WO2007056930A1 - Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants - Google Patents

Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants Download PDF

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WO2007056930A1
WO2007056930A1 PCT/CN2006/002901 CN2006002901W WO2007056930A1 WO 2007056930 A1 WO2007056930 A1 WO 2007056930A1 CN 2006002901 W CN2006002901 W CN 2006002901W WO 2007056930 A1 WO2007056930 A1 WO 2007056930A1
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glucose isomerase
fragment
mgi4
minutes
mutant
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PCT/CN2006/002901
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English (en)
French (fr)
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Jun Wang
Caike Jin
Rongzhao Fu
Dong Shen
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Bioright Worldwide Company Limited
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Priority to EP06805105A priority Critical patent/EP1956083B1/en
Priority to US12/093,859 priority patent/US7704719B2/en
Priority to JP2008540429A priority patent/JP4955692B2/ja
Priority to DE602006019135T priority patent/DE602006019135D1/de
Priority to AT06805105T priority patent/ATE492634T1/de
Publication of WO2007056930A1 publication Critical patent/WO2007056930A1/zh
Priority to US12/723,833 priority patent/US7923222B2/en
Priority to US12/723,918 priority patent/US8067561B2/en

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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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose

Definitions

  • Glucose isomerase mutant its use, and DNA encoding the mutant
  • the present invention relates to the field of molecular biology and biotechnology, and in particular to a method for producing a highly active, or highly viable, and thermotolerant glucose isomerase mutant using a gene mutation technique, a mutant obtained, and use thereof. Background technique
  • Glucose isomerase (E.G. 5.3.1.5, GI for short) or Xylose isomerase is a key enzyme in the pentose fermentation pathway. This enzyme is one of the most important industrial enzyme preparations (Kaneko, et al., Biosci Biotechnol Biochem, 64: 940-947, 2000). The food industry uses this enzyme to prepare fructose syrup.
  • the fructose syrup is produced enzymatically, and the amount of fructose in the product depends on the temperature of the reaction. The higher the temperature, the more the heterogeneous reaction tends to produce fructose.
  • commercial glucose isomerase is mainly from Actinobacillus migralis 04ct «o ?/a «es missouriensis), Bacillus coagulans
  • fructose syrup is carried out at about 60 ⁇ in the industry, and the content of fructose in the product is also low, generally not more than 44% by weight.
  • Fructose syrups containing higher concentrations of fructose are usually produced by chromatographic separation methods, thereby increasing production costs.
  • the present invention improves the glucose isomerase derived from T. saccharolyticum, and obtains a series of highly catalytically active glucose isomerase mutants, which are suitable for producing fructose syrup containing high fructose.
  • the present inventors conducted a large number of in-depth experiments by site-directed mutagenesis of the cc. ⁇ cc/ro/j ⁇ cz ⁇ glucose isomerase gene, followed by screening on MacConkey medium, thereby obtaining A series of glucose isomerase mutants with high catalytic activity or high catalytic activity and heat resistance.
  • a vector plasmid containing a parental glucose isomerase gene is first constructed by molecular biology techniques, and then a site of site-directed mutagenesis and a mutant amino acid are set, and an appropriate primer is synthesized to prepare the parental glucose.
  • the vector plasmid of the isomerase gene is used as a template, and the DNA fragment is PCR-amplified, the amplified DNA fragment is assembled, and the full-length mutant gene is PCR-amplified.
  • a positive clone having glucose isomerase activity was selected by culture by cloning the full-length mutant gene into an appropriate vector and transforming the appropriate host cell. Plasmid DNA was extracted from positive clones and subjected to DNA sequence analysis to determine the introduced mutation. Finally, the enzyme activity of the glucose isomerase with the set site mutation was determined by using D-glucose as a substrate, and the enzyme activity was compared with the enzyme activity of the parent to select a glucose isomerase having a higher affinity than the parent. Catalytically active mutants of the glucose isomerase of the invention.
  • any suitable carrier can be employed.
  • suitable vectors include, but are not limited to, prokaryotic expression vectors pGEMT-Easy, pRSET and pET21; including but not limited to eukaryotic expression vectors pYD1 and pYES2/GS; These include, but are not limited to, the cloning vectors pUC 18/19 and pBluscript-SK.
  • the obtained glucose isomerase mutant gene can be expressed in a prokaryotic or eukaryotic cell, or can be achieved by any other suitable method known in the art. Extracellular expression of prokaryotic or eukaryotic cells.
  • 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 931), saccharolyticum and Streptomyces diastaticus M1033 o the eukaryotic microorganism These include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris (eg, P. pastoris GS 1 15/9891).
  • the invention obtains a glucose isomerase mutant, which adopts the sequence 2 attached in the specification as a reference sequence, and the 139th mutation is phenylalanine (Phe), and the 182th mutation is alanine (Ala).
  • the 187th mutation is a serine (Ser) mutation and the 299th mutation is glutamine (Gln), which also has at least one mutation at the 87th, 217th, 260th, and 276th positions, and D-glucose is a substrate which has a higher glucose isomerase catalytic activity than the parent.
  • the 87th position is methionine (Met) or leucine (Leu); the 217th position is arginine (Arg), or tryptophan (Trp:), or glycine ( Gly); the 260th position is glutamic acid (Glu) or alanine (Ala); and/or the 276th position is glycine (Gly) or threonine (Thr).
  • SEQ ID NO.: 4 in the Sequence Listing shows the amino acid sequence of a glucose isomerase mutant of the present invention, wherein Xaa represents an amino acid at a mutation site.
  • the glucose isomerase mutant of the present invention comprises MGI4-F87L, MGI4-F87M, MGI4-V217R, MGI4-V217W, MGI4-D260E, MGI4-F276G, MGI4-24, listed in Table 2 below. Amino acid sequence of MGI4-25, MGI4-34, MGI4-35.
  • mutants have high catalytic activity.
  • a mutant MGI4-34 having seven point mutations has a specific activity of 769% higher than that of the parent, and remains 50% or more after 26 hours of reaction at 80 °C. vitality.
  • Another mutant with seven point mutations, MGI4-35 was 727% more active than the parent and remained viable at 50% or more after 27 hours of reaction at 80 °C.
  • Glucose isomerase with high catalytic activity or high catalytic activity and heat resistance obtained by the present invention can be used to directly produce fructose syrup having a fructose content of 55% by weight or more, or to produce a fructose pulp having a fructose content of less than 55% by weight.
  • the glucose isomerase mutant of the present invention can be used to produce crystalline fructose having a fructose content of more than 90% by weight.
  • 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 T. saccharolyticum ATCC 49915, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence is 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).
  • GenBank L09699 The difference between the two nucleotides, that is, compared with the nucleotide sequence of the gene bank (GenBank L09699), in the present invention, the parental gene is located at positions 241-242, and the corresponding amino acid sequence at 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 as used in the context of the present application, when it is a nucleotide sequence, refers to the sequence 1 attached to the specification, and when it is an amino acid sequence, it refers to the sequence 2 attached to the specification.
  • the reference sequence and other mutated glucose isomerase sequences are sorted. The comparison can be done manually or by computer (currently there are many computer software available, such as CLUSTALW, AMAS, DIALIGN programs, etc.).
  • site or "position X" as used in the context of the present application means that the glucose isomer is compared when the sequence of the glucose isomerase mutant of the present invention has the greatest homology to the sequence of the reference sequence.
  • the nucleotide or amino acid position in the reference sequence corresponding to each nucleotide or amino acid of the enzyme mutant.
  • glucose isomerase mutant refers to a reference sequence consisting of the amino acid sequence shown in SEQ ID NO: 2, and the mutation at position 139 is phenylalanine (Phe), position 182. Mutation to alanine (Ala), mutation at position 187 to serine (Ser) mutation, and mutation at position 299 to glutamine (Gln), at the 87th and 217th positions, The 260th and 276th positions have at least one mutation, and when the fructose is produced using D-glucose as a substrate, it has an enzyme having higher glucose isomerase catalytic activity than the parent.
  • the glucose isomerase mutant comprises a mutant having the amino acid sequence shown in SEQ ID NO: 4, a conservatively substituted form of SEQ ID NO: 4, a form in which one or several amino acids are added or deleted, and an amino terminal truncation. Form, carboxy-terminal truncated form, and part or all of the sequence repeat of Form 4.
  • FIG. 1 Thermal stability map of parental glucose isomerase and glucose isomerase mutants at 80 C. Among them, MGI4-34 and MGI4-35 represent a glucose isomerase mutant having seven mutation sites, as described in Example 12. detailed description
  • Example 1 Amplification of parental genes and construction of pGEMT-TS
  • T2 (see Table 1).
  • the glucose isomerase parent gene was amplified from T. saccharolyticum ATCC 49915 (available 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 Taq
  • DNA polymerase (Promega, USA), pick a small amount of ⁇ saccharolyticum cells using the inoculating loop, and adjust the reaction volume to 50 ⁇ l with sterile water.
