Izolacija amilaze amilolitičke bakterije mliječne kiseline Lactobacillus amylovorus DSM 20531T ion-izmjenjivačkom kromatografijom

master's thesis

• Title (english): Isolation of amylase produced by amylolytic lactic acid bacterium Lactobacillus amylovorus DSM 20531T by using ion-exchange chromatography
• Author: Ivona Jakša
• 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: 2017-07-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Amilolitička bakterija mliječne kiseline Lactobacillus amylovorus DSM 20531T uzgojena je u MRS podlozi s maltozom kao supstratom u laboratorijskom bioreaktoru s miješalom pri konstantnoj temperaturi, pH vrijednosti suspenzije i broju okretaja miješala. Iz eksperimentalnih podataka procijenjeni biokinetički parametri za rast biomase i proizvodnju mliječne kiseline s pomoću ovog bakterijskog soja jasno pokazuju da je L. amylovorus DSM 20531T izvrstan kandidat za industrijsku proizvodnju ove kiseline. Maltoza inducira proizvodnju vanstanične amilaze u relativno visokim koncentracijama i zadovoljavajuće aktivnosti za provođenje izolacije i pročišćavanja ovog enzima. Proizvedena amilaza je izdvojena taloženjem amonijevim sulfatom i to pri 25, 50 i 75% zasićenja. Većina aktivne amilaze izdvojena je pri 75% zasićenja otopine amonijevim sulfatom, pa su ovako dobiveni peleti korišteni za daljnje pročišćavanje izdvojenog enzima. Pročišćavanje resuspendirane amilaze provedeno je ion-izmjenjivačkom kromatografijom. Od ukupno pet proteinskih pikova odijeljenih tijekom eluiranja proteina s ion-izmjenjivačke kolone pomoću otopine natrijeva klorida, samo tri proteinske frakcije imaju amilolitičku aktivnost. Najmanju specifičnu aktivnost ima proteinska frakcija eluirana pri 0,5 mol L-1 NaCl (47 U mg-1), dok veću specifičnu aktivnost imaju proteinske frakcije eluirane pri 0,4 (62 U mg-1) i 0,6 mol L-1 natrijevog klorida u puferu (263 U mg-1). Sve tri ion-izmjenjivačkom kromatografijom izdvojene frakcije amilaze sadrže nekoliko proteinskih vrpci, kako je to utvrđeno SDS-PAGE metodom, i to molekulskih masa između 50 i 94 kDa.

Abstract (english)

Cultivation of an amylolytic lactic acid bacterium Lactobacillus amylovorus DSM 20531T was conducted in a MRS medium with a maltose as a substrate in a laboratory scale stirred tank bioreactor at a constant temperature, pH value of suspension and a stirrer speed. Biokinetic parameters that were estimated by using experimentally obtained values for the growth and lactic acid production by this bacterial strain clearly shows that L. amylovorus DSM 20531T is an excellent candidate for an industrial production of the acid. Maltose induces production of extracellular amylase at relatively high concentrations and satisfying activity that are suitable for isolation and purification of the enzyme. Produced amylase was precipitated by ammonium sulfate at a final saturation of 25, 50 and 75%. Majority of active amylase was isolated at the final saturation of ammonium sulfate of 75% and, therefore, obtained pelets were used in further purification of the enzyme. Further purification of resuspended enzyme was accomplished by an ion-exchange chromatography. Three out of five protein peaks eluted from the column by using sodium chloride possessed amylolytic activity. The lowest activity was determined for the protein fraction eluted by 0,5 mol L-1 of sodium chloride (47 U mg-1), while higher activity was determined for protein fractions eluted by 0,4 (62 U mg-1) and 0,6 mol L-1 of sodium chloride in buffer (263 U mg-1). All three active protein fractions contain several protein bands with molecular weight between 50 and 94 kDa, as revealed by SDS-PAGE.

• Keywords: amilolitička bakterija mliječne kiseline; Lactobacillus amylovorus; indukcija amilaze; taloženje proteina; ion-izmjenjivačka kromatografija
• Keywords (english): amylolytic lactic acid bacterium; Lactobacillus amylovorus; amylase induction; protein precipitation; ion-exchange chromatography
• Language: croatian
• URN:NBN: urn:nbn:hr:159:509965
• 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: 2017-11-07 11:06:05