WO2006024204A1 - Mutant de glucose isomerase et son utilisation - Google Patents

Mutant de glucose isomerase et son utilisation Download PDF

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Publication number
WO2006024204A1
WO2006024204A1 PCT/CN2004/001357 CN2004001357W WO2006024204A1 WO 2006024204 A1 WO2006024204 A1 WO 2006024204A1 CN 2004001357 W CN2004001357 W CN 2004001357W WO 2006024204 A1 WO2006024204 A1 WO 2006024204A1
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glucose isomerase
mgi
fructose
semen
minutes
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PCT/CN2004/001357
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English (en)
Chinese (zh)
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Jun Wang
Rongzhao Fu
Caike Jin
Dong Shen
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Bioright Worldwide Company Limited
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Publication of WO2006024204A1 publication Critical patent/WO2006024204A1/fr

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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
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)

Definitions

  • the present invention relates to the field of molecular biology and biotechnology, and in particular to a glucose-isomerase mutant having high activity, or high activity and heat resistance, in the determination of fructose Application and kit used. Background technique:
  • the fructose in semen is secreted by the seminal vesicles and is an important nutrient for sperm metabolism. Therefore, the amount of fructose directly affects the vitality of sperm.
  • semen fructose is mainly used for the diagnosis and differential diagnosis of seminal vesicles, vas deferens, prostate diseases and the diagnosis of infertility.
  • the function of fructose secretion from seminal vesicles is regulated by androgen. Therefore, the fructose content of semen can reflect the changes of androgen levels in the body.
  • the determination of semen fructose can be used for the evaluation of male gonad secretion function and sexual function and the diagnosis of related diseases.
  • semen fructose analysis has many important clinical values, due to the limitations of detection methods, semen fructose analysis has not been widely carried out in clinical laboratories.
  • the methods for determining semen fructose are mainly chromatography, chemical method and fructose dehydrogenase method. Chromatography is limited by the instrument, because clinical laboratories rarely have high-performance liquid chromatography and lack of relevant technology.
  • the chemical method uses a compound such as fructose and resorcinol, hydrazine or carbazole to form a color change, and the content is determined by colorimetry, but these chemical color reaction reactions must be in a strongly acidic medium and a high temperature.
  • Glucose isomerase (E.C. 5.3 ⁇ 5, GI for short) catalyzes the production of fructose by glucose, and it can also produce glucose by fructose.
  • the existing glucose isomerase catalyzes the low activity of fructose to produce glucose, and the sensitivity of measuring semen fructose is correspondingly low, which is not sufficient for clinical examination of semen fructose. Therefore, it is currently necessary to first provide a series of glucose isomerases which are resistant to high temperatures and can efficiently convert fructose into glucose, and then use glucose isomerase to efficiently and sensitively detect fructose content.
  • the invention utilizes genetic engineering and protein engineering technology to improve the glucose isomerase derived from Thermoanaerobacterium saccharolyticum, and cultivates a series of high temperature resistant and high efficiency fructose A glucose isomerase that is converted to glucose. These glucose isomerases efficiently convert fructose in semen into glucose, and then measure the amount of glucose produced by the glucose oxidase method, thereby determining the amount of fructose in the semen.
  • the method of the invention is the first new detection method in the world, which overcomes the drawbacks of the existing method for measuring semen fructose, and has the characteristics of simple operation, accurate result, mild reaction condition, low cost, stable reagent and convenient commercial operation.
  • the glucose isomerase mutant of the present invention can also be used for the determination of fructose content in a human body, or in an organ in a body, or other organism, or in a commercial product.
  • An object of the present invention is to provide a glucose isomerase mutant having high activity for catalyzing the activity of fructose to glucose, or having high activity for catalyzing the activity of fructose as glucose and heat.
  • the invention accordingly also provides DNA encoding the glucose isomerase mutants and the use of these mutants for determining fructose content.
  • the present invention includes the following aspects:
  • the present invention provides a glucose isomerase mutant characterized by the sequence 2 (SEQ ID NO.: 2) in the sequence listing as a reference sequence, having a mutation at position 186 and selected from the 139th At least three mutations at position 182, 187 and 299, the tryptophan mutation at position 139 is lysine (Lys;), or serine (Ser), or cysteine ( Cys), or isoleucine (Ile), or threonine (Thr), or asparagine (Asn), or phenylalanine (Phe), the arginine at position 182 is mutated to hydrazine Acid (Pro), or serine (Ser), or alanine (Ala), or isoleucine (Ile), or threonine (Thr), or valine (Val), the 186th position The proline is mutated to threonine (Thr), or alanine (Ala), or aspartic acid (Asp), and the phenyla
  • threonine at position 299 is mutated to isoleucine (Ile), or tyrosine (Tyr), or cysteine Acid (Cys), or methionine (Met), Glutamic acid (Glu), or glutamine (Gln).
