CHYMOSINS A AND B FROM GENETICALLY
MODIFIED MICROORGANISMS
First draft prepared by Dr F.S.D. Lin,
Division of Toxicological Review and Evaluation
Center for Food Safety and Applied Nutrition,
US Food and Drug Administration
1. EXPLANATION
Chymosins A and B derived from genetically modified
microorganisms have not been previously evaluated by the Joint
FAO/WHO Expert Committee on Food Additives.
Chymosin, commonly known as rennin, is the principal milk-
coagulating enzyme present in rennet. Rennet, which has a long and
extensive history of safe use in making cheese and other dairy
products, is commercially prepared by aqueous extraction of dried
fourth stomach of unweaned calves. The aqueous extract contains a
chymosin precursor, prochymosin, which is subsequently converted to
enzymatically active chymosin. Commercial preparations of calf
rennet contain two forms of chymosin, A and B, usually in the
proportion of about 40% of A and 60% of B. Health aspects of rennet
as a food ingredient were reviewed and evaluated at the fifteenth
meeting of the Joint FAO/WHO Expert Committee on Food Additives in
1972 (Annex 1, reference 26).
Biochemically, chymosin (IUB No. 3.4.4.3) is a protein
consisting of a single polypeptide chain of 323 amino acids with
intramolecular disulfide linkages. Chymosins A and B have been
shown to differ only by one amino acid in the polypeptide chain; the
former has an aspartic acid residue at position 286, whereas the
latter has a glycine residue at the same position.
Chymosin is produced intracellularly as preprochymosin.
Preprochymosin is shortened by 16 amino acids during secretion and
appears in the stomach as prochymosin, which, in turn, is activated
to chymosin by cleavage of an additional 42 amino acids.
As a proteolytic enzyme, chymosin hydrolyses a specific bond in
kappa-casein of milk, cleaving it into two peptides, para-kappa-
casein and a macropeptide. In milk, kappa-casein acts as a micelle
stabilizer. After this activity is destroyed by chymosin, milk
coagulation occurs. Chymosin A slightly exceeds chymosin B in
proteolytic activity, whereas chymosin B is more stable at low pH
(< 3.5) than chymosin A.
In recent years recombinant DNA technology has made it possible
to obtain calf chymosin as a fermentation product from
nontoxicogenic and nonpathogenic strains of bacterium, yeast or
filamentous fungus, which have been transformed with a plasmid
vector containing a DNA sequence coding for the chymosin precursor.
Available biochemical evidence has established that the transferred
prochymosin sequence can be expressed correctly in the new host
organisms. The prochymosin product has the same molecular weight as
prochymosin found in calf rennet and it can be cleaved into chymosin
that has the same chemical, physical and functional (enzymatic)
properties as its mammalian counterpart.
The three recombinant chymosins that were reviewed in this
monograph, as well as their respective production organisms are
identified below:
(1) chymosin A from Escherichia coli K-12
(2) chymosin B from Kluyveromyces lactis, and
(3) chymosin B from Aspergillus niger var. awamori.
1.1 Chymosin A produced from Escherichia coli K-12 containing
calf prochymosin A gene
1.1.2 Construction of production strain
E. coli K12 JA198 strain was subjected to several genetic
manipulations to construct the recipient strain for the expression
plasmid carrying the prochymosin A gene. The expression plasmid was
derived from the widely used cloning vector pBR322. cDNA coding for
bovine chymosin A was previously cloned and characterized. The
prochymosin gene was divided into three sections, each terminated by
a unique restriction endonuclease recognition site. Each section
was assembled from several synthetic oligonucleotides synthesized in
an automated DNA synthesizer. Each assembled section was subcloned
into a pBR322 vector, transformed into E. coli, and amplified.
The correctness of each construct was verified by restriction
analysis and by sequencing. The errors found in synthetic
oligonucleotides were corrected by cassette mutagenesis. All three
subcloned sections were assembled together in the correct order to
reconstruct the prochymosin gene, which was inserted into pBR322
vector. The gene was attached to the vector DNA through the
ribosomal binding site and the E. coli tryptophan (trp) promoter.
The created expression vector was transformed into the recipient
strain GE81. The plasmid carries the ampicillin resistance gene as
a selective marker for bacterial transformants carrying the
prochymosin gene (Pfizer Central Research, 1988).