  • 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 in length) was ligated into the vector pGEMT-Easy to obtain plasmid pGEMT-TS. Identification of the parent by DNA sequencing The nucleotide sequence of the glucose isomerase is sequence 1, and the corresponding amino acid sequence is sequence 2.
  • the parental genes in the present invention are located at positions 241-242 of GC, and the corresponding amino acid sequence at position 81 is alanine (Ala), GenBank L09699 corresponding, 241
  • the -242 nucleotide sequence is CG, and the corresponding amino acid sequence at position 81 is arginine (Arg).
  • Example 2 Site-directed mutagenesis of glucose isomerase site 139
  • the T1FR fragment was amplified, and the primer pair 139FF and T2 amplified the FFT2 fragment.
  • 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 139 FF and 400 nM primer T2, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pGEMT-TS, 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
  • 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 T2, 1.5 U Pfu DNA polymerase, 20 ng T1FR fragment and 20 ng FFT2 fragment, adjusted to a volume of 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-W139F. MGI-W139F and carrier pGEMT-Easy The plasmid pGEMT-MGI-W139F was obtained. Plasmid pGEMT-MGI-W139F was transferred to competent bacterial cell E.
  • the primer pair T1 and 182AR were used to amplify the T1AR fragment, the primer pair 182AF and T2, and the AFT2 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 182 AR or 400 nM primer 182AF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, 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.
  • 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 TlAR fragment and 20 n g 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 3 minutes, last 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.
  • the MGI-R182A was ligated to the vector pGEMT-Easy to obtain the plasmid. pGEMT-MGI-R182A.
  • Plasmid pGEMT-MGI-R182A was transferred to competent bacterial cell E. coli HB 101, and clones with glucose isomerase activity were screened on 1% MacConkey plates (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.
  • Example 4 Site-directed mutagenesis of glucose isomerase site 187
  • 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 (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1 % Triton X-100 , 50 ⁇ dATP,
  • the Extraction Gel Kit was recovered to obtain a T1 SR fragment and a SFT2 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 Tl SR fragment and 20 ng SFT2 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-F 187S.
  • the MGI-F187S was ligated to the vector pGEMT-Easy to obtain the plasmid pGEMT-MGI-F187S o. Transfer the plasmid pGEMT-MGI-F187S to competent bacterial cells. E.
  • primer pairs 299QF and 299QR were designed to mutate the Thr(T) at position 299 of the parent amino acid sequence to
  • the primer pair T1 and 299QR were used to amplify the T1 QR fragment, 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 adjust the reaction volume with sterile water to 50 ⁇ l.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95. C 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 KCl, 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 Tl QR 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 for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by 1% agarose gel electrophoresis and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene MGI-T299Q.
  • the MGI-T299Q was ligated to the vector pGEMT-Easy to obtain the plasmid pGEMT-MGI-T299Q. Transfer plasmid pGEMT-MGI-T299Q to competent bacterial cell E.
  • the T1FR fragment was amplified and recovered as in Example 2, and the QFT2 fragment was amplified and recovered as in Example 5.
  • the FFAR fragment was amplified using primer pairs 139FF (see Table 1) and 182AR (see Table 1).
  • the FFAR fragment amplification reaction conditions are: 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM
  • the Extraction Gel Kit was recovered and the FFAR fragment was obtained.
  • the AFSR fragment was amplified using primer pair 182AF (see Table 1) and 187SR (see Table 1).
  • the AFSR fragment 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 182AF and 400 nM 187SR, 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 AFSR fragment.
  • the SFQR fragment was amplified using primer pairs 187SF (see Table 1) and 299QR (see Table 1).
  • the SFQR fragment amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 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 187SF 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 5 minutes. Separated by 1%-agarose gel electrophoresis The QIAquick Extraction Gel Kit was recovered and the SFQR fragment was obtained. 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 TlFR fragment, 20 ng FFAR fragment, 20 ng AFSR fragment, 20 ng SFQR fragment and 20 ng QFT2 fragment, 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 full-length mutant gene MGI-4. MGI-4 was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI-4. Transfer plasmid pGEMT-MGI-3 to competent bacterial cell E.
  • primer pairs 87LF and 87L were designed (see Table 1), and Phe(F) at position 87 of the MGI-4 amino acid sequence was mutated to Leu(L). , obtained mutant MGI4-F87L.
  • Primer pairs T1 and T2 are shown in Table 1.
  • the T1LR fragment was amplified with primer pairs T1 and 87LR, and the LFT2 fragment was amplified with primer pairs 87LF 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 Tl and 400 nM primer 87LR or 400 nM primer 87LF and 400 nM primer T2, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pGEMT-MGI-4, reuse Sterile water to adjust the reaction 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 (QIAGEN, German) to obtain a T1LR fragment and an LFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (H 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton
  • PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles of 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes.
  • MGI4-F87L was ligated to the vector pGEMT-Easy to obtain the plasmid pGEMT-MGI4-F87L.
  • the plasmid pGEMT-MGI4-F87L was transferred to competent bacterial cell E. coli HB 101, and a clone with glucose isomerase activity was screened on a 1% MacConkey plate containing 1% D-xylose and 50 mg/L ampicillin. .
  • the plasmid pGEMT-MGI4-F87L DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the resulting mutant sequence contained five mutations of F87L, W139F R182A, F187S and T299Q.
  • the amino acid sequence of MGI4-F87L is shown in Sequence Listing 5.
  • the mutant MGI4-F87M was constructed in a similar manner, and the primers used are shown in Table 1.
  • the MGI4-F87M mutant sequence contains five mutations of F87M, W139F, R182A, F187S and T299Q.
  • the amino acid sequence of the obtained mutant is shown in Sequence Listing 6.
  • Example 8 Construction of a glucose isomerase five mutation combination MGI4-V217R and MGI4-V217W
  • 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 217RR or 400 nM primer 217RF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, 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 T1RR fragment and an RFT2 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,
  • the reaction volume was adjusted to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles of 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 1% agarose gel electrophoresis and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene.
  • MGI4-V217R The MGI4-V217R was ligated to the vector pGEMT-Easy to obtain the plasmid pGEMT-MGI4-V217R.
  • the plasmid pGEMT-MGI4-V217R was transferred to competent bacterial cell E. coli HB 101, and a clone with glucose isomerase activity was screened on a 1% MacConkey plate containing 1% D-xylose and 50 mg/L ampicillin. .
  • the plasmid pGEMT-MGI4-V217R DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the resulting mutant sequence contained five mutations of W139F, R182A, F187S, V217R and T299Q.
  • the amino acid sequence of MGI4-V217R is shown in Sequence Listing 7.
  • the mutant MGI4-V217W was constructed in a similar manner, and the primers used are shown in Table 1.
  • the MGI4-V217W mutant sequence contains five mutations, W139F, R182A, F187S, V217W and T299Q.
  • the amino acid sequence of the obtained mutant is shown in Sequence Listing 8 of the Sequence Listing.
  • Example 9 Construction of a glucose isomerase five mutation combination MGI4-D260E
  • the primer pair T1 and 260ER were used to amplify the TIER fragment, and the primer pair 260EF and T2 were used to amplify the EFT2 fragment.
  • the amplification reaction conditions were: 20 mM Tris-HCl ( ⁇ 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 260ER or 400 nM primer T2 and 400 nM primer 260EF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, 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.
  • the full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 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 1 ⁇ 11 £ fragment and 20 1 ⁇ £? 2 fragments, 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 full-length mutant gene MGI4-D260E.
  • the MGI4-D260E was ligated to the vector pGEMT-Easy to obtain the plasmid pGEMT-MGI4-D260Eo.
  • the plasmid pGEMT-MGI4-D260E was transferred into the competent bacterial cell E. coli HB 101 in 1% MacConkey plate (containing 1% D-xylose). Clones with glucose isomerase activity were screened on and 50 mg/L ampicillin. The plasmid pGEMT-MGI4-D260E DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing. The resulting mutant sequence contained five mutations, W139F, R182A, F187S, D260E and T299Q. The amino acid sequence of MGI4-D260E is shown in Sequence Listing 9.
  • Example 10 Construction of a glucose isomerase five-mutation combination MGI4-F276G The site-directed mutagenesis technique is described in Ho et al. (Gene 77: 51-59, 1989) and White et al. (PCR Protocol: current methods and applications. Totowa, NJ '.Humana Description of Press, 1993).
  • Primer pair 276GF and 276GR were designed with plasmid pGEMT-MGI-4 (see Example 6) as template, and Phe(F) at position 276 of MGI-4 amino acid sequence was mutated to Gly(G). , obtained mutant MGI4-F276G.
  • Primers T1 and T2 are shown in Table 1.
