Permeabilizacija stanica bakterije Leuconostoc mesenteroides i njihova primjena u proizvodnji alfa-glukozilglicerola

master's thesis

• Title (english): Permeabilization of cells of bacterium Leuconostoc mesenteroides and their application in production of alfa-glucosylglycerol
• Author: Ivan Vučenović
• Mentor: Anita Slavica (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: 2018-07-19, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Gram-pozitivna bakterija mliječne kiseline Leuconostoc mesenteroides znatno bolje raste u kemijski djelomično definiranoj podlozi pri aerobnim ( = 0,24 h-1) nego mikroaerofilnim uvjetima ( = 0,02 h-1). Pri aerobnim uvjetima ova bakterija doseže polovinu eksponencijalne faze rasta kroz 5,0 h, kada je koncentracija i aktivnost saharoza fosforilaze, citoplazmatskog enzima koji katalizira transglukozilaciju, maksimalna. U reakcijskoj smjesi u kojoj je istraživana transglukozilacijska aktivnost saharoza fosforilaze, saharoza je donor glukozila, dok je glicerol akceptor glukozila. Međutim, saharoza i glicerol su supstrati za centralni metabolizam u stanicama ove heterofermentativne bakterije mliječne kiseline s pomoću koje je proizvedena samo mliječna kiselina, dok ostali krajnji proizvodi razgradnje ovih supstrata nisu identificirani HPLC metodom. Primjena ultrazvuka i površinski aktivne tvari Tween-80 pokazale su se kao dobre metode, kojima je moguće permeabilizirati bakterijske stanice ali i utjecati na aktivnost ove bakterije. Nakon provedenih postupaka permeabilizacije stanice se dalje eksponencijalno umnažaju i zadržavaju svoju metaboličku aktivnost. U reakcijskoj smjesi s pomoću permeabiliziranih stanica bakterije L. mesenteroides formira se α-glukozilglicerol, koji se eluira između saharoze i glukoze tijekom HPLC analize uzoraka. Nastavak ovog istraživanja uključuje izolaciju proizvedenog proizvoda i njegovu identifikaciju i kvantifikaciju npr. nuklearnom magnetnom rezonancijom.

Abstract (english)

Gram-positive lactic acid bacterium Leuconostoc mesenteroides grew significantly better in chemically semi-defined medium under aerobic ( = 0.24 h-1) than under microaerophilic conditions ( = 0.02 h-1). Under aerobic conditions this bacterium reached half of the exponential growth phase over 5.0 h when concentration and activity of sucrose phosphorylase, cytoplazmatic enzyme that catalyzes transglucosylation reaction, should be maximal. In reaction mixture for the transglucosylation reaction catalyzed by the sucrose phosporylase, sucrose serves as glucosyl donor while glycerol is glucosyl acceptor. However, sucrose and glycerol are also substrates in central metabolism of this heterofermentative lactic acid bacterium that produced only lactic acid, while other end-products of the two substrates degradation were not identified by HPLC method. Application of ultrasound and surfactant Tween-80 are methods of choice and both methods can be used for cell permeabilization but also in directing metabolic activity of the bacterium. After permeabilization the cells continued to grew exponentially and they retained their metabolic activity. In reaction mixture formation of α- glucosylglycerol was catalyzed by the permeabilized cells of the bacterium L. mesenteroides and formed product eluted between sucrose and glucose during HPLC analysis of withdrawn samples. Next steps in this investigation should include isolation of produced α-glucosylglycerol and its unambiguous identification and quantification by e.g. nuclear magnetic resonance.

• Keywords: heterofermentativna bakterija mliječne kiseline; Leuconostoc mesenteroides; permeabilizacija; mliječna kiselina; transglukozilacija
• Keywords (english): heterofermentative lactic acid bacterium; Leuconostoc mesenteroides; permeabilization; lactic acid; transglucosylation
• Language: croatian
• URN:NBN: urn:nbn:hr:159:382166
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-11-26 13:47:49