Chemistry Reference
In-Depth Information
and they can be cultivated in fermenter to reach a high biomass concentration. Rational
prediction of the best host, based on the enzyme to be produced, can often be made. Homol-
ogous expression (e.g.
Bacillus
enzyme in a
Bacillus
host) is generally more efficient than
heterologous expression (e.g. eukaryotic enzyme in a
Bacillus
host). However, if a new type
of enzyme has to be produced, it is worthwhile to attempt expression in a battery of hosts to
determine the best candidate.
It must be emphasized that the paragraph above refers to genetically modified organisms
(GMOs). Non-GMOs are also used in the production of food enzymes. Often, the food
enzyme manufacturers have a wide range of products as options to meet customers' specific
needs.
3.2.2 GMO versus non-GMO
The main advantage of using non-GMOs for food enzymes production is their larger accep-
tance by the consumer.
4
On the other hand, GMOs offer numerous advantages, both for the
customers and the producer. An enzyme produced by a recombinant organism is generally
cheaper because it can typically be expressed at higher levels compared to non-GMOs. The
final products also tend to have higher purity as GMO strains are engineered to overexpress
the target enzyme per total background host protein.
An enzyme will be labelled as recombinant based on the following guidelines: (1) the
enzyme amino acid sequence is the same as can be found in nature but the organism used to
produce it is non-native,
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(2) the organism used to produce it has been genetically modified
(e.g. stronger promoter in front of the gene
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) and/or (3) the enzyme itself (i.e. its amino acid
sequence) has been engineered.
2
To keep with the idea of minimizing the impact of genetic
engineering on the environment, a recent development in strain construction has been to
remove the antibiotic marker which often accompanies the gene coding for the enzyme of
interest.
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Product labelling is regulated by the Food and Drug Administration (FDA) in the US, and
by the European Union in Europe.
3.2.3 Example: construction of a Bacillus subtilis
production host
In order for an enzyme to be produced by a host organism, the gene coding for the enzyme
of interest has to be introduced into the host, together with other DNA structures allowing
its transcription, translation and secretion. Figure 3.1 shows the minimum gene components
required to produce an enzyme extracellularly. In brief, the promoter initiates transcription,
the ribosome binding site (RBS) is necessary to initiate translation, the signal sequence (S.S.)
targets the protein to the membrane to allow its secretion, the gene X encodes the enzyme of
interest and the terminator marks the end of transcription. Often an antibiotic marker is added
after the gene X to permit the selection of a correct recombinant clone. If the enzyme is to
Promoter
Promoter
Terminator
Terminator
RBS
RBS
S.S.
S.S.
Gene X
Gene X
Fig. 3.1
Typical structure of an expression cassette for enzyme production by microorganisms. RBS:
ribosome binding site; S.S.: signal sequence. Gene X encodes for the enzyme of interest.