  • the glucose isomerase mutant of the present invention has the amino acid sequence set forth in SEQ ID NO.: 4, wherein X at position 139 represents phenylalanine (Phe), X at position 182 Representing alanine (Ala), X at position 186 represents threonine (Thr), X at position 187 represents phenylalanine, and X at position 299 represents glutamine (Gln).
  • the invention provides a DNA comprising a nucleotide sequence encoding a glucose isomerase mutant of the invention.
  • the use of a glucose isomerase mutant of the invention is provided for determining fructose content.
  • the fructose content of a human body fluid or an organ in the body can be determined using the highly catalytically active glucose isomerase mutant of the present invention.
  • the glucose isomerase mutant of the present invention is particularly useful in determining the fructose content of human semen.
  • the present invention provides a method for determining fructose content in human semen, comprising the steps of:
  • the glucose isomerase mutant of the present invention is added to a semen sample to obtain a reaction mixture.
  • step 2 It is also preferred to include a step of removing the protein in the semen sample prior to performing step 2).
  • this step for example, by contacting the semen sample with an ion exchange resin, or by denaturation of the protein in the semen sample by adding a protein denaturant to the semen sample, or by separating the semen sample at 90- This is achieved by heating at 100 °C for 20-40 minutes and then centrifuging.
  • Many ion exchange resins are available in the prior art, for example, Chelex l00.
  • the protein denaturant it is preferred to use protein denaturant which is easily removed from the reaction system from the viewpoint of the degree of influence on the subsequent steps.
  • kits for determining fructose content in human semen, characterized by comprising a glucose isomerase mutant of the invention.
  • kits also include reagents and glucose oxidase for removing proteins from the semen sample to be tested.
  • the reagent for removing the protein in the sample to be tested for semen may be an ion exchange resin or a general protein denaturant. From the standpoint of ease of handling and degree of influence on subsequent steps, the use of an ion exchange resin is preferred.
  • the kit of the invention may further comprise a standard solution of fructose concentration.
  • the function of the standard solution can also be implemented by a computer.
  • FIG. 1 Glucose isomerase mutant MGI-4B polyacrylamide gel electrophoresis.
  • the four lanes from left to right are protein molecular weight standard, bovine serum albumin, crude protein, and partially purified glucose isomerase mutant MGI-4B. (Preparation of crude protein, partially purified glucose isomerase mutant MGI-4B is shown in Example 9)
  • FIG. 2 Parental glucose isomerase and glucose isomerase mutants are thermostable at 80 °C. Among them, MGI-4B represents a glucose isomerase mutant having four mutation sites, see Example 13 for details.
  • Figure 3 shows the standard curve for determination of fructose content.
  • the present invention performs site-directed mutagenesis of the Thermoanaerobacterium saccharolyticum glucose isomerase gene, and then screens for glucose isomerase cloning on MacConkey (DIFCO, USA) medium, thereby obtaining a series of highly efficient catalyzed fructose glucose activities, or highly efficient fructose A glucose isomerase mutant that is glucose active and thermotolerant.
  • one of the mutants, MGI-4B has a specific activity that is 755% higher than that of the parent.
  • the parental gene in the present invention refers to a glucose isomerase derived from Thermoanaerobacterium sachcharolyticum ATCC 49915, the nucleotide sequence of which is shown in SEQ ID NO.: 1 and the amino acid sequence is SEQ ID NO.: 2 Shown.
  • the nucleotide sequence of the parental gene of the present invention is compared with the published gene sequence of T. saccharolyticum glucose isomerase (Lee et al., Journal of General Microbiology, 139: 1227-1234, 1993; GenBank L09699). There are differences in positions 241-242. See the definition of "parent” and the description of example 1 below.
  • suitable vectors include, but are not limited to, prokaryotic expression vectors pGEMT-Easy, pRSET and pET21; including but not limited to eukaryotic expression vectors pYD l and pYES2/GS; including but not limited to cloning Vectors pUC 18/19 and pBluescript-SK.
  • a suitable glucose isomerase in the method of the present invention for preparing a glucose isomerase, can be expressed in a prokaryotic or eukaryotic cell, or can be achieved in a prokaryotic or eukaryotic cell by any other suitable method known in the art. Extracellular expression.
