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

Abstract

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. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50 °C, respectively. Activation energy (Ea) 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 Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (Km) of ECXI-BL21 for D-glucose was calculated as 0.82 mM, while maximum velocity (Vmax) 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, Tm = 75 °C, has great potential for high fructose syrup production used in food and soft drink industries.

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Acknowledgements

Provision of laboratory facilities by University of Veterinary and Animal Sciences (UVAS) Lahore and Institute of Public Health (IPH) Lahore to undertake this research is gratefully acknowledged.

Author information

MMJ conceived and supervised the study. BF designed and performed the experiments, analysed the data and wrote the manuscript. Both authors contributed to the discussion and revision of the manuscript.

Correspondence to Bilqees Fatima.

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The authors declare no conflict of interest related to this article.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Fatima, B., Javed, M.M. Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21. 3 Biotech 10, 39 (2020). https://doi.org/10.1007/s13205-019-2036-6

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Keywords

  • Escherichia coli
  • Electro-elution
  • Kinetics
  • Thin-layer chromatography
  • D-glucose isomerase
  • Inducers