CN109897840B - Polyphosphate-dependent glucokinase variants and uses thereof - Google Patents
Polyphosphate-dependent glucokinase variants and uses thereof Download PDFInfo
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Abstract
The present invention relates to polyphosphate-dependent glucokinase variants. In particular, the present invention relates to variants of polyphosphate-dependent glucokinase that have improved thermostability and enzyme activity, which comprise substitutions of one or more specific amino acid residues. Variants may also optionally include additional changes at other sites.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a polyphosphate dependent glucokinase (polyphosphate glucokinase) variant, a preparation method and application thereof, and a DNA molecule, a vector and a host cell for encoding the variant.
Background
Polyphosphoric acid-dependent glucokinase (PPGK) for short can catalyze glucose to form glucose 6-phosphate by using inorganic polyphosphoric acid as a phosphate donor. Inorganic polyphosphoric acid (Pi)nThe phosphate-based polyphosphate ester is a linear polymer formed by connecting a plurality of phosphate residues through phosphoric anhydride bonds, and has a series of advantages compared with ATP (adenosine triphosphate) and inorganic polyphosphate, such as low price, good thermal stability, strong tolerance to pH (potential of hydrogen), oxidation resistance and the like. The product glucose 6-phosphate can be used as a synthetic precursor of many high-value biochemical finished products, such as stevioside, ginsenoside, fructose 1, 6-diphosphate, inositol and the like. In the medical field, glucokinase can also be used for the quantitative determination of blood glucose.
PPGK from a plurality of different sources is discovered at present, but most of the known PPGK is from mesophilic bacteria, the only PPGK from thermophilic Thermobifida fusca is poor in thermal stability, and the half-life (t) of free enzyme at 50 ℃ is1/2) Only 15min (Liao et al, 2012). At present, no research on the improvement of PPGK thermal stability exists, the application range of PPGK is limited due to poor thermal stability of PPGK, enzymes are easy to inactivate, and the production cost is increased, so that the polyphosphate-dependent glucokinase variant with improved thermal stability is urgently needed to be obtained for producing ATP-independent glucose 6-phosphate and derivatives thereof.
Disclosure of Invention
Aiming at the problem of poor thermal stability of the existing polyphosphate dependent glucokinase, the invention provides a plurality of polyphosphate dependent glucokinase variants and a preparation method thereof, and the thermal stability and half-life period of the variants are improved, so that the service life of the enzyme can be prolonged, the production cost of the enzyme is reduced, and the polyphosphate dependent glucokinase variant has a wide industrial application prospect.
First, the present invention provides a plurality of polyphosphate-dependent glucokinase variants.
Compared with parent polyphosphate-dependent glucokinase, the polyphosphate-dependent glucokinase variant provided by the invention has improved thermal stability, and the denaturation temperature is increased by 1-20 ℃, preferably increased by 10-20 ℃, and more preferably increased by 15-20 ℃ compared with the parent polyphosphate-dependent glucokinase.
The polyphosphate-dependent glucokinase variant comprises at least one substitution of the amino acid residues corresponding to positions F30, R34, T72, V88 and A231 of the polyphosphate-dependent glucokinase derived from Thermobifida fusca strain YX.
Preferably, the substitution of the amino acid residues of the variant at positions F30, R34, T72, V88, a231 is F30L, R34P, T72A, V88M, a231T, respectively.
More preferably, the amino acid residue substitution of the variant comprises the following combinations a) or b) or c):
a)F30L+T72A+A231T；
b)F30L+T72A+V88M+A231T；
c)F30L+R34P+T72A+V88M+A231T。
preferably, the parent polyphosphate-dependent glucokinase is selected from the following sequence characteristics a) or b):
a) derived from actinomycetes, specifically Thermobifida fusca, more specifically Thermobifida fusca strain YX, and has an amino acid sequence SEQ ID NO of 1;
b) an amino acid sequence having at least 50% homology, in particular at least 70% homology, more in particular at least 80% homology with the amino acid sequence shown in SEQ ID No. 1.
In a second aspect, the present invention provides a method for preparing a polyphosphate-dependent glucokinase variant.
