Canan TARİ, Ilknur SEN, Marco A. MATA-GOMEZ, Marco Rito PALOMARES, Melike DİNÇ

Aspergillus sojae Tarafından Üretilen Poligalakturonazın Kısmi Saflaştırılması için Kromatografik Bir Yaklaşım

Bu çalışmanın amacı, A. sojae mutantından poligalakturonaz üretilmesi ve ham ekstraktın kromatografik yöntemlerle kısmi saflaştırılmasıdır. Peptitlerin konfirmasyonu için ilk basamak olarak, jel içinde sindirilmiş sodyum-dodesil-sülfatpoliakrilamid-jel-elektroforezi (SDS-PAGE) jellerinde matriks-yardımlı lazer desorpsiyon/iyonlaştırmalı-uçuş zamanlıkütle spektrometresi (Maldi-TOF MS) analizi yapılmıştır. Poligalakturonaz üretimi için, katı-faz ve derin fermentasyonlarda üç farklı karbon kaynağı kullanılmıştır. Ham ekstrakt ilk olarak iyon değişim kromatografisi (IEXC) ile saflaştırılmıştır ve ardından bunu boyut eleme kromatografisi izlemiştir. Derin [acı portakal kabuğu, şeker pancarı melası ve (NH4)2SO4] ve katı-faz (buğday kepeği, şeker pancarı ve HCl) fermentasyonlarından elde edilen ham ekstraktlar yüksek seviyede poligalakturonaz enzim aktivitesi (sırasıyla 95.22 and 50.27 U/mL) göstermiştir. IEXC toplanmış fraksiyonunun (180, 200 ve 220 mM tuz fraksiyonları) boyut elemesi, en yüksek verimi (%36) ve saflaştırma katını (2.00) göstermiştir. SDS-PAGE'den elde edilen olası poligalakturonaz bantları jel içinde sindirilmiş ve peptit konfirmasyonu için Maldi-TOF-MS ile analiz edilmiştir.

A Chromatographic Approach for Partial Purification of Polygalacturonase Produced by Aspergillus sojae

The aim of this study was to produce polygalacturonase from A. sojae mutant and partially purify the crude extract by chromatographic methods. As a preliminary step for the confirmation of its peptides, matrix-assisted laser-desorptionionization-time-of-flight mass spectrometry (Maldi-TOF MS) analysis was performed on in-gel digested sodiumdodecyl-sulphate-polyacrylamide-gel-electrophoresis (SDS-PAGE) gels. Three different carbon sources were employed in submerged and solid-state fermentations for the production of polygalacturonase. Crude extract was first purified by ion-exchange chromatography (IEXC) and followed further by size exclusion chromatography. Crude extracts obtained from sub-merged [of bitter orange peel, sugar beet molasses and (NH4)2SO4] and solid-state [of wheat bran, sugar beet and HCl] fermentation exhibited high levels of polygalacturonase enzyme activity (95.22 and 50.27 U/mL, respectively). Size exclusion of IEXC pooled fraction (180, 200 and 220 mM salt fractions) revealed the highest yield (36%) and purification fold (2.00). The likely polygalacturonase bands from SDS-PAGE were in-gel digested and analyzed by Maldi-TOF MS in route for peptides confirmation.

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