Dissertation

Purification and characterisation of branching enzyme from saccharomyces cerevisiae

Seecharran, Camille

1999

Overview

BE [(1,4)-a-D-glucan:(I,4)-a-D-glucan 6-glucosyltransferase, EC 2.4.1.18] catalyses a transglycosylation reaction where a branch-point is created by the cleavage of an a-l,4 glycosidic bond to form an a-l,6 glycosidic bond. Branching enzyme (BE) from baker's yeast was purified to near homogeneity by chromatography on DEAE-cellulose, Sephacryl S-200 and Protein Pak Q. Electrophoresis on SDS-PAGE revealed one major band of molecular weight 74 kDa. Three distinct methods for determining BE activity (Phosphorylase Stimulation, Iodine- Binding and Branch-Linkage Assays) were used to characterise the purified protein. The enzyme displayed a temperature optimum between 15-25°C and a broad pH optimum of 6.5-7.5 with maximum activity occurring in phosphate buffer. The enzyme was fully stable after incubation at 20°C for 5 hours. A Km value of 1474 Jlg/ ml for amylose was obtained. Primary structural analysis involving N-terminal sequencing and amino acid composition suggested that yeast BE may share some homology with BEs isolated from other sources. Immunological comparisons between yeast, maize (BEll) and Escherichia coli BE using yeast polyclonal antiserum indicated that the enzymes may share antigenic determinants. However, similar comparisons between yeast BE and E.coli antiserum revealed that the antibody only recognised yeast BE in its denatured conformation. Yeast BE was used to modify potato amylose and amylopectin and wheat starch. The enzyme was capable of introducing additional branch points to these substrates resulting in a displacement of the iodine Amax from 629 nm to 568 nm, from 543 nm to 411 nm and from 632 nm to 568 nm for amylose, amylopectin and wheat starch, respectively. HPAEC-PAD analysis of the branched products produced by yeast BE revealed that predominantly short chains of dp 2 to I? were transferred. At least three BE fractions of higher specific activities were isolated from brewer's yeast hatyested at the late exponential phase, suggesting the expression of more than one BE in Saccharomyces cerevisiae.

• Publisher: ProQuest Dissertations & Theses