Purification, characterization and immobilization of glucose isomerase from Streptomyces albaduncus

BACKGROUND Aldo‐keto isomerizations of D‐xylose and D‐glucose catalyzed by xylose isomerase (XI) is commercially carried out to produce bio‐ethanol and high fructose corn syrup. XI is a metalloenzyme. In this paper, kinetics and thermodynamics of the activity and stability of hyperthermophilic recombinant XI from Thermotoga naphthophila RKU‐10T (TnapXI) were studied in the presence and absence of metal ions. RESULTS Kinetics parameters of recombinant XI from TnapXI were calculated as Km = 0.99 mmol L–1, Vmax = 500 μmol.mg−1.min−1, and kcat = 68.3 min−1 for the isomerization of D‐xylose(aldo)⇌D‐xylulose(keto), whereas for D‐glucose(aldo)⇌D‐fructose(keto) conversion Km = 7.72 mmol L–1, Vmax = 90 μmol.mg−1.min−1, and kcat = 12.4 min−1. Ionization constants were calculated as pKa1 = 6.0 and pKa2 = 7.6, whereas activation constants (Ka) of TnapXI were 0.3, 0.5, and 4.0 mmol L–1 for Co2+, Mn2+, and Mg2+, respectively. Inhibition constant (Ki) was 0.119 mmol L–1 for Ca2+. Q10 was 2.8 and Ea = 82.25 kJ.mol−1. Thermodynamics for isomerization were calculated as ∆H* = 79.2 kJ.mol−1, ∆G* = 83 kJ.mol−1, and ∆S* = −10.5 J.mol−1.K−1. The z‐value was 12.6 °C for apo‐TnapXI and 32.6 °C for holo‐TnapXI. Deactivation parameters were ∆G*(d) = 100 kJ.mol−1, ∆H*(d) = 206 kJ.mol−1, ∆S*(d) = 0.28 kJ.mol−1.K−1 for apo‐TnapXI and t1/2 = 18 min at 95 °C, while ∆G*(d) = 104 kJ.mol−1, ∆H*(d) = 767 kJ.mol−1, ∆S*(d) = 1.8 kJ.mol−1.K−1 for holo‐TnapXI and t1/2 = 65 min at 95 °C. CONCLUSION These noteworthy features make this enzyme an attractive candidate for the industrial production of fuel ethanol and high fructose corn syrup at high temperatures to maximize the product yield. © 2021 Society of Chemical Industry (SCI).

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Kinetics and thermodynamics for aldo/keto conversion of D‐xylose and D‐glucose catalyzed by xylose isomerase from Thermotoga naphthophilaRKU‐10

Fatima

Aftab

Haq

2021

J of Chemical Tech & Biotech

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Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU‐10

Fatima

Aftab

Haq

2016

J. Basic Microbiol.

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A 1.3 kb xyl-A gene encoding xylose isomerase from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10 (TnapXI) was cloned and over-expressed in Escherichia coli to produce the enzyme in mesophilic conditions that work at high temperature. The enzyme was concentrated by lyophilization and purified by heat treatment, fractional precipitation, and UNOsphere Q anion-exchange column chromatography to homogeneity level. The apparent molecular mass was estimated by SDS-PAGE to be 49.5 kDa. The active enzyme showed a clear zone on Native-PAGE when stained with 2, 3, 5-triphenyltetrazolium chloride. The optimum temperature and pH for D-glucose to D-fructose isomerization were 98 °C and 7.0, respectively. Xylose isomerase retains 85% of its activity at 50 °C (t1/2 1732 min) for 4 h and 32.5% at 90 °C (t1/2 58 min) for 2 h. It retains 90-95% of its activity at pH 6.5-7.5 for 30 min. The enzyme was highly activated (350%) with the addition of 0.5 mM Co(2+) and to a lesser extent about 180 and 80% with the addition of 5 and 10 mM Mn(2+) and Mg(2+) , respectively but it was inhibited (54-90%) in the presence of 0.5-10 mM Ca(2+) with respect to apo-enzyme. D-glucose isomerization product was also analyzed by Thin Layer Chromatography (Rf 0.65). The enzyme was very stable at neutral pH and sufficiently high temperature and required only a trace amount of Co(2+) for its optimal activity and stability. Overall, 52.2% conversion of D-glucose to D-fructose was achieved by TnapXI. Thus, it has a great potential for industrial applications.

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Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21

Fatima

Javed

2020

3 Biotech

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Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9 kDa on SDS-PAGE. pH opt. and T opt. of the enzyme were calculated as 7.0 and 50 °C, respectively. Activation energy (E a ) of ECXI-BL21 was 45 kJ/mol. Enzyme was stable from 30 to 55 °C and at pH range 6.0-8.0. ECXI-BL21 (holo) was activated by 10 mM magnesium (35%), 0.5 mM cobalt (20%) and manganese (25%), and 0.5/10 mM Mn 2+ /Mg 2+ (50%) and Co 2+ /Mg 2+ (30%) as compared to ECXI-BL21 (apo) . Catalytic affinity (K m ) of ECXI-BL21 for D-glucose was calculated as 0.82 mM, while maximum velocity (V max ) of the reaction D-glucose (aldo) ⇌ D-fructose (keto) was 108 μmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, T m = 75 °C, has great potential for high fructose syrup production used in food and soft drink industries.

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Purification, characterization and immobilization of glucose isomerase from Streptomyces albaduncus

Cited by 3 publications

References 23 publications

Kinetics and thermodynamics for aldo/keto conversion of D‐xylose and D‐glucose catalyzed by xylose isomerase from Thermotoga naphthophilaRKU‐10

Kinetics and thermodynamics for aldo/keto conversion of D‐xylose and D‐glucose catalyzed by xylose isomerase from Thermotoga naphthophilaRKU‐10

Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU‐10

Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21

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