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

Yıl 2017, , 8 - 16, 15.04.2017

İlknur Sen Marco A. Mata-gomez Marco Rito-palomares Canan Tari , Melike Dinç

https://doi.org/10.24323/akademik-gida.305767

Öz

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-desorption-ionization-time-of-flight mass spectrometry (Maldi-TOF MS)
analysis was performed on in-gel digested sodium-dodecyl-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.

Anahtar Kelimeler

Aspergillus sojae, Polygalacturonase, Maldi-TOF MS, Size exclusion chromatography, Ion exchange chromatography

Aspergillus sojae Tarafından Üretilen Poligalakturonazın Kısmi Saflaştırılması için Kromatografik Bir Yaklaşım

Öz

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-sülfat-poliakrilamid-jel-elektroforezi
(SDS-PAGE) jellerinde matriks-yardımlı lazer desorpsiyon/iyonlaştırmalı-uçuş
zamanlı-kütle 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. 

Anahtar Kelimeler

Aspergillus sojae, Poligalakturonaz, Maldi-TOF MS, Boyut eleme kromatografisi, İyon değişim kromatografisi

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