  • the T1GR fragment was amplified with primer pairs T1 and 276GR, and the GFT2 fragment was amplified with primer pairs 276GF and T2.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 S0 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 276GR or 400 nM primer 276GF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, 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 T1GR fragment and a GFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 50 ⁇ dATP, 50 ⁇ dTTP,
  • 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 isolated by 1% agarose gel electrophoresis and recovered by QIAquick Extraction Gel Kit to obtain a full-length mutant gene.
  • MGI4-F276G The amino acid sequence of MGI4-F276G is shown in Sequence Listing 10 of the Sequence Listing.
  • Example 1 1 Construction of a glucose isomerase six-mutation combination MGI4-24 and MGI4-25 The site-directed mutagenesis technique is described in Ho et al. (Gene 77: 51-59, 1989) and White et al. (PCR Protocol: current methods and applications. Totowa, N ⁇ : Humana Press, 1993).
  • the T1LR fragment was amplified and recovered as in Example 7.
  • the LFAR fragment was amplified and recovered using primer pairs 87LF and 260AR (see Table 1).
  • the LFAR fragment 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
  • PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles of 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 1% agarose gel electrophoresis and recovered by QIAquick Extraction Gel Kit.
  • the AFT2 fragment was amplified and recovered using primer pairs 260AF and T2 (see Table 1).
  • the AFT2 fragment 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 260AF and 400 nM T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, 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 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 TlLR fragment, 20 ng LFAR fragment and 20 ng AFT2 fragment, then adjust the reaction volume to 50 with sterile water 11.
  • 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 full-length mutant gene was obtained by electrophoresis on a 1% agarose gel and using the QIAquick DNA Recovery Kit.
  • MGI4-24 was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI4-24.
  • the plasmid pGEMT-MGI4-24 was transferred to competent bacterial cell E. coli 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-MGI4-24 DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • MGI4-24 The amino acid sequence is shown in Sequence Listing 1 1.
  • the resulting mutant sequence contained six mutations of F87L, W139F, R182A, F187S, D260A and T299Q.
  • the mutant MGI4-25 was constructed in a similar manner.
  • the primer pairs used for the mutant MGI4-25 were T1 and 87LR, 87LF and 276TR, 276TF and T2 (see Table 1), and the resulting mutant sequences contained six mutations of F87L, W139F, R182A, F 187S, F276T and T299Q.
  • the amino acid sequence of the obtained mutant is shown in Sequence Listing 12 of the Sequence Listing.
  • Example 12 Construction of a glucose isomerase seven mutation combination MGI4-34 and MGI4-35. Site-directed mutagenesis techniques are described in Ho et al, (Gene 77: 51-59, 1989) and White et al. (PGR Protocol: current methods and Applications. Totowa, NJ: Humana
  • the T1LR fragment was amplified and recovered as in the Examples.
  • the LFGR fragment was amplified and recovered using primer pairs 87LF and 217GR (see Table 1).
  • the LFGR fragment amplification reaction conditions were: 20 mM Tris-HCl (H 8.8), 10 mM KC1, 10 mM (NH 4 ) 2 S0 4 , 2 mM MgSO 4 , 0.1 % Triton X-100 , 50 ⁇ dATP, 50 ⁇ dTTP, 50 ⁇ dCTP, 50 ⁇ dGTP, 400 nM
  • the jPCR 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 LFGR fragment. The GFTR fragment was amplified and recovered using primer pairs 217GF and 276TR (see Table 1).
  • the GFTR 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 217GF and 400 nM 276TR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, 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
  • the TFT2 fragment amplification reaction conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 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 276TF and 400 nM T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, 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 with a QIAquick Extraction Gel Kit to obtain a TFT2 fragment. The full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH
  • MGI4-34 was ligated to the vector pGEMT-Easy to obtain plasmid pGEMT-MGI4-34. Plasmid pGEMT-MGI4-34 was transferred to competent bacterial cell E. coli HB 101, and clones with glucose isomerase activity were screened on 1% MacConkey plates (containing 1% D-xylose and 50 mg/L ampicillin). .
  • the plasmid pGEMT-MGI4-34 DNA was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing.
  • the MGI4-34 sequence contains seven mutations of F87L, W139F, R182A, F187S, V217G, F276T and T299Q.
  • the amino acid sequence of MGI4-34 is shown in Sequence Listing Sequence 13.
  • the seven mutant MGI4-35 was constructed in a similar manner.
  • the primer pairs used for the mutant MGI4-35 were T1 and 87LR, 87LF and 217GR, 217GF and 260AR, 260AF and T2 (see Table 1), and the resulting mutant sequence contained F87L, W139Fs R182A, F187S, V217G, D260A and T299Q seven mutations. .
  • the amino acid sequence of the obtained mutant is shown in Sequence Listing 14 of the Sequence Listing.
  • the primers used to amplify the parental glucose isomerase (parent) and the glucose isomerase mutants of Examples 1-12 are listed in Table 1 below:
  • MGI4-F276G 276GR 5 ' GA AGTCGTGGCCTGCCA ATGTCGCATGGTTT 3'
  • MGI4-F276T 276TR 5'GAAGTCGTGGGTTGCCAATGTCGCATGGTTT 3'
  • Example 13 Extraction and Purification of Parental Glucose Isomerase
  • glucose isomerase The extraction and purification of glucose isomerase is mainly referred to Lee et ah, Journal of General Microbiology, 87: 1227-1234 (1993).
  • the plasmid pGEMT-TS containing the parental glucose isomerase gene was transformed into competent bacterial cell E. coli HB 101 and cultured on MacConkey plate (containing 1% D-xylose and 50 mg/L ampicillin:) at 37 °C. 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 (pH 6.5), and CoCl fl M g Cl 2 was added to a final concentration of 250 ⁇ M and 5 mM, respectively. The bacterial cells are then lysed with ultrasound.
  • Example 15 Determination of parental glucose isomerase activity
  • Substrate solution A contained 1.0 M D-glucose, 20 mM sodium phosphate, 250 ⁇ CoCl 2 and 5 mM MgCl 2 , pH 6.5.
  • the substrate solution A was taken as 90 ⁇ , and then 10 ⁇ L of the glucose isomerase prepared in accordance with Examples 1 and 13 was added.
  • 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-fructose was determined by the cysteine-carbazole method. For the method of determination, see Dische et al., J. Biol. Chem, 192: 583-587, 1951 and
  • the glucose isomerase mutant activity was measured in the same manner as in Example 15.
  • Table 2 shows the difference in specific activity between each glucose isomerase mutant and the parental glucose isomerase (parent). The difference in specific activity between parental glucose isomerase and glucose isomerase mutant
  • Example 15 The partially purified parental glucose isomerase obtained as in Example 13 was placed in a 4-tube 1.5 ml centrifuge tube. Add 200 ⁇ M enzyme solution to each tube and add 200 ⁇ M mineral oil. Place the centrifuge tube in a water bath at 80 ° C, and take a tube of enzyme solution after 0 hours, 2 hours, 6 hours, and 27 hours, centrifuge (10 ° C, 17800 g, 20 minutes), then take the supernatant, press Example 15 was carried out to determine the specific activity of the glucose isomerase residual protein.
  • Figure 1 shows the thermal stability of the parental glucose isomerase at 80 Torr.
  • Example 18 Determination of the stability of glucose isomerase mutants
  • thermostability determination of the glucose isomerase mutant MGI4-34 (see Examples 12 and 14) or MGI4-35 (see Examples 12 and 14) was the same as in Example 17.
  • Figure 1 shows the thermostability of the glucose isomerase mutant MGI4-34 or MGI4-35 at 80 °C.
  • the parental glucose isomerase activity has a half-life of 4.1 hours at 80 ⁇ ; the MGI4-34 activity has a half-life of 26 hours at 80 ⁇ ; and the MGI4-35 activity has a half-life of greater than 27 hours at 80 °C.
  • Example 19 Solid phase glucose isomerase mutant MGI4-35
  • Example 20 Immobilized cells containing the glucose isomerase mutant MGI4-35 were grown overnight in LB medium containing 50 mg/L ampicillin. E. coli HB101 to OD 6G() carrying pGEMT-MGI4-35 was 7. The bacterial cells were collected by centrifugation. Take 10 g of bacterial cells, add 20 g of 3% sodium alginate solution, mix well, then add the cells from a needle with a diameter of about 0.5 mm into 500 ml of 2% calcium chloride solution, and react at room temperature for 1 hour.
  • the cells were washed with steamed water for 3 times for about half an hour to obtain about 30 g of immobilized cells.
  • the immobilized cell viability assay was as described in Example 16, wherein the enzyme used was 0.01 g of the immobilized cells prepared as above, and the activity of the immobilized cells was measured to be 370 units/g.
  • the invention is not limited by the specific details of the invention described above, and the invention may be varied within the scope of the appended claims. These changes are all within the scope of the invention.

