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    STUDIA BIOLOGIA - Issue no. 1 / 2021  
         
  Article:   SUBCLONING AND EXPRESSION OF RECOMBINANT GLUCOSE-DEHYDROGENASE FROM BACILLUS SUBTILIS.

Authors:  ANDREA POPA, SEBASTIAN EMANUEL GHERMAN, IULIA LUPAN.
 
       
         
  Abstract:  
Published Online: 2021-06-30
Published Print: 2021-06-30
pp. 46-47

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ABSTRACT: Glucose-dehydrogenase (GDH) is an enzyme that catalyzes the oxidation of glucose to gluconate, reducing NAD(P)+ to NAD(P)H + H+. This enzyme is of particular interest due to the fact that it can be used to regenerate the NAD cofactor using a cheap substrate such as glucose. The aim of this study was the fusion of recombinant GDH gene with the His-Tag at the N-terminus. For this purpose, the recombinant GDH gene was subcloned into the pET28a expression vector and expressed in E. coli BL21(DE3) cells. The recombinant protein was expressed both in soluble form (10% of total proteins) and in inclusion bodies. The recombinant GDH was purified by Ni-agarose affinity chromatography and tested for enzymatic activity (glucose and NAD). In conclusion, after subcloning the ORF did not change, the protein was fused with His-tag, and this fusion did not affect the activity or solubility of the enzyme. The results obtained in this study may be used to optimize glucose-dehydrogenase production so that it would increase its biotechnological importance.

Key words: cofactor regeneration; glucose-dehydrogenase; recombina┬Čtion; subcloning; vector
 
         
     
         
         
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