Acrylamide-degrading enzyme

Abstract

This invention is primarily in the field of food and beverage products, such as coffee and coffee substitutes. [Solution] A novel enzyme is provided that can decompose acrylamide at temperatures preferably above 50°C, particularly within the temperature range that occurs in the manufacture of coffee / coffee substitutes, and / or at pH values ​​between pH 4 and pH 7, as is common in the manufacture of coffee / coffee substitutes. Furthermore, a method for decomposing acrylamide from preparations selected from semi-processed and finished products is provided. The present invention also relates to preparations having a reduced acrylamide content compared to preparations not subjected to the method for removing acrylamide according to the present invention, by the enzyme according to the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of production of foods and luxury consumables, particularly coffee and coffee substitute products. The present invention provides a novel enzyme capable of degrading acrylamide, wherein said degradation is preferably performed at a temperature exceeding 50°C, particularly in the temperature range in which the production of coffee / coffee substitute products is carried out, and / or between pH 4 and pH 7, which are standard pH values in the production of coffee / coffee substitute products. Further provided is a method for degrading acrylamide in preparations selected from semi-finished products, as well as acrylamide in preparations selected from finished products. The present invention also relates to a preparation having a reduced acrylamide content compared to a preparation that has not been subjected to the acrylamide removal step of the present invention using the enzyme according to the present invention.

Background Art

[0002] End consumers' demand for consistently safe and safely consumable foods and luxury consumables is constantly increasing, imposing special requirements on producers. In the new EU regulation (EU 2017 / 2158) effective from 2018, acrylamide is classified as a process contaminant and a potential health risk for end consumers. Accordingly, producers of foods and luxury consumables are obligated to maintain or reduce the acrylamide content in foods and luxury consumables below a certain level. In animal experiments, acrylamide has been shown to be carcinogenic and mutagenic. Acrylamide is formed in all processes of stir-frying, roasting and frying of starch-containing raw materials, and is a heating product of the reaction between asparagine and reducing sugars (e.g., glucose and fructose) as part of the so-called Maillard reaction.

[0003] In the manufacture of coffee and coffee substitutes, these processes include roasting, or roasted and ground coffee beans, and extraction of substitutes such as chicory, barley, or rye. During roasting, coffee beans are typically exposed to temperatures ranging from 145°C to 250°C, resulting in complex chemical reactions, including the Maillard reaction, caramelization, and thermal decomposition. These reactions alter the chemical, physical, and sensory properties of the roasted product, which are fundamentally important to the flavor of the beverage. Other substances important to the final product, such as antioxidants, are also formed (Non-Patent Literature 1). Acrylamide is also formed as an undesirable process contaminant by roasting coffee and coffee substitutes (Non-Patent Literature 2). The index values ​​for acrylamide in the new EU regulations are 400 μg / kg for roasted coffee, 850 μg / kg for soluble coffee, 500 μg / kg for coffee substitutes made solely from grains, and 4000 μg / kg for products made from chicory.

[0004] Acrylamide is also formed in numerous other processes in the food and beverage industries. For example, it is formed when potatoes are fried. In particular, it is advantageous, and even necessary, to partially or completely remove acrylamide from semi-processed or finished products in order to obtain a safe final product that meets the requirements of EU Regulation (EU2017 / 2158) and is harmless to consumers.

[0005] Numerous attempts have been made to reduce the acrylamide content in food and beverages, but nearly complete or complete removal of acrylamide at an economically reasonable cost has not been achieved. For example, in Patent Document 1, the acrylamide content is reduced after coffee extraction by absorbing acrylamide using a cationic resin. This process requires an additional step and is time-consuming because absorption is kinetically slow. Furthermore, only 50% of the acrylamide is removed, and the cationic resin represents an additional cost to the process.

[0006] Another method for reducing acrylamide is to reduce its precursors. This is the subject of Patent Document 2, which discloses a process for reducing asparagine and aspartic acid, initiated before the roasting process. Reducing the asparagine content before roasting reduces the acrylamide content of the final product, but alters the flavor profile of the final product. Furthermore, this process is costly for the user because it requires additional new equipment.

[0007] Another approach, adopted by the authors of Patent Document 3, also deals with the enzyme-assisted production of coffee. In this method, the starch in the coffee beans is enzymatically broken down into its individual components to improve the flavor profile of the final product. In this process, due to the high availability of monosaccharides, acrylamide is present in higher amounts than in standard coffee products. This clearly indicates that the timing of the enzymatic treatment is critical to reducing the acrylamide content.

[0008] Patent Document 4 relates to a thermostable amidase isolated from a thermophilic organism, particularly an amidase derived from a thermophilic actinomycete such as Pseudonocardia thermophilia. According to the sequence listing disclosed in the specification, the amino acid sequence of the amidase derived from Pseudonocardia thermophilia is disclosed in Patent Document 4 as Sequence ID No. 3 (corresponding to Sequence ID No. 42 of this application). Subsequently, the genome of Pseudonocardia thermophilia was completely sequenced and deposited as Non-Patent Document 3. The genome contains an amidase locus in the range of 50704-52245, and this protein has been deposited as GenBank SHK14489.1. The sequence alignment of the deposited amidase compared to the wild-type amidase of Sequence ID No. 2 (not according to the present invention) shows 100% identity over the entire length of the protein. However, the amino acid sequence provided in Patent Document 4 appears incorrect, particularly in the sequence portion of the first approximately 100 N-terminal amino acid residues. Since these sequence inaccuracies may be mainly due to the sequencing methods available at the time, Sequence ID No. 3 in Patent Document 4 is incorrect from a modern perspective. Therefore, the correct amino acid sequence of wild-type amidase from Pseudonocardia thermophilia is rather the current (non-inventive) Sequence ID No. 2. Sequence alignment of Sequence ID No. 3 from Patent Document 4 compared with wild-type amidase from Pseudonocardia thermophilia using the current Sequence ID No. 2 shows that only 447 out of 528 positions are identical (identity: 84.7%) and 458 out of 528 positions are similar (similarity: 86.7%), and the alignment is shown in Figure 1.

