WO1990000196A1 - Mutants d'isomerase de xylose - Google Patents

Mutants d'isomerase de xylose Download PDF

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Publication number
WO1990000196A1
WO1990000196A1 PCT/GB1989/000748 GB8900748W WO9000196A1 WO 1990000196 A1 WO1990000196 A1 WO 1990000196A1 GB 8900748 W GB8900748 W GB 8900748W WO 9000196 A1 WO9000196 A1 WO 9000196A1
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lys
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PCT/GB1989/000748
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David Mervyn Blow
Brian Selby Hartley
Kim Henrick
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David Mervyn Blow
Brian Selby Hartley
Kim Henrick
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Application filed by David Mervyn Blow, Brian Selby Hartley, Kim Henrick filed Critical David Mervyn Blow
Publication of WO1990000196A1 publication Critical patent/WO1990000196A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)

Definitions

  • This invention relates to mutants of the enzyme xylose isomerase which is an industrial enzyme used in the industrial conversion of glucose syrups obtained from corn or other starches or by hydrolysis of
  • ketol-isomerase A review of the industrial uses of xylose isomerase appears in the reference Wen-Pin Chen, "Process Biochemistry” August/September 1980, pages 36 to 41 and C. Burke in Microbial Enzymes and
  • xylose isomerase mutants which have improved activity under acidic pH conditions and/or increased thermal stability (with resulting increase in the enzyme life times when used at current processing temperatures of from 60 to 65°C) and/ or affinity for metal ions.
  • Xylose isomerase occurs in numerous bacterial species but, although the enzyme action is similar throughout, there are variations in the composition of the protein and the complex molecular structure of the various forms of the enzyme. For example, in some variants the protein chains are longer than in others, some have dimeric protein chains and others are
  • xylose isomerase One major industrial use of xylose isomerase is in the food industry for conversion of glucose to the sweeter fructose in the form of
  • high-fructose-containing-syrups which has a market for caloric sweeteners used in solution , principally carbonated beverages.
  • the enzyme is derived from the bacterial species of Streptomyces. Bacillus, Lactobacillus,
  • a further problem in the industrial use of natural xylose isomerases is the fact that this enzyme is dependent on the presence of magnesium ion (and also cobalt(II) ion in some species) as an activator. All the natural xylose isomeiocses are inhibited by calcium ion which is present in starch feedstocks.
  • Calcium ions are required for the enzymic activity of alpha- amylase, which is used at about 100°C, pH 6 to 7, to convert starch to dextrose syrups in a batch process.
  • the second step also in batch, converts the resulting soluble dextrins to glucose, catalysed by the enzyme glucoamylase at 60°C and pH 5.5.
  • the final glucose isomerisation is generally carried out at 60oC and pH 7 to 8 on columns of immobilised enzyme because the xylose isomerase is relatively costly and can
  • a further disadvantage of the conventional xylose isomerase step is the pH and temperature at which it is performed. If catalysed to equilibrium a glucose syrup would yield a mixture of 46.5% glucose and 53.5%
  • fructose is much sweeter than glucose, the latter product would be more desirable.
  • the constraints for an 80°C process of the xylose isomerases in current use are that undesirable "browning" reactions occur when sugars are heated at alkaline pH.
  • the operational pH for xylose isomerase is incompatible with other enzymes used in the industrial processes, such as glucoamylase.
  • An object of the present invention is to obviate or mitigate the aforesaid disadvantages.
  • a xylose isomerase mutant in which at least one of the following mutations (Table I) in the naturally occurring protein sequence of the xylose isomerase, using the residue numbering system hereinafter defined, has been made:
  • the xylose isomerase is that derived from Arthobacter or from Streptomyces. Bacillus.
  • Lactobacillus Lactobacillus, Ampullariella and or Arthrobacter.
