There is a good deal of scientific consensus on how to assess the safety of an enzyme preparation
a) Concept of continua
Different enzyme preparations may be similar in some attributes and dissimilar in others. The
relative similarity or equivalence of different enzyme preparations can be determined by comparing
characteristics of the enzymes themselves, the organisms from which they are produced, and the
methods and materials used in the manufacture of the preparation. The importance of any differences
will depend on how they affect the safety and utility of the preparations.
There is a good deal of scientific consensus on how to assess the safety of an enzyme
preparation. However, there is less consensus regarding the criteria by which one decides at what
point an enzyme preparation is different enough from an accepted one that formal review is required
to establish safety. For example, at what point do manufacturing changes or strain modifications
become significant enough to warrant review? At what point is the substantial equivalence of two
enzyme preparations no longer self-evident? This is as much a regulatory question as a scientific one.
Two different batches of the same enzyme purified by the same methods from the same strain of
production organism grown under the same conditions may be considered potentially different if a
small change in activity is significant for its intended use. Alternatively, two different enzymes with
similar functions, but produced by different methods from different species of organisms grown under
different conditions, may be considered substantially equivalent if the differences do not significantly
affect the safety and utility of the preparations. The point at which an enzyme preparation differs
from its accepted counterpart enough to be considered different, and to warrant evaluation, is again as
much a regulatory question as a scientific one.
In the case of the microbial chymosin preparation discussed in the first case study, the
preparation’s functional activity is identical to that of its traditional counterpart, animal rennet.
However, it is produced by a completely different manufacturing method and consequently has
completely different impurities. The United States Food and Drug Administration (FDA) found that
these differences were significant enough to warrant formal review in order to determine whether the
new preparation was substantially equivalent to the traditional one.18
In contrast to the chymosin preparation, the alpha-amylasepreparation discussed in the second
case study below was derived from the same organism as that traditionally used as a source of
alpha-amylase, Bacillus subtilis, albeit from a new strain. The enzyme itself, B. stearothermophilus
alpha-amylase, was independently reviewed and determined to be safe for use in food when derived
from its native host. Additionally, it is functionally similar to the traditional enzyme, differing
principally in its ability to perform at higher temperatures. Thus, in content and activity, the new
preparation is very close to its traditional counterpart. Whether they are close enough that formal
review should not be needed to determine substantial equivalence is a regulatory question.
b) Temporal considerations
Food-use microbial enzyme preparations derived from recombinant organisms are only newly
being developed. At this early stage they may be considered more novel, or worthy of greater
scrutiny, than they will be after a number of such products have been introduced. It is possible, for
example, that the preparation of B. stearothermophilus alpha-amalyse derived from B. subtilis would
not have been treated as a new preparation warranting review had it been introduced at some future
time after a number of similar products had been reviewed.
c) Safety as defined as a “reasonable certainty” of no harm resulting from intended uses under
expected conditions of consumption
It is not feasible to answer all possible questions pertaining to the safety of a new (or traditional,
for that matter) food product. The standard of safety generally considered acceptable is that there is a
reasonable certainty that no harm will result from the intended use of the product under the expected
conditions of consumption.
The intended use of a food-grade enzyme preparation is usually to process food or food
ingredients in a particular way. The enzyme is generally present in the final food product, if at all, at
very low levels.
Commercial food-use enzyme preparations, even when purified, are typically quite impure and
may comprise more cell debris than enzyme. Therefore, in assessing the safety of an enzyme
preparation it is at least as important to review information concerning the production strain, and the
methods and materials used in growing it and purifying the enzyme, as it is to review the
characteristics of the enzyme itself.
In general, when assessing the safety of the enzyme itself one determines the relationship of that
enzyme to other enzymes used in food or food processing. If it is of a type commonly used in food or
food processing and has no unusual properties that warrant concern, then the enzyme itself may be
considered substantially equivalent to other accepted food-use enzymes. Since food-use enzymes are
in (and of) themselves safe, a determination of substantial equivalence generally constitutes a finding
of safety. If the enzyme has unusual properties or is of a type not previously used in food, then
information will be required to show that the enzyme will be safe for its intended use.
