Xylose isomerase is the third most commercially important enzyme used in the production of high fructose corn syrup. It catalyzes the reversible isomerization of D-xylose and D-glucose. Glucose isomerase is a tetrameric protein containing two metal binding sites and uses a hydride shift mechanism to convert glucose to fructose. It is produced commercially using various microorganisms like Bacillus, Streptomyces, and Candida species. Glucose isomerase is immobilized and used for continuous production of high fructose corn syrup from glucose through isomerization in industrial processes.

1. Presented by
Esakki Muthu Lakshmi V
M. Tech 1st yr (IBT)

2. Introduction
 Xylose isomerase
 Third most commercial enzyme
 High fructose corn syrup production
 Reversible isomerization
2

3. Enzyme Nomenclature
IUBMB
Entry
• EC 5.3.1.5
Name
• Accepted Name : D-xylose isomerase; D-xylose
ketoisomerase; D-xylose ketol-isomerase; glucose
isomerase (GI)
• Systematic Name : D-xylose aldose-ketose-isomerase
Class
• Isomerases;
Intramolecular oxidoreductases;
Interconverting aldoses and ketoses, and related compounds
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4. Physicochemical properties
 Intracellular enzyme
 Substrate specificity- broad
 Ribose, rhamnose, arabinose
 Metal ions- Mg2+, Co2+ or Mn2+
 Inhibitors
 Ag2+,Hg2+, Cu2+,Zn2+, Ni2+, Ca2+
 Xylitol, arabitol, sorbitol, mannitol,
lyxose, and Tris
 pH- 7 to 9
 Temperature- 60 to 80 ºC
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5.  Homomeric tetramer
 Molecular weight- 185 kDa
 Subunit- two domains
 Domain 1- (α/β)8
 Domain 2- α5
 2-fold symmetry
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Arthrobacter B3728
Henrick et.al 1989
Three-dimensional structure

6. TIM barrel structure
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Streptomyces rubiginosus
Ref: RCSB PDB

7. Metal ion requirement
 Monomer- 2 metal binding sites
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Bacillus coagulans (Van
Bastelaere et al., 1991)
Incubation condition T=35ºC
pH= 7
Co2+
Kd1= 4*10-8
Kd2= 4*10-7
Mn2+
Kd1= 2*10-8
Kd2= 2*10-7
Increase of affinity
Mg2+
Kd1= <10-6
Kd2= 6*10-5
Kdl = dissociation constant for the high-affinity (structural) metal site; Kd2 = dissociation
constant for the low-affinity (catalytic) metal site.

8. Active site
 His -101 and -271- E.coli isomerase
 His-54 and His-217- s.rubiginosus
 Stabilise open chain substrate (Lee et al. 1990)
 Involved in ring opening
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9. Catalytic mechanism
 proton transfer involving a cis-enediol intermediate
 hydride shift mechanism
 Proton transfer involving a cis-enediol intermediate

10. Contd..
Hydride shift mechanism
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Anion intermediate
Collyer et.al.1990

11. Enzyme Kinetics
D-Xylose;
Mg2+
Km (mM)
0.903
Kcat/Km
(M-1 S-1)
1429
Kcat (S-1)
1.29
D-Glucose;
Mg2+
Km (mM)
145
Kcat (S-1)
0.462
Kcat/Km
(M-1 S-1)
3.2
Bacillus coagulans (Van Bastelaere et al., 1991)
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12. Importance of glucose isomerase
 High fructose corn syrup (HFCS)
 Ethanol production
 Xylitol production
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13. What is HFCS?
 Glucose-fructose syrup (1:1)
 Two formulations- HFCS- 42% & HFCS-55%
HFCS production
 liquefaction of starch by α-amylase
 corn, wheat, tapioca, and rice
 saccharification of starch
 amyloglucosidase and a debranching enzyme
 isomerization of glucose by GI.
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15. Advantages of HFCS as sweetener
 1.3 times sweeter than sucrose and 1.7 times sweeter than
glucose (Bhosale et al. 1996)
 Flavor enhancement
 Cost effective
 High stability
 Low water activity
 Resist crystallisation
 fermentable
 Diabetic sweetener
 Beverage, soft drinks, dairy products, baked food
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16. Production of GI
Sources
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wheat
germ
Actinoplanes
Streptomyces
Bacillus
More than
100 species
Aspergillus
-oryzae
Candida
-utilis
&
Candida
-boidinii