1.1.3 Fermentation
The production strain is grown in an aqueous solution
containing carbohydrates, nitrogen, mineral salts and miscellaneous
inorganic and organic compounds (Pfizer Central Research, 1988).
1.1.4 Recovery
The solid prochymosin is liberated from the producing organism
by cell disruption and harvesting of "inclusion bodies" by
centrifugation or membrane concentration. The harvested inclusion
bodies are washed with phosphate buffer solution containing 1-4 M
urea. The residual E. coli are inactivated by holding at pH less
than 2.0 for at least one hour. The inclusion bodies are then
dissolved by addition of urea to a concentration of 7-9 M and pH
adjustment to 10.0 - 11.0. Subsequently, the solution containing
prochymosin is diluted with a buffer, and the pH is reduced to 8.5-
9.5, followed by a 2-hour period to allow renaturing of the
prochymosin, which is subsequently activated to chymosin by
adjusting the pH to 1.8 - 2.2 and holding for one hour. Following
readjustment of the pH to 5.5 - 6.0, the chymosin is purified via
absorption on a suitable anion-exchange resin followed by elution
with a buffer containing 1 M sodium chloride (Pfizer Central
Research, 1988).
1.1.5 Biochemical characterizations
A number of side-by-side experiments were performed to
demonstrate that the recombinant chymosin is identical to natural
chymosin A, including the following (Pfizer Central Research, 1988):
1. Relative mass by SDS-PAGE (Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis)
2. Isoelectric Point
3. Absorption Spectra
4. Anion-Exchange Chromatography
5. Amino Acid Analysis
6. Amino Terminal Sequencing
7. Peptide Mapping
8. Composition and Sequence of Selected Internal Peptides
9. Kinetic Specificity
10. Heat Stability
2. BIOLOGICAL DATA
2.1 Biochemical Aspects
No available data.
2.1.1 Absorption, distribution, and excretion
No available data.
2.2 Toxicological data
2.2.1 Acute studies
No available data.
2.2.2 Short-term studies
2.2.2.1 Rat
Four groups of 15 male and 15 female Long-Evans rats,
approximately 90 g b.w., were held three weeks, then were given
chymosin in their diet for 36-39 days at daily dose levels of 0,
0.5, 2.0, or 5.0 mg/kg b.w. Treated animals grew normally and
exhibited normal clinical chemistry, haematology and urinalysis
findings. A transient decrease in food consumption and growth was
observed at week 4 of the study in all treatment groups, male and
female. However, normal growth and food consumption continued for
the remainder of the study. Organ weights and the incidence of
gross and microscopic lesions were comparable in control and
treatment groups (Fisher, 1987b).
2.2.2.2 Other relevant safety data
Samples of the chymosin product were tested and found free from
the production organism and expression plasmid (Pfizer Central
Research, 1988).
Chymosin product was tested by Limulus Amoebocycte Lysate
bacterial endotoxin assay, and the levels of endotoxin found were
low and comparable to those reported in US drinking water (Pfizer
Central Research, 1988).
Using in vitro cytotoxicity test the production strain was
shown to produce no detectable levels of "Shiga-like" toxin (SLT).
Although the chymosin product was not tested directly for the
presence of SLT, the absence of SLT was concluded from a feeding
study in Beagle dogs, in which the animals (6/sex/group) were given
15 ml/dog of a formulation containing 0.1 (1.5 mg chymosin/kg
b.w./day) or 0% of the enzyme preparation once daily for 5 days.
Treatment was well tolerated and produced no significant effects on
food consumption or body weight gain. Results from faecal occult
blood determinations were negative, and no unusual emesis, diarrhoea
or other gastrointestinal effects were reported in the treated
animals (Fisher, 1987a).
3. COMMENTS
The Committee reviewed the available data pertaining to the
molecular construction of the chymosin expression plasmid;
characterization of the producing organism; the fermentation
process; biochemical and enzymatic characterization of the
recombinant enzyme; and short-term feeding studies on the finished
enzyme product.
The Committee noted that a well-documented non-pathogenic
strain of E. coli had been used as the host organism for the
expression plasmid (pPFZ87A) which was derived from a widely used
cloning vector (pBR322). The prochymosin A coding sequence, which
was inserted into pBR322 plasmid, was initially synthesized
chemically and shown to be identical to that of the natural calf
gene. The plasmid construct was introduced into the host organism
by standard transformation procedures, and the ampicillin-resistance
gene carried by the plasmid was used as a marker for the selection
of the transformed cells. Genetic stability of the transformed
cells was demonstrated after repeated subculturing.