In assessing the safety of the production organism, one generally focuses on whether it is
pathogenic or produces toxins. The species of production organism should be shown to have a history
of safe food use, or otherwise be shown by scientific information to be safe for such use. The
particular strain used should also be shown to be safe, i.e. to have no new properties that would affect
it as a source of enzyme preparation safe for use in food.19
In assessing the safety of recombinant production organisms, one typically first determines if the
parent organism is acceptably safe for the intended use. If so, one then reviews all steps in strain
construction to ensure that all vectors used are safe and that the inserted DNA does not encode toxic
or otherwise undesirable proteins. The entire segment of cloned DNA, including sequences flanking
the target gene, should be analysed. If the donor organism produces toxins or other undesirable
compounds, data should be provided demonstrating that DNA encoding these substances was not
inadvertently cloned along with the target DNA.
If the safety of the parent organism for use in food processing has not been established, there
would probably have to be substantial information, including results of toxicology tests, to
demonstrate that the modified strain was acceptable for food use.
As discussed below, the microbial chymosin and alpha-amylasepreparations were found to be
safe after evaluation of the production organisms, the enzymes, and the manufacturing processes. The
manufacturing method destroys the production organism and removes the bulk of the cell debris, and
this was an added factor in assuring the safety of the preparation.
d) Concept of substantial equivalence
Microbial enzyme preparations can be considered substantially equivalent to each other if three
conditions are met: the enzymes themselves are substantially equivalent, for example having similar
intended uses and functional properties; the microbes from which they are derived are substantially
equivalent, for example being safe strains of species with a safe history of use as sources of food-use
enzymes; and the manufacturing and purification processes are substantially equivalent. However,
there are as yet no agreed-upon criteria by which substantial equivalence is determined for each of
these parameters.
A new enzyme preparation may be substantially equivalent to an accepted preparation even if the
production organisms and manufacturing methods are not, so long as the differences do not affect the
safe use of the final preparation. The more the new production organisms or manufacturing methods
differ from traditional ones, the more information will be necessary to determine whether the new
preparation is substantially equivalent to the old.
The concept of substantial equivalence can be applied broadly or narrowly. For example, all
enzymes of any type used for food processing might be considered substantially equivalent; or all
carbohydrates might be considered substantially equivalent; or all amylases; or all alpha-amylases;
or all alpha-amylases that have the same functional activities under the same conditions and are
intended for use in the same foods. The preparations of substantially equivalent enzymes might then
be considered substantially equivalent enzymes if they are produced by a safe strain of any microbial
species with a safe history of use in food; or only if they are produced by the same microbial species;
or only if they are native to and produced by the same microbial species. Additionally, the
manufacturing processes might have to meet certain criteria to assure that the final product meets
acceptable specifications before the enzyme preparations would be considered substantially
equivalent.
In the safety evaluation of the two enzyme preparations described below, the term “substantial
equivalence” was nowhere used by the evaluators. However, though not articulated as such, the safety
of the preparations was determined essentially by establishing that each was substantially equivalent
to an accepted preparation.20
In the case of chymosin derived from E. coli K-12, it is obtained from a completely different
source organism and by a completely different method than is its traditional counterpart, animal
rennet. Thus the types of potential impurities differ, and significant characteristics of the preparations
may differ. To determine if the preparations were substantially equivalent, the FDA compared the
enzymatic activities of the preparations and evaluated whether the impurities in the microbial
preparation affected its safe use. As described in Section 3 below, FDA determined that the enzymes
themselves and the functional activity of the enzyme preparations were substantially equivalent, and
that the impurities in the microbial preparation did not affect its safe use. Thus, while the two
preparations are clearly different and have different names, they are substantially equivalent in safety
and function.
In the case of B. stearothermophilus alpha-amylaseobtained from B. subtilis, the Joint
FAO/WHO Expert Committee on Food Additives (JECFA) evaluated the production organism and
determined that the genetic modifications were well-characterised and did not cause it to produce
toxins or other undesirable substances. It could therefore be considered substantially equivalent to
other food-use strains of B. subtilis. JECFA evaluated the enzyme and found that it was the same as
that produced by B. stearothermophilus. JECFA evaluated the manufacturing method and found it
met acceptable standards for producing microbial enzyme preparations.
