Purification and characterisation of branching enzyme from Saccharomyces cerevisiae.
BE [(1,4)-a-D-glucan:(I,4)-a-D-glucan 6-glucosyltransferase, EC 184.108.40.206] 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
Primary structural analysis involving N-terminal sequencing and amino acid
composition suggested that yeast BE may share some homology with BEs isolated from
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.