
- FIG. 2 is an explanatory diagram showing the result of the comparison result shown in FIG. 1 concerning the C-terminal sequence.
- nucleotide sequences represented by the sequences of SEQ ID NOs: 6-10, 12-13, 15-18, 21-26, 28, 30-32, or 35-36 correspond to primers MNC-02, MNC-03, Primer MKA-05, Primer MKA-06, pMKD01, Primer pMN-Bam, pJD01, Primer NCE4—Ne, Primer NCE4—Ce, Primer NCE4—Ns, Primer NCE4—Cs, Primer CBn—Stu, Primer CBc—Xho, Primer CB1—SmSp h, Primer CB1—Bam, Primer CB1—Pst, pCBl—M2, pCBl—M2, Primer STCE1—TNERV, Primer STCE1—TCET22I, Primer STCE1—Tm
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CN2004800364534A CN1890367B (zh) | 2003-12-08 | 2004-12-07 | 耐表面活性剂的纤维素酶及其修饰方法 |
EP04820192A EP1702981B1 (en) | 2003-12-08 | 2004-12-07 | Surfactant-tolerant cellulase and method of converting the same |
DK04820192.5T DK1702981T3 (da) | 2003-12-08 | 2004-12-07 | Tensid-tollerant cellulase og fremgangsmåde til omdannelse deraf |
US10/582,002 US8569033B2 (en) | 2003-12-08 | 2004-12-07 | Surfactant tolerant cellulase and method for modification thereof |
AT04820192T ATE522612T1 (de) | 2003-12-08 | 2004-12-07 | Tensid-tolerante cellulase sowie verfahren zur umwandlung davon |
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US14/031,616 Division US8796005B2 (en) | 2003-12-08 | 2013-09-19 | Surfactant tolerant cellulase and method for modification thereof |
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EP1702981B1 (en) | 2011-08-31 |
US20070099265A1 (en) | 2007-05-03 |
JP4644603B2 (ja) | 2011-03-02 |
ES2371916T3 (es) | 2012-01-11 |
US20140051147A1 (en) | 2014-02-20 |
ES2539955T3 (es) | 2015-07-07 |
US8569033B2 (en) | 2013-10-29 |
US8796005B2 (en) | 2014-08-05 |
CN103103171A (zh) | 2013-05-15 |
EP1702981A4 (en) | 2007-09-26 |
EP2330199A2 (en) | 2011-06-08 |
EP1702981A1 (en) | 2006-09-20 |
ATE522612T1 (de) | 2011-09-15 |
DK2330199T3 (en) | 2015-05-18 |
DK1702981T3 (da) | 2011-10-17 |
EP2330199B1 (en) | 2015-04-08 |
EP2330199A3 (en) | 2012-06-13 |
JPWO2005056787A1 (ja) | 2007-12-06 |
CN1890367B (zh) | 2012-11-14 |
CN1890367A (zh) | 2007-01-03 |
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