Monophosphate (Amp) Enzyme Preparation from a Genetically Modified Strain of Streptomyces Murinus

Home , Adenosine monophosphate, AMP deaminase, Streptomyces murinus

89th JECFA - Chemical and Technical Assessment (CTA), 2020 © FAO 2021

ADENOSINE 5′-MONOPHOSPHATE (AMP) ENZYME PREPARATION FROM A GENETICALLY MODIFIED STRAIN OF STREPTOMYCES MURINUS

Prepared by Stephan Walch, Ph.D., and reviewed by Jannavi R. Srinivasan, Ph.D.

1. Summary

This Chemical and Technical Assessment summarizes data and information on Adenosine 5′-monophosphate (AMP) enzyme preparation produced by a strain of Streptomyces murinus (AMP deaminase enzyme preparation). The dossier for evaluation was submitted to JECFA on 28 Feb 2020. This document also discusses published information relevant to the safety of AMP deaminase enzyme, namely, the S. murinus production organism, manufacturing, specifications, use and use levels in food. This document uses the expressions “AMP deaminase” to refer to the Adenosine 5′-monophosphate deaminase enzyme and its amino acid sequence, and the expression “AMP deaminase enzyme preparation” to refer to the preparation formulated for commercial use. The Committee has not previously evaluated this additive.

AMP deaminase catalyses the hydrolysis of the amine group from AMP, also referred to as 5’-adenylic acid, to produce inosine 5’ monophosphate (IMP), also referred to as 5’-inosinic acid. The AMP deaminase enzyme preparation is used as a processing aid to convert AMP to IMP during the production of yeast autolysate powders, yeast extracts, yeast cell wall products, and flavouring substances from vegetable, animal or microbial origin; it is intended for use at levels up to 1000 milligrams of Total Organic Solids per kilogram of raw material (mg TOS/kg).

Streptomyces murinus is an actinobacterium. Streptomyces species in general are non-pathogenic microorganisms. Streptomyces murinus AE-DNTS, the production strain, was obtained by chemical mutagenesis of the parent strain, S. murinus (NBRC14802) followed by selection of individual colonies. No evidence or indications of pathogenicity of S. murinus are known (M. Stavnsbjerg, 1986).

The AMP deaminase enzyme preparation is manufactured by a controlled aerobic batch fermentation of a pure culture of S. murinus carrying the AMP deaminase gene. The AMP deaminase is secreted into the fermentation broth and separated by a series of filtration steps, followed by concentration and purification. The final enzyme product is a powder that is formulated with food-grade dextrin to the desired activity for commercial use. The AMP deaminase enzyme preparation complies with the General Specifications and Considerations for Enzyme Preparations Used in Food Processing (FAO/WHO, 2006).

AMP deaminase is not known to be allergenic when used in food processing. Examination of the potential for this enzyme to be a food allergen was made by comparing its amino acid sequence to sequences of known allergens contained within the AllergenOnline allergen database using internationally accepted search criteria. No meaningful identity with known allergens was observed. Based on the results obtained, oral intake of AMP deaminase is not anticipated to pose any allergenicity concern.

2. Description

White to dark brown powder; soluble in water.

AMP deaminase from S. murinus (2020) Page 1 of 6 ©FAO 2021

3. Method of manufacture

3.1 S. murinus

The genus Streptomyces was first described by Waksman and Henrici in 1943 and currently includes more than 700 species (http://www.bacterio.net/streptomyces.html). Streptomyces are widely distributed in various environments (Promnuan et al. 2013, Yamamura et al. 2014, Lin et al. 2012), and Streptomyces’ species have been used in food applications (Bourdichon et al., 2012). The taxonomy of S. murinus was confirmed based on macroscopic and microscopic characteristics.

The taxonomy of S. murinus is as follows:

Kingdom: Bacteria Division: Actinobacteria Class: Actinobacteria Order: Actinomycetales Family: Streptomycetaceae Genus: Streptomyces Species: S. murinus

Strains of S. murinus that have been deposited in several public culture collections, including the American Type Culture Collection (ATCC, website) and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, website). S. murinus has been designated as a Safety Level 1 strain. It is not included in the list of pathogens in Annex III of Directive 2000/54/EC on the protection of workers from risks related to exposure to biological agent at work or on the list of pathogens in Belgium (EC, 2000; Belgian Biosafety Server, 2008). While most Streptomyces species are non-virulent, they can produce secondary metabolites, including metabolites with antimicrobial activity. Therefore, Streptomycetes were not eligible when considered for Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) (EFSA, 2014).

S. murinus has been used as source organism to produce glucose isomerase that is intended for use in food processing (Pariza & Johnson, 2001; Codex Alimentarius, 2012; Health Canada, 2020). In Japan, S. murinus is permitted as a source of AMP deaminase enzyme that is intended for use as a food additive or processing aid.

3.2 S. murinus production strain

The S. murinus production strain, AE-DNTS, was obtained by chemical mutagenesis of the parent strain, S. murinus (NBRC14802) followed by selection of individual colonies. The production strain was verified to be S. murinus by comparing the partial sequence of its gyrB gene (DNA gyrase β-subunit gene, approx. 1200 bp) with that of the parent S. murinus NBRC 14802 strain (Hatano, et al, 2003) and of other Streptomyces species (S. graminearus NBRC 15420, S. griseofuscus NBRC 12870, and S. costaricanus NBRC 100773). Based on the results of a homology search using BLAST, the production strain, S. murinus AE-DNTS, showed 100% sequence similarity to S. murinus.

3.3 Fermentation, recovery, and formulation

The seed material for the inoculum of the fermentation for the production of the AMP deaminase enzyme is obtained from cultures of the S. murinus production strain AE-DNTS that meet identity, viability, and microbial purity and productivity criteria of the Working Cell Bank (WCB) under controlled conditions. The manufacture of the AMP deaminase enzyme preparation consists of three steps: pre-culture fermentation, seed fermentation, and main fermentation. Control processes are in place during the fermentation and down-stream processing to prevent the potential for microbial contamination, as are periodic testing for enzyme activity during production. Fermentation is terminated after optimal enzyme production is reached; the AMP deaminase is secreted into the culture medium. The enzyme is separated from the culture medium containing the production organism and fermentation media via a series of filtration steps. The liquid filtered enzyme is AMP deaminase from S. murinus (2020) Page 2 of 6 ©FAO 2021

concentrated and purified. The enzyme concentrate is formulated into the commercial AMP deaminase enzyme preparation by the addition of dextrin. The entire production of AMP deaminase enzyme preparation is carried out in accordance with GMP and the principles of Hazard Analysis and Critical Control Points (HACCP) using raw materials that are appropriate for food-use. The AMP deaminase enzyme preparation is free of antibiotic activity. The AMP deaminase conforms to the General Specifications for Enzyme Preparations used in Food Processing (JECFA, 2006), and the enzyme concentrate is tested to be free from the production organism.

4. Identity and characterization

4.1 AMP deaminase

AMP deaminase belongs to the subcategory of hydrolases that act on carbon-nitrogen bonds, other than peptide bonds in cyclic amidines (BRENDA Comprehensive Enzyme Information System). The enzyme catalyzes the hydrolysis of the adenosine monophosphate to inosine monophosphate, freeing an ammonia molecule in the process. It is classified by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) as follows:

Accepted name: AMP deaminase

Other names(s): adenylic acid deaminase; AMP deaminase; AMP aminase; adenylic deaminase; adenylate deaminase; 5-5’ deaminase; adenosine 5-monophosphate deaminase; 5- adenylate deaminase; adenyl deaminase; 5-adenylic acid deaminase; adenosine monophosphate deaminase; adenylate aminohydrolase; adenylate desaminase; adenosine 5-phosphate aminohydrolase; 5-adenylate deaminase

Reaction: Deamination of adenosine monophosphate (AMP) via hydrolysis to produce inosine 5’-monophosphate (IMP).

Systematic name: AMP aminohydrolase

EC No.: 3.5.4.6

AMP deaminase produced by S. murinus does not possess any other enzymatic activities. The primary sequence of AMP deaminase has been determined to consist of 491 amino acids; its molecular weight, based on SDS- PAGE, is ~60 kDa, with no post-translational modification applied.

AMP deaminase activity is determined spectrophotometrically by treating AMP substrate with AMP deaminase and measuring the difference in absorbance at 265 nm after incubation for 15 minutes. AMP deaminase activity is expressed in deaminase activity units (U). 1,000 U is defined as the amount of the enzyme required to decrease the absorbance by 0.1 when measured at 265 nm for 60 minutes.

4.2 AMP deaminase enzyme preparation

The AMP deaminase enzyme preparation will be marketed as a liquid formulation under the trade name Deamizyme T “Amano”. A representative composition of a commercial liquid formulation of the AMP deaminase enzyme preparation is provided below:

Enzyme activity: 95 000 000 - 183 000 000 U/g Water: 2.4 - 5% Ash: 13 - 15% Protein: 3 - 4% TOS*: 35% *includes dextrin

AMP deaminase from S. murinus (2020) Page 3 of 6 ©FAO 2021

The AMP deaminase enzyme preparation consists of the enzyme and substances from the fermentation process; these constitute proteins, peptides, amino acids, carbohydrates, lipids and salt. The components of fermentation are referred to as Total Organic Solids (TOS).

The TOS content of an enzyme preparation is calculated according to the following equation (NAS/NRC, 1981; FAO/WHO, 2006):

TOS (%) = 100 – (A + W + D)

where A is the % ash, W is the % water and D is the % diluents and/or other formulation ingredients.

The specifications for AMP deaminase preparation include activity (approximately 60 000 000 U/g), lead (≤ 5 mg/kg), total coliforms (≤ 30 CFU/g), Salmonella sp. (absent in 25 g), E. coli (absent in 25 g), antimicrobial activity (absent by test). AMP deaminase enzyme preparation complies with the General Specifications for Enzyme Preparations used in Food Processing as established by the 67th meeting of the Joint Expert Committee on Food Additives (FAO/WHO, 2006).

5. Functional uses

The AMP deaminase enzyme preparation promotes the conversion of AMP (generally without taste) to IMP (umami flavour) to enhance the flavour profile of certain products. The AMP deaminase enzyme is intended for use in yeast processing for cereals and in the production of flavourings of vegetable, animal or microbial origin. Food uses containing flavourings include soups, sauces, stocks, dressings, snack foods, meat-derived foods, bread, crackers and beverages. The AMP deaminase enzyme preparation is used at levels ranging from 10 to 1000 mg TOS per kg raw material.

6. Reaction and fate in food

AMP deaminase catalyses the deamination of AMP to produce IMP.

5’-deaminase

Adenosine monophosphate (AMP) Inosine 5’-monophosphate (IMP)

The enzyme is not expected to have a technological function on the final food and will be denatured or removed. Carry-over of active AMP deaminase to the final food product is expected to be negligible. Deamination activity of AMP deaminase results in the formation of IMP which is a normal constituent of the diet and therefore does not result in the formation of any new substrates that do not already occur in food.

7. References

ATCC, website. http://www.atcc.org/Search_Results.aspx?dsNav=Ntk:PrimarySearch%7cStreptomyces+murinus%7c3%7c,Ny:Tr ue,Ro:0,N:1000552&searchTerms=Streptomyces+murinus&redir=1

Belgian Biosafety Server, 2008. List of bacteria and similar organisms presenting at the wild state a biological risk for immunocompetent humans and/or animals and corresponding maximum biological risk. Brussels: Belgian Biosafety Server, Division of Biosafety and Biotechnology, Scientific Institute of Public Health. https://www.biosafety.be/sites/default/files/h_a_bacteries.pdf

Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J. C., Gerds, M. L., Hammes, J. P., Harnett, J., Huys, G., Laulund, S., Ouwehand, A., Powell, I. B., Prajapati, J. B., Seto, Y., Schure, E. T., Van Boven, A., Vankerckhoven, V., Zgoda, A., Tuijtelaars, S., Hansen, E. B. 2012. Food fermentations: Microorganisms with technological beneficial use. Int. J. Food Micro., 154, 2003, 87-97.

BRENDA Comprehensive Enzyme Information System. https://www.brenda-enzymes.org/enzyme.php?ecno=3.5.4.6

Codex Alimentarius, 2012. AMP deaminase. In: Inventory of substances used as processing aids (IPA), updated list (information document), Joint FAO/WHO Food Standards Programme Codex Committee on Food Additives (CCFA), Forty Fourth Session, Mar. 12-16, 2012, Hangzhou, China. Rome: Codex Alimentarius; 42. (FA/44/ INF/03 http://www.fao.org/tempref/codex/Meetings/CCFA/ccfa44/fa44_inf3e.pdf

DSMZ, website. https://www.dsmz.de/search/bacteria?tx_kesearch_pi1%5Bsword%5D=Streptomyces%20murinus&cHash=ecd82 f77125b438a4d893e223f1e1f63

EFSA, 2014. Statement on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA. 1: Suitability of taxonomic units notified to EFSA until October 2014 (EFSA Panel on Biological Hazards/BIOHAZ) (Question no EFSA-Q-2014-00611). EFSA J. 12(12):3938 [41 pp., plus Appendix]. https://www.efsa.europa.eu/en/efsajournal/pub/3938.

EU, 2000. DIRECTIVE 2000/54/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC). Official Journal, L 262(17 August), 21-45 Belgian classifications for micro-organisms based on their biological risks (Last revision: March 8, 2010). Belgian Biosafety Server. http://www.biosafety.be/RA/Class/ClassBEL.html

FAO/WHO, 2006. General specifications and considerations for enzyme preparations used in food processing. In: Compendium of food additive specifications, volume 4. Analytical methods, test procedures and laboratory solutions used by and referenced in the food additive specifications. FAO/JECFA Monographs 1. Rome, Italy, pp. xxi-xxv.

Hatano, K., Nishii, T., Kasai H. 2003. Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA-DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. Rev, Int. J. Syst. Evol. Microbial., 2003, 53, 1519-1529.

Health Canada, 2020. List of permitted food enzymes. Ottawa: Health Canada. (Lists of Permitted Food Additives, No. 5. https://www.canada.ca/en/health-canada/services/food-nutrition/food-safety/food-additives/lists-permitted/5- enzymes.html

Lin Y.B., Wang X.Y., Fang H, Ma Y.N., Tang J., Tang M., Wei G.H. 2012. Streptomyces shaanxiensis sp. nov. a blue pigment-producing streptomycete from sewage irrigation soil. Int J Syst Evol Microbiol 62:1725–1730.

NBRC Catalogue. http://www.nbrc.nite.go.jp/NBRC2/NBRCCatalogueDetailServlet?ID=NBRC&CAT=00014802 AMP deaminase from S. murinus (2020) Page 5 of 6 ©FAO 2021

NRC/NAS, 1981. The 1978 Enzyme Survey Summarized Data. National Research Council/National Academy of Sciences. Washington, D.C., USA. U.S. Department of Commerce. National Technical Information Service.

Pariza, M. W. and Johnson, E. A. 2001. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century, Regul Toxicol Pharmacol. 33(2):173–86.

Promnuan Y., Kudo T., Ohkuma M., Chantawannakul P. 2013. Streptomyces chiangmaiensis sp. nov. and Streptomyces lannensis sp. nov. isolated from the South-East Asian stingless bee (Tetragonillacollina). Int J Syst Evol Microbiol, 2013, 63:1896–1901.

Stavnsbjerg, M., 1986. Evaluation of the Pathogenic Potential of Streptomyces murinus. Internal Report, Novo Industri A/S, file number F-860144, 1986.

Waksman S.A., Henrici A.T. 1943. The nomenclature and classification of the actinomycetes. J Bacteriol. 46:337–341. http://www.bacterio.net/streptomyces.html

Yamamura H., Ashizawa H., Hamada M., Hosoyama A., Komaki H., Otoguro M., Tamura T., Hayashi Y., Nakagawa Y., Ohtsuki T., Fujita N., Ui S., Hayakawa M. 2014. Streptomyces hokutonensis sp. nov. a novel actinomycete isolated from the strawberry root rhizosphere, J Antibiot. 2014, 67:465–470.