[0009] The final report of Hamburg University of Technology in "Use of Amidase Derived from Extremophilic Microorganisms for Enantioselective Synthesis of Amino Acids and Carboxylic Acids (Non-Patent Document 4)" from December 2005, which is clearly related to Patent Document 4, contains supplementary information. According to the project description, the inventor of Patent Document 4 is a co-investigator of this project. Accordingly, a portion of the protein sequence of amidase isolated from Pseudonocardia thermophilia was sequenced and used to amplify and sequence the putative amidase gene in organisms by PCR. In experiments to produce recombinant amidase by expression in an E. coli host system, the gene sequence amplified by PCR was cloned into the arabinose-induced expression vector pBad-Thio-TOPO. The expression of a 54 kDa protein could be detected by SDS-PAGE, but in enzyme assays using substrates converted by the native protein, Pseudonocardia thermophilia The activity of recombinant amidase derived from thermophilia could not be detected. Therefore, the disclosure in Patent Document 4 is not possible for those skilled in the art.

[0010] Patent document 5 relates to a process for the decomposition of acrylamide using amidase.

[0011] Non-patent document 5 relates to the enzymatic control of acrylamide content in coffee using enzymes derived from Ralstonia eutropha and Geobacillus thermoglucasidasius. Although specific enzyme sequences are not named in this document, the maximum known identity of enzymes derived from Ralstonia eutropha is approximately 36.4% and that derived from Geobacillus thermoglucasidasius is approximately 53.3%, compared to enzymes derived from Pseudonoracdia thermophilia.

[0012] Non-patent document 6 concerns the development and application of a transcription sensor for detecting heterologous production of acrylic acid in E. coli. This paper names an amidase derived from Geobacillus pallidus, which has approximately 14% identity with but is unrelated to an enzyme derived from Pseudonoracdia thermophilia, as RAPc8.

[0013] Non-patent document 7 relates to the cloning of amidase derived from Bacillus stearothermophilus BR388 in Escherichia coli. The amidase derived from Bacillus stearothermophilus BR388 has approximately 14% identity with the enzyme derived from Pseudonoracdia thermophilia, but is unrelated to it.

[0014] Non-patent document 8 concerns the exchange of Thr-103-Ile and Trp-138-Gly in amidase derived from Pseudomonas aeruginosa. Amidase derived from Pseudomonas aeruginosa has approximately 15% identity with enzyme derived from Pseudonoracdia thermophilia, but this is unrelated.

[0015] Non-patent document 9 relates to the undirected evolution of amidase-expressing Methylophilus methylotrophus through proliferation selection and chemical mutation. This amidase has approximately 14% identity with the enzyme derived from Pseudonoracdia thermophilia, but is unrelated to it.

[0016] Non-patent document 10 relates to the physical, biochemical, and immunological characterization of a thermostable amidase derived from Klebsiella pneumoniae. Although no specific enzyme sequence is named in this document, the maximum known identity of the enzyme derived from Klebsiella pneumoniae compared to the enzyme derived from Pseudonoracdia thermophilia is approximately 51.2%.

[0017] In all processes involving enzymes, their use is tied to the precise setting of process variables. Every enzyme has a temperature and pH range (including its optimal value) in which it catalyzes or can catalyze its respective reaction. Using it outside of these ranges usually prevents the enzyme from exhibiting its catalytic function, or from doing so to the desired extent. This is often the case, for example, with non-thermophilic enzymes at temperatures above 42°C, as well as with substantially acidic or clearly basic pH values. Therefore, one of the challenges in enzyme processes is always correctly selecting or modifying the enzyme so that it is suitable for the process conditions.

[0018] The main object of the present invention was to provide suitable enzymes and methods for treating acrylamide-containing preparations, which can reduce the acrylamide content in the preparation, preferably by at least 80% by weight compared to the preparation before treatment. Said enzymes preferably retain their enzymatic activity and / or exhibit high stability even at high temperatures which are common after the steps of hot water treatment, stir-frying and roasting of foods and luxury foods, and / or even at pH values between pH 4 and pH 7. Further objects underlying the present invention arise from the following description and the appended claims. [PRIOR ART DOCUMENT] [PATENT DOCUMENTS]

[0019] [Patent Document 1] European Patent Application Publication No. 3254568A1 [Patent Document 2] International Publication No. 2013 / 005145A1 [Patent Document 3] European Patent No. 1745702B1 [Patent Document 4] International Publication No. 2004 / 083423A1 [Patent Document 5] European Patent Application Publication No. 0272024A2 [Patent Document 6] European Patent No. 1608746B1 [Patent Document 7] International Publication No. 2006 / 040345 [NON-PATENT DOCUMENTS]