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Description

葡萄糖异构酶突变体、 其应用以及编码该突变体的 DNA 技术领域
本发明涉及分子生物学与生物技术领域， 具体地说， 涉及利用 基因突变技术制备高活力、或高活力并耐热的葡萄糖异构酶突变体的 方法、 所获得的突变体及其应用。 背景技术
葡萄糖异构酶（Glucose isomerase , E.G.5.3.1.5 , 简称 GI)或称木 糖异构酶 (Xylose isomerase)是戊糖发酵途径的关键酶。该酶是最重要 的工业酶制剂之一 (Kaneko , et al. , Biosci Biotechnol Biochem , 64:940-947 , 2000)。 食品工业使用该酶制备果葡糖浆。
酶法生产果葡糖浆， 产品中果糖的含量取决于反应的温度。 温 度越高， 异构反应越趋向于果糖的生成。 目前商用葡萄糖异构酶主要 取自密苏里游动放线菌 04ct «o ?/a«es missouriensis)、 凝结芽抱杆菌
(Bacillus co gw/ara)或链霉菌 Streptomyces)。 这些葡萄糖异构酶在高 温 (如高于 65 °C )下稳定性差。 因此， 目前工业上在 60 Ό左右制备果 葡糖浆， 产物中果糖的含量也就较低， 一般不超过 44重量％。 含更 高浓度果糖的果葡糖浆通常依赖层析分离方法生产，由此增加了生产 成本。
世界各地的科学家对从耐热菌中筛选并通过蛋白质工程的方法 培育耐热、 高催化活性的葡萄糖异构酶进行了许多研究。 如 J.G. Zeikus 及实合 者 Thermoanaerobacterium saccharolyticum (下文 ¾s T. saccharolyticum ) 、 Thermotoga neapolitana 等数禾中耐高温菌中 分离并研究了耐高温葡萄糖异构酶 (见 Lee et al. , Journal of General
Microbiology， 87: 1227- 1234， 1993 ； Vieille et al.， Methods Enzymology , 330 :215-24, 2001； Lee et al. , Journal of General Microbiology , 87: 1241 - 1243 , 1993； Scriprapundh et al. , Protein Engineering， 13 :259-265， 2000； Scriprapundh et al.， Protein Engineering , 16:683-690 , 2003； Zeikus et al. , US Patent NO. 5,656,497)。 这些或其它来源的耐高温葡萄糖异构酶之耐热性能均不 如人意， 且活力较低， 尚未见用于工业化生产。 因此， 目前仍存在对 高活力、 或高活力并耐热的葡萄糖异构酶的需求。 发明内容
本发明对来源于 T. saccharolyticum的葡萄糖异构酶进行改良， 获得了一系列高催化活性的葡萄糖异构酶突变体，适宜生产含高果糖 的果葡糖浆。
本发明的目的在于提供高催化活性的葡萄糖异构酶突变体。 本 发明的目的还在于提供使用本发明所述的葡萄糖异构酶突变体直接 生产果糖含量等于或高于 55重量％的果葡糖浆。 本发明的目的还在 于提供使用本发明所述的葡萄糖异构酶突变体生产果糖含量低于 55 重量％的果葡糖浆。
为实现本发明的上述目的， 本发明人进行了大量深入的实验， 通过对 Γ. ^cc/ ro/j^cz^葡萄糖异构酶基因进行定点突变， 随后在 MacConkey 培养基上筛选， 从而取得一系列高催化活性、 或具高催 化活性并耐热的葡萄糖异构酶突变体。具体而言， 先利用分子生物学 技术， 构建含有亲本葡萄糖异构酶基因的载体质粒， 然后设定定点突 变的位点以及突变后的氨基酸， 再合成适当的引物， 以所述的含亲本 葡萄糖异构酶基因的载体质粒为模板， PCR扩增 DNA片段、 装配所 扩增的 DNA片段以及 PCR扩增全长突变基因。通过将该全长突变基 因克隆到适当的载体上并转化适当的宿主细胞，经培养筛选出具有葡 萄糖异构酶活性的阳性克隆。从阳性克隆中提取质粒 DNA，进行 DNA 序列测定分析， 以确定引入的突变。 最后， 以 D-葡萄糖为底物测定 具有所设定位点突变的葡萄糖异构酶的酶活力，将该酶活力与亲本的 酶活力相比较，从而筛选出具有比亲本高的葡萄糖异构酶催化活性的 本发明的葡萄糖异构酶突变体。
在本发明制备葡萄糖异构酶突变体的方法中， 可采用任何适当 的载体。例如，适用的载体包括但不限于原核表达载体 pGEMT-Easy、 pRSET和 pET21 ; 包括但不限于真核表达载体 pYDl和 pYES2/GS; 包括但不限于克隆载体 pUC 18/19和 pBluscript-SK。
在本发明制备葡萄糖异构酶突变体的方法中， 所获得的葡萄糖 异构酶突变体基因可以在原核细胞或真核细胞胞内表达，也可采用本 领域已知的任何其它适当方法实现在原核细胞或真核细胞胞外表达。
在本发明制备葡萄糖异构酶突变体的方法中， 所述载体的微生 物宿主细胞为原核细胞或真核细胞。所述原核微生物包括但不限于大 肠杆菌、 凝结芽孢杆菌、 枯草芽胞杆菌、 巨大芽胞杆菌 (如巨大芽胞 杆菌 ΒΡ931)、Γ. saccharolyticum禾口链霉菌 (女口 Streptomyces diastaticus M1033) o 所述真核微生物包括但不限于酿酒酵母和毕赤巴斯德酵母 (如毕赤巴斯德酵母 GS 1 15/ 9891)。
本发明获得了一种葡萄糖异构酶突变体,以说明书中所附的序列 2为参考序列， 其第 139位突变为苯丙氨酸 (Phe)、第 182位突变为丙 氨酸 (Ala)、 第 187位突变为丝氨酸 (Ser)突变以及第 299位突变为谷 氨酰胺 (Gln)， 其还同时在第 87位、 第 217位、 第 260位、 第 276位 具有至少一个突变，并且以 D-葡萄糖为底物其具有比所述亲本高的葡 萄糖异构酶催化活性。 优选的是， 所述第 87位为甲硫氨酸 (Met)或亮 氨酸 (Leu) ; 所述第 217位为精氨酸 (Arg )、 或色氨酸 (Trp：)、 或甘氨酸 (Gly) ; 所述第 260位为谷氨酸 (Glu)或丙氨酸 (Ala) ; 和 /或所述第 276 位为甘氨酸 (Gly)或苏氨酸 (Thr)。序列表中的 SEQ ID NO. :4显示了一 种本发明的葡萄糖异构酶突变体的氨基酸序列， 其中的 Xaa 代表突 变位点的氨基酸。最优选的是， 本发明的葡萄糖异构酶突变体包含下 文表 2 中所列的 MGI4-F87L、 MGI4-F87M、 MGI4-V217R、 MGI4-V217W、 MGI4-D260E、 MGI4-F276G、 MGI4-24、 MGI4-25、 MGI4-34、 MGI4-35的氨基酸序列。
这些突变体具有高的催化活性。 例如， 在本发明获得的一系列 突变体中， 一个具有七个点突变的突变体 MGI4-34 的比活性较亲本 高 769%， 且在 80 °C反应 26小时后仍保持 50%或以上的活力。 另一 个具有七个点突变的突变体 MGI4-35的比活性较亲本高 727%，且在 80 °C反应 27小时后仍保持 50%或以上的活力。
本发明获得的高催化活性或高催化活性并耐热的葡萄糖异构酶 突变体可用于直接生产果糖含量等于或高于 55重量％的果葡糖浆， 或用于生产果糖含量低于 55重量％的果葡糖桨。 本发明的葡萄糖异 构酶突变体可用于生产果糖含量高于 90重量％以上的结晶果糖。
所述的葡萄糖异构酶突变体可以以未经纯化粗酶形式使用,也可 以是经部分纯化的或完全纯化的形式。如果需要， 还可利用本领域已 知的固化技术将本发明葡萄糖异构酶突变体制成固相酶或固相细胞 形式的固化酶。
定义
本申请文本中所用的术语"亲本"系指来自 T. saccharolyticum ATCC 49915的葡萄糖异构酶， 其核苷酸序列如序列 1所示， 氨基酸 序列如序列 2 所示。 本发明中亲本基因的核苷酸序列与公布的 T. saccharolyticum 葡萄糖异构酶的基因序歹 lj (Lee et al.， Journal of General Microbiology , 139: 1227- 1234， 1993 ; GenBank L09699)相比 有两个核苷酸的差异， 即与基因库的核苷酸序列 (GenBank L09699) 相比， 本发明中亲本基因第 241-242位为 GC , 相应的第 81位氨基 酸序列为丙氨酸 (Ala) , GenBankL09699对应处、 第 241-242位核苷 酸序列为 CG， 相应的第 81位氨基酸序列为精氨酸 (Arg)。
本申请文本中所用的术语"参考序列"， 当其为核苷酸序列时， 是指说明书中所附的序列 1， 当其为氨基酸序列时， 是指说明书中所 附的序列 2。在将参考序列和其它突变的葡萄糖异构酶序列进行排序. 比较时， 可以手工进行， 也可以用计算机进行（目前有许多可供利用 的计算机软件， 例如 CLUSTALW, AMAS, DIALIGN程序等） 。
本申请文本中所用的术语"位点"或"第 X 位"是指当本发明的葡 萄糖异构酶突变体的序列与参考序列的排序对比达最大同源性时，被 比较的葡萄糖异构酶突变体的各核苷酸或氨基酸所对应的参考序列 中的核苷酸或氨基酸位置。
本申请文本所用的术语"葡萄糖异构酶突变体 "是指一种以说明 书中所附序列 2所示氨基酸序列为参考序列，其第 139位突变为苯丙 氨酸 (Phe)、第 182位突变为丙氨酸 (Ala)、第 187位突变为丝氨酸 (Ser) 突变以及第 299位突变为谷氨酰胺 (Gln)， 同时在第 87位、第 217位、 第 260位、 第 276位具有至少一个突变,并且以 D-葡萄糖为底物生成 果糖时其具有比亲本高的葡萄糖异构酶催化活性的酶。 因此， 在本专 利申请中，所述葡萄糖异构酶突变体包括具有序列 4所示氨基酸序列 的突变体、序列 4的保守取代形式、增加或缺失一个或几个氨基酸的 形式、 氨基端截断的形式、 羧基端截断的形式、 以及序列 4的部分或 全部串联重复形式。
在本申请文本所用的氨基酸三字母或单字母表达方式， 采用 IUPAC规定的氨基酸代码 (Eur. J. Biochem., 138 :9-37, 1984)。 附图说明
图 1. 亲本葡萄糖异构酶与葡萄糖异构酶突变体在 80 C的热稳 定性图谱。 其中 MGI4-34和 MGI4-35代表具有七个突变位点的葡萄 糖异构酶突变体， 具体参见实施例 12。 具体实施方式
下列实施例仅用于说明本发明而不应视为限定本发明的范围。实施 例中未注明具体条件者， 按常规条件或制造商建议的条件进行。 实施例 1 : 亲本基因的扩增及 pGEMT-TS的构建
根据基因库公布的基因序列（GenBank L09699)设计引物 T1 和
T2(见表 1)。用引物对 T1和 Τ2从 T. saccharolyticum ATCC 49915(购自 ATCC , USA)中扩增葡萄糖异构酶亲本基因。
扩增条件为： 20 mM Tris-HCl (pH 8.8) , 10 mM KC1 , 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1 % Triton X- 100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl， 400 nM引物 T2， 1.5 U Taq
DNA聚合酶 (Promega, USA), 用接种环挑取少许 Τ· saccharolyticum菌 体， 再用无菌水调反应体积至 50 μ1。
PCR扩增反应程序为： 95°C 3分钟， 40圈循环： 95°C 50秒、 50°C 30秒和 72°C 1分钟， 最后 72°C 10分钟。 将扩增的产物 (长约 1.5 kb) 连接至载体 pGEMT-Easy， 得质粒 pGEMT-TS。 经 DNA测序确定亲本 葡萄糖异构酶的核苷酸序列为序列 1， 相应的氨基酸序列为序列 2。 与 基因库公布的核苷酸序列 (GenBank L09699)相比， 本发明中亲本基因第 241-242 位为 GC， 相应的第 81 位氨基酸序列为丙氨酸 (Ala)， GenBankL09699对应处、 第 241 -242位核苷酸序列为 CG， 相应的第 81 位氨基酸序列为精氨酸 (Arg)。 实施例 2 : 葡萄糖异构酶位点 139之定点突变
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al. (PCR Protocol: current methods and applications. Totowa, N. J.： Humana Press, 1993) 之描述。