Thus, by determining that the enzyme, the production organism, and the manufacturing method
were substantially equivalent to accepted counterparts, JECFA determined that the new enzyme
preparation was safe for its intended use. Depending on the interpretation of substantial equivalence,
one could also conclude that the new enzyme preparation is substantially equivalent to the traditional
B. subtilis preparation, despite the fact that the stearothermophilus enzyme will likely be used with
different substrates because of its ability to digest starches at higher temperatures.
e) Concept of variability
Inapplicable.
f) Concept of sequential review
The first step in evaluating a new enzyme preparation is to compare characteristics of the enzyme
itself, the production organism, and the manufacturing method with those of the closest accepted
counterpart. One can then focus on those characteristics that differ between the new and the old
preparations to determine whether the differences affect the safe use of the new product.
Where the enzyme, the production organism, and the manufacturing method are determined to be
substantially equivalent to those of accepted enzyme preparations, and any new combinations do not
affect the safe use of the product, the new preparation can be accepted as safe. When there are no
accepted counterparts, or where the differences between the accepted and the new are too large to
allow meaningful comparison, additional information is necessary to establish the safety of the
preparation.
g) Evaluation of marker genes in a substantial equivalence determination
Recombinant organisms frequently contain marker genes, some of which may encode resistance
to therapeutically useful antibiotics. Whether the presence of a marker gene in a production organism
affects its substantial equivalence to an accepted safe preparation will depend on a number of
considerations. For example, does the marker gene encode a protein product? If so, at what levels21
would it be expected to be in the food, what is its function, and are there any concerns about its safety
in food at the predicted levels?
For antibiotic resistance marker genes, does the marker gene encode resistance to a clinically
useful form of an antibiotic? If so, does ingestion of the product at the time of therapeutic use of the
antibiotic interfere with the clinical effectiveness of the antibiotic? In general, this would not be
expected to be a concern for enzyme preparations. The preparations are present in very low levels in
the food. Thus, the levels in the food of any constituent of the preparation active against the antibiotic
would almost always be biologically insignificant.
Finally, what is the likely level of horizontal transfer of resistance genes to pathogens in the food
or in the intestinal tract of the consumer? For an enzyme preparation derived from an
antibiotic-resistant microbe to be substantially equivalent to one derived from an antibiotic-sensitive
microbe, the likely level of transfer must be biologically insignificant.
In the case of chymosin derived from E. coli K-12, the level of transfer of the antibiotic
resistance marker was found to be insignificant because the purification method destroyed the
production organism and degraded its DNA to fragments smaller than that of the gene encoding
resistance. In the case of the particular alpha-amylasepreparation described here, there was no intact
antibiotic resistance gene in the production strain.
2. Organism/product: chymosin derived from E. coli K-
Chymosin, also known as rennin, is the principal milk-clotting enzyme present in rennet. Rennet
is derived from the stomach of a variety of animals, must commonly unweaned calves but also kids
and lambs. It has been used for millennia to make cheese. Chymosin is a protease that hydrolyses
one bond in the kappa-casein protein of milk, cleaving it into two peptides. Kappa-casein normally
stabilises nacelles in milk. When kappa-casein is cleaved, the niicelles precipitate into curds. After
removal of the liquid whey, the curds may be processed into cheese or other dairy products such as
frozen dairy desserts.
3. Traditional product evaluation
As discussed in 1.c) above, a new enzyme preparation is evaluated to determine if it is safe for its
intended use. Such an evaluation focuses on characteristics and properties of the enzyme, the
production organism, and the materials and methods used in the manufacturing process.
E. coli-derived chymosin preparation is manufactured by a completely different method than is rennet.
Therefore, it was important to determine whether the change in manufacturing method affected the
safety of the enzyme preparation.
The safety of chymosin derived from E. coli K-12 was established from the following
information. First, the enzyme was shown to be structurally and functionally identical to that of the
chymosin in rennet, and was therefore considered safe as a replacement for the chymosin in rennet.
Data was provided documenting that the prochymosin gene had been cloned and that it was properly
expressed in its microbial hosts to produce functional chymosin.
