Extracelulární enzymové aktivity půdních kvasinek
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Předložená práce se zabývá schopností kvasinek, izolovaných z půdy pod ovocnými stromy a z kontaminované půdy oblasti Pernek jihozápadního Slovenska, produkovat extracelulární enzymové aktivity. Celkem bylo screeningu podrobeno 68 kmenů kvasinek náležících k 45 různým druhům a testována byla schopnost produkce polygalakturonáz, lipáz, celuláz, proteáz, -glukosidáz, -amyláz a schopnost tvorby škrobu podobného polysacharidu. V rámci diplomové práce byla také provedena optimalizace metody pro stanovení chitinázové aktivity u kvasinek. Testována byla vhodnost celkem čtyř metod využívající jednak kultivaci na pevném tak v kapalném médiu. Zároveň byla prověřena vhodnost koloidního a nerozpustného chitinu pro kultivaci kvasinek. Jako nejvhodnější byla vyhodnocena kultivace kvasinek v kapalném médiu s koloidním chitinem a následná detekce chitinázové aktivity Ehrlichovým testem. Tato metoda byla posléze použita pro stanovení extracelulární chitinolytické aktivity všech 68 kmenů kvasinek. Více jak 75 % testovaných kvasinek vykazovalo alespoň jednu enzymatickou aktivitu a pouze deset kmenů bylo zcela enzymaticky neaktivních. Nejčastěji produkovanými enzymy byly extracelulární lipázy, proteázy a -glukosidázy a naopak méně častá byla produkce chitináz a celuláz. Širší spektrum enzymatických aktivit vykazovaly kvasinky izolované z půdy pod ovocnými stromy než kvasinky z kontaminovaných půd. Největší produkce extracelulárních enzymů byla pozorována u Aureobasidium pullulans, CCY 27-1-134, který vykazoval všechny testované aktivity, ale neprodukoval polysacharid podobný škrobu. Druhými největšími producenty byly druhy Cystofilobasidium macerans a Tausonia pullulans. Navíc tyto druhy vykazovaly vnitrodruhovou variabilitu v produkci lipáz a polygalakturonáz. Zástupci rodu Cryptococcus byli nejčastěji producenty lipáz, -amyláz a -glukosidáz, ale schopnost produkce jednotlivými kmeny se poměrně výrazně lišila. Pro všechny testované kmeny rodu Galactomyces byla stanovena produkce polygalakturonáz a všechny kmeny rodu Candida a Cyberlindnera produkovaly -glukosidázy. Pro všechny kmeny druhu Galactomyces candidum (CCY 16-3-2, 16-3-4, 16-3-6), Galactomyces pseudocandidus (CCY 16-3-1), Tausonia pullulans (CCY 30-1-15) a Trichosporon asahii (CCY 30-19-1) byly sestaveny aktivitní křivky produkce polygalakturonáz a růstové křivky v průběhu kultivace (168 hodin). Kmen Galactomyces candidum CCY 16-3-2 byl vyhodnocen jako nejlepší producent termostabilních polygalakturonáz, které by bylo vhodné blíže charakterizovat. Výsledky screeningu poukazují na mnohdy výrazný vnitrodruhový rozdíl v produkci enzymatických aktivit, takže pro biotechnologické využití těchto enzymů je vždy nutné vybrat a charakterizovat přímo daný produkční kmen a nelze v tomto směru očekávat shodné chování ani přes jejich prokázanou příbuznost.
Yeasts form significant and important part of pedosphere microbiota. They keep nature balance, participate in cycles of elements and nutrients, are antagonists of various pathogens and as important decomposers, they produce the whole spectrum of different extracellular enzymes. The aim of this study was to determine the ability of yeasts, isolated from the soil adjacent to the fruit trees in Southwest Slovakia as well as from the contaminated soil (Pernek area, Slovakia), to produce extracellular enzymes. In total, 68 strains belonging to 45 different species were tested for the production of starch-like polysaccharide and for extracellular enzyme activities: polygalacturonases, lipases, proteases, cellulases, chitinases, -glucosidases and -amylases. This work was also focused on optimization of method for the yeast chitinase assay. Four methods were proved; two of them utilized liquid medium with chitin (colloidal and insoluble) as the sole carbon source and two others used solid plate methods with agar medium containing chitin. Based on results, cultivation in colloidal liquid chitin medium, terminated by the chitinase assay according to Ehrlich, was evaluated as the best method for detection of predominant exochitinase activity of yeasts. More than 75 % of tested yeasts exhibited some extracellular activity. Generally, the yeasts isolated from the soil under the fruit trees showed broader spectrum of enzyme activities than those originated from contaminated soils. Lipases, proteases and -glucosidases were found to be the most common activities. Only small proportion of yeasts was able to produce chitinases and/or cellulases. Aureobasidium pullulans, CCY 27-1-134, from the soil adjacent to the apple tree, showed the widest range of activities from all tested strains and it possessed all examined activities. On the other side, it did not produce starch-like polysaccharide. Tausonia (Trichosporon) pullulans and Cystofilobasidium macerans were the second most active producers of extracellular enzymes with variations in production of cellulases and -amylases. Representatives of the former polyphyletic genus Cryptococcus exhibited lipases, -glucosidases, -amylases and they were producers of the starch, but the interspecies differences were also noted. All strains of the genus Galactomyces were positive for polygalacturonases and the genera Candida and Cyberlindnera were positive for -glucosidases. All strains of Galactomyces candidum were tested for the production of polygalacturonases during 168 hours long cultivation on pectin media. Strain CCY 16-3-4 showed very stable growth on this medium and simultaneously exhibited significant amount of extracellular polygalactouronases. It has a potential to be very suitable producer of these enzymes but particular characterization of properties is necessary for its future use. Results of the screening showed that the production of extracellular enzymes is mostly strain-dependent and not species-dependent.