The transformed cells were grown under properly controlled
conditions in media containing ingredients commonly used in the
production of food-grade substances by fermentation. Prochymosin A
was recovered from the producing organisms after disruption and
separation of the cells. Residual cells were inactivated by
acidification, which also served to activate prochymosin A to
chymosin A.
After chromatographic purification, the active enzyme was
formulated with stabilizer and preservatives typically used in
commercial enzyme preparations.
The recombinant enzyme was extensively characterized and shown
to be chemically and functionally identical to the Chymosin A.
The results of studies on the microbiological purity of the
enzyme product indicated the absence of producing organisms, minimal
transfer of endotoxin from the bacterial cell walls, and the
presence of insignificant levels of "shiga-like" toxins. The low-
levels of residual DNA detected in the product consisted of short
fragments without any demonstrable genetic activity.
The Committee noted that in a short-term (one month) study in
rat to which the enzyme product was administered no adverse effects
were observed at the highest dose level of 5 mg of chymosin per kg
of body weight per day.
4. EVALUATION
Taking into account the available safety information and the
extremely low exposure resulting from its use in food production,
the Committee established an ADI "not specified" for the recombinant
chymosin A preparation.
1. EXPLANATION
1.1 Chymosin B produced from kluyvermyces lactis
containing calf prochymosin B Gene
1.1.2 Construction of production strain
To obtain donor DNA, messenger RNA (mRNA) for preprochymosin
was isolated from calf stomach and used to prepare complementary DNA
(cDNA). The preprochymosin cDNA was cloned into pBR322 plasmid,
amplified in E. coli as an intermediate host, and identified by
sequencing. DNA encoding prochymosin was then isolated and used for
the construction of an expression vector on the basis of pUC18
plasmid. The prochymosin-encoding DNA was attached to various DNA
sequences with regulatory or other functions. DNA coding for the
lactase promoter of K. lactis and the prepro-region of the alpha-
factor gene from Saccharomyces cerevisiae was inserted in the
front of the prochymosin-encoding DNA. alpha-Factor is a sex
pheromone of S. cerevisiae that is excreted to the fermentation
medium. The secretion is directed by its prepro-region. Thus, the
prepro-region of the alpha-factor provided prochymosin with the
secretion signal. The lactase transcription termination signal from
K. lactis was positioned downstream from the prochymosin-encoding
DNA and was followed by DNA coding for resistance to G418
aminoglycoside as a selective marker. Before transformation of the
host K. lactis cells, the expression vector was cleaved in the
lactase promoter. Since the lactase promoter of the vector was
identical to the one in the host organism, the vector was integrated
by homologous recombination into the lactase promoter of the host.
Molecular weights of the chymosin derived from K. lactis and the
chymosin from calf rennet, as determined by SDS-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) were found to be identical (Gist-
brocades, 1990).
1.1.3 Fermentation
The fermentation process consists of three steps: laboratory
propagation, inoculum fermentation and primary fermentation. The
production of prochymosin occurs during the primary fermentation.
During the primary fermentation, the production strain is grown
aerobically under aseptic conditions in a fermenter equipped with
suitable devices for pH, oxygen, and temperature controls (Gist-
brocades, 1990).
1.1.4 Recovery of chymosin
After approximately 100 hours of fermentation, the process is
stopped by the addition of sulfuric acid and sodium benzoate. The
yeast cells are killed and prochymosin is activated to chymosin due
to the low pH (ca. 2). The cellular debris is then separated by
filtration. The product is further purified by several cell
filtrations and concentrated by ultrafiltration. The concentrate is
formulated with sodium chloride and sodium benzoate. The product
may also be formulated with glycerol, propyleneglycol or sorbitol.
The formulation may contain from 10,000 to 46,000 MCU (Milk Clotting
Units) per mL of the chymosin enzyme, depending on the market
requirements, and be either liquid or powdered (Gist-brocades,
1990).
1.1.5 Biochemical Characterizations
A number of side-by-side experiments were performed to
demonstrate that the recombinant chymosin is identical to natural
chymosin B, including the following (Gist-brocades, 1990):
1. DNA Sequencing
2. Chromatographic Fractionation
3. Molecular Size
4. Molecular Weight
5. Immunological Identification
6. Sequence and Composition of Amino Acids
2. BIOLOGICAL DATA
2.1 Biochemical aspects
No available data.
