JP5182861B2 - Highly active thermostable chitinase - Google Patents

Description

  The present invention provides a highly active thermostable chitinase.

  Chitin is the next most abundant biomass after cellulose, and is produced by various biological species (shrimp, moths, insects, etc.) on the earth every year for 100 billion tons. N-acetylglucosamine obtained by hydrolyzing chitin is not only a health food additive, but also a raw material for hyaluronic acid that has recently become a hot topic as a moisturizing ingredient, so the cosmetic industry is also drawing attention. It is also expected to be used as a therapeutic drug for dermatitis, osteoarthritis, etc. and is expected to be used in a wide range of industrial fields. Chitinase is an enzyme obtained from plants, microorganisms, etc., and plays an extremely important role in industrial use to hydrolyze chitin to obtain very high added value N-acetylglucosamine as described above. Therefore, its effective use is expected. The development of chitinase is one of the important issues for its effective use.

Chitinases of various origins have been isolated and thermostable chitinases are also known (Patent Documents 1 and 2). We isolated and purified the catalytic domain of chitinase from the hyperthermophile Pyrococcus furiosus chitinase gene, successfully crystallized it, and determined the three-dimensional structure in the crystal by X-ray crystallography. Have applied for a patent that provides the structural information of the domain in the crystal and the crystal of the catalytic domain of the chitinase necessary for enhancing the chitinase catalytic activity using protein engineering techniques (Title of Invention: Patent Document 3) Crystals of the catalytic domain of chitinase that degrades crystalline chitin).

Among the 298 amino acids constituting the catalytic domain, Gln ⁵²⁶ , Asp ⁵²⁴ , Asp ⁵²² are very important for chitinase catalytic activity, as suggested by the inventor in the prior patent (Patent Document 3).
When producing a mutant domain with improved chitinase catalytic ability, it is desirable to modify Gln ⁵²⁶ , Asp ⁵²⁴ , Asp ⁵²² or the vicinity thereof. The prior patent states that the amino acid mutation introduced into the chitinase catalytic domain is 1 to several, for example 1 to 9, preferably 1 to 6, more preferably 1 to 4, particularly 1 to 2. (Patent Document 3).
JP 2001-54381A JP 2006-25701 A JP 2007-312687 A

  An object of the present invention is to provide a highly active thermostable chitinase.

The present inventor created a mutant in which one of the amino acids around the catalytic site (FIG. 1), which is important for high functionality described in the above-mentioned patent application (FIG. 1), was substituted with serine. Analysis revealed that this mutant had about two times the degradation activity against low molecular weight synthetic substrates compared to the wild type.

The present invention relates to the following inventions.
Item 1. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
Item 2. A gene encoding the polypeptide according to Item 1.
Item 3. A chitinase comprising the polypeptide according to Item 1.
Item 4. A gene encoding the chitinase according to Item 3.

According to the present invention, an improved thermostable chitinase having high activity is provided. By using the chitinase of the present invention, it is possible to effectively utilize chitin, which is one of the biomass abundantly present on the earth. In addition, synergistic effects (cooperative work) can be expected by mixing with chitinases with different degradation modes (for example, endo-type enzymes that roughly degrade chitin).
In addition, this research and development was carried out with the catalytic domain of thermostable chitinase, and further improvement in activity can be easily expected by appropriately fusing the substrate binding domain.

  By utilizing the enzyme according to the present invention, it can be expected that the development of a thermostable chitinolytic enzyme will gain momentum.

  Hereinafter, the present invention will be described in detail.