[0020] [Non-Patent Document 1] Jin et al., "Relationship between antioxidants and acrylamide formation", Review, Food Research International, May 2013, Volume 51, Issue 2, pages 611-620 [Non-Patent Document 2] Anese M., Acrylamide in Coffee and Coffee Substitutes, Acrylamide in Food, Acrylamide in Food, 2016, pp. 181-195 [Non-Patent Document 3] GenBank Accession No. FRAP01000003.1 https: / / www.ncbi.nlm.nih.gov / nuccore / FRAP01000003 [Non-Patent Document 4] Project entitled Use of amidases from extremophilic microorganisms for the enantioselective synthesis of amino and carboxylic acids AZ 13107 https: / / www.dbu.de / projekt_13107 / 01_db_2409.html, December 2005 [Non-Patent Document 5] M. Cha, European Food Research and Technology, 236, 2013, pp. 567-571 [Non-Patent Document 6] S. Raghavan et al., Microbe Cell Fact, 18, 2019, p. 139 [Non-Patent Document 7] T. K. Cheong et al., Enzyme and Microbial Technology, February 2000, Vol. 26, Issues 2-4, pp. 152-158 [Non-Patent Document 8] A. Karmali et al., Molecular Biotechnology, 2001, Vol. 17, pp. 211-212 [Non-Patent Document 9] N. J. Silman et al., Journal of General Microbiology, 1991, Vol. 137, pp. 169-178 [Non-Patent Document 10] MSNawaz et al., Journal of Bacteriology, 1996, pp. 2397-2401. [Non-Patent Document 11] F. William Studier, Protein Expression and Purification, 2005, Vol. 41, pp. 207-234. [Non-Patent Document 12] http: / / www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html [Non-Patent Document 13] http: / / www.ebi.ac.uk / Tools / psa / emboss_water / [Non-Patent Document 14] http: / / www.ebi.ac.uk / Tools / psa / [Non-Patent Document 15] Smith, TF & Waterman, MS. "Identification of common molecular subsequences." Journal of Molecular Biology, 1981, Vol. 147(1), pp. 195-197. [Non-Patent Document 16] https: / / www.rcsb.org / [Non-Patent Document 17] https: / / www.rcsb.org / structure / 3a1k [Overview of the project] [Means for solving the problem]

[0021] The problem of the present invention is primarily solved by providing an enzyme (as described herein and especially in the claims), preferably an amidase, that can significantly reduce the amount of acrylamide in the preparation, and this is also true at temperatures and pH values ​​that are unfavorable for many amidases.

[0022] Furthermore, according to a preferred embodiment, the present invention relates to such enzymes, preferably amidases, (as described herein and in particular in the claims) that are catalytically active not only at temperatures above 50°C but also in a pH range of pH 4 to pH 7.

[0023] Furthermore, the present invention provides not only a method for preparing a preparation with a reduced acrylamide content, but also a suitable method for decomposing acrylamide in the preparation using the enzyme according to the present invention (as described herein and in particular in the claims).

[0024] Furthermore, a preparation with a reduced acrylamide content is provided, obtained by the process according to the present invention.

[0025] Details, preferred and alternative embodiments and aspects of the present invention will become apparent from the following description, the appended arrangement, and in particular, the appended claims. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 shows the alignment of sequence number 42 (row "65") and sequence number 2 (row "02"), with identity: 447 / 528 (84.7%), similarity: 458 / 528 (86.7%), gap: 41 / 528 (7.8%), and sequence coverage: 507 / 513 (98.8%). [Modes for carrying out the invention]

