JP2007075046A - Heat-resistant cellulose-binding domain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polypeptide having a heat-resistant cellulose-binding action, to provide a cellulose-binding domain, and to provide a method for producing the same. <P>SOLUTION: This polypeptide is characterized in that (1) Glu<SP>279</SP>and/or Asp<SP>281</SP>are replaced by one or two amino acids selected from a neutral amino acid group consisting of Gln, Asn, Ala, Ser, Thr, Cys and Met, and if necessary, (2) one, several or a plurality of amino acids are replaced, added, deleted or inserted, and that the polypeptide has a cellulose-binding activity in an amino acid sequence having a specific sequence and comprising 101 amino acids from Thr<SP>258</SP>to Ile<SP>358</SP>. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a polypeptide having a heat-resistant cellulose binding action, a cellulose binding domain, and a method for producing the same.

In the present specification and claims, Thr which is the first amino acid of SEQ ID NO: 3
Is described as 258th.

Cellulose accounts for most of the biomass on earth, and its effective use is expected. The development of cellulase is one of the important issues for its effective use.
A cellulose-binding domain is essential for cellulase functional expression, and various cellulose-binding domains are known (Patent Document 1).

  The cellulose binding domain is applied to, for example, laundry detergents and contributes to the improvement of detergency (Patent Documents 2 to 3).

Conventionally known cellulose binding domains are non-heat-resistant, deactivated at high temperatures, and have the disadvantage of poor stability even at temperatures from room temperature to 40 ° C. The domain was sought.
Special table flat 08-509127 Special table 2003-521559 Special table 2003-526699

  An object of the present invention is to provide a heat-resistant cellulose-binding domain.

The present inventor conducted research to convert a thermostable chitin-binding domain derived from the hyperthermophilic bacterium Pyrococcus furiosus into a domain capable of recognizing cellulose, and obtained a thermostable cellulose-binding domain by substituting specific amino acid residues. I found out that

The present invention relates to a polypeptide having the following cellulose-binding activity and a method for producing the same.
1. In the 101 amino acid sequence from Thr ²⁵⁸ to Ile ³⁵⁸ of SEQ ID NO: 3,
(1) Glu ²⁷⁹ and / or Asp ²⁸¹ is substituted with an amino acid selected from the group of neutral amino acids consisting of Gln, Asn, Ala, Ser, Thr, Cys and Met, and if necessary
(2) A polypeptide, wherein one or several or a plurality of amino acids are substituted, added, deleted or inserted, and has a cellulose binding activity.
2. The polypeptide according to Item 1, wherein glutamic acid at position ²⁷⁹ (Glu ²⁷⁹ ) is substituted with Thr, Ala, Asn, or Gln, and aspartic acid at position ²⁸¹ (Asp ²⁸¹ ) is substituted with Asn, Ser, Gln, or Ala. .
3. Item 3. The polypeptide according to Item 2, wherein glutamic acid at position ²⁷⁹ (Glu ²⁷⁹ ) is substituted with Thr, and aspartic acid at position ²⁸¹ (Asp ²⁸¹ ) is replaced with Asn.
4). Item 4. A heat-resistant cellulose-binding domain comprising the polypeptide according to any one of Items 1 to 3.
5. A method for producing a cellulose-binding domain, comprising substituting a neutral amino acid for an acidic amino acid in the surface of the chitin-binding domain interacting with chitin.
6). Item 6. The method according to Item 5, wherein the amino acid substituting Glu ²⁷⁹ and / or Asp ²⁸¹ is selected from the group consisting of Ser, Thr, Ala, Asn and Gln.
7). Item 7. The method according to Item 5 or 6, wherein the chitin-binding domain is derived from the genus Thermococcus or Pyrococcus.
8). Item 4. A DNA encoding the polypeptide according to any one of Items 1 to 3.

  According to the present invention, the functional conversion of the thermostable chitin-binding domain to the thermostable cellulose-binding domain has been successful based on a rational interpretation based on the steric information. The newly developed thermostable cellulose-binding domain also retains chitin-binding ability and can be used for the development of high-performance chitinases and cellulases.

  Since carbohydrate-degrading enzymes such as chitinase and cellulase have a substrate-binding domain that is essential for functional expression, it is expected that the development of thermostable carbohydrate-degrading enzymes will gain momentum by using the domains developed in this study. it can.

  In particular, the heat-resistant cellulose binding domain has been revealed for the first time by the present invention, and its significance is great.

  In one embodiment, the present invention proposes a method for converting a chitin binding domain of a chitinase from a thermostable bacterium into a cellulose binding domain.

  Examples of thermostable bacteria include bacteria belonging to the genus Thermococcus or Pyrococcus, such as Pyrococcus furiosus, Thermococcus litoralis, Pyrococcus sp. KOD1, Thermotoga maritima, etc. Domains can be converted to thermostable cellulose binding domains according to the design method of the present invention.

