The hydrolysis of linalyl acetate and α-terpinyl acetate by yeasts
| dc.contributor.advisor | Smit, M. S. | |
| dc.contributor.advisor | Litthauer, D. | |
| dc.contributor.author | Thomas, Elias | |
| dc.date.accessioned | 2017-04-26T09:06:54Z | |
| dc.date.available | 2017-04-26T09:06:54Z | |
| dc.date.issued | 1999-06 | |
| dc.description.abstract | English: Hydrolysis of esters by means of hydrolases such as proteases (Jones and Beck, 1976), lipases (Santiello et al., 1993) and esterases (Boland et al., 1991) has become a well established method for the resolution of racemic mixtures. The first aim of the present study was to screen the yeast culture collection of the University of the Orange Free State for yeast isolates which can be used for the enantioselective hydrolysis of rac-linalyl acetate and rac-α-terpinyl acetate, which are tertiary alcohol ester terpenes, respectively. We screened 74 yeast strains from 17 genera as well as 29 unclassified isolates. Approximately 16% of the strains screened contained tertiary alcohol hydrolase activity. Whole cell experiments, enzyme purification and characterisation were attempted on one of the hydrolases of interest obtained from Trichosporon sp. UOFS Y-0117. Whole cell experiments on reported optimal hydrolase activity in the presence of 1% maltose in a defined media (YNB). The effect of various co-solvents was also documented with a low concentration of ethanol (2.4% v/v) producing superior hydrolase activity. No toxicity, to the microbe, was observed by rac-linalyl acetate (up to 200mM) due to the cell membrane present. The use of digitonin proved that substrate transport across the cell membrane is not a reaction rate determining step. The re-usability experiment showed a significant decrease in hydrolase activity (ca 30% after 1 cycle) as the same batch of cells was exposed to substrate and product. The results also show an optimal pH of 7.5 and temperature of 30°C which coincides with physiological conditions and literature. Protein purification was attempted on a cell free extract once we determined that the hydrolase was intracellular. Small scale evaluations of different chromatographic resins ranging from ion exchange, hydrophobic interaction and affinity chromatography followed. Large scale experiments with gel filtration resins were also attempted. Purification steps were largely unsuccessful and we decided to continue with a DEAE fraction which produced a superior yield (244%). Characterisation experiments, using the DEAE active fraction, followed in which we explored the effect of rac-linalyl acetate concentrations. Enzyme inhibition and protein denaturation at low rac-linalyl acetate concentrations (detected at ca 65-100mM) is significant compared to whole cells (not detected at 200mM). This hydrolase is also an esterase. Specific amino acid modification reagents results indicate the presence of a serine and histidine amino acid present in the catalytic centre i.e. the hydrolase belongs to the serine hydrolase family. A metal chelating reagent EDTA and various metal cations had no effect on hydrolase activity. pH-stability experiments indicate a pH of 7.5 to be optimal for the retention of hydrolase activity in whole cells and crude enzyme preparation. Thermostability experiments show whole cells are four times more stable than the crude enzyme preparation at 4°C. The energy of inactivation required for activity loss is lower in whole cells (47.43kJ) compared to crude enzyme preparation (91.77kJ). The probability for an event to occur which causes inactivation is relatively low (1.8075 events/h). The converse applies to the crude- enzyme preparation (1.17514 events/h). Thus the crude enzyme is 6x108 less stable than the hydrolase present in the whole cell. | en_ZA |