  The polypeptide of the present invention is encoded by the PF1233 gene in a polypeptide {gene database (http://gib.genes.nig.ac.jp), which is presumed to be the catalytic domain of the hyperthermophile Pyrococcus furiosus-derived chitinase. Polypeptide comprising the amino acid sequence (SEQ ID NO: 1) in which the 486th alanine is substituted with serine in the polypeptide consisting of 298 amino acids corresponding to asparagine No. 409 to phenylalanine No. 706 in the amino acid sequence (SEQ ID NO: 2) It is. Furthermore, a polypeptide having a function as a catalytic domain of chitinase in which one or several amino acids are deleted, substituted or added in the polypeptide of the present invention can be prepared. For example, amino acids exposed on the surface can be mutated without degrading heat resistance. The polypeptide of the present invention can function as a chitinase even if only the catalytic domain is present.

The gene encoding the polypeptide of the present invention is a DNA sequence corresponding to the amino acid codon of the polypeptide. The codon to be used can be appropriately selected depending on the species of the gene that expresses the gene.

  The chitinase containing the polypeptide of the present invention is a chitinase in which one or more amino acids are added to the N-terminus, C-terminus or both of the polypeptide. The one or more amino acids to be added are preferably a substrate binding domain. By fusing the polypeptide with a substrate-binding domain or other functional polypeptide, the activity can be enhanced, or the expressed protein can be released to a medium outside the cell. In addition, the enzyme activity can be improved by exchanging the catalytic domain of an existing chitinase with the polypeptide of the present invention.

The gene encoding the chitinase of the present invention is a DNA sequence corresponding to the amino acid codon of the chitinase. The codon to be used can be appropriately selected depending on the species of the gene that expresses the gene.

The PF1233 gene can be easily produced and obtained by general genetic engineering techniques based on the sequence information [Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); (See Laws I, II, III, edited by the Japanese Biochemical Society (1986))
.

By using the obtained PF1233 gene, an expression plasmid containing a thermostable chitinase catalytic domain as a template for the mutant can be prepared by a conventional genetic engineering technique, and a desired mutation can be introduced into DNA. Then, an expression system such as E. coli can be transformed with this plasmid to express the protein. Moreover, the target polypeptide and chitinase can be purified and isolated according to a conventional method.

It is possible to produce a chitinase containing a polypeptide by ligating a DNA sequence corresponding to a substrate binding domain or the like to the one or both of the genes of the catalytic domain of the thermostable chitinase by a conventional genetic engineering technique. it can.

In one embodiment, the present invention proposes a method for enhancing the catalytic activity of a chitinase derived from a thermostable bacterium, but even a chitinase derived from another microorganism, plant, or animal is shown in the present invention. When an amino acid other than serine is structurally corresponding to the mutation introduction site (486th position), it can be enhanced according to the design method of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, it cannot be overemphasized that this invention is not limited to these Examples.

experimental method
Method for creating a mutant of the catalytic domain The expression plasmid containing the catalytic domain of thermostable chitinase used as a template for the mutant is Mine S, Nakamura T, Hirata K, Ishikawa K, Hagihara Y, Uegaki K. Acta Crystallogr Sect F Struct Biol Cryst Commun 62. 791-793 (2006) was used.
The mutants were created using the following synthetic DNA, QuickChange mutagenesis kit (STARATAGENE)
Was performed.

The synthetic DNA for mutagenesis is shown below (underlined codon after substitution)
A486S (Ala → Ser mutation)
5'-GGAGAAGTAATAATA TCC TTTGGTGGGGCTGTAG-3 '
5'-CTACAGCCCCACCAAA GGA TATTATTACTTCTCC-3 '

M587Y (Met → Tyr mutation)
5'-GTCAATCCAATGACG TAC GATTACTACTGGACTC-3 '
5'-GAGTCCAGTAGTAATC GTA CGTCATTGGATTGAC-3 '

M587A (Met → Ala mutation)
5'-GTCAATCCAATGACG GCG GATTACTACTGGACTC-3 '
5'-GAGTCCAGTAGTAATC CGC CGTCATTGGATTGAC-3 '

D588N (Asp → Asn mutation)
5'-CAATCCAATGACGATG AAC TACTACTGGACTCC-3 '
5'-GGAGTCCAGTAGTA GTT CATCGTCATTGGATTG-3 '