Compared with parent polyphosphate-dependent glucokinase, the polyphosphate-dependent glucokinase variant prepared by the preparation method provided by the invention has improved thermostability, and the denaturation temperature is increased by 1-20 ℃, preferably 10-20 ℃, and more preferably 15-20 ℃ compared with the parent polyphosphate-dependent glucokinase. The method specifically comprises the following steps:
a) selecting a parent polyphosphate-dependent glucokinase;
b) modifying the coding sequence of the parent polyphosphate-dependent glucokinase selected in step a) so that the coding sequence has amino acid residue substitution at the position F30 and/or R34 and/or T72 and/or V88 and/or A231 corresponding to the polyphosphate-dependent glucokinase derived from the Thermobifida fusca strain YX.
c) Selecting a variant having a denaturation temperature which is increased by 1-20 ℃, preferably 10-20 ℃, more preferably 15-20 ℃ compared to the parent polyphosphate-dependent glucokinase.
Preferably, the amino acid substitutions at positions F30, R34, T72, V88, a231 are F30L, R34P, T72A, V88M, a231T, respectively.
More preferably, the substitution of the amino acid comprises the following combinations a) or b) or c):
a)F30L+T72A+A231T；
b)F30L+T72A+V88M+A231T；
c)F30L+R34P+T72A+V88M+A231T。
preferably, the parent polyphosphate-dependent glucokinase has the following sequence characteristics a) or b):
a) derived from actinomycetes, specifically Thermobifida fusca, more specifically Thermobifida fusca strain YX, and has an amino acid sequence SEQ ID NO of 1;
b) an amino acid sequence having at least 50% homology, in particular at least 70% homology, more in particular at least 80% homology with the amino acid sequence shown in SEQ ID No. 1.
In a third aspect, the present invention provides a gene encoding the polyphosphate-dependent glucokinase variant of the first aspect, and an expression vector or a host cell comprising the encoding gene.
In a fourth aspect, the present invention provides the use of a polyphosphate-dependent glucokinase variant for the production of glucose 6-phosphate and derivatives of glucose 6-phosphate.
Glucose and polyphosphoric acid are taken as substrates, the polyphosphoric acid dependent glucokinase variant is adopted, and then Inositol-3-phosphate synthase (Inositol-3-phosphate synthase, EC 5.5.1.4) and Inositol monophosphatase (EC 3.1.3.25) are coupled to prepare a multi-enzyme reaction system, so that Inositol is produced at 70 ℃.
The terms and definitions to which this invention relates
Polyphosphate dependent glucokinase
The parent enzyme is a polyphosphate-dependent glucokinase classified as EC 2.7.1.63 according to enzyme nomenclature. It is a polyphosphate-dependent glucokinase derived from bacteria, such as actinomycetes. In particular to a thermobifida fusca, more particularly to a thermobifida fusca strain YX. SEQ ID NO. 1 shows the polyphosphate-dependent glucokinase amino acid sequence (mature peptide) of Thermobifida fusca strain YX.
Nomenclature for amino acids and alterations thereof
The single letter abbreviations used in the specification and claims for amino acids are used when referring to them. The particular amino acid in the sequence is identified by its one-letter code and its position, for example F30 for Phe (phenylalanine at position 30). The nomenclature used herein to define substitutions is as follows, for example F30L indicates the substitution of L (Leu) for F (Phe) at position 30.
Homology and sequence alignment
The relatedness between two amino acid sequences is described by the parameter "homology (identity)". For the purposes of the present invention, the alignment of two amino acid sequences was determined by using the Needle program version 2.8.0 from the EMBOSS software package (http:// EMBOSS. org). The Needle program performs the global alignment algorithm (global alignment algorithm) described in Needleman, S.B. and Wunsch, C.D. (1970) J.mol.biol.48, 443-453. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5.
The degree of homology between an amino acid sequence of the invention ("invention sequence") and an amino acid sequence referred to in the claims (e.g., SEQ ID NO:1) is calculated by the number of exact matches in the alignment of the two sequences, divided by the shortest of the length of the "invention sequence" or the length of SEQ ID NO: 1. Results are expressed as percent identity.
An exact match occurs when the "invention sequence" and SEQ ID NO:1 have identical amino acid residues at the same positions of overlap (this is represented by "|" in the alignment examples below). The length of the sequence is the number of amino acid residues in the sequence (e.g., amino acids 1-263 of SEQ ID NO:1 are 263 in length).