[0027] A brief explanation of arrays Sequence ID 1 describes the amino acid consensus sequence of the enzyme according to the present invention. Sequence ID 2 describes the amino acid sequence of wild-type amidase derived from Pseudonocardia thermophila (not the present invention). Sequence ID 3 describes the amino acid sequence according to the present invention, which includes a mutation at position 68 compared to Sequence ID 2 (D68N). Sequence ID 4 describes the amino acid sequence according to the present invention, which includes a mutation at position 74 compared to Sequence ID 2 (A74Y). Sequence ID 5 describes the amino acid sequence according to the present invention, which includes a mutation at position 445 compared to Sequence ID 2 (G445A). Sequence ID 6 describes the amino acid sequence according to the present invention, which includes a mutation at position 33 compared to Sequence ID 2 (S33F). Sequence ID 7 describes the amino acid sequence according to the present invention, which includes a mutation at position 33 compared to Sequence ID 2 (S33R). Sequence ID 8 describes the amino acid sequence according to the present invention, which includes a mutation at position 445 compared to Sequence ID 2 (G445S). Sequence ID 9 describes the amino acid sequence according to the present invention, which includes four mutations at positions 33, 74, 445, and 453 compared to Sequence ID 2 (S33R, A74Y, S225T, G445S, A453C). Sequence ID 10 describes the amino acid sequence according to the present invention, which includes five mutations at positions 33, 68, 74, 445, and 453 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445S, A453C). Sequence ID 11 describes the amino acid sequence according to the present invention, which includes five mutations at positions 33, 68, 74, 225, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445A). Sequence ID 12 describes the amino acid sequence according to the present invention, which includes four mutations at positions 68, 74, 445, and 453 compared to Sequence ID 2 (D68N, A74Y, G445S, A453C). Sequence ID 13 describes the amino acid sequence according to the present invention, which includes four mutations at positions 33, 68, 74, and 225 compared to Sequence ID 2 (S33H, D68N, A74Y, S225T). Sequence ID 14 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes 12 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, 453, and 507 (S33R, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453D, A507P). Sequence ID 15 describes the amino acid sequence according to the present invention, which includes 11 mutations at positions 33, 68, 74, 94, 201, 225, 424, 445, 448, 453, and 507 compared to Sequence ID 2 (S33R, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453D, A507P). Sequence ID 16 describes the amino acid sequence according to the present invention, which includes 11 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, and 453 compared to Sequence ID 2 (S33Y, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453D). Sequence ID 17 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes nine mutations at positions 33, 41, 68, 74, 201, 225, 424, 445, and 448 (S33R, W41Y, D68N, A74Y, Y201F, S225T, L424V, G445A, M448H). Sequence ID 18 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes 12 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, 453, and 507 (S33R, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453C, A507P). Sequence ID 19 describes the amino acid sequence according to the present invention, which includes 11 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, and 453 compared to Sequence ID 2 (S33Y, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453N). Sequence ID 20 describes the amino acid sequence according to the present invention, which includes 11 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, and 453 compared to Sequence ID 2 (S33Y, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453Q). Sequence ID 21 describes the amino acid sequence according to the present invention, which includes 11 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, and 453 compared to Sequence ID 2 (S33Y, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453E). Sequence ID 22 describes the amino acid sequence according to the present invention, which includes 11 mutations at positions 33, 41, 68, 74, 94, 201, 225, 424, 445, 448, and 453 compared to Sequence ID 2 (S33Y, W41Y, D68N, A74Y, V94I, Y201F, S225T, L424V, G445A, M448H, A453K). Sequence ID 23 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 445, and 454 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445A, P454N). Sequence ID 24 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 445, and 457 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445A, V457G). Sequence ID 25 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 424, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, L424V, G445A). Sequence ID 26 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 445, and 453 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445A, A453D). Sequence ID 27 describes the amino acid sequence according to the present invention, which includes five mutations at positions 33, 68, 74, 225, and 445 compared to Sequence ID 2 (S33Y, D68N, A74Y, S225T, G445A). Sequence ID 28 describes the amino acid sequence according to the present invention, which includes five mutations at positions 33, 68, 74, 175, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G175A, G445A). Sequence ID 29 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 445, and 507 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445A, A507P). Sequence ID 30 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes six mutations at positions 33, 68, 74, 225, 445, and 453 (S33R, D68N, A74Y, S225T, G445A, A453S). Sequence ID 31 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 94, 225, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, V94I, S225T, G445A). Sequence ID 32 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 317, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, V317I, G445A). Sequence ID 33 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes six mutations at positions 33, 68, 74, 201, 225, and 445 (S33R, D68N, A74Y, Y201F, S225T, G445A). Sequence ID 34 describes the amino acid sequence according to the present invention, which includes five mutations at positions 33, 68, 74, 445, and 448 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445A, M448H). Sequence ID 35 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes six mutations at positions 33, 68, 74, 225, 445, and 453 (S33R, D68N, A74Y, S225T, G445A, A453R). Sequence ID 36 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 221, 225, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, P221G, S225T, G445A). Sequence ID 37 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 217, 225, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, T217R, S225T, G445A). Sequence ID 38 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes six mutations at positions 33, 68, 74, 225, 328, and 445 (S33R, D68N, A74Y, S225T, D328R, G445A). Sequence ID 39 describes the amino acid sequence according to the present invention, which includes five mutations at positions 33, 68, 74, 225, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, G445S). Sequence ID 40 describes the amino acid sequence according to the present invention, which includes six mutations at positions 33, 68, 74, 225, 229, and 445 compared to Sequence ID 2 (S33R, D68N, A74Y, S225T, L229C, G445A). Sequence ID 41 describes the amino acid sequence according to the present invention, which, compared to Sequence ID 2, includes six mutations at positions 33, 41, 68, 74, 225, and 445 (S33R, W41Y, D68N, A74Y, S225T, G445A). Sequence ID 42 corresponds to the protein sequence of Pseudonocardia thermophila disclosed as "Sequence ID 3" in Patent Document 4.

[0028] In a first embodiment of the present invention, an enzyme is provided for reducing the amount of acrylamide in a preparation comprising or consisting of an amino acid consensus sequence according to SEQ ID NO: 1. Herein, the amino acid consensus sequence is not the sequence according to SEQ ID NO: 2, and the enzyme comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a sequence selected from the group consisting of sequences shown in SEQ ID NOs: 3 to 41.

[0029] As described above, Sequence ID No. 1 describes the amino acid consensus sequence of the enzyme according to the present invention. The consensus sequence describes the amino acid sequence that all enzymes according to the present invention possess. Variable positions are indicated by Xaa, representing positions where the enzymes according to the present invention may differ from one another.

[0030] Enzymes are biocatalysts that catalyze specific chemical reactions. In this example, the decomposition of acrylamide by amidase is hydrolytically cleaved, forming acrylic acid and ammonia. The concentrations of acrylic acid and ammonia, or, where applicable, the resulting substances (if acrylic acid or ammonia undergoes further reactions), are very low in coffee preparation and do not adversely affect the end user. For example, ammonia reacts quickly to form harmless ammonium, which does not affect the final product.

