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 culture test. Unpublished report from Laboratory of Pharmacology and Toxicology, Hamburg, F.R.G. 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 var. awamori in mice. Unpublished report, by Microbiological Associates, Inc. Submitted to WHO by Chr. Hansen's Laboratorium, Denmark. FISHER, D.O. (1987a). CP-72, 692. A five day exploratory feeding study in Beagle dogs. Unpublished report No. 86-605-02 from Pfizer Central Research. Submitted to WHO by Pfizer, Inc., Groton, CT, USA. FISHER, D.O. (1987b). CP-72, 692. A one month oral gavage study in Long-Evans rats. Unpublished report No. 86-605-01 from Pfizer Central Research. Submitted to WHO by Pfizer, Inc., Groton, CT, USA. GIST-BROCADES (1990). Submission to WHO by Gist-brocades NV, Delft, Holland. HOORN, A.J.W. (1988). Mutagenicity test on chymosin batch no. 7005 in the Ames Salmonella/microsome reverse mutation assay, p. 532. Unpublished report No. E-9792-0-401 from Hazleton Biotechnologies, Veenendaal, Holland. Submitted to WHO by Gist-brocades NV, Delft, Holland. KELLER, K.A. (1988). Thirteen week dietary toxicity study in rats. Unpublished report No. 540-026 from International Research and Development Corporation. Mattawan, MI, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA. KREUZMANN, J.J. (1988). Delayed contact hypersensitivity study in guinea pigs. Unpublished report No. 88-3198-21 from Hill Top Biolabs, Inc., Cincinnati, OH, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA. LAWLOR, T.E. (1989). Mutagenicity test on chymosin lot P88032FP in the Ames salmonella/microsome reverse mutation assay. Unpublished report No. 20988 from Hazleton Laboratories America, Inc., Kensington, MD, USA. Submitted to WHO by Genencor, Inc., South Francisco, CA, USA. MURLI, H. (1989). Mutagenicity test on chymosin lots P88032FP, P88035FP and P88040FP in an in vitro cytogenetic assay measuring chromosomal aberration frequencies in Chinese hamster ovary (CHO) cells. Unpublished report No. 20990 from Hazleton Laboratories America, Inc., Kensington, MD, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA. MYER, J.R. (1988a). Eye irritation study in rabbits (low volume procedure). Unpublished report No. 540-028 from International Research and Development Corporation. Mattawan, MI, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA. MYER, J.R. (1988b). Primary dermal irritation test in rabbits. Unpublished report No. 540-027 from International Research and Development Corporation. Mattawan, MI, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA. PFIZER CENTRAL RESEARCH (1988). International submission on chymosin. Submitted to WHO by Pfizer, Inc., Groton, CT, USA. VALENTINE, D.C. & LAWLOR, T.E. (1989). Mutagenicity test on chymosin lots P88035FP and P88040FP in the Ames salmonella/ microsome reverse mutation assay. Unpublished report No. 20988 from Hazleton Laboratories America, Inc., Kensington, MD, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA. Van EEKEN, J.J., ABOULWAFA-Van VELTHOVEN, M.J.E., & BIRTHWISTLE, R.D.R. (1986a). An investigation in the rat of the acute oral toxicity of Chymosin. Unpublished report No. 8605 from Gist- brocades NV, Delft, Holland. Submitted to WHO by Gist-brocades, NV, Delft, Holland. Van EEKEN, J.J., ABOULWAFA, M.J.E., & BIRTHWISTLE, R.D.R. (1986b). Short term toxicity study in Wistar rats after addition of Chymosin prepared cheese to the feed. Unpublished report No. 8604 from Gist- brocades NV, Delft, Holland. Submitted to WHO by Gist-brocades, NV, Delft, Holland. Van EEKEN, J.J., ABOULWAFA, M.J.E., & BIRTHWISTLE, R.D.R. (1988c). Ninety-one day toxicity study after oral administration of Chymosin to Wistar rats. Unpublished report No. 8711 from Gist-brocades NV, Delft, Holland. Submitted to WHO by Gist-brocades, NV, Delft, Holland. Van EEKEN, J.J., ABOULWAFA, M.J.E., & BIRTHWISTLE, R.D.R. (1988d). Ninety-one day feeding study in Wistar rats after addition of Chymosin prepared cheese to the feed. Unpublished report No. 8706 from Gist-brocades NV, Delft, Holland. Submitted to WHO by Gist- brocades, NV, Delft, Holland. Van LAMBALGEN, R. (undated). Allergenicity of chymosin tested by passive cutaneous anaphylaxis (PCA) assay. Unpublished report from Gist-brocades NV, Delft, Holland. Submitted to WHO by Gist-brocades NV, Delft, Holland. YOUNG, R.R. (1989). Mutagenicity, test on chymosin lots P88035FP and P88040FP in the mouse lymphoma forward mutation assay. Unpublished report No. 20989 from Hazleton Laboratories America, Inc., Kensington, MD, USA. Submitted to WHO by Genencor, Inc., South San Francisco, CA, USA.
See Also: Toxicological Abbreviations