In the following purely hypothetical comparison examples, the overlap is the amino acid sequence "HTWGER-NL" of sequence 1 or the amino acid sequence "HGWGEDANL" of sequence 2. In this example, the notch is indicated by "-".
Hypothetical comparison example:
sequence 1: HTWGER-NLACMS
|||||||||
Sequence 2: HGWGEDANLMNPT
In a specific embodiment, the percent identity of the amino acid sequence of the polypeptide with or relative to, for example, SEQ ID No. 1 is determined by: i) aligning the two amino acid sequences using the Needle program with the BLOSUM62 substitution matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5; ii) counting the number of exact matches in the alignment; iii) dividing the exact match number by the length of the shortest of the two amino acid sequences, iv) converting the result of the division of iii) into a percentage.
In the hypothetical example above, the exact match number was 6, and the length of the shortest of the two amino acid sequences was 12; the percent identity is therefore 50%.
In particular embodiments of the polyphosphate-dependent glucokinase of the invention, the homology between the parent polyphosphate-dependent glucokinase and the polyphosphate-dependent glucokinase variant may be higher than 80%, for example higher than 85% or 90%, in particular higher than 95%.
Location numbering
The nomenclature used herein to define amino acid positions is based on the amino acid sequence of the mature polyphosphate-dependent glucokinase of Thermobifida fusca YX, which is given in the sequence listing as SEQ ID No. 1. Thus, in this context, the basis for position numbering is SEQ ID NO 1, starting at M1 and ending at A263. (SEQ ID NO:1) as a standard for position numbering and thus as a reference for nomenclature.
Identifying the corresponding position number
As explained above, the amino acid sequence of mature polyphosphate-dependent glucokinase of Thermobifida fusca YX (SEQ ID NO:1) is used herein as a standard for position numbering and thus also for nomenclature.
For additional polyphosphate-dependent glucokinase, particularly polyphosphate-dependent glucokinase variants of the invention, the position corresponding to position D in SEQ ID No. 1 was found by aligning the two sequences as specified previously in the section entitled "polyphosphate-dependent glucokinase polypeptide, percent identity". From the alignment, the position in the sequences of the invention corresponding to position D of SEQ ID NO:1 can be clearly and unambiguously identified (those two positions on top of each other in the alignment).
Some additional, purely hypothetical examples are given below, which were derived from table 1 above, and which comprise, in the third column, a plurality of alignments of two sequences:
referring to table 1, first row, third column: the top row of sequences is the template and the bottom row is the variant. Position number 80 refers to amino acid residue G in the template. Amino acid a occupies the corresponding position in the variant. Thus, this substitution is designated G80A.
TABLE 1
Specific substitution
Variants include, inter alia, one or more substitutions corresponding to the numbering in polyphosphate-dependent glucokinase from thermobifida fusca YX: F30L, R34P, T72A, V88M, a 231T.
Construction method of polyphosphate dependent glucokinase variant
Polyphosphate-dependent glucokinase variants can be obtained by site-directed mutagenesis or locally random mutagenesis at selected sites, i.e., by introducing random amino acid residues at selected sites or over the entire region of a parent polyphosphate-dependent glucokinase, as described, for example, in WO 95/22615. The selected site may be one or more of the sites mentioned above, and the selected region may be a region comprising one or more of such sites. The selected region can be determined by comparison of homologous sequences as described above.
Site-directed mutagenesis
When the nucleotide sequence encoding polyphosphate-dependent glucokinase is isolated and the corresponding site identified, a mutation can be introduced using the synthetic oligonucleotide. These oligonucleotides include nucleotide sequences flanking corresponding sites. In a specific method, a single-stranded gap of nucleotide is generated in a vector carrying the polyphosphate-dependent glucokinase gene, and the nucleotide is a polyphosphate-dependent glucokinase coding sequence. Synthetic nucleotides, carrying the corresponding mutations, are then annealed to homologous portions of the single-stranded nucleotides. The remaining gap was filled with DNA polymerase I (Klenow fragment) and the construct was ligated using T4 ligase.
Another method for introducing mutations in the nucleotide sequence encoding polyphosphate-dependent glucokinase involves 3 steps of generating a PCR fragment including the corresponding mutation, wherein the mutation is introduced in a PCR reaction using a chemically synthesized nucleotide chain as a primer. The nucleotide fragment carrying the mutation can be isolated by cleavage with a restriction enzyme and then reinserted into an expression plasmid.
