# Patent

## Title
Glucose isomerase mutants, DNA thereof and use thereof

## Abstract

This invention provides a series of recombinant Thermoanaerobacterium saccharolyticum glucose isomerases with improved catalytic activity and thermostability obtained by using recombinant techniques. These recombinant glucose isomerases comprise amino acid variation including phenylalanine (Phe) at position 139, alanine (Ala) at position 182, serine (Ser) at position 187, and glutamine (Gln) at position 299, and carry at least one additional mutated amino acid at position 87, position 217, position 260 or position 276, and possess a higher catalytic activity than that of the wild-type when using D-glucose as substrate. These recombinant glucose isomerases can be used for direct production of high fructose corn syrup containing 55.% [wt] or higher concentration of fructose.

## Background
FIELD OF THE INVENTION
The present invention relates to molecular biology and biotechnology, and specifically relates to recombinant glucose isomerases with improved activity or both of improved activity and thermostability, the method of preparing the same using recombinant techniques, and use of the same.
BACKGROUND OF THE INVENTION
Glucose isomerase (E.C.5.3, 1.5, or xylose isomerase) is a key enzyme in the pentose phosphate pathway. It is one of the most important industrial enzymes (Kaneko et al., Bioscience, Biotechnology, and Biochemistry 2000, 64:940-947). In the food industry, it is used for the manufacture of high fructose corn syrup.
The equilibrium of the isomerization of D-glucose to fructose is primarily dictated by the temperature of the reaction. The higher the temperature, the more the fructose in the final reaction mixture. At present, the commercial glucose isomerases come mainly from Actinoplanes missouriensis, Bacillus coagulans or Streptomyces species, and are not stable at high temperature (e.g. temperature above 65° C.). Consequently, the current commercial isomerization is restricted to operate at around 60° C. and the products normally contain no more than 44% of fructose. The high fructose corn syrup containing higher fructose is therefore generated using expensive chromatographic enrichment, leading to higher production cost.
Scientists around the world have been working on the identification of thermostable and highly active glucose isomerases from thermophilic bacteria, and production of the same via protein engineering. J. G. Zeikus and his collaborators isolated and studied thermostable glucose isomerases from thermophilic bacteria, such as Thermoanaerobacterium saccharolyticum (hereinafter referred to as T. saccharolyticum) and Thermotoga neapolitana (Lee et al., Journal of General Microbiology, 139:1227-1234, 1993; Vieille et al., Methods in Enzymology, 330:215-24, 2001; Lee et al., Journal of General Microbiology, 139:1241-1243, 1993; Scriprapundh et al., Protein Engineering, 13:259-265, 2000; Scriprapundh et al., Protein Engineering, 16:683-690, 2003; Zeikus et al., U.S. Pat. No. 5,656,497). Nevertheless, the thermostabilities of the thermostable glucose isomerases from these or other bio-resources are still much to be desired, as the activities of thereof are low, and thus are not applicable to industrial applications. Therefore, glucose isomerase with high activity, or high activity and thermostability remains desirable.
SUMMARY OF THE INVENTION
By using genetic and protein engineering of Thermoanaerobacterium saccharolyticum glucose isomerase, this invention provides a series of glucose isomerases with improved catalytic activity suitable for the production of high fructose corn syrup containing high concentration of fructose.
The objective of the present invention is to provide highly active glucose isomerase mutants. Another objective of the invention is to use the glucose isomerase mutants to directly produce high fructose corn syrup containing 55% or higher concentration of fructose. Still another objective of the invention is to use the glucose isomerase mutants to produce high fructose corn syrup containing less than 55% fructose.
In order to achieve the above-mentioned objects, the inventors of the present invention have conducted extensive experiments. The inventors of the present invention have introduced mutations, by site-directed mutagenesis, into the T. saccharolyticum glucose isomerase gene and obtained a series of highly active or highly active and thermostable glucose isomerase mutants after screening on MacConkey agar. More specifically, the molecular biotechniques being used to generate glucose isomerase mutants include, construction of plasmid carrying the wild-type glucose isomerase gene; design of mutation sites and the amino acids after mutations; design of appropriate primers; PCR amplification of DNA fragments using the wild-type glucose isomerase gene as template; assembly of the DNA fragments; PCR amplification of the full-length glucose isomerase genes containing the mutation(s); cloning of the mutant genes into appropriate vectors; transformation of the vectors containing the genes into appropriate host cells; screening of the transformants for clones carrying desired glucose isomerase mutants; isolation of the plasmid DNA from the positive clones; and carrying out DNA sequencing to verify the mutations. Finally, the activity of the mutated isomerase is assessed using D-glucose as substrate, and mutated isomerase according to the present invention with higher catalytic activity than that of the wild-type is selected.
For the preparation of the novel glucose isomerases in this invention, suitable vectors include but are not limited to prokaryotic expression vectors pGEMT-Easy, pRSET and pET21; include but are not limited to eukaryotic expression vectors pYD1 and pYES2/GS; include but are not limited to cloning vectors pUC18/19 and pBluescript-SK.