以质粒 pGEMT-TS (；见实施例 1)为模板， 设计引物对 139FF 和 139FR(见表 1)， 将亲本氨基酸序列中第 139 位点的 Trp(W)突变为 Phe(F), 获得突变体 MGI-W139F。 弓 |物对 T1和 T2 见实施例 1。
利用引物对 T1和 139FR, 扩增 T1FR片段， 引物对 139FF和 T2， 扩增 FFT2片段。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8) , 10 mM
KC1, 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1 % Triton X-100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM引 物 139FR或 400 nM引物 139FF和 400 nM引物 T2， 1.5 U Pfu DNA聚 合酶 （Promega, USA), 20 ng pGEMT-TS , 再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C
30秒和 72°C 3分钟， 最后 72°C 5分钟。经 1 % 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit (QIAGEN, German) 回收， 得到 T1FR片 段和 FFT2片段。然后扩增全长基因。扩增反应条件为： 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1% Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 弓 I物 Tl和 400 nM T2 , 1.5 U Pfu DNA聚合酶， 20 ng T1FR片段与 20 ng FFT2 片段， 用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit 回 收，得到全长突变基因 MGI-W139F。将 MGI-W139F与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI-W139F。 将质粒 pGEMT-MGI-W139F转入 感受态细菌细胞 E. coli HB 101 , 在 1% MacConkey平板 (含 1% D-木糖 和 50 mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。 从克隆 中提取质粒 PGEMT-MGI-W139F DNA, 经 DNA测序确定引入的点突 变无误。 实施例 3 : 葡萄糖异构酶位点 182之定点突变
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al. (PCR Protocol: current methods and applications. Totowa, N.J. :Humana Press, 1993)之描述。
以质粒 pGEMT-TS (见实施例 1)为模板， 设计引物对 182AF 和 182AR (见表 1)， 将亲本氨基酸序列中第 182 位点的 Arg(R)突变为 Ala(A) , 获得突变体 MGI-R182A。 弓 |物 Tl和 T2 见实施例 1。
利用引物对 T1和 182AR, 扩增 T1AR片段， 引物对 182AF和 T2， 扩增 AFT2片段。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM
KC1, 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1 % Triton X- 100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM引 物 182AR或 400 nM引物 182AF和 400 nM引物 T2， 1.5 U Pfu DNA 聚合酶， 20 ng pGEMT-TS， 再用无菌水调反应体积至 50 μ1。 PCR扩增 反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C
3 分钟， 最后 72°C 5 分钟。 经 1 % 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 T1AR片段和 AFT2片段。 然后扩增全长 基因。扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1% Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM引物 Tl和 400 nM T2, 1.5 U Pfu
DNA聚合酶， 20 ng TlAR片段与 20 ng AFT2 片段， 再用无菌水调反 应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72。C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶 电泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因 MGI-R182A。 将 MGI-R182A 与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI-R182A。将质粒 pGEMT-MGI-R182A转入感受态细菌细胞 E. coli HB 101 , 在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄青 霉素）上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI-R182A DNA, 经 DNA测序确定引入的点突变无误。 实施例 4 : 葡萄糖异构酶位点 187之定点突变
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et ah
(PCR Protocol: current methods and applications. Totowa, N.J.:Humana
Press, 1993)之描述。
以质粒 pGEMT-TS (见实施例 1)为模板， 设计引物对 187SF 和
187SR (见表 1)，将亲本氨基酸序列中第 187位点的 Phe(F)突变为 Ser(S)， 获得突变体 MGI-F 187S。 引物 Tl和 T2 见实施例 1。
利用引物对 T1和 187SR， 扩增 T1 SR片段， 引物对 187SF和 T2， 扩增 SFT2片段。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8) , 10 mM KC1, 10 mM (NH4)2SO4， 2 mM MgS04, 0.1 % Triton X-100 , 50 μΜ dATP,
50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM引 物 187SR或 400 nM引物 T2和 400 nM引物 187SF， 1.5 U Pfu DNA聚 合酶， 20 ng pGEMT-TS， 再用无菌水调反应体积至 50 μ1。 PCR扩增反 应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3 分钟， 最后 72°C 5 分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick
Extraction Gel Kit回收， 得到 T1 SR片段和 SFT2片段。 然后扩增全长 基因。 扩增反应条件为: 20 mM Tris-HCl (pH 8.8) , 10 mM KC1 , 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1 % Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM T2 , 1.5 U Pfu DNA聚合酶， 20 ng Tl SR片段与 20 ng SFT2片段， 再用无菌水调反应 体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50 秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电 泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因 MGI-F 187S。 将 MGI-F187S 与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI-F187S o 将质粒 pGEMT-MGI-F187S转入感受态细菌细胞 E. coli HB 101 ,在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄青 霉素）上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI-F187S DNA, 经 DNA测序确定引入的点突变无误。 实施例 5 : 葡萄糖异构酶位点 299之定点突变
定点突变技术参考 Ho et al. (Gene 77:5 1-59, 1989)和 White et al. (PGR Protocol: current methods and applications. Totowa, NJ.:Humana Press, 1993)之描述。
以质粒 pGEMT-TS (见实施例 1)为模板， 设计引物对 299QF 和 299QR(见表 1)， 将亲本氨基酸序列中第 299 位点的 Thr(T)突变为
Gln(Q), 获得突变体 MGI-T299Q。 弓 |物 Tl和 T2 见实施例 1。
利用引物对 T1和 299QR, 扩增 T1 QR片段， 引物对 299QF和 T2， 扩增 QFT2片段。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1% Triton X-100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM引物 Tl和 400 nM引 物 299QR或 400 nM引物 299QF和 400 nM引物 T2， 1.5 U Pfu DNA聚 合酶， 20 ng pGEMT-TS， 再用无菌水调反应体积至 50 μ1。 PCR扩增反 应程序为： 95°C 3分钟， 35圈循环: 95。C 50秒、 52°C 30秒和 72°C 3 分钟， 最后 72°C 5 分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 T1QR片段和 QFT2片段。 然后扩增全长 基因。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KCl， 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1% Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM引物 Tl和 400 nM T2, 1.5 U Pfu DNA聚合酶， 20 ng Tl QR片段与 20 ng QFT2片段， 再用无菌水调反应 体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50 秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电 泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因 MGI-T299Q。 将 MGI-T299Q 与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI-T299Q。将质粒 pGEMT-MGI-T299Q转入感受态细菌细胞 E. coli HB 101 ,在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄青 霉素）上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI-T299Q DNA, 经 DNA测序确定引入的点突变无误。 实施例 6 : 葡萄糖异构酶四突变组合 MGI-4之构建
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al.