2.1.1 Absorption, distribution, and excretion
No available data.
2.2 Toxicological studies
2.2.1 Acute studies
Species Route LD50 Reference
(mg/kg/b.w.)
Rat oral 5000 van Eeken et al., 1986a
2.2.2 Short-term studies
2.2.2.1 Rat
Groups of 9-20 male and 10-20 female Wistar rats, 89-111 g
b.w., were treated by gavage for 91 days with chymosin at daily dose
levels of 0, 50, 5000 or 1000 mg/kg b.w. The report did not
indicate whether the commercial enzyme preparation or a more
concentrated or purified form of the enzyme was used in the study.
No mortality was observed in experimental animals over the course of
the study. Growth, behaviour and external appearance were normal.
Haematology measurements in treated animals were comparable to
controls. Statistically significant changes were occasionally
observed in clinical chemistry parameters, but the changes were not
dose-related. Absolute and relative organ weights were unaffected
by treatment, except for liver weights in females, which exhibited a
dose-related increase, tabulated as follows:
Increase in Liver Weight
Absolute Relative
14.7% 9.1%
17.6% 13.6%
18.6% 15.9%
However, because the incidence of histopathologic lesions in
livers of treated females was comparable to or less than control
incidence, the increase in liver weight in treated females is not
considered to be toxicologically significant. No treatment effect
was observed on incidence of gross or microscopic lesions in any of
the other tissues that were examined (van Eeken et al., 1988b).
Groups of 5 male and 5 female Wistar rats, 92-117 g b.w., were
maintained for 23 days on a commercial rodent diet, which included a
daily supplement of 5000 mg cheese (prepared with either
conventional or recombinant chymosin). No mortality was observed in
experimental animals over the course of the study. Behavior and
external appearance were normal. Growth of animals consuming diets
supplemented with either of the two cheeses was enhanced up to 15%
compared to controls consuming the standard laboratory diet. No
significant changes in organ weight could be attributed to
treatment. Serum chemistry and haematology values were not affected
significantly by treatment. BUN and glucose were elevated in
animals consuming diets supplemented with either of the two cheeses,
compared to controls consuming the standard laboratory diet, but
these changes were probably related to the increased protein and
caloric intake occurring in animals consuming diets supplemented
with cheese. Incidence of gross and microscopic lesions was not
increased in rats consuming cheese made with the recombinant
chymosin (van Eeken, et al., 1986b).
Groups of 10 male and 10 female Wistar rats, 104-142 g b.w.,
were maintained for 91 days on a commercial rodent diet, which
included a daily supplement of 5000 mg cheese (prepared with either
conventional or recombinant chymosin 0.6-0.9 µg chymosin/5000 mg
cheese). No mortality was observed in experimental animals over the
course of the study. Behavior and external appearance were normal
except for overgrowth of incisors observed in 3/10 females fed
cheese made with recombinant chymosin. Incisor overgrowth has no
toxicological implications for humans.
Rats consuming diets supplemented with cheese (made with either
conventional or recombinant chymosin) grew more rapidly than rats
consuming a standard laboratory diet, as seen in the following
table:
Growth Enhancement Relative to Standard Laboratory Diet
Type of Chymosin Male Female
recombinant 14.6% 19.6%
conventional 19.7% 28.0%
This increased growth may have occurred because the cheese-
supplemented diets tasted better to the animals, leading to
increased food consumption. There are no food consumption data to
substantiate this hypothesis.
Serum chemistry and haematology results were in the normal
range except for occasional statistically significant deviations
that were not dose-related and not considered to be treatment-
related. Organ weights of animals were not affected by consumption
of recombinant chymosin in the cheese supplement. Increased
absolute heart weight and decreased mean relative weight of kidney
and liver was observed in males and females consuming diets
supplemented with either cheese (made with recombinant or
conventional chymosin). The apparent increase in heart weight was a
reflection of the increased overall body weight of cheese-consuming
animals, and could not be related to intake of recombinant chymosin
residues in the cheese. Relative heart weights of cheese-consuming
animals were comparable to relative heart weights of animals
consuming a standard laboratory diet. The changes in relative
kidney and liver weight were related to the cheese diet, rather than
to its content of recombinant chymosin. Incidence of gross and
microscopic lesions was not increased in rats consuming cheese made
with recombinant chymosin (van Eeken, 1988c,d).