For the preparation of the glucose isomerase mutants in this invention, the mutated glucose isomerase gene can be expressed intra-cellularly in prokaryotic or eukaryotic cells, or can be expressed extra-cellularly in prokaryotic or eukaryotic cells by using any other techniques known in the art.
For the preparation of the novel glucose isomerases in this invention, the host cells can be prokaryotic or eukaryotic cells. The prokaryotic cells include but are not limited to E. coli, Bacillus subtilis, Bacillus brevis, Bacillus megaterium (e.g. B. megaterium BP931), T. saccharolyticum and Streptomyces (e.g. S. diastaticus M1033). The eukaryotic cells include but are not limited to Saccharomyces cerevisiae and Pichia pastoris (egg. P. pastoris GS115/9891).
The glucose isomerase mutant according to this invention, using Sequence 2 in the Sequence Listing as the reference sequence, comprises amino acid mutations including phenylalanine (Phe) at position 139, alanine (Ala) at position 182, serine (Ser) at position 187, and glutamine (Gln) at position 299, as well as at least one mutation at position 87, position 217, position 260, or position 276, and using D-glucose as substrate, possesses higher catalytic activity than the wild-type. Preferably, the amino acid at the position 87 is methionine (Met) or leucine (Leu); the amino acid at the position 217 is arginine (Arg), or tryptophan (Trp) or glycine (Gly); the amino acid at the position 260 is glutamic acid (Glu) or alanine (Ala); and/or the amino acid at the position 276 is glycine (Gly) or threonine (Thr). Sequence ID: NO.4 in the Sequence Listing shows the amino acid sequence of a glucose isomerase mutant according to this invention, where Xaa represents the mutated amino acid. The glucose isomerase mutants according to this invention include the amino acid sequences of MGI4-F87L, MGI4-F87M, MGI4-V217R, MGI4-V217W, MGI4-D260E, MGI4-F276G, MGI4-24, MGI4-25, MGI4-34 and MGI4-35 as shown in Table 2.
These glucose isomerase mutants possess high catalytic activity. For example, in a series of glucose isomerase mutants obtained according to this invention, a seven-mutation mutant MGI4-34 possesses a specific activity 769% higher than that of wild-type and still has 50% or more of the original activity after heat treatment at 80° C. for 26 hours. Another seven-mutation mutant MGI4-35 possesses a specific activity 727% higher than that of wild-type, and still has 50% or more of the original activity after heat treatment at 80° C. for 27 hours.
The glucose isomerase mutants with high catalytic activity or high catalytic activity and thermostability obtained according to this invention can be used for direct production of high fructose corn syrup containing 55 wt. % or higher concentration of fructose, or for the production of high fructose corn syrup containing less than 55 wt. % fructose. The described glucose isomerase mutant can be used for the production of crystallized fructose with fructose content of 90 wt. % or higher.
The glucose isomerase mutants can be used in an unpurified crude enzyme form, or in partially purified enzyme form, or as completely purified enzyme preparation. If required, the glucose isomerase mutants can be prepared as immobilized enzyme, or immobilized cells using known immobilization methodologies.
DEFINITIONS
Term “wild-type” as used herein refers to the glucose isomerase from Thermoanaerobacterium saccharolyticum ATCC 49915, with its DNA sequence as Sequence 1 in the Sequence Listing, with its amino acid sequence as Sequence 2 in the Sequence Listing. The DNA sequence of the wild-type glucose isomerase in this invention is different in two nucleotides from the published DNA sequence of glucose isomerase from the same species (Lee et al., Journal of General Microbiology, 139:1227-1234, 1993; GenBank L09699); namely, the nucleotides of the wild-type glucose isomerase in this invention at position 241-242 are GC, coding alanine (Ala) at position 81; while the corresponding nucleotides in GenBank L09699 are CG, coding arginine (Arg) at position 81.
Term “reference sequence” as used herein refers to Sequence 1 in The Sequence Listing when it is a DNA sequence; or Sequence 2 in The Sequence Listing when it is an amino acid sequence. The alignment of the reference sequence and the sequences of the glucose isomerase mutants can be done manually or by computer (e.g. using computer softwares CLUSTALW, AMAS, DIALIGN, etc.).
Term “position” or “position x”, where x is a numeral, as used herein refers to the position of the nucleotide or amino acid of the mutant sequence in the corresponding reference sequence when the alignment between the glucose isomerase mutants of the present invention and the wild-type glucose isomerase reaches maximum in homology.
Term “glucose isomerase mutant” as used herein refers to an enzyme that using Sequence 2 in the Sequence Listing as the reference sequence, comprises the following mutations: phenylalanine (Phe) at position 139, alanine (Ala) at position 182, serine (Ser) at position 187, and glutamine (Gln) at position 299, and contains at least one mutation at position 87, position 217, position 260, or position 276, and has a catalytic activity higher than that of wild-type glucose isomerase in the reaction of fructose generation using D-glucose as substrate. Therefore, in the present invention, the mentioned glucose isomerase mutants include mutants with an amino acid sequence that is the same as Sequence 4 in the Sequence Listing, or is a conservative substitution of Sequence 4, or is Sequence 4 with addition or deletion of one or several amino acids, or is Sequence 4 with amino terminal or carboxyl terminal deletion, or comprises partial or complete repetition of Sequence 4.
IUPAC nomenclature and symbolism for amino acid abbreviations are used in the present invention (European Journal of Biochemistry, 138:9-37, 1984).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the thermal stability of the wild-type glucose isomerase and glucose isomerase mutants MGI4-34 and MGI4-35 containing seven mutations, at 80° C. Details are described in Example 12.