(PCR Protocol: current methods and applications. Totowa, N.J.:Humana Press, 1993)之描述。
按实施例 2扩增和回收 T1FR片段， 按实施例 5扩增和回收 QFT2 片段。用引物对 139FF (见表 1)和 182AR (见表 1)扩增 FFAR片段。 FFAR 片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM
(NH4)2SO4, 2 mM MgSO4， 0.1 % Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 139FF和 400 nM 182AR, 1.5 U Pfu DNA聚合酶， 20 ng pGEMT-TS， 再用无菌水调反应体积至 50 μ1。 PCR 扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟，最后 72°C 5分钟。经 1% 琼脂糖胶电泳分离并用 QIAquick
Extraction Gel Kit回收， 得到 FFAR片段。 用引物对 182AF (见表 1)和 187SR(见表 1)扩增 AFSR片段。 AFSR片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1 % Triton X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 182AF和 400 nM 187SR, 1.5 U Pfu DNA聚合酶， 20 ng pGEMT-TS , 再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 AFSR片段。用引物对 187SF (见表 1)和 299QR (见表 1)扩增 SFQR片段。 SFQR片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， lO mM KCl, 10 mM (NH4)2SO4, 2 mM MgS04， 0.1% Triton X-100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM 187SF和 400 nM 299QR, 1.5 U Pfu DNA聚合酶， 20 ng pGEMT-TS ,再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30 秒和 72°C 3分钟， 最后 72。C 5分钟。 经 1 % -琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收，得到 SFQR片段。然后扩增全长基因。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1， 10 mM (NH4)2SO4， 2 mM MgSO4, 0.1% Triton X-100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM T2, 1.5 U Pfu DNA聚合酶， 20 ng TlFR片段， 20 ng FFAR片段， 20 ng AFSR片段， 20 ng SFQR片段和 20 ng QFT2片段， 再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30 秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到全长突变基因 MGI-4。将 MGI-4 与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI-4。 将质粒 pGEMT-MGI-3转入感受态细菌细胞 E. coli HB 101 , 在 1% MacConkey 平板 (含 1% D-木糖和 50 mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活 性的克隆。 从克隆中提取质粒 pGEMT-MGI-4 DNA， 经 DNA测序确定 引入的点突变无误。 MGI-4序列含有 W139F、 R182A、 F 187S和 T299Q 的四突变。 实施例 7 :葡萄糖异构酶五突变组合 MGI4-F87L和 MGI4-F87M之 构建
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989和 White et al. (PCR Protocol: current methods and applications. Totowa, N.J.: Humana
Press, 1993)之描述。
以质粒 pGEMT-MGI-4 (见实施例 6)为模板， 设计引物对 87LF和 87L (见表 1)， 将 MGI-4 氨基酸序列中第 87 位点的 Phe(F)突变为 Leu(L), 获得突变体 MGI4-F87L。 引物对 T1和 T2 见表 1。
用引物对 T1和 87LR, 扩增 T1LR片段， 用引物对 87LF和 T2， 扩 增 LFT2片段。扩增反应条件为： 20 mM Tris-HCl (pH 8.8), 10 mM KC1， 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1% Triton X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM引物 87LR或 400 nM引物 87LF和 400 nM引物 T2， 1.5 U Pfu DNA聚合酶 (Promega, USA), 20 ng pGEMT-MGI-4 ,再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30 秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit (QIAGEN, German) 回收， 得到 T1LR片 段和 LFT2片段。然后扩增全长基因。扩增反应条件为： 20 mM Tris-HCl ( H 8.8), lO mM KCl , 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1 % Triton
X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 引物 Tl和 400 nM T2 , 1.5 U Pfu DNA聚合酶， 20 ng T1LR片段与 20 ng LFT2 片段， 用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1 % 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit 回 收，得到全长突变基因 MGI4-F87L。将 MGI4-F87L与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI4-F87L。 将质粒 pGEMT-MGI4-F87L转入感 受态细菌细胞 E. coli HB 101 , 在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提 取质粒 pGEMT-MGI4-F87L DNA,经 DNA测序确定引入的点突变无误。 所得突变体序列含有 F87L、 W139F R182A、 F187S和 T299Q 五个突 变。 MGI4-F87L氨基酸序列见序列表序列 5。
按照类似步骤构建突变体 MGI4-F87M , 所用引物见表 1。 MGI4-F87M突变体序列含有 F87M、 W139F、 R182A、 F187S和 T299Q 五个突变。 所得突变体氨基酸序列见序列表序列 6。 实施例 8 :葡萄糖异构酶五突变组合 MGI4-V217R和 MGI4-V217W 之构建
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al. (PCR Protocol: current methods and applications. Totowa, N丄： Humana
Press, 1993)之描述。
以质粒 pGEMT-MGI-4 (见实施例 6)为模板， 设计引物对 217RF和 217RR (见表 1)， 将 MGI-4氨基酸序列中第 217位点的 Val(V)突变为 Arg(R), 获得突变体 MGI4-V217R。 弓 |物 Tl和 T2 见实施例 1。
用引物对 T1和 217RR, 扩增 T1RR片段， 用引物对 217RF和 T2， 扩增 RFT2片段。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1% Triton X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM引 物 217RR或 400 nM引物 217RF和 400 nM引物 T2， 1.5 U Pfu DNA 聚合酶， 20 ng pGEMT-MGI-4， 再用无菌水调反应体积至 50 μ1。 PCR 扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟，最后 72°C 5分钟。经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 T1RR片段和 RFT2片段。 然后扩增全长 基因。 扩增反应条件为: 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100 , 50 μΜ dATP, 50 μΜ dTTP,
50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM T2， 1.5 U Pfu DNA聚合酶， 20 ng T1RR片段与 20 ng RFT2 片段， 再用无菌水调反 应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1 % 琼脂糖胶 电泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因
MGI4-V217R。 将 MGI4-V217R 与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI4-V217R。将质粒 pGEMT-MGI4-V217R转入感受态细菌细 胞 E. coli HB 101 ,在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄 青霉素）上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI4-V217R DNA, 经 DNA测序确定引入的点突变无误。 所 得突变体序列含有 W139F、 R182A、 F 187S、 V217R和 T299Q 五个突 变。 MGI4-V217R的氨基酸序列见序列表序列 7。
按照类似步骤构建突变体 MGI4-V217W , 所用引物见表 1。 MGI4-V217W突变体序列含有 W139F、R182A、F 187S、V217W和 T299Q 五个突变。 所得突变体的氨基酸序列见序列表序列 8。 实施例 9 : 葡萄糖异构酶五突变组合 MGI4-D260E之构建
