Production and characterization of different variants of Myriococcum thermophilum cellobiose dehydrogenase with the aim of improving cofactor binding and kinetic stability

master's thesis

• Title (croatian): Proizvodnja i karakterizacija mutanata Myriococcum thermophilum celobioza dehidrogenaze s ciljem povećanja vezanja kofaktora i kinetičke stabilnosti
• Author: Mario Ilić
• Mentor: Tonči Rezić (mentor)
• Committee member: Blaženka Kos (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2022-02-04, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Biosensors are being extensively used in the healthcare sector but mostly in blood glucose monitoring for early diagnosis of diabetes. The use of enzymes as biological recognition elements is hampered in many ways, e.g., by insufficient communication between enzyme and electrode, low activity and stability. Cellobiose dehydrogenase (CDH) is a flavocytochrome enzyme that in native wild-type form shows efficient direct electron transfer (DET). To alter FAD cofactor binding and kinetic stability, mutations were introduced into the Myriococcum thermophilum CDH gene by site-directed mutagenesis. Variants were expressed in X-33 Pichia pastoris cells, purified and characterized. With an increased specific activity, catalytic efficiency, heme reduction rate and increased melting temperatures in pH range 5.0-8.0 the T306I variant seems to be the most promising variant for further investigation.

Abstract (croatian)

Biosenzori se uvelike koriste u zdravstvenom sektoru, a najveću primjenu pronalaze u praćenju i kontroli šećera glukoze oboljelih od dijabetesa. Uporaba enzima u biosenzorima je ograničena nedovoljno efikasnim direktnim prijenosom elektrona na elektrodu, niskom specifičnom aktivnosti i stabilnosti kroz duži vremenski period. Celobioza dehidrogenaza (CDH) je flavocitokrom koji u svojoj izvornoj formi ima mogućnost direktnog prijenosa elektrona (DET). S ciljem povećanja vezanja FAD koenzima i kinetičke aktivnosti, uvedene su mutacije u CDH gen izoliran iz askomicete Myriococcum thermophilum. Enzimi proizvedeni u soju kvasca Pichia pastoris X-33 su pročišćeni i karakterizirani. Mutant T306I pokazuje zadovoljavajuća svojstva (povećanu specifičnu aktivnost, kinetičku efikasnost, brzinu redukcije hema i temperaturu prijelaza) u području pH vrijednosti 5,0-8,0, stoga je potrebno provesti dodatna istraživanja korištenjem mutanta T306I.

• Keywords: biosensors; Myriococcum thermophilum; cellobiose dehydrogenase; DET; kinetic stability
• Keywords (english): biosenzori; Myriococcum thermophilum; celobioza dehidrogenaza; DET; kinetička stabilnost
• Language: english
• URN:NBN: urn:nbn:hr:159:847876
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-05-27 09:12:10