Usporedba različitih tehnika kultivacije za proizvodnju celobioza dehidrogenaze

master's thesis

• Title (english): Comparing different fermentation cultivation for production of cellobiose dehydrogenase
• Author: Filip Lozančić
• Mentor: Božidar Šantek (mentor)
• 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: 2014-11-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Flavocitokrom celobioza dehidrogenaze (CDH) je gljivični redoks enzim koji sudjeluje u oksidativnoj razgradnji celuloze. CDH je višenamjenski biokatalizator koji se može koristiti kao dio elektrokemijskih osjetila za laktozu i glukozu ili u biokatalizi. U prikazanom radu proučavala se proizvodnja CDH iz gljive Phanerochaete sordida, poznate basidiomicete sa sposobnostima razgradnje lignina. Opaženo je značajno izlučivanje CDH kada gljiva raste na celulozi kao jedinom izvoru ugljika. Međutim, proizvodnja i pročišćavanje je izazov zbog sporog rast gljive i niskog prijelaza enzima u sirovi supernatant (~ 2.6% w / w od ukupnog proteina). Stoga su uspoređene različite strategije uzgoja s ciljem optimizacije prinosa enzima. Istraživanje je provedeno u Erlenmayer tikvicama sa i bez odbijača. Osim toga, wave bioreaktor koji se sastoji od vre će (izgrađene od biorazgradivog materijala) postavljene na tresilici, predložen je kao alternativni sustav za kultivaciju. Rast Phanerochaete sordida u wave bioreaktoru je relativno brz, zbog brzog prijenosa kisika i minimalnih sila smicanja. CDH iz Phanerochaete sordida uspješno je pročišćen uz dva kromatografska postupka. Kinetičke konstante pokazuju izvrsnu specifičnu aktivnost prema laktozi i celobiozi, a gotovo da i nema konverzije glukoze (KM> 3 m), što čini enzim zanimljivim kandidatom za određivanje laktoze i celobioze.

Abstract (english)

The flavocytochrome cellobiose dehydrogenase (CDH) is a fungal redox enzyme involved in oxidative cellulose degradation. CDH is a versatile biocatalyst that can be used as biorecognition element in electrochemical lactose and glucose sensors or in biocatalysis. In thiswork the production of CDH from the fungus Phanerochaete sordida, a well-known basidiomycete with high lignin-degrading abilities, was studied. Significant secretion of CDH was found when the fungus was grown on cellulose as sole carbon source. However, production and purification is challenging due to the slow growth of the fungus and low abundance of the enzyme in crude supernatants (~2.6% w/w of total protein). Therefore, different cultivation strategies were examined in order to optimize enzyme yields. Research was done in conventional shaken flasks with and without baffles. Additionally, the WAVE bioreactorsystem consisting of a disposable cellbag placed on a rocking device was evaluated as alternative cultivation system for filamentous fungi. Fungal growth in the WAVE bioreactor was relatively fast because of high oxygen transfer rate and minimum shear stress for the mycelium. CDH from Phanerochaete sordida was successfully purified by a two-step chromatography procedure. Kinetic constants show excellent specific activity towards lactose and cellobiose, whereas almost no conversion of glucose is observed (KM>3 M), which makes the enzyme an interesting candidate for lactose or cellobiose biosensors.

• Keywords: celobioze dehidrogenaze; Phanerochaete sordida; WAVE bioreactor; određivanje koncentracije biomase pomoću DNA
• Keywords (english): cellobiose dehydrogenase; Phanerochaete sordida; WAVE bioreactor; DNA biomass assay
• Language: english
• URN:NBN: urn:nbn:hr:159:562552
• 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: Access restricted to students and staff of home institution
• Created on: 2017-04-26 12:51:23