  • an Arthrobacter B3728 xylose isomerase having one or more of the following (Table II) mutations:
  • Figure 1 shows the structure of the active site bound Mg 2+ and the inhibitor sorbitol (sorbitol is an acyclic analogue of the open chain structure of glucose which is the chemical configuration upon which the enzyme acts). It is probable that protonation of His219 is responsible for the reduction of activity at lower pH. It has been demonstrated in the case of the protease subtilisin that elimination of a negative charge lying 15 Angstroms away from the catalytic site reduces the pKa of the active site histidine in this enzyme by 0.6 pH units. The mutations of this
  • Figure 3 shows the specific activity of erizyme (0.7 mg/ml) incubated for one hour at 30°C in 10mM
  • Glucose isomerase assays were conducted on 20 microlitre samples at 30oC and pH8 in the presence (o) or absence (o) of urea at the relevant concentration. The bars show % of monomer (inactive) or dimer-tetramer equilibrium mixture (fully active) calculated from Fig. 2b below.
  • Figure: 4 shows the elution profiles of the above samples separated at 22oC on DHEAE-Sephacel columns (9.5 cm x 0.7 cm diameter) equilibrated with
  • urea-buffers of the same composition as the samples and a gradient from zero to 0.5M sodium chloride. So as to compensate for variability in column packing, the
  • Figure 5 shows tracings of "gradient-urea-" run (top to bottom) at 40 mV for 14 hours at pH8.5.
  • the top gel is stained for total protein and the bottom gel for both protein and xylose isomerase activity.
  • the change in mobility between 2M and 5M is consistent with a rapid reversibly equilibrium between tetramers and dimers that retain activity.
  • furanose ring can be bound to the enzyme at the
  • the amino acid residue numbering sequence used herein is based on the sequence of xylose isomerase derived from Arthrobacter strain B3728 and is shown in Fig. 7 herewith.
  • the complete amino acid sequence of this particular enzyme is shown along with complete or partial sequences of xylose isomerases from other bacterial sources.
  • the sequences of the various enzymes have been aligned with the Arthrobacter B3728 enzyme sequence by aligning the highly conserved regions of the sequences in which the desired mutations are most likely to be made.
  • This diagram may be used to identify the locations in the sequences of enzymes from sources other than the Arthrobacter species for which at least a partial sequence, which includes some of the conserved regions, corresponding to the rim of the barrel region of the enzyme structure, is known.
  • nucleotide sequence of the Xy1A gene of Arthrobacter strain B3728 shown in Figure 8 herewith.
  • the invention also provides the nucleotide
  • the invention provides an
  • oligonucleotide comprising a fragment of the sequence shown in Figure 8 and containing at least one mutation.
  • the present invention also provides a host/vector expression system containing one of the mutant genes of this invention.
  • the host may be E. coli or, more preferably, a yeast, preferably Saccharomyces cerevisiae.
  • the conventional strategy for cloning xylose isomerase from Arthrobacter would be to determine a partial amino acid sequence of the purified enzyme, construction of redundant oligo probes and the use of radioactiovely labelled probes to screen a gene library consisting of a partial Sau 3A digest of Arthrobacter ligated into the Bam HI site of pBR322.
  • this conventional strategy failed to yield the gene of interest: instead it gave a gene which showed some sequence homology to the xylose isomerase gene but was much smaller.
  • strain PCI contained a lesion in its gene for xylose isomerase but not in its xylulokinase (Xy1 B) gene or in its inducibility by xylose as shown by the enzyme activities in crude extracts shown in Table IV below.
  • Cell-free extracts were prepared according to Smith (1980). Protein concentration in the extracts was determined by the Bio-Rad (Trade Mark) assay kit.
  • the plasmid is named pAXIl. Site directed mutagenesis; can be carried out on this fragment by standard methods in M13 phage (Zoller M. J. and Smith M. Methods Enzymol. 100, 468 - 500, 1983) or by ligating it into the Sma I site in plasmid pTZ19U (D.A. Mead, E. Szczensna-Skopura and B. Kemper, Protein Engineering. 1, 67 -74, 1986). This allows hybridisation with oligonucleotide primers to be carried out directly on the single strand form of the plasmid, according to the method of these authors.