[0031] Whenever this disclosure refers to the sequence identity of an amino acid sequence in terms of a percentage, such reference means a value that can be calculated using EMBOSS Water Pairwise Sequence Alignments (nucleotides) <Non-Patent Document 12> for nucleic acid sequences, or EMBOSS Water Pairwise Sequence Alignments (proteins) <Non-Patent Document 13> for amino acid sequences. In the case of the local sequence alignment tool provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI), the modified Smith-Waterman algorithm is used (see Non-Patent Documents 14 and 15). Furthermore, when performing pairwise alignment of each of two sequences using the modified Smith-Waterman algorithm, the default parameters currently provided by EMBL-EBI are referred to. These are: (i) for amino acid sequences: matrix = BLOSUM62, gap start penalty = 10, and gap extension penalty = 0.5, and (ii) for nucleic acid sequences: matrix = DNAfull, gap start penalty = 10, and gap extension penalty = 0.5. In addition to the default parameters, when aligning a target sequence (the "query sequence" of the first sequence in EMBOSS) with a reference sequence (the "target sequence" of the second sequence in EMBOSS), the target sequence must be represented by at least 93% (at least 93% "sequence coverage") over the length of a single alignment, and alignments of the target sequence with lower sequence coverage are excluded from determining sequence identity for the purposes of this application. However, the query sequence may be longer than the alignment length, and the sequence represented by the alignment of the query sequence may be greater than or less than 93%. For example, in the alignment of sequence number 2 as the target sequence and sequence number 56 as the query sequence (Figure 1), 507 of the 513 amino acids of the target sequence are present, and therefore the sequence coverage is 507 / 513 (98.8%).

[0032] Therefore, the term "sequence identity" can be used interchangeably with "sequence homology" in the context of the present invention. The latter always refers to the total length of the enzyme according to the present invention relative to the total length of the enzyme for which sequence identity or sequence homology is determined.

[0033] In the context of the present invention, "preparation" means any food, confectionery, or cosmetic product, whether raw, semi-processed, or finished, including, for example, products of fried or deep-fried potatoes, roasted grains or products containing roasted grains, corn products, coffee products such as solid or liquid coffee extracts and green beans, chicory extracts, grain coffee products, coffee substitutes, snacks, wheat products, cosmetics, bakery products or pastries such as cookies, biscuits, rusks, cereal bars, scones, ice cream cones, waffles, crumpets, gingerbread, crispbread, and bread substitutes, pasta, rice, fish products, meat products, grains, beer, nuts, complementary foods for children and infants, hair care products, personal care products, hair care products, and facial care products.

[0034] In the context of the present invention, the enzyme according to the present invention does not include, or is not based on, the sequence of Sequence ID No. 2. Sequence ID No. 2 describes the amino acid sequence of the wild-type enzyme derived from Pseudonocardia thermophila, from which the enzyme according to the present invention is derived.

[0035] A homology model of the amino acid sequence according to Sequence ID No. 2, i.e., the wild-type enzyme from Pseudonocardia thermophila, was constructed. YASARA structure software (version 20.10.4.L.64), along with its included macro hm_build.mcr, was used for this purpose. Default settings were retained. Crystal structures 3A1K, 3A1I, 3IP4, and 2GI3 (Non-Patent Literature 16) were used as templates for the final homology model, which is primarily based on the homodimer structure 3A1K that forms the basis of the amidase from Rhodococcus N-771, existing as a catalytically active homodimer (Non-Patent Literature 17). While we do not wish to be bound by any scientific theory, Sequence ID No. 2, i.e., the amino acid sequence of the wild-type enzyme from Pseudonocardia thermophila, has the folded structure of PDB-type 3A1K. With respect to the Cα atom of the catalyst serine (194), the following amino acid residues have the following spatial distances: A74 (17.1 Å), D68 (21.0 Å), A507 (23.2 Å), G445 (10.6 Å), L424 (11.6 Å), and S33 (21.8 Å). Between these, the following residues have the following spatial distances: G445 to L424 (13.1 Å), G445 to S33 (12.0 Å), D68 to A74 (8.3 Å), and D68 to A507 (16.0 Å). Therefore, the active site is located within a spatial sphere with a radius of approximately 13 Å centered on G445, similar to L424 and S33. Residues D68 and A74 are located in a loop structure within a spatial sphere of radius 4.2 Å (from the geometric centers of both amino acids), or within a spatial sphere of radius 6.5 Å centered on the Cα atom of A74. All distances mentioned refer to the Cα atom of the amino acid.

[0036] Furthermore, according to a preferred embodiment, in the context of the present invention, it is generally not necessary to use wild-type enzymes derived from Pseudonocardia thermophila. That is, according to one preferred embodiment, the sequence according to SEQ ID NO: 1 is not only independent of the sequence according to SEQ ID NO: 2, but also generally independent of the sequence of wild-type amidase derived from Pseudonocardia thermophila.

[0037] Other (thermally stable) amidases derived from Pseudonocardia thermophila, for example, are described in Patent Document 6, but these are not enzymes according to the present invention. Furthermore, Patent Document 6 does not indicate that the enzymes described herein can be advantageously used in the field of food or beverages, in the sense of the present invention, not particularly under the temperature and / or pH conditions described herein.

