Enzyme Technology
Enzyme engineering
A most exciting development over the last few years is the application
genetic engineering techniques to enzyme technology. A full description this
burgeoning science is beyond the scope of this text but some suitable references
are given at the end of this chapter. There are a number of properties which may
be improved or altered by genetic engineering including the yield and kinetics
of the enzyme, the ease of downstream processing and various safety aspects.
Enzymes from dangerous or unapproved microorganisms and from slow growing or
limited plant or animal tissue may be cloned into safe high-production
microorganisms. In the future, enzymes may be redesigned to fit more
appropriately into industrial processes; for example, making glucose isomerase
less susceptible to inhibition by the Ca2+ present in the starch
saccharification processing stream.
The amount of enzyme produced by a microorganism may be increased by
increasing the number of gene copies that code for it. This principle has been
used to increase the activity of penicillin-G-amidase in Escherichia coli. The
cellular DNA from a producing strain is selectively cleaved by the restriction
endonuclease HindIII. This hydrolyses the DNA at relatively rare sites
containing the 5'-AAGCTT-3' base sequence to give identical 'staggered' ends.
[8.4]
intact DNA cleaved DNA
The total DNA is cleaved into about 10000 fragments, only one of which
contains the required genetic information. These fragments are individual cloned
into a cosmid vector and thereby returned to E. coli. These colonies containing
the active gene are identified by their inhibition of a 6-amino-penicillanic
acid-sensitive organism. Such colonies are isolated and the penicillin-G-amidase
gene transferred on to pBR322 plasmids and recloned back into E. coli. The
engineered cells, aided by the plasmid amplification at around 50 copies per
cell, produce penicillin-G-amidase constitutively and in considerably higher
quantities than does the fully induced parental strain. Such increased yields
are economically relevant not just for the increased volumetric productivity but
also because of reduced downstream processing costs, the resulting crude enzyme
being that much purer.
Figure 8.1. The protein engineering cycle. The process starts with the
isolation and characterisation of the required enzyme. This information is
analysed together with the database of known and putative structural effects of
amino acid substitutions to produce a possible improved structure. This
factitious enzyme is constructed by site-directed mutagenesis, isolated and
characterised. The results, successful or unsuccessful, are added to the
database, and the process repeated until the required result is obtained.
Another extremely promising area of genetic engineering is protein
engineering. New enzyme structures may be designed and produced in order to
improve on existing enzymes or create new activities. An outline of the process
of protein engineering is shown in Figure 8.1. Such factitious enzymes are
produced by site-directed mutagenesis (Figure 8.2). Unfortunately from a
practical point of view, much of the research effort in protein engineering has
gone into studies concerning the structure and activity of enzymes chosen for
their theoretical importance or ease of preparation rather than industrial
relevance. This emphasis is likely to change in the future.
As indicated by the method used for site-directed mutagenesis
(Figure 8.2),
the preferred pathway for creating new enzymes is by the stepwise substitution
of only one or two amino acid residues out of the total protein structure.
Although a large database of sequence-structure correlations is available, and
growing rapidly together with the necessary software, it is presently
insufficient accurately to predict three-dimensional changes as a result of such
substitutions. The main problem is assessing the long-range effects, including
solvent interactions, on the new structure. As the many reported results would
attest, the science is at a stage where it can explain the structural
consequences of amino acid substitutions after they have been determined but
cannot accurately predict them. Protein engineering, therefore, is presently
rather a hit or miss process which may be used with only little realistic
likelihood of immediate success. Apparently quite small sequence changes may
give rise to large conformational alterations and even affect the
rate-determining step in the enzymic catalysis. However it is reasonable to
suppose that, given a sufficiently detailed database plus suitable software, the
relative probability of success will increase over the coming years and the
products of protein engineering will make a major impact on enzyme technology.
Much protein engineering has been directed at subtilisin (from
Bacillus amyloliquefaciens), the principal enzyme in the detergent enzyme preparation,
Alcalase. This has been aimed at the improvement of its activity in detergents
by stabilising it at even higher temperatures, pH and oxidant strength. Most of
the attempted improvements have concerned alterations to:
- the P1 cleft,
which holds the amino acid on the carbonyl side of the targeted peptide bond;
- the oxyanion hole (principally Asn155), which stabilises the tetrahedral
intermediate;
- the neighbourhood of the catalytic histidyl residue
(His64),
which has a general base role; and
- the methionine residue (Met222) which
causes subtilisin's lability to oxidation.
It has been found that the effect of
a substitution in the P1 cleft on the relative specific activity between
substrates may be fairly accurately predicted even though predictions of the
absolute effects of such changes are less successful. Many substitutions,
particularly for the glycine residue at the bottom of the P1 cleft
(Gly166),
have been found to increase the specificity of the enzyme for particular peptide
links while reducing it for others. These effects are achieved mainly by
corresponding changes in the Km rather than the Vmax. Increases in relative
specificity may be useful for some applications. They should not be thought of
as the usual result of engineering enzymes, however, as native subtilisin is
unusual in being fairly non-specific in its actions, possessing a large
hydrophobic binding site which may be made more specific relatively easily (e.g., by reducing its size). The inactivation of subtilisin in bleaching solutions
coincides with the conversion of Met222 to its sulfoxide, the consequential
increase in volume occluding the oxyanion hole. Substitution of this methionine
by serine or alanine produces mutants that are relatively stable, although
possessing somewhat reduced activity.
Figure 8.2. An outline of the process of site-directed mutagenesis, using a
hypothetical example. (a) The primary structure of the enzyme is derived from
the DNA sequence. A putative enzyme primary structure is proposed with an
asparagine residue replacing the serine present in the native enzyme. A short
piece of DNA (the primer), complementary to a section of the gene apart from the
base mismatch, is synthesised. (b) The oligonucleotide primer is annealed to a
single-stranded copy of the gene and is extended with enzymes and nucleotide
triphosphates to give a double-stranded gene. On reproduction, the gene gives
rise to both mutant and wild-type clones. The mutant DNA may be identified by
hybridisation with radioactively labelled oligonucleotides of complementary
structure.
An example of the unpredictable nature of protein engineering is given by
trypsin, which has an active site closely related to that of subtilisin.
Substitution of the negatively charged aspartic acid residue at the bottom of
its P1 cleft (Asp189), which is used for binding the basic side-chains of
lysine or arginine, by positively charged lysine gives the predictable result of
abolishing the activity against its normal substrates but unpredictably also
gives no activity against substrates where these basic residues are replaced by
aspartic acid or glutamic acid.
Considerable effort has been spent on engineering more thermophilic enzymes.
It has been found that thermophilic enzymes are generally only 20-30 kJ more
stable than their mesophilic counterparts. This may be achieved by the addition
of just a few extra hydrogen bonds, an internal salt link or extra internal
hydrophobic residues, giving a slightly more hydrophobic core. All of these
changes are small enough to be achieved by protein engineering. To ensure a more
predictable outcome, the secondary structure of the enzyme must be conserved and
this generally restricts changes in the exterior surface of the enzyme. Suitable
for exterior substitutions for increasing thermostability have been found to be
aspartate ® glutamate, lysine ®
glutamine, valine ® threonine, serine ®
asparagine, isoleucine ® threonine, asparagine ®
aspartate and lysine ® arginine.
Such substitutions have a fair probability of success. Where allowable, small
increases in the interior hydrophobicity for example by substituting interior
glycine or serine residues by alanine may also increase the thermostability. It
should be recognised that making an enzyme more thermostable reduces its overall
flexibility and, hence, it is probable that the factitious enzyme produced will
have reduced catalytic efficiency.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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