  • the microbial host cell of the vector is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic microorganism includes, but is not limited to, Escherichia coli, Bacillus coagulans, Bacillus subtilis, Bacillus megaterium (such as Bacillus megaterium ⁇ 93 1), ⁇ . ⁇ cc ⁇ rt ⁇ t, and Streptomyces (such as Strepto ⁇ diastaticus M 1033) ).
  • the eukaryotic microorganisms include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris (e.g., P. pastoris GS 1 15/9891).
  • the present invention obtains a glucose isomerase mutant characterized in that the amino acid sequence represented by the sequence 2 (SEQ ID NO.: 2) in the sequence listing is used as a reference sequence, at least one amino acid difference exists, and fructose is the bottom.
  • the glucose isomerase catalytic activity is at least 50-150% higher, preferably at least 150-250% higher, more preferably at least 250% higher than the parent.
  • the sequence 2 in the sequence listing is used as a reference sequence, having a mutation of the 186th proline to the other 19 natural amino acids and at least three of the 139th selected from the 139th to the other 19 natural amino acids , 182th arginine to 19 other natural amino acids, 187th phenylalanine to 19 other natural amino acids and 299th threonine to 19 other natural ammonia Mutation of the base acid, and with fructose as a substrate, it has at least 50% higher glucose isomerase catalytic activity than the parent.
  • the tryptophan at position 139 is mutated to lysine (Lys), or serine (Ser), or cysteine (Cys), or isoleucine (Ile), or threonine (Thr , or asparagine (Asn), or phenylalanine (Phe);
  • the arginine at position 182 is mutated to proline (Pro), or serine (Ser), or alanine (Ala) Or isoleucine (Ile), or threonine (Thr), or valine (Val);
  • the proline at position 1 86 is mutated to threonine (Thr), or alanine ( Ala), or aspartic acid (Asp);
  • the phenylalanine at position 187 is mutated to glycine (Gly), or serine (Ser), or alanine (Ala), or valine (Pro) And/or the threonine mutation at position 299 is isoleucine (I
  • mutants have high efficiency in catalyzing the activity of fructose as glucose and have heat resistance.
  • a mutant MGI-4B having four point mutations has a specific activity of 755% higher than that of the parent, and remains 50% or more after 16 hours of reaction at 80 ° C. vitality.
  • the high catalytic activity or high catalytic activity and heat-resistant glucose isomerase mutant obtained by the present invention can be used for determining the fructose content in an organ, or other organism, or commodity in a human body, or in a body.
  • the glucose isomerase mutant may be used in the form of a crude enzyme which has not been purified, or may be in a partially purified or completely purified form. If desired, the glucose isomerase mutant of the present invention can also be made into a solid phase enzyme or a solid phase cell solidified enzyme using a curing technique known in the art.
  • parent refers to a glucose isomerase from Thermoanaerobacterium saccharolyticum ATCC 49915 having a nucleotide sequence as shown in SEQ ID NO: 1, and an amino acid sequence as shown in SEQ ID NO: 2.
  • the nucleotide sequence of the parental gene of the present invention is compared with the published gene sequence of T. saccharolyticum glucose isomerase (Lee et al., Journal of General Microbiology, 139: 1227-1234, 1993; GenBank L09699).
  • the parental genes are located at positions 241-242 of GC, and the corresponding amino acid sequence of position 81 is alanine (Ala).
  • GenBank L09699 corresponds to the nucleotide sequence of 241-242, and the corresponding amino acid sequence of 81 is arginine (Arg).
  • reference sequence when it is a nucleotide sequence, refers to sequence 1 in the sequence listing, and when it is an amino acid sequence, it refers to sequence 2 in the sequence listing.
  • reference sequence and the mutated glucose isomerase sequence are sorted and compared, they can be performed manually or by computer (currently there are many computer software available, such as CLUSTALW, AMAS, DIALIGN program, etc.).
  • glucose isomerase mutant refers to a sequence in which the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing is used as a reference sequence, at least one amino acid difference is present, and the ratio of fructose is used as a substrate.
  • the glucose isomerase mutant comprises a conservative substitution form, addition or deletion of other positions other than positions 139, 182, 186, 187 and 299 in the amino acid sequence shown in SEQ ID NO: 2.
  • Example 1 Amplification of parental genes and construction of pGEMT-TS
  • Primers T1 and T2 were designed based on the gene bank (GenBank L09699) gene sequence (see Table 1).