D636N (Asp → Asn mutation)
5'-CCAATGATAGGAGTAAAT AAC GACAAGAGTGTG-3 '
5'-CACACTCTTGTC GTT ATTTACTCCTATCATTGG-3 '

D524N (Asp → Asn mutation)
5'-GCCACTTACTTGGACTTT AAC ATAGAAGCCGG-3 '
5'-CCGGCTTCTAT GTT AAAGTCCAAGTAAGTGGC-3 '

D524A (Asp → Ala mutation)
5'-GCCACTTACTTGGACTTT GCG ATAGAAGCCGG-3 '
5'-CCGGCTTCTAT CGC AAAGTCCAAGTAAGTGGC-3 '

E526Q (Glu → Asn mutation)
5'-GGACTTTGACATA CAA GCCGGTATCGATGC-3 '
5'-GCATCGATACCGGC TTG TATGTCAAAGTCC-3 '

E526A (Glu → Ala mutation)
5'-GGACTTTGACATA GCG GCCGGTATCGATGC-3 '
5'-GCATCGATACCGGC CGC TATGTCAAAGTCC-3 '

Preparation of transformants containing chitinase catalytic domain
In a 1.5 ml tube, 0.04 ml (20,000,000 cfu / mg) of competent cells of E. coli Rosetta (DE3) strain (Novagen) and 0.003 ml of plasmid DNA solution containing the above-mentioned catalytic domain gene (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, 0.25 ml of SOC medium was added to the tube and cultured with shaking at 37 ° C. for 1 hour. 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 mutant chitinase catalytic domain The resulting transformant was inoculated into LB medium containing ampicillin, cultured at 37 ° C until the absorbance at 600 nm reached 0.5, and then IPTG (isopropyl-bD to increase the expression level. -thiogalactopyranoside) was added (final concentration: 1 mM) and the cells were further cultured for 19 hours. 10 min at 8,000 rpm
Bacteria were collected by centrifugation. BugBuster solution (NOVAGEN) to 10g of collected cells
100 ml was added and the cells were sonicated at 90 W for 30 minutes. The disrupted bacterial solution was centrifuged at 15,000 rpm for 30 minutes, and the supernatant was collected. Column chromatography was carried out using a metal chelate column HiTrap-Chelating (manufactured by GE Healthcare) equilibrated with BugBuster solution (10 ml of 100 mM NiCl ₂ was added in advance and Ni was bound). Elution is 25 mM with 0.5 M imidazole.
A linear gradient of trishydroxymethylaminomethane, 500 mM NaCl (pH 8.5) was used. PreScission Protease (manufactured by GE Healthcare) was added to the obtained target fraction, put into a dialysis tube (fraction size Mw.3500), and 4 ° C. with 25 mM Trishydroxyaminomethane, 25 mM NaCl (pH 8.5) solution. Dialysis was performed for 16 hours.

After completion of the dialysis, again, 25 mM tris (hydroxymethyl) aminomethane, and 25 mM NaCl (pH 8.5) (manufactured by GE Healthcare) was equilibrated with metal chelate column HiTrap chromatography Chelating column (previously 100 mM NiCl ₂ was added 10 ml Ni The fraction that passed through the column was collected, and the anion exchange resin HiTrapQ (manufactured by GE Healthcare) equilibrated with 25 mM Trishydroxymethylaminomethane, 25 mM NaCl (pH 8.5) buffer. The collected fraction was added to the column, 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.

Activity measurement of mutants The enzyme activity was determined by using p-nitrophenyl-Di-N-acetyl-β-chitobioside (Seikagaku Corporation) as a measurement substrate and quantifying the amount of p-nitrophenol released by the enzymatic reaction. All reactions were carried out at 50 ° C. p-Nitrophenol was quantified by mixing 50 μl of the reaction solution with 0.2 M sodium carbonate solution pH 10 and measuring the absorbance at 405 nm. The reaction was performed under the following reaction conditions.