The chitin-binding surface of chitin-binding domain 2 (ChBD2) derived from Pyrococcus furiosus is structurally concentrated on one side as shown in FIG. 1a. Further investigation of the surface charge of this domain revealed that there was a negative electrostatic potential formed by two acidic residues (E279 and D281) at one location on the chitin-binding surface (FIG. 1b). When considering the interaction between ChBD2 and the substrate chitin (N-acetylglucosamine), the acetamide group (NHCOCH _{3 in} N-acetylglucosamine)
NH) and the carboxyl group of these acidic residues are considered to form hydrogen bonds. Such an interaction is seen in the complex of lysozyme and its substrate (including N-acetylglucosamine) (FIG. 2a). That is, the present inventor considered that these two acidic amino acids are responsible for specific recognition of chitin. On the other hand, the difference between chitin and cellulose is that this acetamide group is only changed to a hydroxyl group in cellulose (FIG. 2b).

  Therefore, the present inventor considered that the substrate recognition of this domain can be controlled by substituting the two acidic amino acids (E279 and D281) with other amino acids.

Examples of amino acids introduced in place of acidic amino acids include neutral amino acids with low hydrophobicity, such as Gln, Asn, Ala, Ser, Thr, Cys, Met, etc., preferably Gln, Asn, Ala, Ser, Thr, Cys, more preferably Gln, Asn, Ala, Ser, Thr. Gln is more preferably substituted for Glu, and Asn is more preferably substituted for Asp.

In other positions, the amino acid sequence of SEQ ID NO: 3 is 1 or several or plural, for example 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5. , Especially 1-3 amino acids may be substituted, added, deleted or inserted.

  The method for imparting cellulose-binding activity to the chitin-binding domain obtained in the present invention can be suitably applied to the chitin-binding domain of chitinase derived from thermostable archaea such as Thermococcus or Pyrococcus, but other microorganisms, The same applies to chitinase domains of chitinases derived from plants and animals.

As a method for introducing mutations such as substitution, addition, deletion, and insertion into the chitin-binding domain, DNA encoding the domain can be obtained by, for example, cytospecific mutagenesis (Methods in Enzymology, 154, 350, 367-382 (1987). ); 100, 468 (1983); Nucleic
Acids Res., 12, 9441 (1984)), chemical synthesis means such as the phosphate triester method and phosphate amidite method (for example, using a DNA synthesizer) (J. Am. Chem. Soc 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981)). Codon selection can be determined by taking into account the codon usage of the host.

In one preferred embodiment of the present invention, the strategy for introducing mutations for the purpose of converting a chitin binding domain into a cellulose binding domain is as follows: 1) Maintaining solubility while reducing the hydrophilicity of the substrate binding surface. 2) Select polar amino acids that can interact with hydroxyl groups. 3) Maintaining the size of the amino acid after mutation. In accordance with this policy, for example, design mutants in which E279 is replaced with threonine and D281 is substituted with serine or asparagine (ChBD2TN, ChBD2TS), and mutants in which both amino acids are substituted with alanine (ChBD2AA) to reduce hydrophilicity. (Fig. 3).

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Reference example 1
Prescission Protease recognition to express a large amount of the predicted chitin-binding domain of the gene corresponding to ChBD in the E. coli polypeptide (corresponding to leucine 258 to 358 isoleucine) Amplification was carried out by PCR using synthetic DNA containing a base sequence encoding the sequence. Incorporation into an expression vector was carried out by transforming into E. coli (XL-1Blue) (NOVAGEN) using a LIC cloning kit (NOVAGEN) to prepare a ChBD expression vector.
Transformants were selected using colony formation on LB agar plates containing (0.05 mg / ml ampicillin) as an indicator. A ChBD gene-containing plasmid was extracted from the transformant by the alkaline method.

Example 1
Experimental method Sample preparation
-Preparation method of ChBD2 mutant Using the ChBD2 expression vector obtained in Reference Example 1 as a template, the synthetic DNA (SEQ ID NO: 1-2) shown in Fig. 3a was used to create a mutant (Fig. 3b).

The mutant was prepared using Quick change mutagenesis kit (STARATAGENE).

・ Creation of transformant containing ChBD2 mutant gene
In a 1.5 ml tube, 0.04 ml (2,000,0000 cfu / mg) competent cell of E. coli Rosetta (DE3) strain (manufactured by Novagen) and 0.003 ml of the ChBD2 gene-containing plasmid DNA solution prepared above ( Plasmid DNA (8.4 ng) was added and allowed to stand in ice for 30 minutes, followed by heat shock at 42 ° C. for 30 seconds. Next, add 0.25 ml of SOC medium into the tube and add 1 at 37 ° C.
Cultured with shaking. Subsequently, it apply | coated to the LB agar plate containing an ampicillin, and the transformant was obtained by culture | cultivating at 37 degreeC overnight.