DESCRIPTION OF THE PREFERRED EMBODIMENT
The examples presented below are for illustration of the invention only and are not intended to be regarded as the limitation of the invention. In the following examples, conventional practice or manufacturers' suggestion/protocol was followed in cases where the conditions were not specified.
Example 1
Amplification of Wild-Type Glucose Isomerase and Construction of pGEMT-TS
Primers T1 and T2 (Table 1) were designed based on the sequence of GenBank L09699 and used to amplify the wild-type glucose isomerase gene from T. saccharolyticum ATCC 49915 (ATCC, USA).
The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1, 400 nM primer T2, 1.5 U Taq DNA polymerase (Promega, USA), a loopful of T. saccharolyticum colony, and the total volume was adjusted to 50 μl with sterile distilled water.
The PCR amplification program for the reaction was: 95° C., 3 min; then 40 cycles of 95° C., 50 sec, 50° C., 30 sec, 72° C., 1 min; and finally 72° C., 10 min. The amplified PCR product, about 1.5 kb in length, was ligated into vector pGEMT-Easy to generate pGEMT-TS. The pGEMT-TS was sequenced to determine the DNA sequence of the wild-type glucose isomerase as Sequence 1 in the Sequence Listing and the corresponding amino acid sequence as Sequence 2 in the Sequence Listing. The DNA sequence of the wild-type glucose isomerase is different from that of the published DNA sequence of a glucose isomerase from the same species (GenBank L09699) where the nucleotides of the wild-type glucose isomerase in this invention at position 241-242 are GC, coding alanine (Ala) at the amino acid position 81; while the corresponding nucleotides in GenBank L09699 are CG, coding arginine (Arg) at the amino acid position 81.
Example 2
Site-Directed Mutagenesis of Trp139 of Wild-Type Glucose Isomerase
The site directed mutagenesis was done as 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.
With pGEMT-TS (Example 1) as template, the Trp (W) at position 139 of the wild-type glucose isomerase was mutated to Phe (F) to generate glucose isomerase mutant MGI-W139F by PCR amplification using primers 139FF and 139FR (Table 1) and universal primers T1 and T2 (Example 1).
Fragment T1FR was amplified using primer pair T1 and 139FR. Fragment FFT2 was amplified using primer pair 139FF and T2. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 139FR (for fragment T1FR) or 400 nM primer T2 and 400 nM primer 139FF (for fragment FFT2), 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pGEMT-TS, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragment T1FR and fragment FFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit (QIAGEN, German). The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1FR and 20 ng fragment FFT2, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI-W139F was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MG-W139F, generated after ligation of MGI-W139F into pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/l ampicillin. Plasmid pGEMT-MGI-W139F DNA was then isolated from the positive clones and sequenced.
Example 3
Site-Directed Mutagenesis of Arg182 of Glucose Isomerase
The site directed mutagenesis was done as 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.
Using pGEMT-TS (Example 1) as template, the Arg (R) at position 182 of the wild-type glucose isomerase was mutated to Ala (A) to generate glucose isomerase mutant MGI-R182A by PCR amplification with site-directed primers 182AF and 182AR (Table 1) and universal primers T1 and T2 (Example 1).
Fragment T1AR was amplified using primer pair T1 and 182AR. Fragment AFT2 was amplified using primer pair 182AF and T2. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 182AR or 400 nM primer T2 and 400 nM primer 182AF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragment T1AR and fragment AFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1AR and 20 ng fragment AFT2, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI-R182A was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI-R182A, generated after ligation of MGI-R182A into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/l ampicillin. Plasmid pGE-MT-MGT-R182A DNA was then isolated from the positive clones and sequenced.
Example 4
Site-Directed Mutagenesis of Phe 187 of Glucose Isomerase
The site directed mutagenesis was done as 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.
Using pGEMT-TS (Example 1) as template, the Phe (F) at position 187 of the wild-type glucose isomerase was mutated to Ser (S) to generate glucose isomerase mutant MGI-F187S by PCR amplification with site-directed primers 187SF and 187SR (Table 1) and universal primers T1 and T2 (Example 1).
Fragment T1SR was amplified using primer pair T1 and 187SR. Fragment SFT2 was amplified using primer pair 187SF and T2. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 187SR or 400 nM primer T2 and 400 mM primer 187SF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragment T1SR and fragment SFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1SR and 20 ng fragment SFT2, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI-F187S was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI-F187S, generated after ligation of MGI-F187S into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI-F187S DNA was then isolated from the positive clones and sequenced.
Example 5
Site-Directed Mutagenesis of Thr299 of Glucose Isomerase
The site directed mutagenesis was done as 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.
Using pGEMT-TS (Example 1) as template, the Thr (T) at position 299 of the wild-type glucose isomerase was mutated to Gln (Q) to generate glucose isomerase mutant MGI-T299Q by PCR amplification with site-directed primers 299QF and 299QR (Table 1) and universal primers T1 and T2 (Table 1).