1) Measurement of enzyme activity over time shown in Fig. 2
100 mM sodium acetate pH 4.8
Wild-type chitinase (14.1nM) or mutant (6.9nM)
0.96 mM p-nitrophenyl-Di-N-acetyl-β-chitobioside

2) Measurement of substrate saturation curve shown in Fig. 3
100 mM sodium acetate pH 5.5
Wild-type chitinase (14.1nM) or mutant (6.9nM)
Measure reaction curves up to 60 minutes at various p-nitrophenyl-Di-N-acetyl-β-chitobioside concentrations, calculate reaction rates, and plot reaction rates against substrate concentrations

3) Table 1 Comparison of wild-type and mutant-type kinetic parameters K _m (Michaelis-Menten constant) representing the affinity between the enzyme and the substrate according to the Michaelis-Menten equation from the substrate saturation curve measured according to FIG. The k _cat (molecular activity) indicating how many substrates are catalyzed per protein molecule per second was calculated.

4) Comparison of activity between other mutant types and wild type shown in Fig. 4
100 mM sodium acetate pH 5.5
Each enzyme was added to a reaction solution of 0.48 mM p-nitrophenyl-Di-N-acetyl-β-chitobioside so as to have the following enzyme concentration and reacted for 15 minutes.
For wild-type and mutant chitinases (A486S, M587Y, D588N, D636N, D524N) (final concentration 14 nM) and mutants (M587A, D524A, E526Q, E526A) (final concentrations 1200, 1200, 2200, 2200 nM) . The relative value of each mutant is shown as 100% wild type.

Analysis of reaction rate parameter As shown in FIG. 2, it was found that the time course of the enzyme reaction was clearly higher in the mutant type than in the wild type. However, it is necessary to distinguish whether this phenomenon is due to enhanced substrate binding (K _m ) or enhanced reaction efficiency (k _cat ) in terms of enzyme reaction. Therefore, in order to know which effect is stronger, the reaction rate depending on the substrate concentration shown in FIG. 3 was plotted (substrate saturation curve), and enzyme reaction parameters were obtained. Table 1 shows the results, the wild type, K _m indicating the affinity between the variants both substrates without changes significantly, found that k _cat showing the reaction efficiency is enhanced more than twice It was. From this, it can be concluded that the introduction of mutation has succeeded in the development of an enzyme with greatly enhanced degradation activity of the mutant against a low molecular weight synthetic substrate. Other amino acids that form the catalytic site (524D, 526E, 587M, 588D, 636D
Mutation was carried out on (see FIG. 1), and the change in activity was examined (FIG. 4). As a result, with the exception of 486A, the other amino acid mutations showed no activity enhancement, and it was found that all the amino acids subjected to the mutation caused a decrease in activity. From the above analysis results, it was found that A486S was the only amino acid variant that was effective in enhancing the activity in this study. However, it is clear from this experiment that the amino acid positions (524D, 526E, 587M, 588D, 636D) whose activity has been reduced by mutation play an important role in the activity. There is still a possibility that the activity can be enhanced by other amino acid mutations or by introducing mutations into these amino acids at a plurality of positions.

FIG. 3 is a configuration diagram of amino acids around the catalytic site of chitinase. The spatial arrangement of Gln526, Asp524, Ala486, M587, D588, and D636 is shown. It is a figure showing the time change of an enzyme activity in an Example. It is a figure showing a substrate saturation curve in an Example. In an Example, it is a figure showing the comparison of the enzyme activity of another mutant type and a wild type.

Claims (4)

Polypeptide comprising an amino acid sequence shown in SEQ ID NO: 1. A gene encoding the polypeptide of claim 1. A chitinase comprising the polypeptide of claim 1. A gene encoding the chitinase according to claim 3.

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