  • the glucose isozyme parent gene was amplified from T. saccharolyticum ATCC 49915 (purchased from ATCC, USA) using primer pairs T1 and ⁇ 2.
  • the amplification conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl, 400 nM primer T2, 1.5 U Pfu DNA polymerase (Promega, USA), pick a small amount of T. saccharolyticum cells with an inoculating loop, and adjust the reaction volume with sterile water. Up to 50 ⁇ 1.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 40 cycles of cycle: 95 ° C for 50 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute, and finally 72 ° C for 10 minutes.
  • the amplified product (about 1.5 kb long) was ligated into the vector pGEMT-Easy (Promega, USA) to obtain plasmid pGEMT-TS.
  • the plasmid pGEMT-TS was extracted using a rapid plasmid preparation kit (Marligen Bioscience, USA), and the nucleotide sequence of the parental glucose isomerase was determined by DNA sequencing as Sequence Listing 1 and the corresponding amino acid sequence was Sequence Listing 2.
  • the parental genes in the present invention are at positions 241 to 242 of GC, and the corresponding amino acid sequence at position 81 is alanine (Ala).
  • the nucleotide sequence of 241-242 corresponding to GenBank L09699 is CG, and the corresponding amino acid sequence of 81 is arginine (Arg).
  • primer pairs 139FF and 139FR were designed, and the Trp(W) at position 139 in the parent amino acid sequence was mutated to Phe(F) to obtain mutant MGI. -W139F.
  • Primer pairs Tl and T2 are shown in Example 1.
  • the T1FR fragment was amplified, the primer pair 139FF and T2, and the FFT2 fragment was amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM primer 139FR or 400 nM primer 139FF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjust the reaction volume with sterile water Up to 50 ⁇ 1.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit (QIAGEN, German) to obtain a T1FR fragment and an FFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM T2, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng TIFR fragment and 20 ng FFT2 fragment, adjusted to 50 ⁇ l with sterile water .
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes.
  • the mixture was separated by 1% agarose gel electrophoresis and recovered by QIAquick Extraction Gel Kit DNA recovery kit to obtain a full-length mutant gene MGI-W139F.
  • MGI-W139F was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-W139F.
  • the plasmid pGEMT-MGI-W139F was transferred to competent bacterial cell HB101, and a clone having glucose isomerase activity was selected on a 1% MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin).
  • the plasmid pGEMT-MGI-W139F DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • mutants MGI-W139K, MGI-W139S, MGI-W139C, MGI-W139L MGI-W139T and MGI-W139N were constructed in a similar manner, and the primers used are shown in Table 1.
  • the site-directed mutagenesis technique is mainly described by Ho et al. (Gene 77: 51-59, 1989) and White et al. (PCR Protocol: current methods and applications. Totowa, N.J.: Humana Press, 1993).
  • primer pairs 182AF and 182AR were designed, and Arg(R) at position 182 of the parent amino acid sequence was mutated to Ala(A) to obtain mutant MGI. -R182A. See Fig. 1 for the T1 and T2.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer T l and 400 nM primer 182AR or 400 nM primer 182AF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then sterilized with sterile water Volume to 50 ⁇ l.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a T1AR fragment and an AFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM T2, 1.5 U Pfu DNA polymerase, 20 ng T1AR fragment and 20 ng AFT2 fragment, and the reaction volume was adjusted to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene MGI-R182A. MGI-R182A was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-R182A.
  • the plasmid pGEMT-MGI-R182A was transferred to competent bacterial cell HB101, and a clone having glucose isomerase activity was selected on a 1% MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin).
  • the plasmid pGEMT-MGI-R182A DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • mutants MGI-R182P, MGI-R182S, MGI-R182K MGI-R182T and MGI-R182V were constructed in a similar manner, and the primers used are shown in Table 1.
  • the T1TR fragment was amplified, the primer pair 186TF and T2, and the TFT2 fragment was amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM primer 186TR or 400 nM primer T2 and 400 nM primer 186TF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjust the reaction volume to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a T1TR fragment and a TFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4) 2 mM MgSO 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM T2, 1.5 U Pfu DNA polymerase, 20 ng T1TR fragment and 20 ng TFT2 fragment, and the reaction volume was adjusted to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene MGI-V186T. MGI-V 186T was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-V186T.
  • the plasmid pGEMT-MGI-V186T was transferred to competent bacterial cell HB101, and a clone having glucose isomerase activity was selected on a 1% MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin).
  • the plasmid pGEMT-MGI-V186T DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the mutants MGI-V186A and MGI-V186D were constructed in a similar manner, and the primers used are shown in Table 1.