Construction of expression vector containing nucleotide sequence encoding polyphosphate-dependent glucokinase variant
According to the present invention, the nucleotide sequence encoding the variant obtained by the above-described method or any other method alternative in the art may be expressed enzymatically using an expression vector typically comprising a promoter, an operator, a ribosome binding site, a translation initiation signal, and optionally a control sequence for a repressor gene or a different activator gene.
Expression vector
The recombinant expression vector carrying the variant encoding polyphosphate-dependent glucokinase of the present invention may be any vector which can be conveniently subjected to genetic recombination, and its choice will often depend on the host cell into which it is to be introduced. The expression vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and then replicated along with the chromosome(s) into which it has been integrated. Suitable expression vectors include pET20b, pET28a, and the like.
Promoters
In an expression vector, the nucleotide sequence must be operably linked to a suitable promoter sequence. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Suitable promoters which may direct the transcription of the nucleotide sequence encoding a polyphosphate-dependent glucokinase variant of the present invention are, in particular, the promoter of the lac operon of Escherichia coli, the dagA promoter of the agarase (agarase) gene of Streptomyces coelicolor, the alpha-amylase (amyL) gene promoter of Bacillus licheniformis (Bacillus licheniformis), the maltogenic amylase gene (amyM) promoter of Bacillus stearothermophilus (Bacillus stearothermophilus), the alpha-amylase gene (amyQ) promoter of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the xylA and xylB gene promoters of Bacillus subtilis (Bacillus subtilis), and the like, when bacteria are used as hosts.
The expression vectors of the invention also include a suitable transcription terminator, and the termination sequence may suitably be derived from the same source as the promoter.
The expression vector used may also comprise a nucleotide sequence which allows the expression vector to replicate in the host cell in question. Examples of such sequences are the replication start sites for plasmids pET20b, pET28a, pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ 702.
The expression vector used may also comprise a selectable marker, for example a gene the product of which complements a defect in the host cell, for example the dal genes from B.subtilis or B.licheniformis, or confers resistance to antibiotics, for example ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the expression vectors used may comprise Aspergillus (Aspergillus) selection markers, such as amdS, argS, niaD and sC, markers conferring hygromycin resistance, or selection by co-transformation, such as described in WO 91/17243.
The methods used to ligate the nucleotides encoding polyphosphate-dependent glucokinase variants, promoters, terminators, and other elements of the present invention, and to insert them into suitable expression vectors having the information necessary for replication are well known to those skilled in the art.
Host cell
The host cells used in the present invention, which comprise the nucleotide construct encoding the PPGK mutant of the present invention or the expression vector as defined above, are conveniently used as host cells for the recombinant production of the polyphosphate-dependent glucokinase variant of the present invention. The host cell may be conveniently transformed with the nucleotide construct of the present invention encoding the variant by integrating the nucleotide construct (in one or more copies) into the host chromosome. Such integration is generally considered to be advantageous because the nucleotide sequence is more likely to be stably maintained in the cell. Integration of the nucleotide construct into the host chromosome may be carried out according to conventional methods, for example, using homologous or heterologous recombination. Alternatively, the cell may be transformed with the above-described expression vector depending on the type of host cell.
The host cell of the invention may be a cell of a higher organism, e.g.a mammalian or insect cell, or a cell of a lower organism, especially a microbial cell, e.g.a bacterial or fungal (including yeast) cell.
Examples of suitable bacteria are gram-negative bacteria such as E.coli, gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus (Bacillus lentus), Bacillus brevis (Bacillus brevis), Bacillus thermophilus, Bacillus alkalophilus (Bacillus alkalophilus), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus coagulosus), Bacillus circulans (Bacillus circulans), Bacillus lautus (Bacillus lautus), Bacillus megaterium (Bacillus megaterium), Bacillus thuringiensis (Bacillus thuringiensis), Streptomyces lividans (Streptomyces lividans), Streptomyces murinus (Streptomyces murinus). Transformation of the bacteria can be accomplished by protoplasts or competent cells according to well known methods.
The yeast organism is preferably selected from a strain of Saccharomyces (Saccharomyces) or Schizosaccharomyces (Schizosaccharomyces), such as Saccharomyces cerevisiae.