定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al. (PCR Protocol: current methods and applications. Totowa, N.J.:Humana Press, 1993)之描述。 以质粒 pGEMT-MGI-4 (见实施例 6)为模板， 设计引物对 260EF和 260ER (见表 1)， 将 MGI-4氨基酸序列中第 260位点的 Asp(D)突变为 Glu(E) , 获得突变体 MGI4-D260E。 引物 T1和 T2 见表 1。
用引物对 T1和 260ER, 扩增 TIER片段， 用引物对 260EF和 T2， 扩增 EFT2片段。 扩增反应条件为： 20 mM Tris-HCl (ρΗ 8.8)， 10 mM
KC1, 10 mM (NH4)2SO4， 2 mM MgSO4, 0.1% Triton X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM引物 Tl和 400 nM引 物 260ER或 400 nM引物 T2和 400 nM引物 260EF , 1.5 U Pfu DNA聚 合酶， 20 ng pGEMT-MGI-4， 再用无菌水调反应体积至 50 μ1。 PCR扩 增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C
3 分钟， 最后 72°C 5 分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 TIER片段和 EFT2片段。 然后扩增全长 基因。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KCl， 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1% Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM引物 Tl和 400 nM T2, 1.5 U Pfu
DNA聚合酶， 20 1^ 11£1 片段与 20 1^ £?了2片段， 再用无菌水调反应 体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50 秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电 泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因 MGI4-D260E。 将 MGI4-D260E 与载体 pGEMT-Easy 连接， 得质粒 pGEMT-MGI4-D260Eo将质粒 pGEMT-MGI4-D260E转入感受态细菌细 胞 E. coli HB 101，在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄 青霉素）上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI4-D260E DNA, 经 DNA测序确定引入的点突变无误。 所 得突变体序列含有 W139F、 R182A、 F 187S、 D260E和 T299Q 五个突 变。 MGI4-D260E的氨基酸序列见序列表序列 9。 实施例 10 : 葡萄糖异构酶五突变组合 MGI4-F276G之构建 定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al. (PCR Protocol: current methods and applications. Totowa, N. J. '.Humana Press, 1993)之描述。
以质粒 pGEMT-MGI-4 (见实施例 6)为模板， 设计引物对 276GF和 276GR (见表 1)， 将 MGI-4氨基酸序列中第 276位点的 Phe(F)突变为 Gly(G), 获得突变体 MGI4-F276G。 引物 T1和 T2 见表 1。
用引物对 T1和 276GR, 扩增 T1GR片段， 用引物对 276GF和 T2， 扩增 GFT2片段。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8) , 10 mM KCl, 10 mM (NH4)2S04， 2 mM MgSO4， 0.1% Triton X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM引 物 276GR或 400 nM引物 276GF和 400 nM引物 T2， 1.5 U Pfu DNA聚 合酶， 20 ng pGEMT-MGI-4， 再用无菌水调反应体积至 50 μ1。 PCR扩 增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3 分钟， 最后 72°C 5 分钟。 经 1 % 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 T1GR片段和 GFT2片段。 然后扩增全长 基因。 扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KCl, 10 mM (NH4)2SO4， 2 mM MgS04， 0.1% Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP,
50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM T2， 1.5 U Pfu DNA聚合酶， 20 ng TlGR片段与 20 ng GFT2片段， 再用无菌水调反应 体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50 秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电 泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因
MGI4-F276G 0 将 MGI4-F276G 与载体 GEMT-Easy 连接， 得质粒 pGEMT-MGI4-F276Go将质粒 pGEMT-MGI4-F276G转入感受态细菌细 胞 Ε· coli HB 101，在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄 青霉素）上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI4-F276G DNA, 经 DNA测序确定引入的点突变无误。 所 得突变体序列含有 W139F、 R182A, F 187S、 F276G和 T299Q 五个突 变。 MGI4-F276G的氨基酸序列见序列表序列 10。 实施例 1 1 : 葡萄糖异构酶六突变组合 MGI4-24和 MGI4-25之构建 定点突变技术参考 Ho et al. (Gene 77:51-59, 1989)和 White et al. (PCR Protocol: current methods and applications. Totowa, N丄: Humana Press, 1993)之描述。
按实施例 7扩增和回收 T1LR片段。用引物对 87LF和 260AR(见表 1)扩增和回收 LFAR片段。 LFAR片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8), 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgSO4， 0.1% Triton
X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 87LF和 400 nM 260AR, 1.5 U Pfu DNA聚合酶， 20 ng pGEMT-MGI-4 , 再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit 回收， 得到
LFAR片段。用引物对 260AF和 T2(见表 1)扩增和回收 AFT2片段。 AFT2 片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8), 10 mM KC1， 10 mM (NH4)2SO4， 2 mM MgSO4， 0.1% Triton X-100, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 260AF和 400 nM T2, 1.5 U Pfu DNA 聚合酶， 20 ng pGEMT-MGI-4, 再用无菌水调反应体积至 50 μ1。 PCR 扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟，最后 72°C 5分钟。经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit 回收， 得到 AFT2片段。 然后扩增全长基因。 扩增反 应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100,, 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl和 400 nM T2， 1.5 U Pfu DNA聚 合酶， 20 ng TlLR片段， 20 ng LFAR片段和 20 ng AFT2片段， 再用无 菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循 环： 95°C 50秒、 52°C 30秒和 72°C 3分钟，最后 72°C 5分钟。经 1% 琼 脂糖胶电泳分离并用 QIAquick DNA 回收试剂盒， 得到全长突变基因
MGI4-24。 将 MGI4-24 与载体 pGEMT-Easy 连接 ， 得质粒 pGEMT-MGI4-24。将质粒 pGEMT-MGI4-24转入感受态细菌细胞 E. coli HB101 , 在 1% MacConkey平板 (含 1% D-木糖和 50 mg/L氨苄青霉素) 上筛选出具葡萄糖异构酶活性的克隆。 从克隆中提取质粒 pGEMT-MGI4-24 DNA,经 DNA测序确定引入的点突变无误。 MGI4-24 的氨基酸序列见序列表序列 1 1。 所得突变体序列含有 F87L、 W139F、 R182A、 F 187S、 D260A和 T299Q六个突变。
按照类似步骤构建突变体 MGI4-25。 突变体 MGI4-25所用引物对 为 T1和 87LR、 87LF和 276TR、 276TF和 T2(均见表 1)， 所得突变体 序列含有 F87L、 W139F、 R182A、 F 187S、 F276T和 T299Q六个突变。 所得突变体的氨基酸序列见序列表序列 12。 实施例 12 : 葡萄糖异构酶七突变组合 MGI4-34和 MGI4-35之构建 定点突变技术参考 Ho et al, (Gene 77:51-59, 1989)和 White et al. (PGR Protocol: current methods and applications. Totowa, N.J.:Humana
Press, 1993)之描述。
按实施例 Ί扩增和回收 T1LR片段。用引物对 87LF和 217GR (见表 1)扩增和回收 LFGR片段。 LFGR片段扩增反应条件为： 20 mM Tris-HCl ( H 8.8), 10 mM KC1, 10 mM (NH4)2S04， 2 mM MgSO4， 0.1 % Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP , 400 nM