[0038] The enzyme according to the present invention can be obtained by starting from a wild-type enzyme and performing one or more steps of mutation (preferably directed, or non-directed, or directed and non-directed). Directed mutations are targeted changes to one or more DNA bases in the enzyme gene, resulting in one or more targeted effects on the amino acid sequence. In contrast, non-directed mutations are random mutations in a portion of the entire DNA sequence that are not precisely selected. Following non-directed mutations, the resulting protein is examined to determine whether it has the desired properties. The modified enzyme may be a wild-type enzyme. This was the case in the considerations and studies that led to the present invention. In the context of the present invention, a wild-type enzyme is understood to be a naturally occurring, unmodified enzyme isolated from nature as either a functional enzyme or its sequence, and therefore neither the sequence nor the functional enzyme has been modified by human hands. In one embodiment, the enzyme may be the product of repeated non-directed mutations. In another embodiment, the enzyme may be the product of repeated directed mutations.

[0039] In the research that formed the basis of this invention, more than 2000 different mutants were generated starting from the wild-type enzyme, all of which had various mutations in different parts of the enzyme gene. Then, the activity, as well as the stability, of these mutants was tested at various pH values ​​and temperatures. Based on this, in the subsequent mutation steps, the enzyme was modified to result in increased activity and stability compared to the wild-type enzyme. The amino acid residues related to catalysis remained unchanged; in particular, the three catalytic residues of the enzyme, consisting of lysine at position 95, serine at position 170, and serine at position 194, remained unchanged.

[0040] In the context of the present invention, the enzyme according to the present invention generally applies that, in its amino acid sequence, one, some, or all, preferably all, of the following three positions remain unmuted: the 95th position of the sequence according to the present invention as described herein is preferably lysine, and / or the 170th position of the sequence according to the present invention as described herein is preferably serine, and / or the 194th position of the sequence according to the present invention as described herein is preferably serine, more preferably the 95th position is lysine, the 170th position is serine, and the 194th position is serine.

[0041] In the context of this invention, stability defines the residual activity of an enzyme after a certain period of time under various environmental conditions such as pH and temperature, while activity refers to the enzyme's ability to catalyze the cleavage of amide bonds by hydrolysis per unit time. Appropriate mutagenesis methods, as well as the necessary conditions and reagents, are well known to those skilled in the art. Mutations occur at the gene level, for example, through base exchange (substitution), deletion (removal), or addition. These mutations have various effects on the amino acid sequence of the resulting protein. In the case of substitutions, so-called "nonsense" mutations may occur, causing premature cessation of protein biosynthesis and leaving the resulting protein dysfunctional. In so-called "missense" mutations, only the encoded amino acid is changed, and these mutations result in functional changes to the resulting protein, and in the best case, may improve the stability or activity of the resulting protein. In general nomenclature, amino acid substitution mutations are designated based on their position and the substituted amino acid, for example, A143G. This notation means that the amino acid alanine has been replaced with guanine at position 143 of the N-terminal to C-terminal amino acid sequence.

[0042] In a preferred embodiment of the present invention, the amino acid sequence (i.e., an amino acid sequence included in or constituting an enzyme according to the present invention, having sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the sequence according to SEQ ID NO: 1, or with respect to the sequence according to SEQ ID NO: 1, which is not the sequence according to SEQ ID NO: 2) has at least one, more, or all of the positions selected from the group consisting of positions 33, 41, 68, 74, 94, 175, 201, 217, 221, 225, 229, 317, 328, 424, 445, 448, 453, 454, 457, and 507, which does not correspond to the amino acid at the corresponding position in the amino acid sequence according to SEQ ID NO: 2.

[0043] In another embodiment of the present invention, the amino acid sequence has at least one, more, or all of the following positions, selected from the group consisting of positions 33, 68, 74, 201, 225, 424, 445, 448, and 453, amino acids other than the amino acids at the corresponding positions of the amino acid sequence according to SEQ ID NO: 2.

[0044] In another embodiment of the present invention, The 33rd position of the amino acid sequence is arginine, tyrosine, histidine, or phenylalanine, and / or The 41st position is tyrosine, and / or The 68th position is asparagine, and / or The 74th position is tyrosine, and / or The 94th position is isoleucine, and / or The 175th position is alanine, and / or The 201st position is phenylalanine, and / or The 217th position is arginine, and / or The 221st position is glycine, and / or The 225th position is threonine, and / or The 229th position is cysteine, and / or The 317th position is isoleucine, and / or The 328th position is arginine, and / or The 424th position is valine, and / or The 445th position is arginine or serine, and / or The 448th position is histidine, and / or The 453rd position is aspartic acid, cysteine, asparagine, glutamine, glutamic acid, lysine, arginine, or serine, and / or The 454th position is asparagine, and / or The 457th position is glycine, and / or The 507th position is occupied by proline. In another embodiment of the present invention, The 33rd position of the amino acid sequence is arginine or tyrosine, and / or The 68th position is asparagine, and / or The 74th position is tyrosine, and / or The 201st position is phenylalanine, and / or The 225th position is threonine, and / or The 424th position is valine, and / or The 445th position is alanine, and / or The 448th position is histidine, and / or The 453rd position is either aspartic acid or cysteine.

[0045] From the preferred amino acids at the above positions, the following preferred embodiments or amino acid substitutions occur when starting from the wild-type sequence according to SEQ ID NO: 2.

[0046] In one embodiment of the present invention, the amino acid sequence has at least one amino acid substitution selected from the group consisting of S33R, S33Y, S33H, and S33F, particularly preferably the amino acid substitution S33R or S33Y.

[0047] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution W41Y.

[0048] In a preferred embodiment of the present invention, the amino acid sequence has at least one amino acid substitution D68N.

[0049] According to another preferred embodiment of the present invention, the amino acid sequence has at least one amino acid substitution A74Y.