Fragment T1QR was amplified using primer pair T1 and 299QR. Fragment QFT2 was amplified using primer pair 299QF and T2. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 299QR or 400 nM primer T2 and 400 nM primer 299QF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragment T1QR and fragment QFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 M dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1QR and 20 ng fragment QFT2, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI-T299Q was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI-T299Q, generated after ligation of MGI-T299Q into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI-T299Q DNA was then isolated from the positive clones and sequenced.
Example 6
Generation of Glucose Isomerase Mutant MGI-4 Containing Four Mutations
The site directed mutagenesis was done as 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.
Fragments T1FR and QFT2 were amplified and recovered in accordance with Examples 2 and 5, respectively. Fragment FFAR was amplified using primer pair 139FF (Table 1) and 182AR (Table 2) on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 139FF and 400 nM primer 182AR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR product, fragment FFAR, was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Fragment AFSR was amplified using primers 182AF and 187SR (Table 1) at the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 182AF and 400 nM primer 187SR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and firmly 72° C., 5 min. The fragment AFSR was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Fragment SFQR was amplified using primers 187SF and 299QR (Table 1) at the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 187SF and 400 nM primer 299QR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The fragment SFQR was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1FR and 20 ng fragment FFAR, 20 ng fragment AFSR, 20 ng fragment SFQR and 20 ng fragment QFT2 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI-4 was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI-4, generated after ligation of MGI-4 into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI-4 DNA was then isolated from the positive clones and sequenced. The sequence of the MGI-4 contains four mutations including W139F, R182A, F187S and T299Q.
Example 7
Generation of Glucose Isomerase Mutants MGI4-F87L and MGI4-F87M Containing Five Mutations
The site directed mutagenesis was done as 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.
Using pGEMT-MGI-4 (Example 6) as template, the Phe (F) at position 87 of the MGI-4 was mutated to Leu (L), generating mutant MGI4-F87L by PCR amplification with site-directed primers 87LF and 87LR (Table 1) and universal primers T1 and T2 (Table 1).
Fragments T1LR and LFT2 were amplified by primer pairs of T1 and 87LR, and 87LF and T2 respectively on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 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, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragments T1LR and LFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit (QIAGEN, German). The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1LR and 20 ng fragment LFT2 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI4-F87L was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI4-F87L, generated after ligation of MGI4-F87L into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI4-F87L DNA was then isolated from the positive clones and sequenced. MGI4-F87L sequence contains five mutations of F87L, W139F, R182A, F187S and T299Q. Amino acid sequence of MGI4-F87L is shown as Sequence 5 in the Sequence Listing.
MGI4-F87M was constructed using similar procedures. The primers used are shown in Table 1. The mutant MGI4-F87M contains five mutations of F87M, W139F, R182A, F187S and T299Q. Amino acid sequence of MGI4-F87M is shown as Sequence 6 in the Sequence Listing.
Example 8
Generation of Glucose Isomerase Mutants MGI4-V217R and MGI4-V217W Containing Five Mutations
The site directed mutagenesis was done as 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.
Using pGEMT-MGI-4 (Example 6) as template, the Val (V) at position 217 of the MGI-4 was mutated to Arg (R), generating mutant MGI4-V217R by PCR amplification with site-directed primers 217RF and 217RR (Table 1) and universal primers T1 and T2 (Table 1).
Fragments T1RR and RFT2 were amplified by primer pairs of T1 and 217RR, and 217RF and T2 respectively on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μL dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 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, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragments T1RR and RFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1RR and 20 ng fragment RFT2 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI4-V217R was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI4-V217R, generated after ligation of MGI4-V217R into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI4-V217R DNA was then isolated from the positive clones and sequenced. The sequence of the mutant contains five mutations of W139F, R182A, F187S, V217R and T299Q. Amino acid sequence of MGI4-V217R is shown as Sequence 7 in the Sequence Listing.
MGI4-V217W was constructed using similar procedures. The primers used are shown in Table 1. The mutant MGI4-V217W contains five mutations of W139F, R182A, F187S, V217W and T299Q. Amino acid sequence of MGI4-V217R is shown as Sequence 8 in the Sequence Listing.
Example 9
Generation of Glucose Isomerase Mutant MGI4-D260E Containing Five Mutations
The site directed mutagenesis was done as 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.
Using pGFMT-MGI-4 (Example 6) as template, the Asp (D) at position 260 of the MGI-4 was mutated to Glu (E), generating mutant MGI4-D260E by PCR amplification with site-directed primers 260EF and 260ER (Table 1) and universal primers T1 and T2 (Table 1).
Fragments T1ER and EFT2 were amplified by primer pairs of T1 and 260ER, and 260EF and T2 respectively on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 260ER or 400 mM primer 260EF and 400 mM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragments T1ER and EFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 mM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1ER and 20 ng fragment EFT2 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI4-D260E was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI4-D260E, generated after ligation of MGI4-D260E into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI4-D260E DNA was then isolated from the positive clones and sequenced. The sequence of the mutant contains five mutations of W139F, R182A, F187S, D260E and T299Q. Amino acid sequence of MGI4-D260E is shown as Sequence 9 in the Sequence Listing.