  • the T1 SR fragment was amplified, the primer pair 187SF and T2, and the SFT2 fragment was amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH4) 2SO4, 2 mM MgSO 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM primer 187SR or 400 nM primer T2 and 400 nM primer 187SF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjust the reaction volume to 50 ⁇ l with sterile water .
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a T1 SR fragment and a SFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions are: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO4, 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM bow
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene MGI-F 187S. MGI-F 187S was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-F 187S.
  • the plasmid pGEMT-MGI-F 187S was transferred to competent bacterial cell HB 101, and a clone having glucose isomerase activity was selected on a 1% MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin).
  • the plasmid pGEMT-MGI-F 187S DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the mutants MGI-F 187G, MGI-F 187P and MGI-F 187A were constructed in a similar manner, and the primers used are shown in Table 1.
  • the T1 QR fragment was amplified, the primer pair 299QF and T2, and the QFT2 fragment was amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM primer Tl and 400 nM primer 299QR or 400 nM primer 299QF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjusted the reaction volume with sterile water Up to 50 ⁇ 1.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a T1QR fragment and a QFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM T1 and 400 nM ⁇ 2, 1.5 U Pfu DNA polymerase, 20 ng T1QR fragment and 20 ng QFT2 fragment, and the reaction volume was adjusted to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C 30 Seconds and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene MGI-T299Q. MGI-T299Q was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-T299Q.
  • the plasmid pGEMT-MGI-T299Q was transferred to competent bacterial cell HB101, and a clone having glucose isomerase activity was selected on a 1% MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin).
  • the plasmid pGEMT-MGI-T299Q DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the mutants MGI-T299I, MGI-T299Y, MGI-T299C, MGI-T299M and MGI-T299E were constructed in a similar manner, and the primers used are shown in Table 1.
  • Example 7 Construction of a Glucose Isomerase Four Mutant Combination MGI-4B
  • the T1FR fragment was amplified and recovered as in Example 2, and the QFT2 fragment was amplified and recovered as in Example 6.
  • the FFAR fragment was amplified and recovered using primer pairs 139FF (as in Example 2) and 182AR (as in Example 3).
  • the FFAR fragment amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM C1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM 139FF and 400 nM 182AR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjust the reaction volume to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain an FFAR fragment.
  • the AFTR fragment was amplified with primer pairs 182AF (as in Example 3) and 186TR (as in Example 4).
  • the AFTR fragment amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM 182AF and 400 nM 186TR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjust the reaction volume to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain an AFTR fragment.
  • the TFQR fragment was amplified with primer pairs 186TF (as in Example 4) and 299QR (as in Example 6).
  • the TFQR fragment amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), l O mM KCl, 10 mM, 2 mM MgS0 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP , 50 ⁇ dGTP, 400 nM 186TF and 400 nM 299QR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and then adjust the reaction volume to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a TFQR fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1 % Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP , 50 ⁇ dCTP, 50 ⁇ dGTP, u 400 nM primer Tl and 400 nM T2, 1.5 U Pfu DNA polymerase, 20 ng TIFR fragment, 20 ng FFAR fragment, 20 ng AFTR fragment, 20 ng TFQR fragment and 20 ng QFT2 fragment, and then adjust the reaction volume with sterile water to 50 ⁇ 1.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by electrophoresis on a 1% agarose gel and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene MGI-4B. MGI-4B was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-4B.
  • the plasmid pGEMT-MGI-4B was transferred to competent bacterial cell HB101, and a clone having glucose isomerase activity was selected on a 1% MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin).
  • the plasmid pGEMT-MGI-4B DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the MGI-4 sequence contains four mutations of W139F, R182A, V186T and T299Q.