The host cell may also be a filamentous fungus, for example a strain of Aspergillus selected (Aspergillus), especially Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium, for example Fusarium oxysporum (Fusarium oxysporum), Fusarium graminearum (Fusarium graminearum), Fusarium graminearum (Fusarium cerealis) or Fusarium venenatum (Fusarium venenatum).
In a particular embodiment of the invention, the host cell is a protease deficient or protease negative (minus) strain.
Culture expression of polyphosphate-dependent glucokinase variants
The medium used to culture the host cells may be any medium that is conventional, suitable for growth of the host cells in question and capable of expressing the polyphosphate-dependent glucokinase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published cultures (e.g., as described in catalogues of the American Type Culture Collection).
The polyphosphate-dependent glucokinase variant expressed in the host cell is purified and collected by collecting the cells, disrupting the cells, collecting the supernatant, and purifying by a well-known method such as a chromatographic method, for example, a nickel ion column.
The polyphosphate-dependent glucokinase variant secreted by the host cell can be conveniently recovered from the culture medium by well-known methods, including separating the cells from the medium by centrifugation or filtration, precipitating the protein components of the medium by salts, such as ammonium sulfate, and then using chromatographic methods, such as ion exchange chromatography, affinity chromatography, and the like.
Experimental Material
Strains and plasmids
Coli TOP10 was used to prepare recombinant plasmids.
pET28a-PtacPpgk is an expression plasmid containing tac and T7 double promoters, and is used for constructing a polyphosphate-dependent glucose kinase variant library in Escherichia coli T0P10 and expressing the polyphosphate-dependent glucose kinase variant in Escherichia coli BL21(DE 3).
Culture medium
LB medium is shown in Table 2
TABLE 2
| 10g/L | Peptone (Peptone) |
| 5g/L | Yeast extract (Yeast extract) |
| 10g/L | Sodium chloride |
Polyphosphoric acid dependent glucokinase activity determination method
The production of glucose 6-phosphate was measured in 50mM HEPES buffer (pH7.5) containing 4mM magnesium ion, 5mM glucose, and 1mM sodium hexametaphosphate, and the enzyme activity was measured for 5 minutes. 1IU polyphosphate-dependent glucokinase activity is defined as the amount of enzyme used to produce 1. mu. mol of glucose 6-phosphate per minute.
Drawings
FIG. 1 is a comparison of the results of the thermostability test at 55 ℃ for wild-type and mutant enzyme proteins.
FIG. 2 shows the results of the determination of the specific enzyme activities of wild-type and variant enzyme proteins at 25-85 ℃.
FIG. 3 shows the circular dichroism measurement of T for wild-type and variant enzyme proteinsmThe result of (1).
Example 1 construction of a mutant library of polyphosphate-dependent glucokinase Using error-prone PCR method
An error-prone PCR technique was used to introduce nucleotide mutations into the polyphosphate-dependent glucokinase gene ppgk in vitro. The error-prone PCR reaction conditions were as follows: 1 ng/. mu.l of pET28a-PtacPpgk (carrying the polyphosphate-dependent glucokinase gene ppgk from Thermobifida fusca YX), 0.2mM dATP, 0.2mM dGTP, 1mM dCTP, 1mM dTTP, 5mM MgCl2，0.05mM MnCl20.4. mu.M primers (MPPGK-IF and MPPGK-IR), and 0.05U/. mu.l Taq polymerase. PCR amplification conditions: 5min at 94 ℃; 94 ℃ for 30s, 56 ℃ for 30s, 68 ℃ for 45s, 18 cycles; 5min at 68 ℃.
The expression vector backbone was amplified using high fidelity PCR. The reaction conditions for high fidelity PCR were as follows: 1 ng/. mu.l of pET28a-Ptac-ppgk (carrying the polyphosphate-dependent glucose kinase gene ppgk from Thermobifida fusca), 0.2mM of various dNTPs, 0.5. mu.M primers (MPPGK-VF and MPPGK-VR), and 0.02U/. mu. l Q5DNA polymerase. PCR amplification conditions: 1min at 98 ℃; 30 cycles of 98 ℃ for 20s, 60 ℃ for 20s, 72 ℃ for 72 s; 5min at 72 ℃.
TABLE 3
Wherein the MPPGK-IF and the MPPGK-VR are end phosphorylation primers, and the two primers have an overlapping region of 28 bp.