Three lines of evidence were used to show that the correct gene had been cloned. The cloned
DNA was digested with restriction enzymes, and the resulting fragments were found to be the sizes22
predicted by the DNA sequence of the prochymosin gene. The cloned DNA, and RNA synthesised
from it, were found to hybridise appropriately with the calf prochymosin gene. Finally, the sequence
of the cloned DNA was found to correspond to the amino acid sequence of the prochymosin protein.
The cloned prochymosin gene produced chymosin of the expected size and biological activity.
Cloned chymosin was shown to have the same molecular weight as chymosin derived from calf
rennet, as demonstrated by SDS polyacrylamide electrophoresis. Cloned chymosin was also shown to
have the same functional activity as chymosin derived from calf-rennet, as demonstrated by milk
clotting assays performed under various conditions of temperature, salt concentration and pH.
Second, the production organism, E. coli K-12, was found to be safe as a source of chymosin,
based primarily on published evidence demonstrating that E. coli K-12 is non-pathogenic and
non-toxigenic. Such evidence includes published studies showing that E. coli K-12 does not colonise
the gut of man or other animals after being fed at high concentrations (109 to 1010 viable organisms
per ingestion), that the K-12 strain has been widely used as a laboratory organism for 30 years with no
reported incidents of illness, that it does not produce toxins that cause illness upon ingestion, and that
it is deficient in virtually all characteristics necessary for pathogenesis. Additionally, non-pathogenic
strains of E. coli are a part of the normal flora of the gastrointestinal tract of man, where they are
found at 106 to 101 organisms per gram of intestinal contents.
Third, the fermentation and purification methods were shown not to introduce any unsafe
substances into the preparation and to remove the bulk of the cellular materials from it. All the
chemicals used in the fermentation and purification are approved for use in food. By removing the
bulk of the microbial material from the final product, the purification process yielded a preparation
having acceptably low levels of endotoxin. Endotoxin is a component of the cell wall of E. coli of
potential concern for people with certain intestinal tract disorders. The endotoxin levels in the
chymosin preparation are comparable to those in US drinking water.
The purification method was also shown to destroy the E. coli and degrade its DNA, thereby
adding another level of safety assurance and eliminating the possibility that the antibiotic resistance
gene present in the vector could be transferred at a biologically significant level to pathogens in the
consumer or on food in contact with the enzyme preparation. Data were provided demonstrating that
the preparation did not contain sufficient DNA of a quality capable of transforming
transformation-competent cells to permit detectable transformation of such cells. In addition, no
DNA fragments larger than 200 bases were detected when assayed by radiolabelled hybridisation after
gel electrophoresis. For comparison, the coding sequence of the antibiotic resistance gene carried by
the production strain is 858 bases long.
As corroborative evidence of safety, two short-term feeding studies were conducted with the
enzyme preparation: a five-day feeding study in dogs and a one-month gavage study in rats. No
adverse results were observed in these studies at any dose tested.
Based on the information described above and the fact that consumers would be exposed to it at
relatively low levels, the US FDA concluded that the chymosin preparation is safe for its intended use
as replacement for rennet.
4. Database available for traditional evaluation
5. Novel component(s)/product
Microbial chymosin differs from its traditional counterpart, rennet, in its impurities because it is
obtained from a different source organism and by different manufacturing methods. In all other
aspects, such as activity, function, use, and active component, the two preparations are substantially
equivalent, in fact are identical.
6. Additional evaluation procedures
The chymosin enzyme preparation was subjected to safety evaluation because it is manufactured
by a completely different method from that of its traditional counterpart, animal rennet. It was not
subjected to review simply because it is derived from a recombinant organism. The parts of the
review that could be considered specific for a recombinant organism were the review of the antibiotic
resistance marker and the review of the strain construction, including information concerning vectors
and intermediate strains. Non-recombinant micro-organisms used to produce enzymes for food use
have not had antibiotic markers and have not been subject to extensive strain construction.
7. Rationale for additional evaluation procedures
Chymosin preparation is obtained from a different source organism and by a different
manufacturing process than is rennet. Any time there are significant changes in the source and
manufacturing method of a product, there are likely to be changes in types of impurities. Therefore,
specifications written for one manufacturing method may not be appropriate for a different
manufacturing method. It is also important to determine whether any significant characteristics
affecting the use of the product are changed, that is, whether in fact the new product is substantially
equivalent to the traditional product.
According to dbtbiosafety