2.2.3 Special study on genotoxicity
The chymosin preparation was tested for potential mutagenicity
in the standard Ames test. Negative results were obtained with and
without microsomal activation (Hoorn, 1988).
2.2.4 Special study on immune response
Groups of 10 male and 10 female young adult guinea pigs (376-
525 g b.w.) were treated dermally with chymosin preparation for 6
hr, once/wk for three weeks (the induction phase). Two weeks after
the last exposure in the induction phase, test animals were dermally
challenged with chymosin at a previously untreated site.
Concurrently, control animals (5 male, 5 female) never previously
exposed to the test material were also challenged. The next day,
application sites were depilated, rinsed, examined and scored for
the presence of erythema.
The incidence and severity of erythema in test animals was
comparable to that observed in the control group. No response of
grade 1 severity was observed in test or control animals, although
an increased incidence of positive response (7/20) occurred in test
animals, compared to controls (1/10) (Kreuzmann, 1988).
Antisera were raised in 10 guinea pigs (Dunkin/Hartley/
Pirbright, 360-436 g.) by intradermal injection of 0.1 ml chymosin
(5 µg protein/ml) divided over 4 sites on the back. Two boosters of
0.1 ml (50 µg protein/ml) were given at 2 week intervals. Seven
control animals received comparable injections of saline. Sera were
collected via puncture of the retro-orbital sinus at immunization
(pre-immune serum), at each booster shot, and two weeks after the
last booster.
Non-exposed animals were administered freshly prepared serial
dilutions of sera in a saline (50 µl volume) by intradermal
injection along each side of the back. Each recipient animal
received 6 test samples in duplicate: one saline control and 5 sera
from different donor animals. Four hours after the injection of
sera, a solution of rennet or recombinant chymosin (150 µg protein)
together with a 2% solution of Evan's blue (1 ml/kg b.w.) was
injected intravenously. Thirty minutes later, areas of the
"blueing" reactions at the intradermal injection sites were
estimated.
Guinea pigs remained healthy throughout the treatment period,
and growth was normal. Preimmune sera and saline controls were
negative in the PCA assay, as was recombinant chymosin, but
conventional calf rennet gave a positive allergenic response with
sera from 7/10 donors. The PCA titers increased with time. Sera
taken after 4 weeks and 6 weeks (2 boosters) were positive, but 6
week sera were positive at higher dilutions (1:32, 1:128) and with
larger areas of response than sera taken after 4 weeks (1 booster).
Scattered positive reactions were observed for 1:8 dilutions of 2/10
week-6 chymosin sera, but these responses were considered anomalous.
It was concluded that recombinant chymosin poses less of an
allergenic hazard than conventional chymosin (van Lambalgen,
undated).
2.2.5 Other relevant safety data
The genetic stability of the production strain with respect to
the inserted DNA was confirmed by a Southern blot analysis after 6,
50 and 100 generations.
The ampicillin resistance marker normally present in the pUC18
vector was not expressed in the production strain.
The chymosin preparation was tested for the presence of
recombinant DNA and chymosin production organism. No recombinant
DNA was detected above the detection limit of 2 ng/ml product, and
there were no viable K. lactis cells present in the final
preparation (Gist-brocades, 1990).
3. COMMENTS
The Committee reviewed the available data pertaining to the
molecular construction of the chymosin expression plasmid,
characterization of the producing organism, the fermentation
process, biochemical and enzymatic characterization of the
recombinant enzyme and various toxicological studies of the finished
enzyme product.
The host organism for the chymosin expression plasmid was
originally isolated from dairy products, and is a known source of
the commercial lactase preparation. It is neither toxicogenic nor
pathogenic for humans.
Plasmid pUC18 was used as the cloning vector for the
prochymosin B coding sequence. The sequence was generated by a
procedure involving several steps, starting with the isolation and
purification of preprochymosin mRNA from the calf stomach. A
complementary DNA (cDNA) sequence to the preprochymosin mRNA was
prepared, cloned into plasmid pBR322, amplified in Escherichia coli
as an intermediate host, and identified by DNA sequencing. The
isolated prochymosin sequence was inserted into plasmid pUC18, and
the expression plasmid was integrated into the host genome by
standard transformation procedures. The transformed cells were
identified by their resistance to G418 aminoglycoside.