| dc.description.abstract | Afrikaans: Die hidrolise van ester bindings deur middel van proteases (Jones and Beck, 1976), lipases (Santiello et al., 1993) en esterases (Boland et al., 1991) word dikwels gebruik om rasemiese mengsels te skei. Die eerste doel van hierdie studie was om organismes wat positief vir tersiêre alkohol ester hidrolase aktieweteit toets, te vind deur middel van 'n siftingsproses. Eksperimente is gedoen op giste van die gisversameling van die Universiteit van die Oranje Vrystaat vir die omskakeling van rasemiese mengsels van linaliel asetaat en terpiniel asetaat as substrate. Ons het 74 gisisolate van 17 genera en 29 onklassifiseerde isolate getoets vir die bogenoemde aktiviteite. Sowat 16% van die isolate het positief getoets vir hierdie hidrolase aktiwiteit. Eksperimente met heel selle is eers doen, gevolg deur gedeeltelike ensiemsuiwering en -karakterisering van een van hierdie hidrolases in die gis Trichosporon sp. UOVS Y-0117 gevind. Heel sel eksperimente het getoon dat maksimale hidrolase aktiwiteit in die teenwoordigheid van 1% maltose in 'n gis-stikstof medium geïnduseer word. Verskillende mede-oplosmiddels het getoon dat lae konsentrasies ethanol (2.4% v/v) die hoogste hidrolase aktiwiteit bewerkstellig. Geen newe-effekte op die gis is gesien nie, selfs by hoe konsentrasies linaliel asetaat (200mM), waarskynlik as gevolg van die beskermende teenwoordigheid van die selmembraan. Digitonien, 'n middel wat selmembrane deurlaatbaar maak, het ook geen effek op die reaksie spoed gehad nie. Proewe om die moontlike hergebruik van heel selle het getoon dat In noemenswaardige verlies in hidrolase aktiwiteit plaasvind na een siklus (ca 30% afname) wanneer dieselfde selle aan substraat en produk blootgestel word. 'n Optimale pH van 7.5 en 'n optimale temperatuur van"30°Cwas ook gevind. " Eksperimente is op klein skaal gedoen op In selvrye ekstrak om te bepaal watter chromatografieharse (ioonuitruiling, hidrofobiese en affiniteit) vir die suiwering van die hidrolase gebruik kon word en uitsluitingschromatografie media is ook getoets. Suiweringstappe was meestalonsuksesvol en ons het besluit om met 'n aktiewe fraksie na DEAE ioonuitruilingschromatografie voort te gaan. Karakterisering van die aktiewe DEAE fraksie, het getoon dat ensiem inhibisie en protein inaktivering plaasvind by lae konsentrasies linaliel asetaat (65- 100mM) in vergelyking met heelselle (geen effek tot by 200mM). Spesifieke aminosuur modifikasie reagense het getoon dat In serien en histidien in die katalitiese setel teenwoordig is en dus is hierdie hidrolase deel van die serine hidrolase familie. EDTA en verskillende metaalkatione het geen beduidende effek op hidrolase aktiwiteit gehad nie. pH eksperimente dui daarop dat In pH van 7.5 optimaal was om hidrolase aktiwiteit te behou in heel selle en selvrye ensiem ekstrak. Thermiese stabiliteit eksperimente dui daarop dat die ensiem in die heel selle baie meer stabiel is as die selvrye ensiem ekstrak. Die aktiveringsenergie vir deaktivering van die ensiem is laer in heelselle (47.43kJ) in vergelyking met selvrye ensiem ekstrak (91.77kJ). Die preeksponensiële faktor vir inaktivering in heelselle was 1.807X105 UU(1. Vir die selvrye ensiem ekstrak is hierdie waarde 1.175X1014 UU(1. Dus is die selvrye ensiem ekstrak 6x108 keer minder stabiel in vergelyking met die hidrolase in die heel selle. | af |
| dc.description.sponsorship | Foundation for Research and Development | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/6166 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Hydrolase | en_ZA |
| dc.subject | Yeasts | en_ZA |
| dc.subject | Tertiary alcohol ester | en_ZA |
| dc.subject | Linalyl acetate | en_ZA |
| dc.subject | Linalool | en_ZA |
| dc.subject | Esterase | en_ZA |
| dc.subject | Thermostability | en_ZA |
| dc.subject | Characterisation | en_ZA |
| dc.subject | Acetates | en_ZA |
| dc.subject | Dissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 1999 | en_ZA |
| dc.title | The hydrolysis of linalyl acetate and α-terpinyl acetate by yeasts | en_ZA |
| dc.type | Dissertation | en_ZA |