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Pridobivanje glikozid hidrolaze 10 iz metagenoma komposta in ugotavljanje njenih lastnosti
ID Zupančič, Viktor (Author), ID Vodovnik, Maša (Mentor) More about this mentor... This link opens in a new window

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Abstract
Metagenomski pristop iskanja novih biotehnološko zanimivih encimov temelji na pregledu celokupne DNA kompleksnih okoljskih vzorcev, čemur sledi kloniranje in izražanje tarčnih genov v dobro poznanih in z vidika gojenja nezahtevnih ekspresijskih sevih. Med iskane encime uvrščamo tudi glikozid hidrolaze, ki razgrajujejo kompleksne rastlinske polisaharide. V magistrski nalogi smo se osredotočili na izolacijo in karakterizacijo produkta izbranega gena (xyl6) z zapisom za domnevno glikozid hidrolazo iz družine 10 (GH10), identificiranega v metagenomu komposta. Izolirali smo encim z molekulsko maso približno 40 kDa, z aktivnostjo na substratih bukovem ksilanu in arabinoksilanu. Temperaturni optimum delovanja izoliranega encima na bukovem ksilanu v 50 mM K-fosfatnem pufru s pH 8 je pri temperaturi 50 °C. Ugotovili smo, da kovinski ioni Co2+, Fe3+, Mn2+ ali Ca2+ v koncentracijah 5 mmol/L povečajo aktivnost preučevanega encima, v koncentracijah 10 mmol/L pa jo zmanjšajo. Aktivnost encima popolnoma inhibirajo ioni Cu2+ v koncentraciji 10 mmol/L. Aktivnost tarčnega encima se zmanjša tudi v prisotnosti NaCl v koncentracijah nad 100 mmol/L, ob prisotnosti 10 % (v/v) metanola, etanola ali acetona ter 20 mM EDTA ali SDS. Prav tako encim ni stabilen pri temperaturah nad 50 °C ter v kislih pogojih (pod pH 6). Povečano aktivnost preučevanega encima smo zaznali v prisotnosti reducentov, 2 mM DTT ali 10 mM ß-merkaptoetanola. Ugotavljali smo tudi kinetične parametre tarčnega encima pri optimalnih pogojih delovanja na bukovem ksilanu in ocenili vrednosti Vmax na 787,2 ± 12,9 μmol/min/mg, Km na 1,8 ± 0,3 mg/mL in kcat na 526,2 ± 17,4 s-1. Pokazali smo tudi, da je ksilanaza Xyl6 sposobna razgraditi tudi s hemicelulozo bogate materiale, kot so pšenični ali ječmenovi otrobi, zaradi česa je potencialno biotehnološko zanimiv encim.

Language:Slovenian
Keywords:encimi, glikozid hidrolaza, GH10, ksilanaza, heterologno izražanje, biokemijska karakterizacija encimov, ksilan, metagenom komposta
Work type:Master's thesis/paper (mb22)
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2019
Publisher:[V. Zupančič]
UDC:604.4:577.152.3:575.112
COBISS.SI-ID:5041784 This link opens in a new window
Publication date in RUL:04.04.2019
Views:848
Downloads:57
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Secondary language

Language:English
Title:Production and characterization of novel glycoside hydrolase 10 from compost metagenome
Abstract:
Metagenomic approach seeks novel enzymes beneficial to microbial biotechnology through the screening of metagenomes from complex environments, with crucial steps: cloning and the expression of selected genes in unpretentious bacterial or other types of strains. Glycoside hydrolases degrade complex plant polysaccharides and belong to a group of one of the most demanded industrially relevant enzymes. In this thesis we focused on isolation and characterization of putative GH10 identified in compost metagenome (xyl6). We successfully isolated xylanase with molecular mass approx. 40 kDa and activity against beechwood xylan and arabinoxylan. Recombinant xylanase revealed optimal xylanolytic activity against beechwood xylan at 50 °C and pH 8. Xylanolytic activity of Xyl6 increased in the presence of 5 mM and decreased in the presence of 10 mM Co2+, Fe3+, Mn2+ or Ca2+. The latter concentration of Cu2+ completely inhibited the enzyme activity. Enzyme activity also decreased when exposed to NaCl above 100 mmol/L. Exposure to 10 % (v/v) methanol, ethanol or acetone, as well as 20 mM EDTA or SDS was shown to decrease xylanolytic activity of Xyl6. Furthermore, enzyme was not stable at a temperature above 50 °C or in acidic conditions (below pH 6). Xylanolytic activity increased when exposed to reducing agents such as 2 mM DTT or 10 mM ß-mercaptoethanol. The recombinant xylanase exhibited Km and Vmax values of 1,8 ± 0,3 mg/mL and 787,2 ± 12,9 μmol/min/mg for beechwood xylan at optimal conditions, and turnover number (kcat) value of 526,2 ± 17,4 s-1. Enzyme also proved successful in the degradation of natural hemicellulose-rich substrates, such as wheat or barley bran, thus has great potential for future use in industrial applications.

Keywords:enzymes, glycoside hydrolase, GH10, xylanase, heterologous expression, biochemical characterization of enzymes, xylan, compost metagenome

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