  • the MGI-4B sequence is shown in Sequence Listing 5 (SEQ ID NO.: 5). Primers used to amplify parental glucose isomerase (parent) and Examples 1-7 glucose isomerase mutants are listed in Table 1 below:
  • 139CF 5, AAGTTTTGTGCGGTACCGCAAATCTTTTCT 3
  • 139CR 5, TGCGGTACCGCACAAAACTTTTGTCTTGCT 3
  • MGI-W139I 139IF 5' AAGTTTTGATTGGTACCGCAAATCTTTTCT 3'
  • MGI-W139T 139TF 5' AAGTTTTGACAGGTACCGCAAATCTTTTCT 3,
  • MGI-W139N 139NF 5' AAGTTTTGAACGGTACCGCAAATCTTTTCT 3'
  • MGI-W139F 139FF 5 AAAAGTTTTGTTTGGTACCGCAAATCTTTTCTC 3,
  • MGI-R182R 182RF 5 AGCTTGGCGGCGAAAACTACGTATTTTGGG 3'
  • 182RR 5, GTAGTTTTCGCCGCCAAGCTCCTTAGTAAT 3
  • MGI-R182S 182SF 5' AGCTTGGCTCAGAAAACTACGTATTTTGGG 3' 182SR: 5' GTAGTTTTCTGAGCCAAGCTCCTTAGTAAT 3'
  • MGI-R182A 182AF 5' GGAGCTTGGCGCGGAAAACTACGTATTTTGGGG 3'
  • MGI-R182I 182IF 5' AGCTTGGCATTGAAAACTACGTATTTTGGG 3'
  • MGI-R182T 182TF 5' AGCTTGGCACAGAAAACTACGTATTTTGGG 3'
  • MGI-R182V 182VF 5' AGCTTGGCGTGGAAAACTACGTATTTTGGG 3'
  • MGI-V186T 186TF 5' AAAACTACACCTTTTGGGGTGGAAGAGAAGG 3,
  • MGI-V186A 186TF 5 AAAACTACGCATTTTGGGGTGGAAGAGAAGG 3,
  • MGI-V186D 186TF 5' AAAACTACGATTTTTGGGGTGGAAGAGAAGG 3'
  • MGI-V187G 187GF 5'
  • MGI-V187P 187PF 5 ACTACGTACCGTGGGGTGGAAGAGAAGGGT 3,
  • MGI-V187A 187AF 5 ACTACGTAGCATGGGGTGGAAGAGAAGGGT 3,
  • MGI-V187S 187SF 5' ACTACGTAAGCTGGGGTGGAAGAGAAGGGT 3,
  • MGI-T299I 299IF 5' GACGCAAATATTGGCGACATGCTTTTAGGAT 3'
  • MGI-T299C 299CF 5 GACGCAAATTGCGGCGACATGCTTTTAGGAT 3,
  • glucose isomerase The extraction and purification of glucose isomerase is mainly referred to Lee et al., Journal of General Microbiology, 139: 1227-1234 (1993).
  • Containing glucose isomerase gene the parent plasmid pGEMT- TS transformed HB101 competent bacterial cells, and purified on MacConkey plates (containing 1% D- xylose and 50 m g / L ampicillin) on 37 cultured for 36 hours. Individual clones were inoculated for 16 hours in 5 ml LB liquid medium (containing 50 mg/L ampicillin). The cells were collected by centrifugation and suspended in 1 ml of 20 mM sodium phosphate buffer ( ⁇ 6.5), and CoCl 2 and MgCl 2 were added to a final concentration of 250 ⁇ M and 5 mM, respectively. The bacterial cells are then lysed with ultrasound.
  • Centrifuge (1 (TC, 17, 800 g, 15 min) and collect the supernatant for crude protein extraction.
  • the crude protein was heat treated at 80 ° C for 10 minutes and centrifuged (10 ° C, 17, 800 g, 15 min).
  • the supernatant is a partially purified glucose isomerase that can be used to determine the activity of the enzyme and determine the fructose content of the semen.
  • the extraction and purification of the glucose isomerase mutant MGI-4B was identical to that of Example 8, except that the plasmid used was pGEMT-MGI-4B o.
  • the partially purified glucose isomerase is shown in Figure 1.
  • the pH of the substrate solution was adjusted to pH 6.5 with 1.0 M of fructose, 20 mM sodium phosphate buffer solution (pH 6.5), a final concentration of 250 ⁇ M of CoCl 2 and a final concentration of 5 mM MgCl 2 . .
  • the substrate solution SA 90 ⁇ l was taken, and then 10 ⁇ l of the glucose isomerase prepared according to Example 8 was added, and the reaction was carried out at 80 ° C for 10 minutes. The reaction was placed on ice to stop the reaction.
  • the reaction product D-glucose was determined by the D-glucose oxidase method (see Trinder, Ann. Clin.
  • Enzyme protein concentration was determined using a Coomassie 3 ⁇ 4 Plus Protein Assay Reagent Kit (PIERCE, USA) in combination with SDS polyacrylamide gel electrophoresis.
  • One unit enzyme specific activity is defined as the amount of enzyme required to convert one micromole of fructose to glucose per minute under the above conditions.