[0050] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution V94I.

[0051] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution G175A.

[0052] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution Y201F.

[0053] According to another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution T217R.

[0054] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution P221G.

[0055] In another preferred embodiment of the present invention, the amino acid sequence has at least one amino acid substitution S225T.

[0056] According to another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution L229C.

[0057] In yet another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution V317I.

[0058] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution D328R.

[0059] According to another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution L424V.

[0060] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution selected from the group consisting of G445A and G445S, preferably the amino acid substitution G445A.

[0061] According to another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution M448H.

[0062] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution selected from the group consisting of A453D, A543C, A453N, A453Q, A453E, A453K, A453R, and A453S, particularly preferably the amino acid substitution A453D or A453C.

[0063] According to another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution P454N.

[0064] According to yet another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution V457G.

[0065] In another embodiment of the present invention, the amino acid sequence has at least one amino acid substitution A507P.

[0066] It will be obvious to those skilled in the art that, as described herein, one or more of the amino acid substitutions, or amino acids preferably present at each of these positions, can be combined with further substitutions not described herein, or with amino acids other than those specified in SEQ ID NO: 1, in any way desired to obtain the enzyme according to the present invention (in this regard, see “sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the sequence according to SEQ ID NO: 1” as described in the present invention). In the context of the present invention, it is preferable that these amino acid substitutions are outside the functional region, particularly outside the catalytic region (see above).

[0067] While Patent Document 7 already mentions the use of other enzymes (not according to the present invention) in food manufacturing processes, their effectiveness in the production of acrylamide-reduced products was not demonstrated there. Furthermore, the enzyme according to the present invention makes it possible to efficiently and rapidly decompose acrylamide in preparations. This is particularly advantageous in large-scale continuous or semi-continuous processes such as those in the food and beverage industries, as it ensures a short residence time in the enzymatic treatment, as well as rapid further treatment of perishable semi-processed products. Much more preferably and advantageously, since the decomposition of acrylamide is an essential step in obtaining a safe and harmless final food product, the enzyme according to the present invention can be used in the production of coffee products / coffee substitutes.

[0068] According to preferred embodiments of the present invention, the enzyme is an amidase. Amidases, or amide hydrolases, are a class of enzymes that catalyze the hydrolysis of amide bonds. Amidases are abundant in nature and are used in conventional products such as detergents and household cleaning products. The use of at least one amidase has been shown to be advantageous in the context of the present invention because it cleaves the amide bond of acrylamide by a very simple mechanism that does not require additional agents or cofactors.

[0069] In a more preferred embodiment, the enzyme is suitable to be catalytically active up to a temperature of 50°C or higher, preferably at least 60°C, more preferably at least 70°C, and even more preferably at least 80°C, and / or used at such temperatures in the context of the present invention. In the context of the present invention, catalytic activity means that detectable cleavage of the amide bond of acrylamide occurs. Since most non-heat-resistant proteins and enzymes lose their activity at temperatures above 42°C, activity up to 80°C is particularly preferred in the context of the present invention. Advantageously, with the enzyme according to the present invention, it is possible to obtain catalytic activity of the enzyme even at temperatures up to 80°C. Such high temperatures are required in many processes in the food and beverage sector, as critical cooking, boiling, and processing steps are carried out at temperatures above 50°C, and sometimes above 80°C.

[0070] In yet another preferred embodiment, the enzyme is suitable for exhibiting catalytic activity in the pH range of 4 to 7 and / or is used in such a pH range within the context of the present invention. According to a further preferred embodiment, the enzyme exhibits catalytic activity in the range of (at least) 4 to 6.5, preferably 4.5 to 5.5. Of course, the enzyme may also exhibit catalytic activity outside these pH ranges, or may be used within such ranges within the scope of the present invention. pH plays a crucial role in the stability and activity of the enzyme. pH above or below the optimal pH usually results in partial or complete loss of activity. It is therefore all the more surprising that the enzyme according to the present invention can be efficiently used in the acrylamide decomposition process in preparations with a slightly acidic pH.

[0071] In one embodiment, the enzyme exhibits catalytic activity for at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours, up to a temperature of 50°C or higher, preferably at least 60°C, more preferably at least 70°C, more preferably at least 80°C, in the range of (at least) pH 4 to pH 7, preferably in the range of pH 4 to pH 6.5, more preferably in the range of pH 4.5 to pH 5.5.

[0072] In a more preferred embodiment, the enzyme exhibits catalytic activity up to a temperature of 50°C or higher, preferably at least 60°C, more preferably at least 70°C, and even more preferably at least 80°C, in a pH range of (at least) 4 to 7, preferably 4 to 6.5, and more preferably 4.5 to 5.5. Of course, the enzyme may also exhibit catalytic activity outside these pH ranges up to the temperatures mentioned and can therefore be used in such ranges.

[0073] In connection with the investigations that form the basis of this invention, it was surprisingly possible to identify enzymes that possess or retain their catalytic activity even in acidic pH ranges and at high temperatures. The use of such enzymes is quite advantageous in the decomposition of acrylamide in the production of coffee / coffee substitute products, for example, in the treatment of extracts from roasted coffee / coffee substitute products. After extraction, the temperature of such preparations is often above 50°C, even above 70°C, or even above 80°C. It is well known that such extracts have a slightly acidic pH. Naturally occurring enzymes often do not exhibit sufficient catalytic activity under these conditions. Therefore, it is all the more advantageous to use the enzymes according to the present invention in such preparations to efficiently decompose acrylamide under these conditions.

