This thesis investigates the functional consequences of neutralising the negative charges on the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by binding to and inactivating the host RM system. The aim of this study was to analyse the effect of reducing the negative charge on the ocr molecule by mutating the acidic residues of the protein. The ocr molecule (117 residues) is replete with Asp and Glu residues; each monomer of the homodimer contains 34 acidic residues. Our strategy was to begin with a synthetic gene in which all the acidic residues of ocr had been neutralised. This so called ‘positive ocr’ (or pocr) was used as a template to gradually reintroduce codons for acidic residues by adapting the ISOR strategy proposed by D.S.Tawfik. After each round of mutagenesis an average of 4-6 acidic residues were incorporated into pocr. In this fashion a series of mutant libraries in which acidic residues were progressively introduced into pocr was generated. A high-throughput in vivo selection assay was developed and validated by assessing the antirestriction behaviour of a number of mutants of the DNA mimic proteins wtOcr and Orf18 ArdA. Further to this, selective screening of the libraries allowed us to select clones that displayed antirestriction activity. These mutants were purified and in vitro characterisation confirmed these mutants as displaying the minimum number of acidic residues deemed critical for the activity of ocr. This in vitro process effectively simulated the evolution of the charge mimicry of ocr. Moreover, we were able to tune the high-throughput assay to different selection criteria in order to elucidate various levels of functionality and unexpected changes in phenotype. This approach enables us to map the “in vitro” evolution of ocr to identify acidic residues that are required for protein expression, solubility and function proceeding to a fully functional antirestriction protein.

The in vivo detection of enzyme activity is a significant biomarker in tumorigenesis. Assessment of enzyme activity relative to enzyme concentration can serve as quite an accurate measurement of several disease states. Chemical Exchange Saturation Transfer (CEST) MRI is a non-invasive imaging technique that can be used to evaluate enzyme activity. Compared to other contrast agents CEST MRI agents have a slower chemical exchange rate and thus have greater specificity for detecting the intended biomarker. Chapter 1 provides an overview of the advances made in the field of molecular imaging for detection of cancer biomarkers. The molecular mechanism of each technique is explained with specific examples and advantages as well as disadvantages of each technique. Chapter 2 investigates the specific example of detection of an enzyme, γ-glutamyl transferase (GGT) in ovarian cancer tumor models using a catalyCEST MRI contrast agent. This chapter discusses the step-by step evaluation of the non-metallic contrast agent, from synthesis to evaluation of its catalytic efficiency with Michaelis Menten kinetics studies and finally in vivo GGT detection in ovarian tumor models of OVCAR-8 and OVCAR-3. Chapter 3 investigates the enzyme, Kallikrein-6 and its detection in HCT116 colon cancer tumor model. In addition to enzyme detection, enzyme inhibition using Antithrombin III inhibitor has also been explored within in vitro media and in vivo HCT116 tumor model. Chapter 4 introduces the catalyCEST agent for detection of sulfatase enzyme. This chapter discusses the synthesis of this agent and its ability to detect sulfatase in bacterial cell suspension and mammalian cell suspension. These examples portray catalyCEST MRI as a platform technology for enzyme activity detection. Finally in Chapter 5 future ideas have been proposed to improve the in vivo detection and broaden the applications of catalyCEST MRI in the field of enzyme studies.

No description available.