And after the error-prone PCR amplification product and the expression vector skeleton are subjected to gel cutting recovery by an agarose recovery kit, performing fusion PCR on the error-prone PCR product and the expression vector skeleton, purifying and connecting the PCR product by using a DNA purification kit to obtain an expression vector containing a variant gene, and transforming the expression vector to E.coli TOP10 competent cells. The library of mutants expressing polyphosphate-dependent glucokinase was obtained by plating on LB plates (containing 100. mu.g/. mu.L kanamycin) containing 1.2% plant gel Phytagel.
Example 2 screening of high-thermostability polyphosphate-dependent glucokinase Positive variants Using plates
The screening process used a double plate method. Culturing the plate containing the mutant library at 37 deg.C overnight, heat treating the plate at different temperatures for 60min to lyse cells on the plate and release the target protein, and simultaneously release the target protein from Escherichia coli, which may be associated with glucose, polyphosphate and NADP+The enzymes that react and the PPGK, which is not sufficiently heat stable, are inactivated, degrading the background NAD (P) H. After the plate was cooled to room temperature, the plate was covered with 0.5% agarose gel, and the agarose was dissolved using 50mM HEPES buffer (pH7.5) while adding 5mM glucose, 1mM polyphosphoric acid, 1U/ml glucose 6-phosphate dehydrogenase, 10. mu.M Phenazine Methosulfate (PMS), 50. mu.M tetranitrotetrazolium blue (TNBT) and 1mM NAD+. Only blue-black spots can appear around the variants with high thermostability. The expression vector of the broken thallus can be directly extracted by using a trace plasmid extraction kit. And (3) performing multi-round high-throughput screening on the mutant protein in the library by adjusting the heating temperature, the heating time and the color development time, and realizing the directed evolution of the thermostable polyphosphate-dependent glucokinase. The following polyphosphate-dependent glucokinase variants with improved thermostability and activity were obtained by measuring thermostability and catalytic activity of the screened variants, as shown in Table 4, wherein the amino acid sequence of the 4-1 variant is shown in SEQ ID NO: 2.
TABLE 4
| 1-1 | A231T |
| 2-1 | F30L+T72A+A231T |
| 3-1 | F30L+T72A+V88M+A231T |
| 4-1 | F30L+R34P+T72A+V88M+A231T |
Example 3: determination of the half-Life t of polyphosphate-dependent glucokinase at 55 deg.C1/2
10. mu.g/ml polyphosphate-dependent glucokinase wild-type and variants thereof were incubated at 55 ℃ for various times, removed, placed on ice for 10 minutes and the residual activity of the enzyme at 25 ℃ was determined. By first order deactivation constant kdThe half-lives t of the different variants at 55 ℃ were determined1/2. The variants described in example 2 above were all found to have an increased half-life at 55 ℃ compared to the wild type, and the results are shown in FIG. 1 and Table 5.
TABLE 5
Example 4: stability determination by circular dichroism
The purified polyphosphate-dependent glucokinase wild-type and variants described in example 2 were investigated for thermostability by circular dichromatic chiralscan (Applied Photophysics, Surrey, UK) at pH7.5 (50mM phosphate buffer). 0.2mg/mL of sample and reference solution (approximately 0.3mL) were thermally pre-equilibrated (thermolly pre-equibrate) at 20 ℃ for 10 minutes with a constant programmed heating rateThe enzyme solution was heated (1 ℃ C./min) and the change in ellipticity at 220nm was measured. Determination of the temperature of thermal denaturation, T, by means of the change in the mean residue ellipticitymThe results are shown in FIG. 3 and Table 6.
TABLE 6
Example 5: inositol production by glucose and polyphosphoric acid at 70 DEG C
A multi-enzyme reaction system was prepared using variant 4-1 provided in example 2 coupled with Inositol-3-phosphate synthase (EC 5.5.1.4) derived from Archaeoglobus fulgidus and Inositol monophosphatase (Inositol monophosphate, EC3.1.3.25) derived from Thermotoga maritima to produce Inositol at 70 ℃ using glucose and polyphosphate as substrates.
The reaction was carried out in a 1-ml reaction system containing 100mM HEPES buffer (pH7.5), 10g/L glucose, 20mM divalent magnesium ion, 12.5. mu.g/ml polyphosphate-dependent glucokinase, 1U/ml inositol-3-phosphate synthase, and 1U/ml phytase. The reaction was carried out at 70 ℃ for a total of 24 hours.