Purification of ChBD2 mutant In order to increase the expression level of ChBD2 after inoculating the obtained transformant into LB medium containing ampicillin and culturing at 37 ° C. until the absorbance at 600 nm reaches 0.5. IPTG (Isopropyl-bD-thiogalactopyranoside) was added and further cultured for 19 hours. The culture was collected by centrifuging at 8,000 rpm for 10 minutes. 100 ml of BugBuster solution (NOVAGEN) was added to 10 g of the collected bacterial cells, and the bacterial cells were ultrasonically crushed for 30 minutes at an output of 90 W. The disrupted bacterial solution was centrifuged at 15,000 rpm for 30 minutes, and the supernatant was collected. Metal chelate column HiTrap-Cleating (Amersham Biosciences) column equilibrated with the same buffer (100 mM NiCl2 in advance)
Was added to the column and Ni was bound). Elution is 25 mM trishydroxymethylaminomethane containing 0.5 M imidazole, 500 mM NaCl (pH 8.5)
A linear gradient was used.

The obtained sample fraction was dialyzed overnight against 20 mM Tris-Cl, 25 mM NaCl (pH 8.5), and equilibrated with 20 mM Tris-Cl, 25 mM NaCl (pH 8.5) buffer. A dialysis sample was added to a column (Amersham Bioscience) and ion exchange chromatography was performed. The fraction containing the target protein contained a homogeneous sample that gave a single band by SDS-polyacrylamide gel electrophoresis. The protein concentration was calculated from the absorption at 280 nm.

Analysis of interaction between cellulose powder and mutant protein <br/> Solution of 1 nmole of each mutant protein in 20 mM Tris-Cl, pH 8.5 containing 1 mg of cellulose powder (Avicel) / chitin fine powder (insoluble in water) And allowed to stand at room temperature for 2 hours. Thereafter, the mixture was centrifuged at 10000 g for 10 minutes to remove cellulose and proteins bound to cellulose, the protein remaining in the supernatant was quantified, and the protein remaining in the supernatant was quantified. The protein was quantified by measuring absorption at 280 nm.

Results and discussion
・ As a result of investigating how chitin-binding ability changed due to mutagenesis with regard to the binding between chitin and each mutant, it was found that the TN mutant had the same chitin-binding ability as wild-type ChBD2 (FIG. 4a). . This is thought to be a complement of the carbonyl group of asparagine.

-Regarding the binding between cellulose and each mutant It was examined how the binding between cellulose and the mutant was changed (Fig. 4b). In the wild type, the binding to cellulose was very weak, but the mutant introduced according to the above-mentioned policy had an increased binding to cellulose. The TN mutant increased its binding power almost 10 times.

A: The interaction surface of ChBD2 and chitin, red shows a very strong interaction, and blue shows a fairly strong interaction. B: shows the electrostatic potential distribution of ChBD2. Red indicates a negatively charged place and blue indicates a positively charged place. The β sheet portion is indicated by a blue line. A: Interaction pattern of aspartic acid and NAG (acetylglucosamine) located in the active site of lysozyme B: Comparison of molecular structure of cellulose and chitin A: Synthetic DNA used to create the mutant Sequence B: Position where the mutation was introduced. The amino acid sequence of ChBD2 is indicated by a single letter code, and the position of the mutation and the amino acid after the mutation are indicated by arrows. The box arrow indicates the β strand. A: Comparison of the affinity of wild type and mutant (ChBD2TN) for chitin B: Comparison of affinity of wild type and wild type (ChBD2TN) for cellulose

Claims (8)

In the 101 amino acid sequence of Thr ²⁵⁸ to Ile ³⁵⁸ of SEQ ID NO: 3, (1) Glu ²⁷⁹ and / or Asp ²⁸¹ is selected from the group of neutral amino acids consisting of Gln, Asn, Ala, Ser, Thr, Cys and Met Substituted with an amino acid, and if necessary
(2) A polypeptide, wherein one or several or a plurality of amino acids are substituted, added, deleted or inserted, and has a cellulose binding activity. 2. The poly of claim 1, wherein glutamic acid at position ²⁷⁹ (Glu ²⁷⁹ ) is substituted with Thr, Ala, Asn or Gln, and aspartic acid at position ²⁸¹ (Asp ²⁸¹ ) is substituted with Asn, Ser, Gln or Ala. peptide. The polypeptide according to claim 2, wherein glutamic acid at position ²⁷⁹ (Glu ²⁷⁹ ) is substituted with Thr, and aspartic acid at position ²⁸¹ (Asp ²⁸¹ ) is substituted with Asn. A heat-resistant cellulose-binding domain comprising the polypeptide according to claim 1. A method for producing a cellulose-binding domain, comprising substituting a neutral amino acid with an acidic amino acid in the surface of the chitin-binding domain that interacts with chitin. The method according to claim 5, wherein the amino acid substituting Glu ²⁷⁹ and / or Asp ²⁸¹ is selected from the group consisting of Ser, Thr, Ala, Asn and Gln. The method according to claim 5 or 6, wherein the chitin-binding domain is derived from the genus Thermococcus or Pyrococcus. DNA encoding the polypeptide according to any one of claims 1 to 3.

Images