  • the primers are designed to produce codon changes resulting in the amino acid replacements shown in Table I.
  • the following mutant genes have been constructed via the synthetic oligonucleotides listed, and the expected codon changes have been confirmed by DNA sequencing of the plasmid:-
  • Oligonucleotide GCC.TTC.ATC.GAC.CGT.CTG.GAG.CAC.
  • Oligonucleotide C.GGG.CGC.AAG.GGC.AGC.G
  • Oligonucleotide TTC.GTC.ATG.GAA.GGC.CCC.C
  • Oligonucleotide A.CGC.GGC.AAG.ATC.TTC.C
  • pAXI2 contains the sequence shown in Fig 8 ligated into site pTZ19U. N.G.
  • the enzyme for industrial use would be produced in E.coli. since this would be doubtfully food-compatible. It is preferable that the mutant enzyme be substituted in
  • Arthrobacter Strain B3728 for the natural Xy1 A and used in an immobilised enzyme process is achieved by a technique of 'gene disruption' using the shuttle vector pCG2100 which has been constructed for this purpose.
  • the properties of the improved enzyme of this invention are such that it is admirably suited to system in which it is expressed in yeast.
  • Yeast is of course food compatible and there exist many vectors for Saccharomyces cerevisiae that allow high expression of foreign proteins. Since the yeast can be produced inexpensively and since its functions will be destroyed by the high-temperature (80oC) of the target progress, the cells will have xylose isomerase activity as their sole enzymic function.
  • isomerases listed in Table 1 in a batch process or in a continuous immobilised cell process is an important embodiment of this invention.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
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  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
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  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des mutants d'isomérase de xylose ayant une activité améliorée dans des conditions de pH acide et/ou une meilleure stabilité thermique. On peut en outre accroître l'affinité pour des ions métal.
PCT/GB1989/000748 1988-07-04 1989-07-04 Mutants d'isomerase de xylose WO1990000196A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8815902.5 1988-07-04
GB888815902A GB8815902D0 (en) 1988-07-04 1988-07-04 Xylose isomerase mutants

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0436502A2 (fr) * 1990-01-04 1991-07-10 Genencor International, Inc. Glucose isomérases avec un optimum de pH changé
EP0440273A3 (en) * 1990-01-04 1992-01-22 Gist-Brocades N.V. Novel glucose isomerases having altered substrate specificity
WO1992007069A1 (fr) * 1990-10-19 1992-04-30 Novo Nordisk A/S Nouvelle enzyme d'isomerisation
US5266475A (en) * 1991-09-19 1993-11-30 Michigan Biotechnology Institute Glucose isomerases with improved affinity for D-glucose
US5268280A (en) * 1990-10-19 1993-12-07 Novo Nordisk A/S Method for glucose isomerization using xylose isomerase purified from Thermotoga Maritima and Thermotoga Neapolitana
WO2000061733A1 (fr) * 1999-04-09 2000-10-19 Forskarpatent I Syd Ab Xylose isomerase presentant des proprietes ameliorees
EP1578937A2 (fr) * 2002-11-06 2005-09-28 Diversa Corporation Xylose isomerases, acides nucleiques les codant et leur methodes de fabrication et d'utilisation
CN102443578A (zh) * 2011-12-08 2012-05-09 江南大学 一种葡萄糖异构酶突变体及其应用
US9951326B2 (en) 2015-07-13 2018-04-24 MARA Renewables Corporation Enhancing microbial metabolism of C5 organic carbon