Example 10
Generation of Glucose Isomerase Mutant MGI4-F276G Containing Five Mutations
The site directed mutagenesis was done as 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.
Using pGEMT-MGI-4 (Example 6) as template, the Phe (F) at position 276 of the MGI-4 was mutated to Gly (G), generating mutant MGI4-F276G by PCR amplification with site-directed primers 276GF and 276GR (Table 1) and universal primers T1 and T2 (Table 1).
Fragments T1GR and GFT2 were amplified by primer pairs of T1 and 276GR, and 276GF and T2 respectively on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 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, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR products, fragments T1GR and GFT2, were separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was then assembled on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1GR and 20 ng fragment GFT2 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutant MGI4-F276G was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit, Plasmid pGEMT-MGI4-F276G, generated after ligation of MGI4-F276G into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI4-F276G DNA was then isolated from the positive clones and sequenced. The sequence of the mutant contains five mutations of W139F, R182A, F1875, F276G and T299Q. Amino acid sequence of MGI4-F276G is shown as Sequence 10 in the Sequence Listing.
Example 11
Generation of Glucose Isomerase Mutants MGI4-24 and MGI4-25 Containing Six Mutations
The site directed mutagenesis was done as 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.
Fragment T1LR was amplified and recovered as in Example 7. Fragment LFAR was amplified with primers pair 87LF and 260AR (Table 1) and recovered. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 87LF and 400 nM primer 260AR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR product, fragment LFAR, was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Fragment AFT2 was amplified with primer pair of 260AF and T2 (Table 1) on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 260AF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The fragment AFT2 was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full length glucose isomerase gene was amplified at the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1LR, 20 ng fragment LFAR and 20 ng fragment AFT2, the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutated gene MGI4-24 was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI4-24, generated after ligation of MGI4-24 into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI4-24 DNA was then isolated from the positive clones and sequenced. Amino acid sequence of MGI4-24 is shown as Sequence 11 in the Sequence Listing. The sequence of the mutant contains six mutations of F87L, W139F, R182A, F187S, D260A and T299Q.
MGI4-25 was constructed using similar procedures. The primer pairs T1 and 87LR, 87LF and 276TR, 276TF and T2 used are shown in Table 1. The mutant contains six mutations of F87L, W139F, R182A, F187S, F276T and T299Q. Amino acid sequence of the mutant is shown as Sequence 12 in the Sequence Listing.
Example 12
Generation of Glucose Isomerase Mutants MGI4-34 and MGI4-35 Containing Seven Mutations
The site directed mutagenesis was done as 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.
Fragment T1LR was amplified and recovered as in Example 7. Fragment LFGR was amplified with primers pair 87LF and 217GR (Table 1) and recovered. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 87LF and 400 nM primer 217GR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The PCR product, fragment LFGR, was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Amplification of fragment GFTR was carried out on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 217GF and 400 nM primer 276TR, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4 and the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The fragment GFTR was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Fragment TFT2 was amplified with primer pair 276TF an T2 (Table 1) on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer 276TF and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-MGI-4, the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The fragment TFT2 was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. The full-length glucose isomerase gene was amplified on the following condition: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer T2, 1.5 U Pfu DNA polymerase, 20 ng fragment T1LR, 20 ng fragment LFGR, 20 ng fragment GFTR and 20 ng fragment TFT2, the total volume was adjusted to 50 μl with sterile distilled water. The PCR amplification program for the reaction was: 95° C., 3 min; then 35 cycles of 95° C., 50 sec, 52° C., 30 sec, 72° C., 3 min; and finally 72° C., 5 min. The full-length mutated gene MGI4-34 was separated on 1% agarose gel and recovered using QIAquick Gel Extraction Kit. Plasmid pGEMT-MGI4-34, generated after ligation of MGI4-34 into vector pGEMT-Easy, was transformed into competent E. coli HB101 and the transformants were screened for glucose isomerase activity on 1% MacConkey plates containing 1% D-xylose and 50 mg/L ampicillin. Plasmid pGEMT-MGI4-34 DNA was then isolated from the positive clones and sequenced. The sequence of the mutant contains seven mutations of F87L, W139F, R182A, F187S, V217G, F276T and T299Q. Amino acid sequence of MGI4-34 is shown as Sequence 13 in the Sequence Listing.
MGI4-35 was constructed using similar procedures. The primer pairs T1 and 87LR, 87LF and 217GR, 217GF and 260AR, 260AF and T2 used are shown in Table 1. The mutant contains seven mutations of F87L, W139F, R182A, F187S, V217G, D260A and T299Q. Amino acid sequence of the mutant is shown as Sequence 14 in the Sequence Listing.