According to the difference of the retention time, HPLC can be used for distinguishing inositol and glucose in the reaction solution; the mobile phase of the HPLC was 5mM dilute sulfuric acid.
The final inositol concentration obtained by using the polyphosphoric acid-dependent glucokinase variant 4-1 reaction is 9.98g/L, and the conversion rate relative to glucose is 99.8%; the final inositol concentration obtained by using wild type polyphosphoric acid-dependent glucokinase reaction is 0.52g/L, and the conversion rate is 5.20%.
The detailed description of the polyphosphate-dependent glucokinase variant and the construction method and application thereof, which have been described above with reference to examples, is illustrative and not restrictive, and several examples may be cited within the scope of the present invention, so that variations and modifications thereof without departing from the general concept of the present invention should fall within the scope of the present invention.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> polyphosphate-dependent glucokinase variants and uses thereof
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Met Ala Ser Arg Gly Arg Val Gly Leu Gly Ile Asp Ile Gly Gly Ser
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Gly Ile Lys Gly Ala Pro Val Asp Leu Asp Arg Gly Thr Phe Val Val
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Asp Arg Val Lys Ile Ala Thr Pro Gln Pro Ala Thr Pro Glu Ala Val
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Ala Ala Val Val Ala Glu Ile Val Thr Ala Phe Ala Asp Asp Val Pro
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Gln Asp Ala Pro Leu Gly Val Thr Phe Pro Ala Val Ile Gln His Gly
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Val Ala Arg Ser Ala Ala Asn Val Asp Arg Ser Trp Ile Gly Thr Asn
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Val Glu Glu Leu Leu Ser Ala Val Thr Gly Arg Arg Val Leu Val Val
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Asn Asp Ala Asp Ala Ala Ala Met Ala Glu His Arg Tyr Gly Ala Ala
115 120 125
Ser Gly Val Asp Gly Val Val Leu Leu Thr Thr Leu Gly Thr Gly Ile
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Gly Thr Ala Val Leu Val Asp Gly Val Leu Leu Pro Asn Thr Glu Phe
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Gly His Leu Glu Ile Asp Gly Tyr Asp Ala Glu Thr Arg Ala Ser Ala
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Ser Ala Lys Glu Arg Glu Asn Leu Ser Tyr Lys Glu Trp Ala Glu Glu
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Arg Leu Gln Arg Tyr Tyr Ser Val Ile Glu Asp Leu Leu Trp Pro Asp
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Leu Ile Val Val Gly Gly Gly Val Ser Arg Lys Ala Asp Lys Phe Leu
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Pro His Leu Arg Leu Arg Ala Pro Ile Val Pro Ala Lys Leu Arg Asn
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Thr Ala Gly Ile Val Gly Ala Ala Val Leu Ala Ala Glu Arg Leu Gly
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Gly Asp Arg Val Ser Ala
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Gly Ile Lys Gly Ala Pro Val Asp Leu Asp Arg Gly Thr Leu Val Val
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Asp Pro Val Lys Ile Ala Thr Pro Gln Pro Ala Thr Pro Glu Ala Val
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Ala Ala Val Val Ala Glu Ile Val Thr Ala Phe Ala Asp Asp Val Pro
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Gln Asp Ala Pro Leu Gly Val Ala Phe Pro Ala Val Ile Gln His Gly
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Val Ala Arg Ser Ala Ala Asn Met Asp Arg Ser Trp Ile Gly Thr Asn
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Val Glu Glu Leu Leu Ser Ala Val Thr Gly Arg Arg Val Leu Val Val
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Asn Asp Ala Asp Ala Ala Ala Met Ala Glu His Arg Tyr Gly Ala Ala
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Ser Gly Val Asp Gly Val Val Leu Leu Thr Thr Leu Gly Thr Gly Ile
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Gly Thr Ala Val Leu Val Asp Gly Val Leu Leu Pro Asn Thr Glu Phe
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Gly His Leu Glu Ile Asp Gly Tyr Asp Ala Glu Thr Arg Ala Ser Ala
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Ser Ala Lys Glu Arg Glu Asn Leu Ser Tyr Lys Glu Trp Ala Glu Glu
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Arg Leu Gln Arg Tyr Tyr Ser Val Ile Glu Asp Leu Leu Trp Pro Asp
195 200 205
Leu Ile Val Val Gly Gly Gly Val Ser Arg Lys Ala Asp Lys Phe Leu
210 215 220
Pro His Leu Arg Leu Arg Thr Pro Ile Val Pro Ala Lys Leu Arg Asn
225 230 235 240
Thr Ala Gly Ile Val Gly Ala Ala Val Leu Ala Ala Glu Arg Leu Gly
245 250 255
Gly Asp Arg Val Ser Ala
260
Claims (9)
1. A variant polyphosphate-dependent glucokinase, wherein said variant is a substitution of an amino acid residue in a parent polyphosphate-dependent glucokinase of the thermobifida fusca strain YX at a position as follows:
a) A231T; or
b) F30L + T72A + a 231T; or
c) F30L + T72A + V88M + a 231T; or
d） F30L+R34P+T72A+V88M+A231T；
The amino acid sequence of the parent polyphosphoric acid-dependent glucokinase is a sequence SEQ ID NO. 1.