No description available.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Reliable energy resources could be considered as one of the cornerstones of the prosperity of the human race. The growing human population is constantly exerting more pressure on the world’s natural resources, which include natural fossil fuels that are non‐renewable. There are concerns regarding the use of fossil fuels due to its growing scarcity and its negative impact on the environment. There is thus a growing need in the world for energy sources that are renewable, more or less carbon neutral and therefore with a minimum environmental impact. Renewable energy is currently being harnessed from the wind, water and sun, but to a limited extent. These forms of natural resources are very attractive for the production of renewable energy, but these technologies are difficult to apply in the current transportation sector. Biofuels provide an alternative to the current use of liquid fossil fuels and it could be able to sustain the current fleet of automobiles worldwide in the intermediate to long term with minimal adjustment to the engines of these vehicles. Extensive research has been done on the production processes for biofuels. Previous processes included the use of high temperatures and acids that further increased the total production cost and thus making biofuels less attractive as an alternative energy source. Recent research has suggested a wide range of organic materials as substrate for the production of biofuels, which include lignin, hemi‐cellulose, cellulose and starch. Processes based on hemi‐cellulose, cellulose and lignin as substrate are still in its early research stages and commercial application of these processes will only occur over the medium‐ to long‐term. Starch is a very good alternative source for the production of biofuels, but there is a need for a microbial system for the conversion of starch to bio‐ethanol in a single step, referred to as Consolidated Bioprocessing (CBP). This would reduce the overall production cost of bio‐ethanol and thus making starch‐based substrates more attractive as an alternative energy source. The cost saving will be mainly due to the elimination of the pre‐treatment of raw starch at high temperatures and the addition of enzymes for the liquefaction and saccharification of starch to simple sugars. However, as there is no currently no known microbial organism known that can produce the required enzymes (i.e. amylases) as well as ferment the resulting sugars to ethanol, heterologous expression of these enzymes in a host strain able to ferment sugars could provide the best alternative system. In the first part of this study, 36 fungal strains known for the production of amylases were screened and compared for the highest extracellular enzyme activity on raw corn starch. The best two candidates, i.e. Aspergillus tubingensis (T8.4) and Mucor cincinelloides (1180), were then further evaluated to determine which organism has the highest efficiency when combined with a Saccharomyces cerevisiae laboratory strain. In fermentation experiments, A. tubingensis (T8.4) in combination with S. cerevisiae Y102 yeast strain resulted in the highest yield of ethanol. Literature on A. tubingensis is limited compared with other Aspergillii and it was previously accepted that A. tubingensis has the highest homology with Aspergillus niger. However, other reports – including the present study ‐ found that A. tubingensis is closer related to other Aspergillus spp. with regard to its amylolytic enzymes. The α‐amylase gene of A. tubingenis has a homology of 99.00% with that of Aspergillus kawachii whereas the glucoamylase gene has a homology of 99.26% with that of Aspergillus shirousami. In the second part of this study, two recombinant S. cerevisiae strains were constructed to express the wild type A. tubingensis α‐amylase (Atamy) and glucoamylase (Atglu), respectively. The combination of the two recombinant yeast strains was able to completely hydrolyse and also utilize raw corn starch for the production of bio‐ethanol, with a yield of 11.04 g/l of ethanol, which translates to 98% of the theoretical yield from starch with a 52% conversion of the total raw starch. This rate of conversion is lower than other reports which indicated up to 82% and 96% of the theoretical yield of ethanol from raw and soluble starch, respectively, by α‐ and glucoamylase. Furthermore, the combined expressed of the two genes was much more effective than when only one of the two genes