Improvement of β-Glucosidase Activity by Amino Acid Mutations in Active Sites

Nalçaoğlu A., Karaoğlan M., Salah Zade N., Güler H. İ., İnan Bektaş K., Beldüz A. O.

Molbiyokon'22 8th International COngress of the Molecular Biology Association of Turkey, İstanbul, Turkey, 9 - 12 June 2022, pp.202-203

  • Publication Type: Conference Paper / Summary Text
  • City: İstanbul
  • Country: Turkey
  • Page Numbers: pp.202-203
  • Karadeniz Technical University Affiliated: Yes


Background/aim: β-Glucosidases are important members of the family of glycoside hydrolases that can de-glycosylate many polyphenolic compounds. β-Glucosidases are found in all living organisms and can be used in many fields. This enzyme have implementations in animal feeds, and bioprocesses such as bioethanol and pharmaceutical production and as a bioflavourings in the food industry. There are mutational studies to increase the activity of the enzyme, which is used in various fields. For example, mutations in conserved residues of glucose-sensitive β-glucosidase from Bgl1317 resulted in enhanced glucose tolerance. The aim of this study is to facilitate the use of this enzyme in industrial areas with the mutations of amino acids in its active sites.
Materials and methods: The β-glucosidase gene (bgl) from Anoxybacillus ayderensis A9 was amplified using specific primers for recombinant enzyme. The PCR product was digested and ligated into pET28(+) and the recombinant plasmid pET28(+)/bgl was transformed into E. coli BL21. The sequence of the gene was confirmed by DNA sequencing. The E.coli BL21 cells containing pET-28a-bgl were grown at OD600=0.8. A final concentration of 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to induce the enzyme expression. The recombinant protein was purified by Ni2+NTA affinity chromatography. β-Glucosidase activity was assayed using pNPG as substrate. Enzymatic assays, determination of kinetic parameters and glucose tolerance were performed.
Results: T220A/L221S/N222S/G226Q/V275A mutations were created in β-glucosidase enzyme. The kinetic parameter values using pNPG as substrate were Km(wildtype) (0.28 mM), Km(mutant)(0,26 mM) Vmax(wildtype) (43.8 μmol/min/mg),Vmax(mutant) (61,5 μmol/min/mg) kcat(wildtype) (38.43 s−1) kcat(mutant) (53.9 s−1) and kcat/Km(wildtype) (135.5 s−1mM−1), kcat/Km(wildtype)(208.7s−1mM−1). The comparisons of characterization results of wild-type and mutant enzymes were revealed that the optimum pH level of the mutant was decreased (from pH 8 to pH 6), and there were changes in their interaction with solvents and metals. Its optimum temperature and interactions with detergents and chemicals were found to be compatible with wild type.
Conclusion: It is aimed to facilitate the use of β-glucosidase with mutation studies, which is applied in various industrial fields. The kinetic parameters of the enzyme have improved changes were obtained with specific mutations. In addition, as a result of enzyme characterization, changes in solvent and detergent parameters were observed. The results obtained will be evaluated and different improvements will be made with different mutations.