  • Example 11 Glucose isomerase mutant MGI-4B assay for fructose activity
  • the glucose isomerase mutant MGI-4B activity was determined in the same manner as in Example 10. The results showed that the glucose isomerase mutant MGI-4B was 755% more active than the parental glucose isomerase to fructose.
  • Example 8 The partially purified parental glucose isomerase obtained in Example 8 was placed in 7 1.5 ml centrifuge tubes. Add 200 ⁇ l of enzyme solution to each tube and add 200 ⁇ M of mineral oil. The tube was placed in an 80 C water bath, and one tube of enzyme solution was taken out after 0 hours, 2 hours, 4 hours, 8 hours, 16 hours, 32 hours, and 72 hours, and centrifuged (1 (TC, 17, 800 20 minutes). After the supernatant was taken, the residual protein and residual specific activity of the glucose isomerase were determined as described in Example 10. Figure 2 shows the parental glucose isomerase at 80 ( 'C's heat stable.
  • Example 13 Determination of Thermal Stability of Glucose Isomerase Mutants
  • the thermal stability of the glucose isomerase mutant MGI-4B was determined in the same manner as in Example 12.
  • Figure 2 shows the thermostability of the glucose isomerase mutant MGI-4B at 80 °C.
  • the parental glucose isomerase has a half-life of 3. 5 hours at 80 ° C; MGI-4B is 16 hours.
  • SB prepared substrate solution containing sodium phosphate -MgCl 2 - CoCl 2 buffer solution [20 mM sodium phosphate (pH 6. 5), 250 ⁇ M CoCl 2, 5 mM MgCl 2] , and 2 M D- fructose to adjust pH to 6. 5.
  • the substrate solution SB was diluted with the same sodium phosphate-MgCl 2 -CoCl 2 buffer solution to contain D-fructose 1. 8 M, 1. 6 M, 1. 4 M, 1. 2 M, 1. 0 M, 0 8 M, 0. 4 M, 0. 2 M, 0. 1 M, 0.05 M and 0.025 M.
  • the glucose isomerase mutant MGI-4B biochemical constant was determined in the same manner as in Example 14.
  • Table 2 shows the comparison of the biochemical constants of the glucose isoformase mutant MGI-4B with the parental glucose isomerase.
  • the reaction product D-glucose was determined by the D-glucose oxidase method as described in Example 10. Take a solution of 20 ⁇ , and add 600 ⁇ of glucose oxidase reagent sputum (provided by the glucose kit of Shanghai Kehua-Dongling Diagnostics Co., Ltd., containing ⁇ 1.0 U/ml peroxidase and 0.1 M Phosphate buffer, pH 6.
  • glucose oxidase reagent sputum (provided by the glucose kit of Shanghai Kehua-Dongling Diagnostics Co., Ltd., containing ⁇ 15 U/ml glucose oxidase, 0.5 mM 4-aminoantipyrine and 0.1 mM phosphate buffer, pH 6. 8), mixed and placed in a 37 ° C water bath for 7 minutes. After the reaction was completed, the 0D S6 of the reactant was measured. Light absorption value. Use fructose concentration for 0D Sfi . The value is used as a standard curve. The results are listed below in Figure 3.
  • the Y axis represents the standard fructose concentration and the X axis represents the 0D 56 value of a standard fructose concentration.
  • the 0D is 0. 24; when the standard fructose concentration is 8 mM, its 0D 56fl is 0. 49.
  • Example 17 MGI-4B is used to determine the amount of fructose in semen (1)
  • the standard fructose curve is as described in Example 16.
  • the control tube was replaced with water using MGI-4B, and the standard tube was replaced with a 10 mM fructose standard solution.
  • the semen sample is heated at 95 ° C for 30 minutes, centrifuged (15000 ⁇ , 15 minutes), the supernatant is taken, and the following table is used.
  • the tubes were then reacted at 75 ° C for 30 minutes and rapidly cooled to room temperature.
  • the reaction product D-glucose was measured as in Example 10, and each tube 0D 560 was obtained.
  • the fructose concentration of semen is calculated according to the following formula:
  • Semen concentration of fructose (the measured sample tube 0D 56 .- control tube measured 0D 56.) ⁇ standard tube 0D 56Q x concentration measured fructose standard tube.
  • the semen sample used in this example is a semen of a 30-year-old male, and the measured 0D 56Q of the sample tube, the control tube, and the standard tube are 0. 854, 0. 012, and 0. 654, respectively, and the fructose content of the semen is:
  • the standard fructose curve is as described in Example 16.
  • the control tube was replaced with water using MGI-4B, and the standard tube was replaced with a 10 mM fructose standard solution.