were expressed, with a yield of 0.32 g/l ethanol for only Atamy and 2.52 g/l ethanol for Atglu. This proved that the combination of the A. tubingensis genes were best suited for the production of biofuels from raw starch. This also proved that the concept of constructing an amylolytic yeast strain capable of raw starch hydrolysis and fermentation was indeed feasible. / AFRIKAANSE OPSOMMING: Betroubare energiebronne kan as een van die boublokke vir die vooruitgang van die mensdom beskou word. Die groeiende menslike populasie is gedurig besig om meer druk op die wêreld se natuurlike hulpbronne te plaas, insluitende nie‐hernubare fossielbrandstowwe. Daar is kommer rakende die gebruik van fossielbrandstowwe weens ‘n afname in die beskikbaarheid en die negatiewe impak wat dit op die omgewing het. Daar is dus ‘n groeiende behoefte in die wêreld vir ‘n hernubare, min of meer koolstof‐neutrale energiebron wat ‘n minimale omgewingsimpak sal hê. Hernubare energie word tans tot ‘n beperkte mate uit wind, water en die son verkry. Hierdie vorms van natuurlike energie hulpbronne is baie aanloklik vir die vervaardiging van hernubare energie, maar hierdie tegnologië is moeilik toepasbaar in die huidige vervoersektor. Biobrandstowwe voorsien ‘n alternatief vir die huidige gebruik van fossielbrandstowwe en kan moontlik die huidige voertuigvloot wêreldwyd oor die medium‐ tot langtermyn onderhou met minimale enjinaanpassings van hierdie voertuie. Deeglike navorsing is alreeds op die vervaardigingsprosesse vir biobrandstowwe gedoen. Vorige prosesse het die gebruik van hoë temperature en sure ingesluit wat produksiekostes verder verhoog en gevolglik die gebruik van biobrandstowwe as ‘n alternatiewe energiebron minder aantreklik gemaak het. Onlangse navorsing het die gebruik van organiese materiaal as substraat vir die produksie van biobrandstowwe voorgestel, wat lignien, hemi‐sellulose, sellulose en stysel insluit. Prosesse met die gebruik van hemi‐sellulose, sellulose en lignien as substraat is nog in die beginfase van ontwikkeling en kommersialisering van hierdie prosesse sal eers oor die medium‐ tot langtermyn plaasvind. Stysel is ‘n baie goeie alternatiewe bron vir die produksie van biobrandstowwe, maar ‘n mikrobiese sisteem word vir die omskakeling van stysel in bio‐etanol in ‘n enkele stap benodig, bekend as gekonsolideerde bioprosessering (GBP). Dit sal die algemene produksiekoste van bio‐etanol verlaag en dus styselsubstrate as ‘n alternatiewe energiebron meer aantreklik maak. Die kostebesparing sal hoofsaaklik realiseer omdat die vooraf‐behandeling van rou stysel byhoë temperature en die toevoeging van ensieme vir die vervloeiing en versuikering van stysel tot eenvoudige suikers, uitgeskakel word. Aangesien daar tans geen bekende mikrobe organisme is wat die nodige ensieme (nl. amilases) kan produseer en ook die suikers wat daardeur vrygestel is, na etanol kan fermenteer nie, kan die heteroloë uitdrukking van hierdie ensieme in ‘n gasheer‐ras wat die suikers kan fermenteer, moontlik die beste alternatief verskaf. In die eerste deel van hierdie studie is 36 fungi rasse wat bekend is vir hul amilase produksie geevalueer en met mekaar vergelyk vir die hoogste ekstrasellulêre ensiemaktiwiteit op rou mieliestysel. Die beste twee kandidate, naamlik Aspergillus tubingensis en Mucor cincinelloides, is verder ge‐evalueer om te bepaal watter organisme het die hoogste effektiwiteit in kombinasie met ‘n Saccharomyces cerevisiae laboratorium gisras. In fermentasie‐eksperimente het A. tubingensis in kombinasie met S. cerevisiae Y102 gisras die hoogste etanol opbrengs gelewer. Inligting rakende A. tubingensis is beperk relatief tot ander Aspergillii en dit was voorheen aanvaar dat A. tubingensis die hoogste homologie met Aspergillus niger het. Ander verslae – insuitende die huidige studie ‐ het egter gevind dat A. tubingensis nader verwant aan ander Aspergillus spp. in terme van amilolitiese ensieme is. Die α‐amilase geen van A. tubingensis het ‘n homologie van 99.00% met dié van Aspergillus kawachii en die glukoamilase ‘n homologie van 99.26% met dié van Aspergillus shirousami getoon. In die tweede gedeelte van hierdie studie is twee rekombinante S. cerevisae gisrasse gekonstrueer om onderskeidelik die α‐amilase (Atamy) en glukoamilase (Atglu) van A. tubingensis uit te druk. Die kombinasie van die twee rekombinante gisrasse was in staat om die volledige hidrolise en benutting van rou mieliestysel vir die produksie van bio‐etanol deur te voer met ‘n opbrengs van 11.04 g/l wat gelykstaande is aan 98% van die teoretiese opbrengs vanaf stysel met ‘n omskakeling van 52% van die totale rou stysel. Hierdie omskakelingskoers is laer as ander studies wat onderskeidelik 82% en 96% van die teoretiese opbrengs van rou en oplosbare stysel vir α‐ en glukoamilase getoon het. Verder was die kombinasie van die twee gene meer effektief as wanneer slegs een gebruik is, met ‘n 0.32 g/l opbrengs vir Atamy en 2.52g/l vir Atglu. Hierdie het bewys dat die kombinasie van die A. tubingensis meergeskik vir die produksie van bio‐etanol was. Dit het ook bewys dat die beginsel van ‘n amilolitiese gisras wat in staat is om rou stysel te hidroliseer en te fermenteer, inderdaad moontlik is.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: A 240-day growth study was conducted to determine the suitability of soybean meal (SBM) as an alternative protein source to fish meal (FM) in the diet of commercially produced South African abalone (Haliotis midae). The suitability of SBM was determined by a close evaluation of the following key factors: feed water stability, morphological impact on the abalone intestine and the effect on the growth performance of abalone. The study was comprised of two phases: a fish meal replacement phase (Phase A) and an enzyme treatment phase (Phase B). Diets used in Phase A consisted of a control fish meal diet (Control=22%FM, 0%SBM), a fish meal-soybean meal diet (FMSBM=20%FM, 15%SBM), a soybean meal-low diet (SBMlow=0%FM, 15%SBM) and a soybean meal diet (SBM=0%FM, SBM30%). In Phase B, the FM diet and SBM diet were used as basal diets (FME0 and SBME0). These diets were then treated with three commercial enzyme products, namely, a β- glucanase (FME1 and SBME1), xylanase (FME2 and SBME2) and α-D-galactosidase (FME3 and SBME3). Subsequently, all three enzymes were combined to make two treatments (FME123 and SBME123). With regard to the gut morphology and growth trials, a thirteenth energy enhanced commercial animal protein-free diet (ECO) was used. In Phase A (fish meal replacement), the findings revealed that water stability did not differ significantly between treatments. In Phase B (enzyme treatment) however, the water stability of β- glucanase treated feeds was significantly lower than that of the control FM diet. It was also observed that in comparison to the control FM diet, soybean meal based diets have a significantly greater effect on intestinal morphology. With reference to Phase A (fish meal replacement), by the end of the 240 day growth trial period, it was evident that animals fed on the commercial (ECO) diet were significantly heavier than those given the control FM diet. With regard to final length in mm, feed conversion ratio (FCR) and specific growth rate (SGR) for mass and length, no differences between the treatments were noted. It was also found that the condition of the ECO fed animals was significantly better in comparison to the other treatment fed animals. No significant differences were observed between the FM and three FMreplaced diets however. With reference to Phase B (enzyme treatment), it was noted that once again, after the 240 day period, abalone fed on the ECO diet were significantly heavier in terms of their final weight when compared to those fed on the other diets. As in Phase A, no differences in FCR and SGR for mass and length were observed. Measurements of the animals’ final length (as observed on day 240) revealed that those fed on the ECO diet were significantly longer than those given the FME1, SBME1 and SBME3 diets. At the end of the trial, abalone fed on the ECO diet were also in significantly better condition than those fed on the SBM, FME3 and FME123 diets. In terms of production performance, no significant difference was found between the SBM diets and FM diets and enzyme supplementation did not significantly increase the production performance either. The results of this study therefore show that SBM has great potential to be used as a FM-replacement diet. The improved performance of the ECO diet was expected due to its energy content. / AFRIKAANSE OPSOMMING: ‘n Groeistudie is gedoen met die perlemoen (Haliotis midae) oor ʼn tydperk van 240 dae om die geskiktheid van sojaboonoliekoek (SBM) as ‘n alternatiewe proteïenbron ter vervanging van vismeel (FM) in die rantsoen te evalueer. Geskiktheid van SBM is getoets aan die hand van waterstabiliteit van voer, morfologie van die spysverteringskanaal en die invloed daarvan op groei van die perlemoen. Die studie het uit twee fases bestaan naamlik ‘n vismeel (FM) vervangingsfase (Fase A) gevolg deur ‘n ensiem behandelingsfase (Fase B). Die diëte wat gebruik was sluit in ’n Kontrole dieet wat slegs vismeel as proteïenbron bevat (Kontrole = 22%FM, 0%SBM), ‘n 2de dieet wat beide vismeel en sojaboonoliekoekmeel bevat (FMSBM =20%FM, 15% SBM), ‘n 3de dieet wat ‘n lae vlak sojaboonoliekoekmeel bevat (SBMlow =0%FM, 15%SBM) en 4de dieet met ʼn hoër sojaboonoliekoek vlak (SBM = 0%FM, 30% SBM). Die basale diëte van Fase B was dieselfde as die FM en SBM diëte van Fase A (FME0 en SBM0) met die verskil dat dit met kommersiële ensieme behandel is. Die onderskeie behandelings was gedoen met β-glukanase (FME1 en SBME1), xylanase (FME2 en SBME2) en α-D-galactosidase (FME3 en SBME3) asook ‘n kombinasie van die drie ensieme (FME123 en SBME123). ‘n Addisionele behandeling bestaande uit ‘n kommersiële diereproteïenvrye dieet (ECO) is as bygevoeg as kontrole vir die histologie gedeelte van die proef. Tydens Fase A is gevind dat waterstabiliteit van die onderskeie diëte nie betekenisvol verskil het nie. Tydens Fase B het ensiembehandeling met β-glukanase egter aanleiding gegee tot betekenisvolle laer waterstabiliteit van FME1 en SBME1 diëte in vergelyking met die FM dieet. Histologiese ontledings het getoon dat die SBM diëte ‘n groter negatiewe effek op die morfologie van die spysverteringkanaal gehad het as die kontrole FM dieet. Fase A het getoon dat die ECO dieet beter groeiresultate opgelewer het as die FM dieet, in terme van liggaamsmassa en kondisiefaktor van die perlemoen. Finale skulplengte (mm), voeromsetverhouding (VOV) en spesifieke groeitempo (SGT) vir massa en lengte was egter nie betekenisvol verskillend vir enige van die behandelings nie. Geen betekenisvolle verskille is ook gevind tussen die FM en enige van die FM vervangingsdiëte nie. Resultate vir Fase B het getoon dat diere wat die ECO dieet gevoer is betekenisvol swaarder was as diere wat ander voere gevoer is. Geen betekenisvolle verskille is waargeneem vir VOV en SGT van massa en lengte nie. Finale lengte van die diere wat ECO gevoer is was langer as die van die FME1, SBME1 en SBME3 diëte. Die ECO diere het ook in betekenisvol beter kondisiefaktor vertoon as diere wat SBM, FME3 en FME123 diëte gevoer is. Geen betekenisvolle verskille in produksie parameters is opgemerk tussen die FM en SBM diëte nie en die toevoeging van ensieme het ook nie ‘n betekenisvolle invloed gehad nie. Die gevolgtrekking is dat sojaboonoliekoekmeel suksesvol aangewend kan word vir die vervanging van vismeel in perlemoen diëte.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Xylanolytic accessory enzymes produced by the endo-p-l,4-xylanase overproducing, colour-variant strain of the euascomycetous fungus Aureobasidium pullulans, NRRL Y-2311-1, were studied. a- Glucuronidase activity was only induced during cultivation on carbon sources containing both xylose and glucuronic acid. An a-glucuronidase was partially purified from the supernatant of A. pullulans cultivated on birchwood glucuronoxylan. The enzyme had an apparent mobility on SDS-PAGE of 170 kDa, and after deglycosylation its mobility shifted to 118 kDa, indicating an extensively decorated protein. Maximal activity was measured at pH 3 in McIlvaine's phosphate-citrate buffer and at 40°C, and the enzyme was stable for 3 h at 40°C. The enzyme displayed substrate inhibition, and Km- and Kj-values were calculated as 3.3 ± 0.29 mM and 9.8 ± 3.8 mM for aldotriouronic acid and 29.5 ± 7.6 mM and 29.0 ± 7.8 for aldobiouronic acid respectively. PCR methods were used to clone the genes encoding an a-glucuronidase and an a-Larabinofuranosidase of A. pullulans NRRL Y-2311-1. The deduced amino acid sequence of the aglucuronidase encoding gene, aguA, shared greater than 60% identity with fungal glucuronidases and between 34% and 42% identity with bacterial a-glucuronidases, and it is member of family 67 of the glycoside hydrolases. The aguA gene encodes a protein of 836 amino acids with a putative secretion signal of 15 amino acids, resulting in a mature protein with a predicted molecular weight of 91 kDa. The gene was expressed in S. cerevisiae Y294 under control of the ADH2 promoter and terminator. The heterologous a-glucuronidase was purified to homogeneity using Ni-chelate affinity chromatography, and it had an electrophoretic mobility of 120 kDa on SDS-PAGE. The enzyme was maximally active at 65°C and between pH 5 and pH 6. The enzyme was stable at 45°C, lost half of its activity after 22.5 minutes at 55°C, and had a half-life of 5.6 min at 65 °C. It was stable