Chemistry Reference
In-Depth Information
very unstable. When the enzyme unfolded at the denaturing temperature, the position of the
catalytic base was shifted from the hydrophobic interior provided by 12th and 13th helixes
to the surface. The sites (amino acid residues) that needed to be altered for engineering
the enzyme in order to increase its thermostability were determined. According to previous
studies, which showed that the introduction of disulphide bonds between flexible regions
most likely contributes to thermostability, the mutants were obtained that formed disulphide
bonds across the 11th helix and increased the enzyme's thermostability. Exchanging several
Gly residues near the 12th and 13th helixes into Ala, in order to increase the hydrophobicity
of this region, also resulted in increased thermostability.
The food enzyme industry continually tries to improve the total process of hydrolyzing
starch to high fructose syrup. This process involves the actions of at least three enzymes: an
α
-amylase for liquefying starch, the above-mentioned glucoamylase for releasing glucose
units and a glucose isomerase (xylose isomerase to be precise) to convert glucose to fructose.
These enzymes are naturally occurring and have pH optima at 5.5-6.0, 4-4.5 and 7.0-8.0,
respectively. Therefore, traditionally these three steps have to be performed separately, and
between reactions the pH has to be adjusted for the best performance of each enzyme. This
causes an unnecessary increase of salt concentration and extra procedures have to be followed
for removal or purification. Since the tertiary structures of several glucoamylase enzymes
were determined, protein engineering was a convenient method for changing the enzyme
into a more suitable biocatalyst. Fang and Ford reported on engineering the glucoamylase
from
A. awamori
to alter its pH optimum.
28
The enzyme has two Glu residues (at 179
and 400) at the catalytic site. One of these acts as the catalytic acid, the other as the
catalytic base. The pH dependence of an enzyme is determined by the ionization of the
catalytic groups that are influenced by various interactions around their microenvironments.
With this enzyme, modifications of Glu179 and Glu400 which affect their ionization state
(indicated by pK1 and pK2, respectively) will likely change its pH optimum. By comparing
the structure of the enzyme with that from other sources, such as
Arxula adeninivorans
,
S. cerevisiae
,
S. diastaticus
and
Saccharomycopsis
, it was found that the amino acid at
position 411 near the catalytic site was different in enzymes from different microorganisms:
some enzymes had a Gly at this position, whereas a Ser was found in the enzymes from other
sources. Since the enzymes from other sources have pH optima more or less shifted to the
alkaline side, S411 was estimated to play an important role in determining the pH optimum
of the enzyme. For that reason, this residue was chosen as the target site for mutagenesis.
Mutants created include S411G, S411A, S411C, S411H and S411D with the serine residue
being replaced with Gly, Ala, Cys, His and Asp, respectively. S411G, S411A and S411C
were created in order to eliminate the hydrogen bonds between SER411 and Glu400 of
the catalytic base so that the carboxylate ion form is destabilized, which results in an
increased pK1. The mutants with His and Asp as replacement of serine aimed at eliminating
the hydrogen bond and introducing positive or negative charges. As a result, S411H and
S411D both showed an increased pK1 but decreased pK2. The former could be explained
as that the positive charge of histidine stabilizes the electrostatic interactions between that
of histidine and the catalytic acid of Glu179, the latter due to increased size of the side
chain. Electrostatic force is also considered to have some effects on the pH property of the
enzyme.
These examples show that understanding of protein structures is important in determining
the sites for mutation and site-directed mutagenesis is an efficient way to improve an enzyme's
property to suit better to industrial applications.
