Proizvodnja i karakterizacija alfa-amilaze bakterije Lactobacillus amylovorus DSM 20531T

master's thesis

• Title (english): Production and characterization of alfa -amylase produced by bacterium Lactobacillus amylovorus DSM 20531T
• Author: Deni Kostelac
• 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: 2016-07-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Amilolitička bakterija mliječne kiseline Lactobacillus amylovorus DSM 20531T uzgojena je modificiranoj MRS podlozi, u koju je umjesto glukoze dodana maltoza (20,0 g L-1). Uzgoj u MRS podlozi s maltozom (MRS-mal20) proveden je u laboratorijskom bioreaktoru s miješalom (VK = 6,0 L) pri konstantnoj temperaturi (40±0,2°C) i uz održavanje pH vrijednosti suspenzije u bioreaktoru (pH 5,5±0,2). L. amylovorus DSM 20531T odmah nakon nacjepljivanja počinje transportirati maltozu, hidrolizira je i razgrađuje isključivo do mliječne kiseline. Istovremeno s razgradnjom maltoze i homofermentativnom proizvodnjom mliječne kiseline odvija se sinteza biomase (eksponencijalni rast) i to sve do t = 6,0 h bioprocesa, kada bakterija ulazi u stacionarnu fazu rasta. Nakon toga se usporavaju potrošnja maltoze, sinteza bakterijske biomase i proizvodnja mliječne kiseline. Do kraja bioprocesa (t = 24,0 h) koncentracija maltoze je opala do 6,95 g L-1 i proizvedeno je 11,34 g L-1 mliječne kiseline tako da je koeficijent konverzije maltoze u mliječnu kiselinu (YP/S) iznosio samo 0,87 g g-1. Soj L. amylovorus DSM 20531T sintetizira α-amilazu i transportira je izvan stanice. Izolacija α-amilaze provedena je afinitetnom kromatografijom i to iz supernatanta uzoraka izuzetih pri kraju eksponencijalne i stacionarne faze rasta. Propuštanjem otopine α-ciklodekstrina kroz kolonu eluirane su i separirane na baznoj liniji različite frakcije α-amilaze. SDS-PAGE metodom utvrđeno je da se frakcija 1 (F1) sastoji od nekoliko proteinskih vrpci koje imaju molekulsku masu <30,0 kDa.

Abstract (english)

Cultivation of amylolytic lactic acid bacterium Lactobacillus amylovorus DSM 20531T was conducted in modified MRS medium in which maltose (20.0 g L-1) was added instead of glucose. The cultivation of the bacterium in MRS medium with maltose (MRS-mal20) was carried out in a laboratory scale stirred tank bioreactor (working volume of 6.0 L) at constant temperature (40±0.2°C) and maintained pH value (pH of 5.5±0.2). Transport of maltose into bacterial cells started immediately after inoculation, transported maltose was hydrolysed and further degraded to lactic acid exclusively. Simultaneously with maltose utilization and lactic acid production bacterial biomass was synthesised (exponential growth) until t = 6.0 h of bioprocess, when bacterium entered stationary growth phase. After this time point utilization of maltose, bacterial biomass synthesis and lactic acid production were slowed down. Until the end of the bioprocess (t = 24.0 h) concentration of maltose decreased to 6.95 g L-1 and 11.34 g L-1 of lactic acid was produced. Resulting substrate to product conversion coefficient (YP/S) was only 0.87 g g-1. Strain L. amylovorus DSM 20531T can synthesise α-amylase and transport it to extracellular space. Isolation of the α-amylase was done by affinity chromatography. Supernatants of samples withdrawn at the end of exponentially and stationary growth phases were used in the isolation. Different α-amylase fractions were eluted and base-line separated by α-cyclodextrin solution. Fraction 1 (F1) consisted of several protein bands with molecular weight <30.0 kDa, as determined by SDSPAGE.

• Keywords: maltoza; Lactobacillus amylovorus; α-amilaza; afinitetna kromatografija; SDS-PAGE.
• Keywords (english): maltose; Lactobacillus amylovorus; α-amylase; affinity chromatography; SDS-PAGE
• Language: croatian
• URN:NBN: urn:nbn:hr:159:290022
• 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: 2017-04-25 08:39:40