The transformed yeast cells were cultured in medium containing
food-grade substances commonly used in the fermentation process.
During cultivation, prochymosin B was secreted into the medium.
Fermentation was stopped by acid treatment, which also served to
activate prochymosin B to chymosin B. After cellular debris was
removed by filtration, the chymosin B-containing filtrate was
further purified by several cell filtrations and concentrated by
ultrafiltration. The enzyme concentrate was formulated with
materials normally applied in the cheese industry.
The recombinant enzyme was extensively characterized and shown
to be biochemically, enzymically and immunologically identical to
bovine chymosin B. The purity of the recombinant enzyme was
reported to be much higher than that of rennet extract. There was
no evidence of antimicrobial activity. In addition, there were no
other proteases, detectable residues of recombinant DNA or viable
yeast cells present in the purified enzyme preparation.
Various standard toxicological tests on the recombinant enzyme
preparation were reviewed by the Committee. In an acute toxicity
study in rats given a single oral dose of 5 g/kg b.w. of the enzyme
preparation no evidence of toxicity was observed. In studies in
rats in which cheese prepared with the recombinant chymosin was
given in the diet at a level of 5 g per day for 21 or 91 days, no
treatment-related effects were noted. In another study
incorporation of the recombinant enzyme preparation into the diet at
dose levels up to 1 g/kg b.w./day for 90 days did not cause any
adverse effects in rats. In vitro genotoxicity studies indicated
that recombinant chymosin B is not mutagenic.
4. EVALUATION
On the basis of the available safety information and in view of
the extremely low exposure from its use in food production, the
Committee established an ADI "not specified" for the recombinant
chymosin B preparation.
1. EXPLANATION
1.1 Chymosin B produced from Aspergillus niger var awamori
containing calf prochymosin B gene
1.1.2 Construction of production strain
The host strain for recombinant DNA was derived from A.
awamori NRRL 3112 (also referred to as A. niger var. awamori).
A glucoamylase-overexpressing mutant obtained from the NRRL
3112 strain was subjected to mutagenesis and selection in order to
isolate two auxotrophic mutants pyrG (deficient in orotidine-5'-
monophosphate decarboxylase; requiring uridine for growth) and argB
(deficient in ornithine transcarbamylase; requiring arginine for
growth). These strains were crossed and a double mutant pyrG argB
was isolated from the progeny and designated as GC12. The purpose
of these modifications was to provide selectable markers for the
subsequent genetic manipulations.
In the next step, the GC12 strain was modified by replacing its
gene coding for aspergillopepsin A, an extracellular aspartic
proteinase that can degrade chymosin and cause off-flavors in
cheese, with the A. nidulans argB gene. A linear DNA fragment
containing 5' and 3' flanking regions from the aspergillopepsin A
gene and the A. nidulans argB gene was used to transform the GC12
strain. The transformants were selected for the loss of the
arginine auxotrophy and screened for an aspergillopepsin-deficient
phenotype. A strain designated GCdeltaAP4 was selected for the
transformation with the expression vector.
The expression vector designated pGAMpR was constructed on the
basis of pBR322 plasmid. The vector contains the A. awamori
glucoamylase promoter and glucoamylase coding region fused in frame
with bovine prochymosin cDNA and followed with the A. niger
glucoamylase terminator. The vector also contains the pyr4 gene
from Neurospora crassa which can complement the pyrG mutation in
the host strain and allow for the selection of transformants in
which the requirement for uridine was lost. The pBR322 sequences
retained in the expression vector contain the gene coding for
ampicillin resistance. However, this gene is not expressed in the
production strain.
The aspergillopepsin-deficient strain GCdeltaAP4 was
transformed with the expression vector. A transformant producing
the highest levels of chymosin (based on an immunoassay) was
selected for chymosin production and designated as strain 4-1. The
analysis of cellular DNA with Southern blotting showed that the
glucoamylase-prochymosin expression unit has integrated into the
host genome probably in multiple copies. The tests for mitotic
stability of the integrated DNA showed that the production strain is
capable of losing the glucoamylase-prochymosin sequences at a
frequency of 10-5. A sporadic formation of morphological variants
of the production strain not related to mitotic instability was also
observed. These variants retained the capability to produce
chymosin (Chr. Hansen's, 1989).
1.1.3 Fermentation and recovery
The production organism is grown in several stages to build up
the inoculum for large-scale production. After inoculation to the
fermenter, the cells are grown aerobically under the proper
conditions of pH, temperature, nutrient composition, etc. After
chymosin reaches a desired level in the fermentation broth, the
fermentation is stopped and the fungal cells are inactivated and
separated from the liquid. Chymosin is then recovered from the
broth by one of two methods: (1) the broth is filtered, followed by
chromatographic purification and concentration of chymosin; or (2)
the chromatographic step is preceded by extraction of chymosin from
the fermentation broth. Chymosin eluted from the chromatographic
column is formulated to the commercial strength (Chr. Hansen's,
1989).
1.1.4 Biochemical characterizations
A number of side-by-side experiments were performed to
demonstrate that the recombinant enzyme is identical to calf
chymosin B, in the following respects:
1. Relative mass by SDS-PAGE (Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis)
2. Isoelectric point
3. Enzymatic activity as a function of pH and temperature
4. Other kinetic parameters
5. Amino acid composition
6. Amino terminal sequencing
7. Immunological characteristics
8. Stability during storage
9. Peptide map
The recombinant chymosin B differs from calf chymosin B in that
10% of the recombinant enzyme is glycosylated (Chr. Hansen's 1989).
2. BIOLOGICAL DATA
2.1 Biochemical aspects
No available data.
2.1.1 Absorption, distribution, and excretion
No available data.
2.2 Toxicological studies
2.2.1 Acute studies
No available data.
2.2.2 Short-term tests
2.2.2.1 Mouse
Mice that were fed a single dose of a preparation of the
production organism were observed for signs of toxicity for a period
of 4 weeks before they were sacrificed and necropsied. No toxic
effects were noted in these animals, indicating that the production
organism is non-pathogenic after oral ingestion (David, 1989).
2.2.2.2 Rat
Four groups of 20 male and 20 female Charles River CD rats, 43
days old, were maintained for 13 weeks on a diet containing 0, 10,
40.5 or 162 ppm of lyophilized chymosin. Average compound intake
during the study was 0, 0.7, 2.7, or 10.6 mg/kg b.w./day for male
groups and 0, 0.8, 3.4 or 13.2 mg/kg b.w./day for female groups.
Under conditions of the study, test animals fed recombinant chymosin
in their diet for 13 weeks suffered no significant adverse effects.
No compound-related deaths were reported. Growth and food
consumption were normal, as were haematology results. Mean serum
cholesterol levels were slightly elevated in treated males, but
standard deviations were large and the elevation is not considered
to be biologically significant. Other serum chemistry parameters
were unaffected by treatment. Mean urinalysis volumes were large in
some treatment groups, including controls, but the effect was not
dose-related. In these groups, some individual urine volumes
approached 50 ml, a volume which would not normally be expected from
rats during the overnight collection period. The urinalysis summary
sheet suggested that water spillage might account for the unusually
large individual urine volumes. Some of the other urinalysis
parameters, such as pH and specific gravity, may have been affected
by the large urine volumes, which makes the urinalysis results
difficult to interpret.
Organ weights and incidence of gross and microscopic lesions
were not affected by treatment. In females, the incidence of
chronic focal inflammation in the liver was increased in high-dose
females (6/20) compared to controls (2/20), but severity was slight
and the lesion was not considered to be toxicologically significant.
Also in high-dose females, the incidence of kidney lesions was
increased (5/20) compared to controls (2/20), but 3 of the lesions
were related to mineralization (compared to one such lesion in
controls), and were considered nutritional in origin, rather than
treatment-related. Incidence of the lesions not involving
mineralization was low and not considered treatment-related (Keller,
1988).
2.2.3 Special studies on genotoxicity
Test System Test Object Concentration of Results Reference
Chymosin (Source)
Ames test (1) S. typhimurium 1-150 µl/plate Negative Lawlor, 1989
TA98, TA100,
TA1535, TA1537,
TA1538
Ames test (1) S typhimurium 1-150 µl/plate Negative Valentine,
TA98, TA100, 1989
TA1535, TA1537,
TA1538
Chromosomal Chinese hamster 1250-5000 µg/ml Negative Murli, 1989
aberration (2) ovary cells
Forward Mouse lymphoma 0.5-5.0 µl/ml Negative Young, 1989
Mutation (3)
(1) Assays were conducted with and without rat liver S-9 fraction.
Chymosin additions were expressed by volume (µl);
concentrations were not specified. Positive controls included
sodium azide (10 µg/plate), 2-nitrofluorene (10 µg/plate),
quinacrine mustard (5 µg/plate) and 2-anthramine (2.5 µg plate)
(2) Assays were conducted both with and without metabolic
activation. Chymosin additions were expressed by volume (µl);
concentrations were not specified. Positive controls included
cyclophamide (20-25 µg/ml, metabolic activation) and mitomycin
C (0.25-0.50 µg/ml, non-activated system).
(3) Assays were conducted both with and without metabolic
activation. Chymosin additions were expressed by volume;
concentrations were not specified. Positive controls included
3-methylcholanthrene (2.5-4.0 µg/ml, metabolic activation) and
ethylmethanesulfonate (0.25-0.40 µg/ml, non-activated system).
(4) Addition of 20 µl of Chymosin completely inhibited cell growth
and cell division, but no morphological changes were seen in
the nucleus or cytoplasm. No adverse effects were observed on
cells at additions of 2 µl or less. Chymosin additions were
expressed by volume (µl); concentrations were not specified.
2.2.4 Other relevant safety data
The enzyme preparation was tested for other enzyme activities
that might be present (peptidase, lipase, amylase, aspergillopepsin,
etc.); in all cases, the activities were either not detected or
detected at very low levels.
Tests were also conducted on the enzyme preparation to
demonstrate the absence of antimicrobial activities, PEG residues,
production organism, and mycotoxins (ochratoxin, aflatoxin, T-2
toxin, etc.) (Chr. Hansen's, 1989).
3. COMMENTS
The Committee reviewed the available data pertaining to the
molecular construction of the chymosin expression plasmid;
characterization of the producing organism; the fermentation
process; biochemical, immunological and enzymatic characterization
of the recombinant enzyme and the various toxicological studies of
the finished enzyme product.
The host organism, GC delta APr4 strain of Aspergillus niger
var. awamori was derived from the parent strain NRRL3112 after a
series of genetic manipulations. The host organism was auxotrophic
and required uridine and arginine for growth, and those properties
served as selectable markers for subsequent genetic manipulations.
The gene coding for aspergillopepsin A, an extracellular aspartic
proteinase that degrades chymosin and causes "off" flavour in
cheese, was also absent.
The expression plasmid (pGAMpR) was constructed on the basis of
pBR322 plasmid, a widely used cloning vector, the prochymosin B gene
sequence was obtained from calf stomach tissue.
The constructed expression plasmid was integrated into the host
genome by standard transformation procedures. The potential
pathogenicity of the resultant chymosin-B-producing organism was
studied in mice for 4 weeks following the administration of a single
oral dose. No evidence of pathogenicity was observed.
The cells were grown under properly controlled conditions in
media containing ingredients commonly used in the production of
food-grade substances by fermentation. The cells secreted chymosin
B in its mature form, which was recovered from the fermentation
broth by liquid-liquid extraction with polyethylene glycol, after
removal of the cells. The resultant crude enzyme was further
purified by chromatography. The purified chromosome B was
formulated to the commercial strength with ingredients typically
used in commercial enzyme preparations.
The recombinant chymosin B was extensively characterized and
shown to be enzymatically and immunologically identical to calf
chymosin B. The recombinant enzyme differed from calf chymosin B
only in the degree of glycosylation, but was otherwise biochemically
identical.
The chymosin B preparation was tested for other enzyme
activities that could be present; in all cases, the activities were
either not detected or detected at very low levels. Results from
additional studies indicated the absence of mycotoxins, of
antimicrobial activity, of residues of polyethylene glycol and of
producing organisms.
In a subchronic (90-day) feeding study in rats, no adverse
effects were noted at levels up to 10 mg of chymosin preparation kg
b.w./day. Negative results were also obtained in a series of
standard mutagenicity and clastogenicity tests.
4. EVALUATION
Taking into account the available safety information and the
extremely low intake resulting from its use in food production, the
Committee established an ADI "not specified" for the recombinant
chymosin B preparation.
5. REFERENCES
ANONYMOUS (1988). Testing of Maxiren 15 TL, lot no. 151/8/LRE, and
animal rennet, lot no. FH3, for cytotoxic properties in a cell
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CHR. HANSEN'S LABORATORIUM (1989). Submission to WHO by Chr.
Hansen's Laboratorium, Denmark.
DAVID, R.M. (1989). Acute oral toxicity study of Aspergillus niger
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