Characterisation, cloning and heterologous expression of the α-glucuronidase from Aureobasidium pullulans

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.