TABLE 1







The Primers Used for Amplification of



Wild-type Glucose Isomerase and the


Mutants in Examples 1-12








Product
Primer Pair













Wild-type
T1:
5′AGCCTAGGTTAATTAACTTTAAG





AAGGAGATATACATATGAATAAATA




TTTTGAGA 3′


 



T2:
5′ATAAGCTCAGCGGCGCGCCTTAT




TCTGCAAACAAATAC 3′


 


Mutant MGI-W139F
139FF:
5′AAAAGTTTTGTTTGGTACCGCAA




ATCTTTTCTC 3′



139FR:
5′TTGCGGTACCAAACAAAACTTTT




GTCTTGCTGG 3′


 


Mutant MGI-R182A
182AF:
5′GGAGCTTGGCGCGGAAAACTACG




TATTTTGGGG 3′



182AR:
5′CGTAGTTTTCCGCGCCAAGCTCC




TTAGTAATCT 3′


 


Mutant MGI-F187S
187SF:
5′ACTACGTAAGCTGGGGTGGAAGA




GAAGGGT 3′



187SR:
5′CCACCCCAGCTTACGTAGTTTTC




GCGGCCA 3′


 


Mutant MGI-T299Q
299QF:
5′TGACGCAAATCAAGGCGACATGC




TTTTGGGATG 3′



299QR:
5′GCATGTCGCCTTGATTTGCGTCG




ATTGATCCTA 3′


 


Mutant MGI4-F87L
87LF:
5′GAAGCAGCACTGGAGTTTTTTGA




TAAGATAA 3′



87LR:
5′AAAAACTCCAGTGCTGCTTCTAC




CCTTGCTTTC 3′


 


Mutant MGI4-F87M
87MF:
5′GAAGCAGCAATGGAGTTTTTTGA




TAAGATAA 3′



87MR:
5′AAAAACTCCATTGCTGCTTCTAC




CCTTGCTTTC 3′


 


Mutant MGI4-V217G
217GF:
5′ACATGGCTGGCGACTATGCAAAG




GAAATCG 3′



217GR:
5′GCATAGTCGCCAGCCATGTGCAA




AAATCTT 3′


 


Mutant MGI4-V217W
217WF:
5′ACATGGCTTGGGACTATGCAAAG




GAAATCG 3′



217WR:
5′GCATAGTCCCAAGCCATGTGCAA




AAATCTT 3′


 


Mutant MGI4-V217T
217TF:
5′ACATGGCTACCGACTATGCAAAG




GAAATCG 3′



217TR:
5′GCATAGTCGGTAGCCATGTGCAA




AAATCTT 3′


 


Mutant MGI4-D260E
260EF:
5′ACGACCTTGAAAAATATTTCAAA




GTAAATA 3′



260ER:
5′AAATATTTTTCAAGGTCGTATTT




TCTCAAG 3′


 


Mutant MGI4-D260A
260AF:
5′ACGACCTTGCGAAATATTTCAAA




GTAAATA 3′



260AR:
5′AAATATTTCGCAAGGTCGTATTT




TCTCAAG 3′


 


Mutant MGI4-F276G
276GF:
5′ACATTGGCAGGCCACGACTTCCA




ACATGAGC 3′



276GR:
5′GAAGTCGTGGCCTGCCAATGTCG




CATGGTTT3′


 


Mutant MGI4-F276T
276TF:
5′ACATTGGCAACCCACGACTTCCA




ACATGAGC 3′



276TR:
5′GAAGTCGTGGGTTGCCAATGTCG




CATGGTTT 3′









Example 13
Isolation and Purification of Wild-Type Glucose Isomerase
The isolation and purification of wild-type glucose isomerase were carried out in accordance with Lee et al., Journal of General Microbiology, 139:1227-1234, 1993.
Plasmid pGEMT-TS transformed E. coli HB101 cells were incubated on MacConkey plate containing 1% D-xylose and 50 mg/L ampicillin at 37° C. for 36 hours. A single colony from the plate was inoculated and cultivated in 5 ml LB supplemented with 50 mg/L ampicillin for 16 hours. The bacterial cells were pelleted, resuspended in 1 ml 20 nM sodium phosphate buffer (pH 6.5), added CoCl2 and MgCl2 to final concentrations of 250 μM and 5 mM respectively, disrupted using ultrasonication and centrifuged at 17,800 g for 15 min at 10° C. to collect the supernatant as crude protein. The crude protein was heated at 80° C. for 10 min and centrifuged at 17,800 g for 15 min at 10° C. to remove the precipitate. The resultant partially purified glucose isomerase was used in the subsequent assays and for preparation of high fructose corn syrup.
Example 14
Isolation and Purification of Glucose Isomerase Mutants
The isolation and purification of glucose isomerase mutant MGI4-35 were carried out as described in Example 13, except the plasmid used was pGEMT-MGI4-35. Other glucose isomerase mutants were also isolated and purified as described in Example 13.
Example 15
Activity Assay of Wild-Type Glucose Isomerase with D-Glucose as Substrate
Stock substrate solution A containing 1.0 M D-glucose, 20 mM sodium phosphate buffer, 250 μM CoCl2 and 5 mM MgCl2, pH 6.5 was prepared, Ninety μl of the stock substrate solution A were mixed with 10 μl of the glucose isomerase prepared as described in Examples 1 and 13, incubated at 80° C. for 10 min and quenched on ice immediately. The D-fructose formed was measured by the cysteine-carbazole method (Dische et al., Journal of Biological Chemistry, 192:583-587, 1951; and Nakamura, Agricultural and Biological Chemistry, 32:701-706, 1968). Protein concentration was determined using Coomassie® Plus Protein Assay Reagent Kit (Pierce, USA) and SDS-PAGE. One unit of enzyme activity is defined as the amount of enzyme that is used to produce 1 μmole of fructose from D-glucose per min under the assay condition. Table 2 below shows the relative specific activity of wild-type glucose isomerase.
Example 16
Activity Assay of Glucose Isomerase Mutants
The activity of glucose isomerase mutant was measured as described in Example 15. Table 2 below shows the comparison of the relative specific activities of wild-type glucose isomerase and the mutants.