  • For the determination take about 0.7 ml of the semen sample to be tested and place it in a small test tube containing about 0.5 g of ion exchange resin Chelex 100 (BioRad), mix, centrifuge for 10 minutes (15000 ⁇ , 15 minutes), and take the supernatant for determination. , operate according to the table method listed in Example 17.
  • the tubes were then reacted at 75 ° C for 30 minutes and rapidly cooled to room temperature.
  • the reaction product D-glucose was measured as in Example 10, and each tube 0D 56U was obtained .
  • the fructose concentration of semen was calculated in the same manner as in Example 17.
  • the semen specimen used in this example was a semen of a 28-year-old male, and the measured sample tube, control tube, and standard tube were 0D 56 .
  • Example 19 MGI-4B is used to determine the content of fructose in semen (3)
  • the semen specimen used in this example was a semen of a 38-year-old male, and the measured sample tube, control tube, and standard tube were 0D 56 .
  • the semen fructose content is 1.02, 0.015 and 0.658, respectively, and the semen fructose content is: (1.02-0,015) ⁇ 0.658x102.15.27mMo
  • the present invention is not limited by the above specific description, and the present invention can be made within the scope of the claims. Various changes. These changes are all within the scope of the invention.

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Abstract

L'invention concerne une série de glucose isomérases de Thermoanaerobacterium saccharolyticum, produites par génie génétique, à résistance élevée, à activité catalytique marquée, qui s'utilisent pour déterminer la teneur en lévulose. Lesdites glucose isomérases présente une mutation en position 186 et au moins trois mutations en position 139, 182, 187 et 229 de la séquence parent 2, qui convertissent le substrat lévulose en glucose. Lesdites glucose isomérases peuvent par conséquent s'utiliser pour déterminer la teneur en lévulose du corps humain ou d'un organe précis du corps ou de tout autre organisme ou de produits.
PCT/CN2004/001357 2004-09-02 2004-11-26 Mutant de glucose isomerase et son utilisation WO2006024204A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1956083A1 (fr) * 2005-11-18 2008-08-13 Bioright Worldwide Company Limited Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants
CN114703170A (zh) * 2022-04-26 2022-07-05 浙江大学 一种d-阿洛酮糖3-差向异构酶的热稳定性突变体及其高通量筛选方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966702B (zh) * 2005-11-18 2011-12-14 百瑞全球有限公司 葡萄糖异构酶突变体的应用
CN102443578B (zh) * 2011-12-08 2013-10-16 江南大学 一种葡萄糖异构酶突变体及其应用
CN104745563A (zh) * 2015-03-05 2015-07-01 浙江工业大学 葡萄糖异构酶、基因、突变体、工程菌及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1156182A (zh) * 1995-11-27 1997-08-06 中国科学技术大学 Sm33gi的突变酶gig138p及一种提高gi热稳定性的突变方案
CN1213003A (zh) * 1997-09-30 1999-04-07 中国科学技术大学 一种葡萄糖异构酶的247位单突变体酶和138位,247位双突变体酶及其构建方法
CN1418949A (zh) * 2001-11-14 2003-05-21 中国科学技术大学 表达m1033gi的突变酶gig138p的基因工程菌株m1033wz及其构建方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1156182A (zh) * 1995-11-27 1997-08-06 中国科学技术大学 Sm33gi的突变酶gig138p及一种提高gi热稳定性的突变方案
CN1213003A (zh) * 1997-09-30 1999-04-07 中国科学技术大学 一种葡萄糖异构酶的247位单突变体酶和138位,247位双突变体酶及其构建方法
CN1418949A (zh) * 2001-11-14 2003-05-21 中国科学技术大学 表达m1033gi的突变酶gig138p的基因工程菌株m1033wz及其构建方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 28 October 1993 (1993-10-28), LEE Y.E., XP003002163, accession no. NCBI Database accession no. (L09699) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1956083A1 (fr) * 2005-11-18 2008-08-13 Bioright Worldwide Company Limited Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants
EP1956083A4 (fr) * 2005-11-18 2009-02-25 Geneharbor Hong Kong Technolog Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants
CN114703170A (zh) * 2022-04-26 2022-07-05 浙江大学 一种d-阿洛酮糖3-差向异构酶的热稳定性突变体及其高通量筛选方法
CN114703170B (zh) * 2022-04-26 2023-11-28 浙江大学 一种d-阿洛酮糖3-差向异构酶的热稳定性突变体及其高通量筛选方法

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