Fine structural analysis of the fungal polysaccharide pullulan elaborated by Aureobasidium pullulans, CH-1 strain

Abstract

The structure of pullulan, the extracellular α-D-glucan elaborated by the yeast-like fungus Aureobasidium pullulans, may be described as a linear α-D-glucan consisting of maltotriosyl repeat units connected terminally by (1->6)-α-D-glucosidic bonds. Occasionally some of maltotriosyl residues are replaced by higher oligosaccharide units, most frequently with maltotetraosyl residues. Using the susceptibility of pullulan CH-1 (obtained from strain CH-1 of Aureobasidium pullulans) to hydrolysis catalysed by porcine alpha-amylase, the polysaccharide was cleaved and the fragments obtained fractionated by gel-permeation chromatography. The heterogenous size of the fragments indicates that there is no apparent regular distribution of tetrasaccharide units in the pullulan chain. Enzymatic digestion of pullulan CH-1 using pullulanase, followed by gel-permeation chromatography of the resulting digest confirmed these results as did preparative paper chromatography and CI mass spectrometry of the separated components, i.e., that maltotetraosyl units (about 7 %) are building units of pullulan CH-1.

Pululan, ekstracelularni polisaharid koji proizvodi kvascu slična gljiva Aureobasidium pullulans, jeste linearni glukan čiju strukturu pretežno čine polimaltotriozil ostatci povezani međusobno α-(1->6)-glikozidnim vezama. Varijacije u strukturi ovog polisaharida mogu nastati usled zamene maltotrioznih ostataka na pojedinim mestima polimernog lanca oligosaharidnim fragmentima dužeg niza, najčešće maltotetraoznim ostacima. U ovom radu ispitan je pululan koji proizvodi A. pullulans, soj CH-1, enzimskom hidrolizom sa pankreasnom α-amilazom i pululanazom, sa ciljem da se utvrdi da li i u kom odnosu ovaj polisaharid sadrži oligosaharidne fragmente, kao i da se odredi dužina oligosaharidnog niza, odnosno broj monosaharidnih ostataka koji ulaze u njegov sastav. Primenom uobičajenih analitičkih metoda (gel-filtraciona hromatografija, metilaciona analiza, preparativna hromatografija kombinovana sa masenom spektrometrijom metilovanih oligosaharidnih alditola), utvrđeno je da su maltotetraozne jedinice (oko 7 %) integralni deo polisaharidnog lanca pululana CH-1. Raspored ovih jedinica je nasumičan.