at pH 4 and pH 6, and had a half-life of 17 min at pH 8. The enzyme had Km-values in the millimolar range for the series from aldobiouronic acid to aldopentaouronic acid. It had the highest catalytic efficiency on aldobiouronic acid and the catalytic efficiency decreased with increasing chain-length of the oligosaccharide substrate. The deduced amino acid sequence of the a-L-arabinofuranosidase gene, ab/A, shared between 69% and 76% identity with family 54 c-arabinofuranosidases. The gene encodes a polypeptide of 498 amino acids with a putative signal peptide of 20 amino acids resulting in a mature protein with a calculated molecular weight of 49.9 kDa. It was expressed in S. cerevisiae Y294 and the heterologous enzyme was purified to homogeneity by gel filtration. It's size estimated by gel filtration was 36 kDa, and it had an apparent mobility of 49 kDa on SDS-PAGE. It showed maximal activity at 55°C and between pH 3.5 and pH 4. It was stable at 50°C and between pH 4 and pH 5. The enzyme had a Km for p-nitrophenyl c-arabinofuranoside of 3.7 ± 0.36 mM and a Vrnax of 34.8 ± 1.1 U/mg protein. It displayed 0.2 U/mg activity against p-nitrophenyl ~-xylopyranoside. / AFRIKAANSE OPSOMMING: Hierdie studie het gefokus op xilanolitiese ensieme van die endo-I3-1,4-xilanase oorproduserende, kleur-variante ras van die euaskomiseet Aureobasidium pullulans, NRRL Y-2311-1. 0:- Glukuronidase-aktiwiteit is slegs geïnduseer tydens groei op koolstofbronne wat beide xilose en glukuronsuur bevat. u-Glukuronidase is gedeeltelik uit die supernatant van A. pullulans gekweek op berkehout glukuronoxilaan gesuiwer. Die ensiem se elekroforetiese mobiliteit met SDS-PAGE was 170 kDa en na deglikosilering het dit verskuif na 118 kDa, beduidend van 'n swaar geglikosileerde ensiem. Maksimum aktiwiteit is gemeet by pH 3 in McIlvaine se sitraat-fosfaat buffer en by 40°C. Die ensiem was stabiel by 40°C tydens 'n 3-uur inkubasie. Substraat inhibisie is bespeur, en die ensiem se Km- en Kj-waardes vir aldotriouronsuur was onderskeidelik 3.3 ± 0.29 mM en 9.8 ± 3.8 mM en vir aldobiouronsuur was die waardes onderskeidelik 29.5 ± 7.6 mM en 29.0 ± 7.8 mM. PKR metodes is benut om die gene vir u-glukuronidase en cc-arabinofuranosidase te kloneer. Die afgeleide aminosuurvolgorde van die c-glukuronidase geen, aguA, was meer as 60% identies aan swam cc-glukuronidases, en tussen 34% en 42% identies aan bakteriële u-glukuronidases, en dit is 'n lid van familie 67 van die glikosied hidrolases. Die aguA geen kodeer vir 'n proteïen van 836 amienosure met 'n sekresiesein van 15 amienosure, wat die produksie van 'n volwasse protein met 'n molekulêre gewig van 91 kDa tot gevolg het. Die geen is uitgedruk in S. cerevisiae Y294 onder beheer van die ADH2 promoter en termineerder. Ni-chelaat affiniteitschromatografie is gebruik om die heteroloë cc-glukuronidase te suiwer. Die elektroforetiese mobiliteit van die suiwer ensiem was 120 kDa met SDS-PAGE. Die ensiem het maksimale aktiwiteit by 65°C en tussen pH 5 en pH 6 getoon. Die ensiem was stabiel vir twee ure by 45°C, het die helfte van sy aktiwiteit binne 22.5 minute by 55°C verloor, en het 'n halfleeftyd van 5.6 minute by 65°C gehad. Dit was stabiel by pH 4 en pH 6 vir twee ure, en het 'n halfleeftyd van 17 minute by pH 8 gehad. Die ensiem het millimolaar Km-waardes getoon vir die substraatreeks vanaf aldobiouronsuur tot aldopentaouronsuur. Dit het die hoogste katalitiese effektiwiteit vir aldobiuronsuur gehad en die katalitiese effektiwiteit het afgeneem met toenemende lengte van die oligosakkaried substraat. Die afgeleide amienosuurvolgorde van die c-t-arabinofuranosidase geen, abfA, was tussen 69% en 76% identies aan familie 54 u-t-arabinofuranosidases. Die geen kodeer vir 'n proteïen van 498 amienosure met 'n seinpeptied van 20 aminosure, wat lei tot die produksie van 'n volwasse proteïen met 'n berekende molekulêre massa van 49.9 kDa. Die geen is uitgedruk in S. cerevisiae Y294 en die heteroloë ensiem is gesuiwer deur gel filtrasie. Die ensiem se geskatte molekulêre gewig met gel filtrasie was 36 kDa, en die ensiem se mobiliteit op SDS-PAGE was 49 kDa. Dit het maksimum aktiwiteit getoon by 55°C, en tussen pH 3.5 en pH 4. Dit was stabiel vir twee ure by 50°C en tussen pH 4 en pH 5. Die ensiem se Km vir p-nitrofeniel c-t-arabinofuranosied was 3.7 ± 0.36 mM en die Vmax was 34.8 ± 1.1 U/mg proteïen. Die ensiem het aktiwiteit teen p-nitrofenie1 I3-D-xilopiranosied van 0.2 U/mg getoon.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Botany / Master / Master of Science