17. Production of GI
 Inducer
 Xylose- expensive
 Mutant strain of b.coagulans- glucose
 Nitrogen sources
 Peptone, yeast extract, ammonium salts (b.coagulans)
 corn steep liquor, soy fluor
 pH- 7 to 8
 Temperature
 Arthrobacter, Streptomyces sp- 30ºC
 Thermophilic Bacillus sp- 50 to 60ºC
 Metal ions- Co2+, Mg2+, Mn2+
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18. Mutant development
 Mutants with following properties
 increased thermostability
 lower pH optimum
 altered metal cation preference
 shift in substrate specificity from xylose to glucose.
 Mutation of Trp139 in Clostridium thermosulfurogenes
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19. Immobilization of GI
Cell free immobilisation
 DEAE- cellulose
 Porous alumina
Whole cell immobilisation
 Physical entrapment in polymeric materials
 Chemical entrapment in membrane and cross linking
with glutaraldehyde
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20. Immobilized enzyme kinetics
Free
enzyme
(batch
reactor)
KG
0.25
mol/L
KF
0.26
mol/L
Kd
0.89
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immobilized
enzyme
(Stirred
batch
reactor)
KG
0.17
mol/L
KF
0.23
mol/L
Kd
1.25
KG, KF are michaelis constants for glucose and fructose respectively
Kd is equilibrium constant

21. Commercial producers
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Bhosale et.al.1996

22. Reference
 Bhosale, S. H., Rao, M. B., & Deshpande, V. V. (1996). Molecular and
industrial aspects of glucose isomerase. Microbiological Reviews, 60(2),
280–300.
 Collyer, C. a, & Blow, D. M. (1990). Observations of reaction intermediates
and the mechanism of aldose-ketose interconversion by D-xylose isomerase.
Proceedings of the National Academy of Sciences of the United States of
America, 87(4), 1362–1366.
 Kuyper, M., Harhangi, H., Stave, a, Winkler, a, Jetten, M., Delaat, W.,
Pronk, J. (2003). High-level functional expression of a fungal xylose
isomerase: the key to efficient ethanolic fermentation of xylose by
Saccharomyces cerevisiae FEMS Yeast Research, 4(1), 69–78.
 Seyhan Tükel, S., & Alagöz, D. (2008). Catalytic efficiency of immobilized
glucose isomerase in isomerization of glucose to fructose. Food Chemistry,
111(3), 658–662.
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23.  Sukumar, M., Jeyaseelan, A., Sivasankaran, T., Mohanraj, P., Mani, P.,
Sudhakar, G., … Susee, M. (2013). Production and partial characterization
of extracellular glucose isomerase using thermophilic Bacillus sp. isolated
from agricultural land. Biocatalysis and Agricultural Biotechnology, 2(1),
45–49.
 Hartley, B. S., Hanlon, N., Jackson, R. J., & Rangarajan, M. (2000).
Glucose isomerase : insights into protein engineering for increased
thermostability, 1543.
 Sayyed, R. Z., Shimpi, G. B., & Chincholkar, S. B. (2010). Constitutive
production of extracellular glucose isomerase by an osmophillic Aspergillus
sp. under submerged conditions. Journal of Food Science and Technology,
47(5), 496–500. http://doi.org/10.1007/s13197-010-0084-3
 Seyhan Tükel, S., & Alagöz, D. (2008). Catalytic efficiency of immobilized
glucose isomerase in isomerization of glucose to fructose. Food Chemistry,
111(3), 658–662. http://doi.org/10.1016/j.foodchem.2008.04.035
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