CN108034649A (zh) * 2018-01-04 2018-05-15 浙江工业大学 一种葡萄糖异构酶突变体及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4410627A (en) * 1982-06-30 1983-10-18 Nabisco Brands, Inc. Glucose isomerase process
WO1989001520A1 (fr) * 1987-08-11 1989-02-23 Cetus Corporation Muteines de xylose isomerase procaryotique et procede d'accroissement de la stabilite proteique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4410627A (en) * 1982-06-30 1983-10-18 Nabisco Brands, Inc. Glucose isomerase process
WO1989001520A1 (fr) * 1987-08-11 1989-02-23 Cetus Corporation Muteines de xylose isomerase procaryotique et procede d'accroissement de la stabilite proteique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Protein Engineering, Volume 1, No. 3, April 1987, P.-C. SHAW et al.: "Protein Engineering of Arthrobacter Glucose Isomerase", page 264 see Abstract No. 154 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5384257A (en) * 1990-01-04 1995-01-24 Gist-Brocades, N.V. Glucose isomerases with an altered pH optimum
US5310665A (en) * 1990-01-04 1994-05-10 Gist-Brocades, N.V. Glucose isomerases having altered substrate specificity
EP0436502A2 (fr) * 1990-01-04 1991-07-10 Genencor International, Inc. Glucose isomérases avec un optimum de pH changé
AU642688B2 (en) * 1990-01-04 1993-10-28 Genencor International, Inc. Novel glucose isomerases with an altered PH optimum
EP0440273A3 (en) * 1990-01-04 1992-01-22 Gist-Brocades N.V. Novel glucose isomerases having altered substrate specificity
EP1264883A3 (fr) * 1990-01-04 2002-12-18 Genencor International, Inc. Glucose isomerases avec une spécificité changée pour le substrat
US5340738A (en) * 1990-01-04 1994-08-23 Gist-Brocades, N.V. Modified prokaryotic glucose isomerase enzymes with altered pH activity profiles
EP1264883A2 (fr) * 1990-01-04 2002-12-11 Genencor International, Inc. Glucose isomerases avec une spécificité changée pour le substrat
EP0436502A3 (en) * 1990-01-04 1992-03-18 Gist-Brocades N.V. Novel glucose isomerases with an altered ph optimum
WO1992007069A1 (fr) * 1990-10-19 1992-04-30 Novo Nordisk A/S Nouvelle enzyme d'isomerisation
US5219751A (en) * 1990-10-19 1993-06-15 Novo Nordisk A/S Novo Alle, Xylase isomerase purified from thermotoga maritima and thermotoga neapolitana
US5268280A (en) * 1990-10-19 1993-12-07 Novo Nordisk A/S Method for glucose isomerization using xylose isomerase purified from Thermotoga Maritima and Thermotoga Neapolitana
US5266475A (en) * 1991-09-19 1993-11-30 Michigan Biotechnology Institute Glucose isomerases with improved affinity for D-glucose
US6475768B1 (en) 1999-04-09 2002-11-05 Forskarpatent I Syd Ab Xylose isomerase with improved properties
WO2000061733A1 (fr) * 1999-04-09 2000-10-19 Forskarpatent I Syd Ab Xylose isomerase presentant des proprietes ameliorees
EP1578937A2 (fr) * 2002-11-06 2005-09-28 Diversa Corporation Xylose isomerases, acides nucleiques les codant et leur methodes de fabrication et d'utilisation
EP1578937A4 (fr) * 2002-11-06 2008-07-02 Verenium Corp Xylose isomerases, acides nucleiques les codant et leur methodes de fabrication et d'utilisation
CN102443578A (zh) * 2011-12-08 2012-05-09 江南大学 一种葡萄糖异构酶突变体及其应用
US9951326B2 (en) 2015-07-13 2018-04-24 MARA Renewables Corporation Enhancing microbial metabolism of C5 organic carbon
US10662418B2 (en) 2015-07-13 2020-05-26 MARA Renewables Corporation Enhancing microbial metabolism of C5 organic carbon
CN108034649B (zh) * 2018-01-04 2020-08-21 浙江工业大学 一种葡萄糖异构酶突变体及其应用
CN108034649A (zh) * 2018-01-04 2018-05-15 浙江工业大学 一种葡萄糖异构酶突变体及其应用

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