87LF和 400 nM 217GR， 1 ,5 U Pfu DNA聚合酶， 20 ng pGEMT-MGI-4， 再用无菌水调反应体积至 50 μ1。 jPCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 LFGR片段。用引物对 217GF 和 276TR (见表 1)扩增和回收 GFTR片段。 GFTR片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8)， 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgS04， 0.1% Triton X- 100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 217GF和 400 nM 276TR, 1.5 U Pfu DNA聚合酶， 20 ng pGEMT-MGI-4， 再用无菌水调反应体积至 50 l。PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C
30秒和 72°C 3分钟， 最后 72°C 5分钟。经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收，得到 GFTR片段。用引物对 276TF 和 T2(见表 1)扩增和回收 TFT2片段。 TFT2片段扩增反应条件为： 20 mM Tris-HCl (pH 8.8), lO mM KCl, 10 mM (NH4)2SO4， 2 mM MgS04, 0.1% Triton X-100 , 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM 276TF和 400 nM T2， 1.5 U Pfu DNA聚合酶， 20 ng pGEMT-MGI-4, 再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit回收， 得到 TFT2片段。 然后扩增全长基因。扩增反应条件为： 20 mM Tris-HCl (pH
8.8)， 10 mM KC1, 10 mM (NH4)2SO4, 2 mM MgSO4 , 0.1% Triton X- 100， 50 μΜ dATP, 50 μΜ dTTP, 50 μΜ dCTP, 50 μΜ dGTP, 400 nM引物 Tl 和 400 nM T2， 1.5 U Pfu DNA聚合酶， 20 ng TlLR片段， 20 ng LFGR 片段， 20 ng GFTR片段和 20 ng TFT2片段， 再用无菌水调反应体积至 50 μ1。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C
30秒和 72°C 3分钟， 最后 72°C 5分钟。经 1% 琼脂糖胶电泳分离并用 QIAquick Extraction Gel Kit 回收， 得到全长突变基因 MGI4-34。 将 MGI4-34与载体 pGEMT-Easy连接， 得质粒 pGEMT-MGI4-34。 将质粒 pGEMT-MGI4-34转入感受态细菌细胞 E. coli HB 101，在 1% MacConkey 平板 (含 1% D-木糖和 50 mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活 性的克隆。 从克隆中提取质粒 pGEMT-MGI4-34 DNA， 经 DNA测序确 定引入的点突变无误。 MGI4-34序列含有 F87L、 W139F、R182A、F 187S、 V217G、 F276T和 T299Q七个突变。 MGI4-34的氨基酸序列见序列表 序列 13。
按照类似步骤构建七突变体 MGI4-35。 突变体 MGI4-35所用引物 对为 T1和 87LR、 87LF和 217GR、 217GF和 260AR、 260AF和 T2 (均 见表 1)， 所得突变体序列含有 F87L、 W139Fs R182A、 F187S、 V217G、 D260A和 T299Q七突变。 所得突变体的氨基酸序列见序列表序列 14。 扩增亲本葡萄糖异构酶 (亲本)与实施例 1-12 之葡萄糖异构酶突变 体所用的引物如下表 1所列：
Figure imgf000021_0001
ΐ挲
T06Z00/900ZND/X3d 0C69S0/.00Z OAV 突变体 276GF: 5'ACATTGGCAGGCCACGACTTCCAACATGAGC 3'
MGI4-F276G 276GR: 5 ' GA AGTCGTGGCCTGCCA ATGTCGCATGGTTT 3'
突变体 276TF: 5'ACATTGGCAACCCACGACTTCCAACATGAGC 3'
MGI4-F276T 276TR: 5'GAAGTCGTGGGTTGCCAATGTCGCATGGTTT 3' 实施例 13 : 亲本葡萄糖异构酶的提取与纯化
葡萄糖异构酶的提取与纯化主要参考 Lee et ah , Journal of General Microbiology, 87: 1227- 1234(1993)。
将含亲本葡萄糖异构酶基因的质粒 pGEMT-TS转化感受态细菌细 胞 E. coli HB 101 , 在 MacConkey平板 (含 1 %D-木糖和 50 mg/L氨苄青 霉素：)上、 37 °C培养 36 小时。 接种单个克隆在 5 ml LB液体培养基 (含 50 mg/L氨苄青霉素)中培养 16小时。 离心收集菌体， 并悬浮于 l ml 20 mM磷酸钠缓冲液 (pH 6.5)中，加 CoCl fl MgCl2至终浓度分别为 250 μΜ 和 5 mM。 然后用超声波裂解细菌细胞。 离心 (10 °C， 17800 g, 15分钟） 并收集上清液为粗提蛋白。粗提蛋白经 80°C热处理 10分钟后，离心 (10 。C , 17800 g, 15分钟)去沉淀， 即为部分纯化的葡萄糖异构酶， 可用于 酶活性的测定和制备果葡糖浆。 实施例 14 : 葡萄糖异构酶突变体的提取与纯化
葡萄糖异构酶突变体 MGI4-35的提取与纯化与实施例 13同， 其中 所用质粒为 pGEMT-MGI4-35。其它葡萄糖异构酶突变体也照实施例 13 所述提取与纯化。 实施例 15 : 亲本葡萄糖异构酶活性的测定
底物溶液 A含 1.0 M的 D-葡萄糖、20 mM磷酸钠、250 μΜ 的 CoCl2 和 5 mM 的 MgCl2， pH6.5。 取底物溶液 A 90 μΐ, 然后加入 10 μΐ按实 施例 1和 13制备的葡萄糖异构酶。 反应于 80°C进行 10分钟。 将反应 物置冰上以终止反应。 反应产物 D-果糖通过半胱氨酸一咔唑法测定。 测定方法参见 Dische et al.， J. Biol. Chem, 192:583-587 , 1951 以及
Nakamura, Agr. Biol. Chem.32:701-706 , 1968。用 Coomassie® Plus Protein Assay Reagent Kit (PIERCE, USA)并结合 SDS聚丙烯酰胺凝胶电泳测 定酶蛋白浓度。一单位酶比活性定义为在上述条件下每分钟转化一微摩 尔 D-葡萄糖为果糖所需的酶量。 表 2显示亲本葡萄糖异构酶的相对比 活性。 实施例 16: 葡萄糖异构酶突变体活性的测定
葡萄糖异构酶突变体活性测定与实施例 15同。表 2 显示各葡萄糖 异构酶突变体与亲本葡萄糖异构酶 (亲本)比活性的差异。 亲本葡萄糖异构酶与葡萄糖异构酶突变体比活性之差异
Figure imgf000023_0001
实施例 17: 亲本葡萄糖异构酶热稳定性测定
将按实施例 13 获得的部分纯化的亲本葡萄糖异构酶置于 4管 1.5 ml离心管中。 每管离心管分别加入 200 μΐ酶溶液， 并加入 200 μΐ矿物 油。 将离心管置于 80°C水浴中， 0小时、 2小时、 6小时、 27小时后分 别取出一管酶液， 离心（10°C， 17800 g, 20 分钟)后， 取上清液， 按实 施例 15来测定葡萄糖异构酶残余蛋白的比活性。 图 1显示亲本葡萄糖 异构酶在 80Ό的热稳定性。 实施例 18 : 葡萄糖异构酶突变体热稳定性测定
葡萄糖异构酶突变体 MGI4-34(见实施例 12和 14)或 MGI4-35(见实 施例 12和 14)热稳定性测定与实施例 17同。 图 1显示葡萄糖异构酶突 变体 MGI4-34或 MGI4-35在 80°C的热稳定性。 如图所示， 亲本葡萄糖 异构酶的活性在 80Ό的半衰期为 4.1小时； MGI4-34的活性在 80Ό的 半衰期为 26小时； MGI4-35的活性在 80°C的半衰期大于 27小时。 实施例 19: 固相化葡萄糖异构酶突变体 MGI4-35
主要参考 Ge， et al , Appl. Biochem. Biotechnol. 69:57-69, 1998。 具体步骤是： 取固相酶载体氯化聚苯乙烯乙基胺 (成都化工研究所提供) 颗粒 100克， 加 10 mM磷酸盐缓冲液 (pH 8.0) 1升和部分提纯之葡萄糖 异构酶突变体 MGI4-35(按实施例 12和 14所述制备) 8克， 室温（22°C ) 搅拌 （60-120转 /分） 反应 18小时。 抽滤收集固相酶， 以水洗涤三次， 得固相酶 107克。 固相酶活力测定如实施例 16所述， 其中所用酶为如 上制备的固相酶 0.01克， 测得该固相酶的活性为 820单位 /克。 实施例 20: 固定化含葡萄糖异构酶突变体 MGI4-35的细胞 在含 50mg/L 氨苄青霉素的 LB 培养液中过夜生长携有 pGEMT-MGI4-35的 E. coli HB101至 OD6G()为 7。 离心收集菌体细胞。 取菌体细胞 10克， 加 3%海藻酸钠溶液 20克， 充分搅拌混合， 然后将 菌体细胞从管径约 0.5 mm的针头滴入 500 ml 2%氯化钙溶液中， 室温 反应 1小时， 用蒸熘水浸泡洗涤 3次， 每次约半小时， 得固定化细胞约 30克。 固定化细胞活力测定如实施例 16所述， 其中所用酶为如上制备 的固定化细胞 0.01克， 测得固定化细胞的活性为 370单位 /克。 本发明不受上述具体文字描述的限制， 本发明可在权利要求书所 概括的范围内做各种改变。 这些改变均在本发明的范围之内。

Claims

权 利 要 求 书
1. 一种葡萄糖异构酶突变体， 其中以说明书中所附的序列 2为 参考序列， 其第 139位突变为苯丙氨酸 (Phe)、 第 182位突变为丙氨 酸 (Ak)、 第 187位突变为丝氨酸 (Ser)突变以及第 299位突变为谷氨 酰胺 (Ghi)， 其特征在于还具有选自第 87位、 第 217位、 第 260位、 第 276位的至少一个突变，并且以 D-葡萄糖为底物其具有比亲本高的 葡萄糖异构酶催化活性。
2. 权利要求 1所述的葡萄糖异构酶突变体，其中第 87位突变为 甲硫氨酸 (Met)或亮氨酸 (Leu)。
3. 权利要求 1所述的葡萄糖异构酶突变体， 其中第 217位的氨 基酸突变为精氨酸 (Arg)、 或色氨酸 (Trp)、 或甘氨酸 (Gly)。
4. 权利要求 1所述的葡萄糖异构酶突变体， 其中第 260位的氨 基酸突变为谷氨酸 (Glu)或丙氨酸 (Ala)。
5. 权利要求 1所述的葡萄糖异构酶突变体， 其中第 276位的氨 基酸为甘氨酸 (Gly )或苏氨酸 (Thr)。
6. 权利要求 1 所述的葡萄糖异构酶突变体， 其具有序列表中 SEQ ID NO. :5至 SEQ ID NO. : 14之一所示的氨基酸序列。
7. 权利要求 1-6任意一项所述的葡萄糖异构酶突变体的应用， 其中所述的葡萄糖异构酶突变体用于将 D-葡萄糖转化为果糖。
8. 权利要求 7所述的葡萄糖异构酶突变体的应用， 其中所述的 应用为制备果葡糖浆。
9. 权利要求 8所述的应用， 其中所述的果葡糖桨的果糖含量为 等于或高于 55重量％。
10. 一种 DNA, 其含有编码权利要求 1-6任意一项所述的葡萄 糖异构酶突变体的核苷酸序列。
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