2. The variant according to claim 1, wherein the parent polyphosphate-dependent glucokinase is derived from actinomycetes.
3. The variant according to claim 2, wherein the parent polyphosphate-dependent glucokinase is derived from thermobifida fusca.
4. A method for preparing a polyphosphate-dependent glucokinase variant, comprising:
a) selecting a parent polyphosphoric acid-dependent glucokinase, wherein the amino acid sequence of the parent polyphosphoric acid-dependent glucokinase is a sequence SEQ ID NO. 1;
b) modifying the coding sequence of the selected parent polyphosphate-dependent glucokinase in the step a), so that the coding sequence of the parent polyphosphate-dependent glucokinase has amino acid residue substitutions at the following positions of the polyphosphate-dependent glucokinase derived from the Thermobifida fusca strain YX:
i) A231T; or
Ii) F30L + T72A + a 231T; or
Iii) F30L + T72A + V88M + A231T; or
ⅳ） F30L+R34P+T72A+V88M+A231T；
c) The selective denaturation temperature is 10 to 20 times higher than that of parent polyphosphate dependent glucokinaseoA variant of C.
5. The method as claimed in claim 4, wherein the denaturation temperature of step c) is increased by 15-20% compared with that of parent polyphosphate-dependent glucokinaseoA variant of C.
6. The method according to claim 4, wherein the polyphosphate-dependent glucokinase is derived from actinomycetes.
7. The method according to claim 6, wherein the polyphosphate-dependent glucokinase is derived from Thermobifida fusca.
8. A gene encoding the variant according to any one of claims 1 to 3 or the variant produced by the method according to any one of claims 4 to 7, and an expression vector or host cell comprising the encoding gene.
9. Use of a variant according to any of claims 1 to 3 or a variant produced by a method according to any of claims 4 to 7 for the production of glucose 6-phosphate or a glucose 6-phosphate derivative.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102803482A (en) * | 2009-06-03 | 2012-11-28 | 丹尼斯科美国公司 | Cellulase variants with improved expression, activity and/or stability, and use thereof |
| TW201732039A (en) * | 2016-02-29 | 2017-09-16 | Ｃｊ第一製糖股份有限公司 | New polyphosphate-dependent glucokinase and method for preparing glucose 6-phosphate using the same |
-
2017
- 2017-12-08 CN CN201711290013.7A patent/CN109897840B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102803482A (en) * | 2009-06-03 | 2012-11-28 | 丹尼斯科美国公司 | Cellulase variants with improved expression, activity and/or stability, and use thereof |
| TW201732039A (en) * | 2016-02-29 | 2017-09-16 | Ｃｊ第一製糖股份有限公司 | New polyphosphate-dependent glucokinase and method for preparing glucose 6-phosphate using the same |
Non-Patent Citations (3)
| Title |
|---|
| One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP;Liao H等;《Appl Microbiol Biotechnol》;20110716;1109-1117 * |
| 多聚磷酸相关蛋白结构及生物学功能;黄金玲等;《中国生物化学与分子生物学报》;20140220(第02期);12-21 * |
| 联合葡萄糖激酶与乙酸激酶催化的ATP再生系统酶法合成葡萄糖-6-磷酸;颜炳坤;《中国优秀硕士学位论文全文数据库 基础科学辑》;20140915(第9期);A006-105 * |
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