TABLE 2







The Specific Activities of Wild-type Glucose Isomerase and the Mutants










SEQ ID NO. of the



Enzyme
Amino Acid Sequence
Relative Specific Activity





Wild-type
SEQ ID NO.: 2 
100


MGI4-F87L
SEQ ID NO.: 5 
271


MGI4-F87M
SEQ ID NO.: 6 
346


MGI4-V217R
SEQ ID NO.: 7 
609


MGI4-V217W
SEQ ID NO.: 8 
151


MGI4-D260E
SEQ ID NO.: 9 
560


MGI4-F276G
SEQ ID NO.: 10
365


MGI4-24
SEQ ID NO.: 11
501


MGI4-25
SEQ ID NO.: 12
652


MGI4-34
SEQ ID NO.: 13
869


MGI4-35
SEQ ID NO.: 14
827









Example 17
Thermostability of Wild-Type Glucose Isomerase
Two hundred μl of the partially purified wild-type glucose isomerase obtained as described in Example 13 were added to each of four microfuge tubes, and overlaid with 200 μl mineral oil. The tubes were placed in an 80° C. water bath. One of the four tubes was removed from the water bath at a time interval of 0 h, 2 h, 6 h and 27 h, and centrifuged at 17,800 g for 20 min at 10° C. The residual protein and the glucose isomerase activity of the supernatants were determined as described in Example 15. FIG. 1 shows the thermostability of wild-type glucose isomerase at 80° C.
Example 18
Thermostability of Glucose Isomerase Mutants
The thermostability of glucose isomerase mutants MGI4-34 or MGI4-35 (see Examples 12 and 14) was measured as described in Example 17 and was shown in FIG. 1. As shown in FIG. 1, the half-life of the activity of wild-type glucose isomerase at 80° C. was 4.1 hours, that of MGI4-34 was 26 hours and that of MGI4-35 was greater than 27 hours.
Example 19
Immobilization of Glucose Isomerase Mutant MGI4-35
The immobilization procedure was carried out in accordance with Ge et al., Applied Biochemistry and Biotechnology, 69:17-29, 1998. Briefly, 100 g of immobilization carrier (ethylamine polystyrene hydrochloride particles, provided by Chengdu Institute of Chemical Engineering), were mixed with 8 g of the partially purified glucose isomerase mutant MGI4-35 prepared as described in Examples 12 and 14 in 1 L of 10 mM phosphate buffer (pH 8.0) and stirred (60-120 rpm/mm) at room temperature (22.degree. C.) for 18 hours. The resultant immobilized enzyme was collected by filtration and washed with water three times to obtain 107 g immobilized enzyme. The activity of the immobilized enzyme measured as described in Example 16 using 0.01 g of the immobilized enzyme, was 820 units/g.
Example 20
Immobilization of E. coli Cells Carrying Glucose Isomerase Mutant MGI4-35
E. coli HB101 cells carrying pGEMT-MGI4-35 were grown in LB broth containing 50 mg/L ampicillin to OD600 of 7. Ten g of the cells, collected by centrifugation, were mixed well with 20 g of 3% sodium alginate, squeezed through a needle of 0.5 mm in diameter into 500 ml of 2% CaCl2 solution. The mixture was allowed to react for 1 hour at room temperature and washed three times by soaking in distilled water for half an hour each. The resultant immobilized cells of approximate 30 g were measured for glucose isomerase activity as described in Example 16 using 0.01 g of the immobilized cells. The activity was 370 units/g.
This invention is not limited by the detailed description in the Examples above. Various modifications can be made by those skilled in the art without departing from the scope of the invention.

## Claims

1. An isolated glucose isomerase mutant comprising the amino acid sequence of SEQ ID NO: 2, wherein the amino acid at position 139 is mutated to phenylalanine (Phe), the amino acid at position 182 is mutated to alanine (Ala), the amino acid at position 187 is mutated to serine (Ser), the amino acid at position 299 is mutated to glutamine (Gln) and the glucose isomerase mutant has at least one mutation at position 87, position 217, position 260, or position 276, and the glucose isomerase mutant has a higher catalytic activity than that of a wild-type glucose isomerase when using D-glucose as substrate.

2. The glucose isomerase mutant according to claim 1, wherein, the amino acid at position 87 is mutated to methionine (Met) or leucine (Leu).

3. The glucose isomerase mutant according to claim 1, wherein, the amino acid at position 217 is mutated to arginine (Arg), tryptophan (Trp) or glycine (Gly).

4. The glucose isomerase mutant according to claim 1, wherein, the amino acid at position 260 is mutated to glutamic acid (Glu) or alanine (Ala).

5. The glucose isomerase mutant according to claim 1, wherein, the amino acid at position 276 is glycine (Gly) or threonine (Thr).

6. The glucose isomerase mutant according to claim 1, wherein, the glucose isomerase mutant further comprises any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 14.