Yeasts form significant and important part of pedosphere microbiota. They keep nature balance, participate in cycles of elements and nutrients, are antagonists of various pathogens and as important decomposers, they produce the whole spectrum of different extracellular enzymes. The aim of this study was to determine the ability of yeasts, isolated from the soil adjacent to the fruit trees in Southwest Slovakia as well as from the contaminated soil (Pernek area, Slovakia), to produce extracellular enzymes. In total, 68 strains belonging to 45 different species were tested for the production of starch-like polysaccharide and for extracellular enzyme activities: polygalacturonases, lipases, proteases, cellulases, chitinases, -glucosidases and -amylases. This work was also focused on optimization of method for the yeast chitinase assay. Four methods were proved; two of them utilized liquid medium with chitin (colloidal and insoluble) as the sole carbon source and two others used solid plate methods with agar medium containing chitin. Based on results, cultivation in colloidal liquid chitin medium, terminated by the chitinase assay according to Ehrlich, was evaluated as the best method for detection of predominant exochitinase activity of yeasts. More than 75 % of tested yeasts exhibited some extracellular activity. Generally, the yeasts isolated from the soil under the fruit trees showed broader spectrum of enzyme activities than those originated from contaminated soils. Lipases, proteases and -glucosidases were found to be the most common activities. Only small proportion of yeasts was able to produce chitinases and/or cellulases. Aureobasidium pullulans, CCY 27-1-134, from the soil adjacent to the apple tree, showed the widest range of activities from all tested strains and it possessed all examined activities. On the other side, it did not produce starch-like polysaccharide. Tausonia (Trichosporon) pullulans and Cystofilobasidium macerans were the second most active producers of extracellular enzymes with variations in production of cellulases and -amylases. Representatives of the former polyphyletic genus Cryptococcus exhibited lipases, -glucosidases, -amylases and they were producers of the starch, but the interspecies differences were also noted. All strains of the genus Galactomyces were positive for polygalacturonases and the genera Candida and Cyberlindnera were positive for -glucosidases. All strains of Galactomyces candidum were tested for the production of polygalacturonases during 168 hours long cultivation on pectin media. Strain CCY 16-3-4 showed very stable growth on this medium and simultaneously exhibited significant amount of extracellular polygalactouronases. It has a potential to be very suitable producer of these enzymes but particular characterization of properties is necessary for its future use. Results of the screening showed that the production of extracellular enzymes is mostly strain-dependent and not species-dependent.
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Citation
PAVLATOVSKÁ, B. Extracelulární enzymové aktivity půdních kvasinek [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2017.
Document type
Document version
Date of access to the full text
Language of document
cs
Study field
Potravinářská chemie a biotechnologie
Comittee
prof. RNDr. Ivana Márová, CSc. (předseda)
doc. RNDr. Alena Španová, CSc. (místopředseda)
doc. Ing. Bohuslav Rittich, CSc. (člen)
doc. Ing. Pavel Diviš, Ph.D. (člen)
doc. Ing. Stanislav Obruča, Ph.D. (člen)
RNDr. Renata Mikulíková, Ph.D. (člen)
Date of acceptance
2017-05-30
Defence
1. Diplomantka seznámila členy komise s náplní a cílem diplomové práce.
2. Byly přečteny posudky na diplomovou práci.
3. Diplomantka akceptovala všechny připomínky oponenta a na všechny otázky odpověděla v plné šíři.
Diskuse
doc. Diviš: Může enzymová aktivita nabývat záporných hodnot ?
doc. Španová: Jak byly připraveny čisté kultury ?
doc. Obruča: Čím byla kontaminovaná půda ? Průmyslové využití chytinas ?
prof. Márová: Využití výsledků ?
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení