Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The production, purification and functional characterisation of feruloyl esterase from Aureobasidium pullulans were set as the primary objectives of this study. A further objective was to investigate a possible co-operative effect with other selected lignocellulolytic enzymes on substrates relevant to industry. In a comprehensive review, feruloyl esterases from various micro-organisms were compared both functionally and with regard to their primary structure, where applicable. Feruloyl esterases show intriguing differences in substrate specificity and sequence structure. Enzymes that are closely related regarding their amino acid sequence exhibit different substrate specificities. Sequence similarities can be found with a range of other enzyme families, including serine esterases, acetyl xylan esterases, lipases, tannases, glycosyl hydrolases and xylanases. More data on the three dimensional structure of feruloyl esterases as well as an examination of all available feruloyl esterases with the same substrates is necessary before structure-function relationships can be established and before the feruloyl esterases can be organized into discrete families based on ancestral origins. The highest production levels of feruloyl esterase by A. pullulans are achieved when grown on birchwood xylan. Expression was not repressed when glucose or xylose was present in the medium. However, free ferulic acid supplemented to the medium affected fungal growth and therefore did not increase feruloyl esterase activity. It is also suggested that the synthesis of feruloyl esterase is independently regulated from xylanase synthesis. Feruloyl esterase from A. pullulans acts on a- and l3-naphthyl acetate, as well as naphthol AS-D chloroacetate as substrates. Feruloyl esterase from A. pullulans was purified to homogeneity using ultrafiltration with high molecular weight cut-off, anion exchange, hydrophobic interaction and ultimately gel filtration chromatography. With a molecular weight of 210 kDa, the enzyme is the largest of the feruloyl esterases reported to date. Kinetic data was produced using both synthetic and natural substrates. A. pullulans feruloyl esterase shows properties similar to other fungal feruloyl esterases, especially from Aspergillus niger cinnamic acid esterase and Penicillium funiculosum feruloyl esterase B. The N-terminal sequence of A. pullulans feruloyl esterase was identified, but no similarities to known enzyme families were found. Peptide mass mapping did not reveal structural information. In an effort to evaluate the significance of feruloyl esterase from A. pullulans in the degradation of lignocellulose, dissolving pulp and sugar cane bagasse were selectively treated using feruloyl esterase and hemicellulolytic enzymes. The enzymatic degradation reaction was monitored using microdialysis sampling, anion exchange chromatography, online desalting and mass spectrometry. It has been shown, that feruloyl esterase activity together with xylanase activity releases monosaccharides from both substrates. Sugars of higher degree of polymerisation were not released, giving evidence for the recalcitrance of the material. The fibre architecture of the substrates was apparently not accessible to the enzymes and therefore complete hydrolysis was hindered. / AFRIKAANSE OPSOMMING: Die produksie, suiwering en funksionele karakterisering van feruloïel esterase afkomstig van Aureobasidium pullulans was die primêre doelwitte van hierdie studie. 'n Verdere doelwit was om vas te stelof daar 'n kooperatiewe effek met ander geselekteerde lignosellulitiese ensieme op substrate wat industrierelevant is, bestaan. Die feruloïel esterase van verskillende mikro-organismes is vanuit die oogpunt van funksie en primêre struktuur omvattend met mekaar vergelyk, waar toepaslik. Interessante verskille tussen die substraat spesifisiteit en volgordestruktuur van feruloïel esterase kan waargeneem word. Ensieme wat nou aanmekaar verwant is wat hul aminosuurvolgorde betref, het duidelik verskillende substraatspesifiteite. Volgordeverwantskap kan in 'n reeks van ander ensiemfamilies, insluitende serienesterase, asetielxilaanesterase, lipases, tannases, glikosielhidrolases en xilanases vasgestel word. Meer inligting oor die driedimensionele struktuur van feruloïel esterase asook 'n analise van al die beskikbare feruloïel esterase met dieselfde substrate is nodig voordat struktuur-funksie verwantskappe vasgestel kan word en voordat die feruloïel esterases in eie families op die grond van huloorsprong georganiseer kan word. Die hoogste produksie vlakke deur feruloïel esterase van A. pullulans word bekom deur dit op berkhoutxilaan te groei. Ekspressie was nie onderdruk wanneer glukose of xilose in die medium aanwesig was nie. Wanneer vrye feruliensuur by die medium bygevoeg is, is die fungale groei beïnloed en het die feruloïel esterase aktiwiteit nie vermeerder nie. Dit word ook voorgestel dat die sintese van feruloïel esterase onafhanklik deur xilanase sintese gereguleer word. Feruloïel esterase van A. pullulans reageer op a- en f3- naftolasetaat, asook naftol AS-D chloroasetaat as substrate. Feruloïel esterase van A. pullulans is tot homogeniteit deur ultrafiltrering met .n hoë molekulêre gewiggrens, anioonuitruiling, hidrofobiese interaksie en eindelik gelfiltrasie-chromatografie gesuiwer. Met 'n molekulêre gewig van 210 kDa, is die ensiem die grootste van die feruloïel esterases tot dusver beskryf. Kinetiese data is met behulp van sintetiese en natuurlike substrate geproduseer. A. pullulans feruloïel esterase het eienskappe wat vergelykbaar is aan die van ander fungal feruloïel esterases, veral die wat afkomstig is van Aspergillus niger sinnamiensuur esterase en Penicillium funiculosum feruloïel esterase B. Die N-terminale volgorde van A. pullulans feruloïel esterase is identifiseer maar geen ooreenkoms aan bekende ensiemfamilies kon vasgestel word nie. Peptiedmassakaartering kon ook geen strukturele inligting gee nie. Oplosbare pulp en suikerrietbagasse is geselekteerd met behulp van feruloïel esterase en lignosellulitiese ensieme behandel om die belang van feruloïel esterase van A. pullulans in die afbraak van lignosellulose vas te stel. Die hidroliese-reaksie is deur mikrodialise monsterneming, anioonuitruilingschromatografie, oplyn ontsouting en massaspektrometrie gemonitor. Wanneer die aktiwiteit van feruloïel esterase met die van xilanase gekombineer is, is monosakkariede deur albei substrate afgeskei. Suikers met 'n hoër graad van polimerisering is nie afgeskei nie, wat 'n bewys van die materiaal se weerstandbiedendheid is. Dit het geblyk asof die vesel-argitektuur van die verbruikte substraat nie toeganklik was vir ensieme nie en dus is algehele hidroliese verhinder.

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.

Cette étude vise à produire deux enzymes amylolytiques (α-amylase et pullulanase) thermostables par des levures contaminant le blé récolté dans des zones semi arides et arides (Biskra - Sahara, Sud Algérien) et capables d’hydrolyser à la fois les liaisons α-1-4 et α-1-6 de polysaccharides comme l’amidon et le pullulane, molécules d’intérêt industriel. Après isolement et caractérisation de colonies levuriennes, la méthode de plate-test-agar permet d’isoler des souches amylolytiques et de montrer que la souche L7 est la plus performante dans la production enzymatique parmi une douzaine de souches de levures productrices d’α-amylase et de pullulanase thermostables. L’identification des souches, basée sur les caractères morphologiques, les tests biochimiques et la biologie moléculaire a permis de répartir la population comme suit : 50% Clavispora lusitaniae (forme anamorph Candida lusitaniae), 25%, Pichia guilliermondii, 8% Pichia carribbicca, 8% Meyerozyma guilliermondii et 8% Rhodotorula rubra. Par sa richesse en amidon, le biotope du blé est favorable à la survie des levures amylolytiques. La majorité de ces souches dont la souche L7 est productrice de pseudo ou vrai mycélium et est tolérante à certains paramètres comme la température, la salinité, les stress osmotique et éthanolique. La souche de levure L7, Clavispora lusitaniae ABS7, semble être la plus performante dans la production d’enzymes thermostables. Son identification moléculaire a montré deux bandes avec l’endonucléase HAE III alors que les autres souches de la même espèce de Clavispora lusitaniae (L5, L9, L10, L11 et L12) présentent une seule bande. En conditions optimales (agitation 136,56 rpm, température 54,14°C, amidon 2,66g/l, extrait de levure 0,365g/l, sels 8, 75ml/l et oligo-éléments 4,3ml/l en erlenmeyers de 250 ml), la production maximale atteint les valeurs suivantes : 13456,36±300 UI pour l’ α-amylase et 12611, 6±154 UI pour la pullulanase. Ces performances sont en accord étroit avec la prédiction du modèle statistique évaluée à 13231UI pour l’α-amylase et 12825,5 UI pour la pullulanase. La production optimisée a pratiquement doublé par rapport à la production avant l’optimisation (6639,16 UI pour l’α-amylase et 6308,5 UI pour la pullulanase). En conditions optimales et en fermenteur de 2 L, la production maximale pour les deux enzymes de la levure Clavispora lusitaniaeABS7 est obtenue au bout de 28 h avec un optimum de croissance obtenu à 40 heures. La production des deux enzymes n’est donc pas associée à la croissance. La production maximale des deux enzymes s’effectue à pH 8. pic protéique. L’élution sur DEAE-cellulose confirme la présence des deux activités dans la même fraction. Les deux enzymes sont donc présentes sur la même molécule. L’α-amylase et la pullulanase sont purifiées avec un taux de purification de 50,5 et 44,6 respectivement et des rendements respectifs de 23,9% et 21,1%. L’extrait purifié montre une seule bande sur le gel de SDS-PAGE avec un poids moléculaire estimé à 75KDa et une activité amylolytique contenant à la fois les activités α-amylasique (indépendante de Ca2+) et pullulanasique (une métalloenzyme à calcium). La souche de la levure Clavispora lusitaniae ABS7 possède donc une enzyme amylolytique avec deux sites actifs. La CCM révèle une enzyme qui hydrolyse l’amidon en maltose et glucose et le pullulane en maltotriose, maltose et glucose, ce qui montre que l’enzyme est saccharifiante et correspond à une pullulanase de type II (amylopullulase). L’optimisation de l’immobilisation de l’enzyme a permis l’amélioration de l’activité: α-amylasique à 4907,75 UI (rendement 72,3 %) et celle de la pullulanase à 4491,83 UI (rendement 70,1%) avec un pH optimum de 8,5. Il ressort de notre étude que l’amylopullulanase type II libre de Clavispora lusitaniae ABS7 est thermostable puisqu’elle résiste à un traitement thermique de 75°C pendant 3 heures d’incubation et conserve 88% de son activité initiale. / This study aims to produce two amylolytic enzymes (α-amylase and pullulanase) by thermostable wheat contaminant yeast harvested in semi arid and arid zones (Biskra, Sahara, Algeria SUD) and capable of hydrolyzing both the α links 1-4 and 1-6 of polysaccharides such as starch and pullulan, molecules of industrial interest. After isolation and characterization of levuriennes colonies, the test method of agar-plate allows to isolate amylolytic strains and show, that the L7 strain is the most effective, in the enzymatic production of the 12 yeast strains producing α-amylase and pullulanase the thermostable. The identification of strains, based on morphological, biochemical tests and molecular biology has helped spread the population as follows: 50% Clavispora lusitaniae (anamorph form Candida lusitaniae), 25%, Pichia guilliermondii, 8% carribbicca Pichia, 8% Meyerozyma guilliermondii and 8% Rhodotorula rubra. By its high starch, the wheat biotope is favorable to the survival of amylolytic yeasts. Most of these strains, including the strain L7, is producer, pseudo or true mycelium and is tolerant to certain parameters such as temperature, salinity, osmotic stress and ethanolic stress. The yeast strain Clavispora lusitaniae ABS7 (L7) seems to be the most efficient in the production of thermostable enzymes. Its molecular identification showed two bands with the endonuclease HAE III while other strains of the same species Clavispora lusitaniae (L5, L9, L10, L11 and L12) have a single band. In optimal conditions (agitation 136.56 rpm, temperature 54.14 ° C, starch 2,66g / l, yeast extract 0,365g / l, salts 8 75ml / l and trace elements 4,3ml / liter Erlenmeyer flasks into 250 ml), the maximum production reached: 13456.36 ± 300 IU for the α-amylase and 12611, 6 ± 154 IU for pullulanase. This performance is in close agreement with the prediction of the statistical model 13231UI evaluated for α-amylase and 12825.5 IU for pullulanase. The optimized production almost doubled compared to production before optimization (6639.16 IU for the α-amylase and pullulanase for 6308.5 IU). In optimal conditions, and 2 L fermenter, the maximum production for the two enzymes of Clavispora lusitaniae ABS7 obtained after 28 hours, with an optimum of growth obtained at 40 hours. The production of both enzymes is thus not associated with growth. The maximum production of both enzymes is obtained at pH 8. The kinetics are characterized by an increase in carbohydrate and a substance spooning the wall of the fermenter, probably an exo-polysaccharide. The chromatographic profile on Sephacryl S200 reveals two α-amylase and pullulanase activities eluted along with the protein peak. Elution DEAE cellulose confirms the presence of both activities in the same fraction. Both enzymes are present on the same molecule. The α-amylase and pullulanase were purified with a purification rate of 50.45 and 44.59 respectively and respective yields of 23.88% and 21.11%. The purified enzyme showed a single band on SDS-PAGE gel with a molecular weight estimated at 75 KDa and an amylolytic activity containing both the α-amylase activities (independent of Ca2+) and pullulanase (a calcium metalloenzyme). The strain of the yeast Clavispora lusitaniae ABS7 therefore has an amylolytic enzyme with two active sites. TLC reveals an enzyme which hydrolyzes starch into maltose and glucose and pullulan into maltotriose, maltose and glucose, which shows that the saccharifying enzyme, and corresponds to a pullulanase type II (amylopullulase). The optimization of the immobilization of the enzyme enabled the improvement of the activity: α-amylase to 4907.75 IU (yield 72.3%) and pullulanase to 4491.83 IU (yield 70, 1%) with a pH optimum of 8.5. It appears from our study that amylopullulanase type II free is thermostable to heat treatment of 75 ° C for 3 hours of incubation, and retains 88% of its original activity.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21819号 / 農博第2332号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5191(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM