Safety evaluation of the food enzyme protein‐glutamine γ‐glutamyltransferase from the genetically modified Bacillus licheniformis strain NZYM‐TR

TL;DR

This study evaluates the safety of a food enzyme produced by a genetically modified bacterium and concludes it is safe for use in food manufacturing.

Contribution

The paper provides a safety assessment of a novel food enzyme from a genetically modified Bacillus licheniformis strain.

Findings

The enzyme production strain meets the qualified presumption of safety (QPS) criteria.

Dietary exposure estimates are below levels of concern for European populations.

No homology to known allergens was found, though a low risk of allergic reactions cannot be excluded.

Abstract

The food enzyme protein‐glutamine γ‐glutamyltransferase (EC 2.3.2.13) is produced with the genetically modified Bacillus licheniformis strain NZYM‐TR by Novozymes A/S. The production strain meets the requirements for the qualified presumption of safety (QPS). The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in eight food manufacturing processes. Dietary exposure was estimated to be up to 0.651 mg total organic solids (TOS)/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the protein‐glutamine γ‐glutamyltransferase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. A risk of contribution to coeliac disease cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme preparation.

Parameters	Unit	Batches
		1	2	3
Protein‐glutamine γ‐glutamyltransferase activity	TGHU(A)/g a	963	917	1160
Protein	%	8.7	7.3	10.6
Ash	%	0.3	0.3	0.3
Water	%	41.0	43.6	47.6
Glycerol (excipient)	%	50.0	49.0	42.0
Total organic solids (TOS) b	%	8.7	7.1	10.1
Activity/TOS ratio	TGHU/mg TOS	11.1	12.9	11.5

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 37

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of dairy products
Production of cheese	Milk	0.7–6.6
Production of fermented dairy products	Milk	0.7–6.6
Processing of meat and fish products
Production of modified meat and fish products	Meat and fish	4.2–30.5
Processing of cereals and other grains
Production of baked products	Flour	0.4–5.7
Production of cereal‐based products other than baked	Flour	0.4–5.7
Production of brewed products	Cereals	0.4–4.2
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Plant materials (e.g. soy, nuts)	0.8–8.6
Production of meat and fish alternatives c	Plant/fungal proteins	4.2–30.5

TABLE 3. Summary of the estimated dietary exposure to food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.053–0.208 (12)	0.137–0.295 (15)	0.110–0.244 (19)	0.053–0.109 (21)	0.041–0.076 (22)	0.032–0.065 (23)
Min–max 95th percentile (number of surveys)	0.166–0.651 (11)	0.252–0.568 (14)	0.203–0.443 (19)	0.115–0.218 (20)	0.082–0.166 (22)	0.071–0.137 (22)

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
No consumption data is available for fish alternatives, as fish alternatives are not available as FoodEx2 category	−
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

‘Food enzyme preparation’ means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;
• there is a reasonable technological need;
• its use does not mislead the consumer.

All food enzymes currently on the EU market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the Union list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7(2) of Regulation (EC) No 1332/2008^1^ on food enzymes.

On 25 June 2024, a new application was introduced by the applicant ‘Novozymes A/S’ for the authorization of the food enzyme Transglutaminase from a genetically modified Bacillus licheniformis (strain NZYM‐TR).

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of the following food enzyme: Transglutaminase from a genetically modified Bacillus licheniformis (strain NZYM‐TR), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings.3

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme transglutaminase from a genetically modified B. licheniformis strain NZYM‐TR.

In the technical dossier, the applicant named this food enzyme as transglutaminase and indicated that its catalytic activity is described by EC 2.3.2.13 according to the IUBMB classification system. Therefore, the EFSA scientific opinion reports this food enzyme in accordance with the IUBMB classification system (EC 2.3.2.13) and the IUBMB accepted name protein‐glutamine γ‐glutamyltransferase is used throughout the opinion.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme transglutaminase from a genetically modified B. licheniformis strain NZYM‐TR.

Additional information was requested during the risk assessment phase. See https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00545.

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20024 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 30 April to 21 May 2025.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureProtein‐glutamine γ‐glutamyltransferaseSystematic nameProtein‐glutamine: amine γ‐glutamyltransferaseSynonymsTransglutaminase; Factor XIIIa; fibrinoligaseIUBMB NoEC 2.3.2.13CAS No80146‐85‐6EINECS No616‐952‐0

Protein‐glutamine γ‐glutamyltransferases catalyse the acyl‐transfer reaction between the γ‐glutamyl group of a glutamine residue and the ε‐amino group of lysine in proteins, resulting in intra‐ and inter‐molecular cross‐linking of proteins. In the absence of amino substrates, transglutaminases catalyse the hydrolysis of the γ‐carboxyamide group of the glutaminyl residue, resulting in deamidation.

The food enzyme under assessment is intended to be used in eight food manufacturing processes: processing of dairy products for the production of (1) cheese and (2) fermented dairy products; (3) processing of meat and fish products for the production of modified meat and fish; processing of cereals and other grains for the production of (4) baked products, (5) cereal based products other than baked and (6) brewed products; processing of plant‐ and fungal‐derived products for the production of (7) plant‐based analogues of milk and milk products and (8) meat and fish alternatives.

Source of the food enzyme

3.1

The protein‐glutamine γ‐glutamyltransferase is produced with the genetically modified bacterium B. licheniformis strain NZYM‐TR, which is deposited at the ■■■■■.6 The production strain was identified as B. licheniformis by whole genome sequence analysis with an average nucleotide identity (ANI) of 99.48% with the type strain B. licheniformis DSM 13.7

The species B. licheniformis is included in the list of organisms for which the qualified presumption of safety (QPS) may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity are verified for the specific strain used, and the genetic modifications do not raise concerns (EFSA, 2007; EFSA BIOHAZ Panel 2022).8 The production strain B. licheniformis NZYM‐TR was found to be non‐cytotoxic to VERO cells using a lactate dehydrogenase assay.9 The WGS of the production strain was interrogated for the presence of AMR genes using two regularly updated databases with thresholds of 80% identity and 70% coverage. No gene of concern was identified.10 In addition, the genetic modifications are considered safe (see Section 3.1.4). Therefore, the production strain meets the requirements for the QPS approach and is considered safe.11

Characteristics of the parental and recipient microorganisms

3.1.1

The parental strain is B. licheniformis ■■■■■.12

Characteristics of introduced sequences

3.1.2

The sequence encoding the protein‐glutamine γ‐glutamyltransferase ■■■■■.13

Description of the genetic modification

3.1.3

The purpose of the genetic modification was to enable the production strain to synthesise protein‐glutamine γ‐glutamyltransferase. ■■■■■.14

■■■■■.15 ■■■■■.

■■■■■.

■■■■■.16

Safety aspects of the genetic modification

3.1.4

The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.

The production strain B. licheniformis strain NZYM‐TR differs from the parental strain in its capacity to produce protein‐glutamine γ‐glutamyltransferase. ■■■■■

No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,17 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.18

The production strain is grown as a pure culture using a typical industrial medium in a submerged, fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.19 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.20

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The protein‐glutamine γ‐glutamyltransferase is a single polypeptide chain of ■■■■■ amino acids.21 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■.22 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.23 A consistent protein pattern was observed across all batches. The gel showed a protein band migrating between the protein markers of ■■■■■ in all batches, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.

The applicant's in‐house determination of protein‐glutamine γ‐glutamyltransferase activity is based on the formation of an iso‐peptide bond between the amino group of 6‐aminohexanoic acid and the acyl group of N‐benzyloxycarbonyl‐l‐glutaminyl‐glycine (Z‐Gln‐Gly) (reaction conditions: ■■■■■). The released ammonia is measured by a subsequent reaction that oxidises NADH to NAD^+^; the resulting decrease in absorbance is detected spectrophotometrically at 340 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in TransGlutaminase Hydrolase Units (A) (TGHU(A)/g).24 One TGHU(A) is defined as the amount of enzyme that produces 0.0417 μmol of ammonia per minute under the conditions of the assay.25

To determine the pH and temperature optima and thermostability profile of the enzyme, a different activity assay was used, substituting 6‐aminohexanoic acid by hydroxylamine as substrate. The food enzyme has a temperature optimum around 50°C (pH 6.0) and a pH optimum around 7.0 (37°C). Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (pH 6.0). The enzyme showed no residual activity at 60°C.26

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme preparation were provided for three batches intended for commercialisation (Table 1).27 The mean total organic solids (TOS) of the three batches was 8.6% and the mean enzyme activity/TOS ratio was 11.8 TGHU(A)/mg TOS.

Purity

3.3.3

The lead content in the three batches was below 0.5 mg/kg28 ^,^ 29 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme preparation complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).30 No antimicrobial activity was detected in any of the tested batches.31

The Panel considered that the information provided on the purity of the food enzyme was sufficient.

Viable cells and DNA of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme preparation was demonstrated in three independent batches analysed in triplicate. ■■■■■. No colonies were produced. A positive control was included.32

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected with primers that would amplify ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme.33

Toxicological data

3.4

As the production strain qualifies for the QPS approach of safety assessment and no issue of concern arising from the production process of the food enzyme was identified (see Sections 3.1, 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021).

Allergenicity

3.4.1

The allergenicity assessment considered only the food enzyme and not additives, carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the protein‐glutamine γ‐glutamyltransferase produced with the B. licheniformis strain NZYM‐TR was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found in the AllergenOnline and WHO/IUIS allergen nomenclature databases.34

No reports on oral or respiratory sensitisation or elicitation reactions of the protein‐glutamine γ‐glutamyltransferase under assessment have been published.35

A case report has shown that protein‐glutamine γ‐glutamyltransferase can cause occupational asthma (Sander et al., 2020). However, several studies have shown that individuals respiratorily sensitisised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004).

Protein‐glutamine γ‐glutamyltransferases have been implicated in the pathogenesis of coeliac disease, as an autoantigen. In addition, due to their functional similarities with endogenous protein‐glutamine γ‐glutamyltransferases, those from microbial origin may cross link with gluten proteins, enhancing their immunogenicity (Di Sabatino et al., 2012; Lerner et al., 2025; Lerner & Matthias, 2020).

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the protein‐glutamine γ‐glutamyltransferase under assessment, but the risk of contribution to coeliac disease cannot be excluded.

■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201136), is used as raw material. In addition, ■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low. The Panel concluded that a risk of contribution to coeliac disease cannot be excluded.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in eight food manufacturing processes at the recommended use levels summarised in Table 2.

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 37

In all of the food manufacturing processes, the protein‐glutamine γ‐glutamyltransferase catalyses the cross‐linking between glutamine and lysine residues, modifying the physical properties (e.g. breaking strength and moisture retention) of the foods and impacts their sensory attributes such as mouthfeel.38

In the production of cheese, the food enzyme is added to milk together with the starter culture and rennet during the gelling and coagulation.39 The food enzyme–TOS remain in the final foods.

In the production of fermented dairy products, the food enzyme is added to milk before the fermentation.40 The food enzyme–TOS remain in the final foods.

In the production of modified meat and fish products, the food enzyme is added during the mixing.41 The food enzyme–TOS remain in the final foods.

In the production of baked products and in the production of cereal‐based products other than baked, the food enzyme is added to flour during dough making.42 The food enzyme–TOS remain in the final foods.

In the production of brewed products, the food enzyme is added to cereals during the mashing.43 The food enzyme–TOS remain in the brewed products.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to plant materials before or during the coagulation in the manufacturing of cheese alternatives,44 and before the fermentation in the manufacturing of dairy alternatives.45 The food enzyme–TOS remain in these plant‐based foods.

In the production of meat and fish alternatives, the food enzyme is added to plant proteins during the hydration.46 The food enzyme–TOS remain in the final foods.

Based on the data provided on thermostability (see Section 3.3.1) and the downstream processing within the respective food manufacturing processes, the Panel considered that the food enzyme is inactivated in the majority of the food manufacturing processes listed in Table 2. However, it may remain in its active form in cheese and in cheese alternatives, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool47 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023). The applicant provided a list of the FoodEx categories that should be considered for the exposure estimation to the production of meat and fish alternatives, together with their respective technical conversion factors.48 This list has been revised by the Panel and the final outcome that was used for the exposure calculation is presented in Appendix C.

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme–TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.651 mg TOS/kg bw per day in infants at the 95th percentile.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.

The conservative approach applied to estimate the dietary exposure to the food enzyme–TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

Margin of exposure

3.6

Since no toxicological assessment was considered necessary by the Panel, a margin of exposure was not calculated.

CONCLUSIONS

4

Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the production process, the Panel concluded that the food enzyme protein‐glutamine γ‐glutamyltransferase produced with the genetically modified B. licheniformis strain NZYM‐TR does not give rise to safety concerns under the intended conditions of use.

The Panel considered the food enzyme free from viable cells of the production organism and recombinant DNA.

DOCUMENTATION AS PROVIDED TO EFSA

5

Dossier and additional information can be accessed at https://open.efsa.europa.eu/dossier/FEN‐2024‐27030.

ABBREVIATIONSAMRantimicrobial resistanceANIaverage nucleotide identitybwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLOQlimit of quantificationOECDOrganisation for Economic Cooperation and DevelopmentPCRpolymerase chain reactionQPSqualified presumption of safetyRBSribosomal binding siteTGHU(A)TransGlutaminase Hydrolase Units (A)TOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00545

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Henk Van Loveren, Laurence Vernis and Holger Zorn.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.

Supporting information

APPENDIX A: Dietary exposure estimates to the food enzyme–TOS in details