[0074] According to a preferred embodiment of the present invention, the enzyme contains or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a sequence selected from the group consisting of sequences by SEQ ID NOs: 3 to 41, preferably from sequences by SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, and 22, and particularly preferably from sequences by SEQ ID NOs: 19, 20, 21, and 22.

[0075] In another preferred embodiment of the present invention, the enzyme is a modified amidase derived from Pseudonocardia thermophila. Pseudonocardia thermophila is an organism characterized by its distribution in somewhat high-temperature environments such as fecal deposits or warm springs. This organism belongs to the thermostable prokaryotes and can grow at temperatures of 40-50°C. Due to adaptation to warmer environments, its enzyme is also more heat-resistant, but it starts from the wild type which does not exceed 50°C. Since Pseudonocardia thermophila is not inherently acid-tolerant, it is remarkable that tolerance in the acidic pH range could be achieved by starting with the enzyme of the organism Pseudonocardia thermophila.

[0076] Another aspect of the present invention relates to a method for decomposing acrylamide, preferably by reducing the amount of acrylamide in a preparation, comprising or including the following steps. (i) A step of providing an enzyme of the present invention (as described herein, preferably, preferred, or particularly advantageous). (ii) A step of providing a mixture, preferably a preparation, containing acrylamide, and adding the enzyme of step (i). (iii) Incubating the mixture obtained as a result of step (ii) at a temperature preferably in the range of 40°C to 80°C, more preferably in the range of 45°C to 75°C, for at least 20 minutes. (iv) Optionally, the incubated mixture resulting from step (iii) is heated to a temperature of at least 90°C and maintained at a temperature above 90°C for at least 15 minutes so that enzyme inactivation occurs, and optionally, the mixture is cooled. A step to obtain a product with a lower acrylamide content than the mixture or preparation provided in step (ii).

[0077] Incubation as used in this invention means that the mixture provided in step (ii) remains at a constant temperature for a predetermined time. Temperature retention as used in this invention means that the temperature of the incubated mixture in step (iii) does not change or change significantly for a specified period. Slight temperature fluctuations are acceptable in this regard and can be appreciated by those skilled in the art.

[0078] In one embodiment of the present invention, the enzyme can be recombinantly produced by suitable expression organisms and conditions well known to those skilled in the art. Furthermore, the enzyme may be unpurified, partially purified, or highly purified as a lysate. Suitable purification steps are known to those skilled in the art.

[0079] In further embodiments, the enzyme according to the present invention may exist as a solution or be immobilized. Suitable immobilization steps are well known to those skilled in the art.

[0080] Inactivation refers to the loss of enzyme activity caused by extremely high temperatures, as well as the unfolding of the enzyme's amino acid chain. In yet another embodiment, the inactivated enzyme can then be removed from the preparation by methods commonly used by those skilled in the art, such as filtration, absorption, or adsorption.

[0081] In one embodiment of the method of the present invention, the acrylamide contained in the mixture or preparation initially provided is the product of the Maillard reaction. The Maillard reaction can be observed, for example, during the frying or stir-frying of food and manifests as typical browning. The Maillard reaction is also essential in the roasting of coffee products to obtain a typical roasted flavor. The product of the Maillard reaction is acrylamide, which is cleaved by going through the process of the present invention using the enzyme of the present invention.

[0082] According to another preferred embodiment of the method of the present invention, the acrylamide contained in the preparation provided is a product of the Maillard reaction, the preparation is a preparation that provides pleasure or nutrition, a cosmetic preparation, or a semi-processed product for preparations of such preparations, preferably, herein, the preparation is selected from the group consisting of fried or deep-fried potato products, roasted grains or products containing them, corn products, coffee products such as solid or liquid coffee extracts and green beans, chicory extracts, grain coffee products, coffee substitutes, snacks, wheat products, cosmetics such as baked goods and pastries such as biscuits, cookies, rusks, cereal bars, scones, ice cream cones, waffles, crumpets, gingerbread, crispbread, and bread substitutes, pasta, rice, fish products, meat products, grains, beer, nuts, complementary foods for children and infants, hair care products, personal care products, hair care products, and facial care products.

[0083] The difference between semi-processed and finished products lies in the degree of processing. Semi-processed products are all products that undergo further processing steps. These include, for example, roasted coffee extract, dough, green coffee beans, and potato products. Finished products, on the other hand, are not processed any further and are packaged in their original form and delivered to consumers. Examples of finished products include instant coffee, ready-to-use coffee powder, chips, and noodles.

[0084] According to a more preferred embodiment of the method of the present invention, the acrylamide content of the resulting product is <2000 μg / kg, preferably <850 μg / kg, and particularly preferably <500 μg / kg, based on the total weight of the product. Whenever a value is referred to as less than ("<") in this disclosure, this range of values ​​preferably also includes 0, as far as is possible and useful. For example, the expression <2000 μg / kg is a range of values ​​from 0 to 2000 μg / kg.

[0085] In further embodiments of the method according to the present invention, the acrylamide content may be reduced by 60%, preferably 65%, particularly preferably 70%, even more preferably 75%, particularly preferably 80%, even more preferably 85%, particularly preferably 90%, even more preferably 95%, and very particularly preferably 100% compared to a preparation not subjected to the steps according to the present invention.

[0086] Another aspect of the present invention relates to a method for preparing a pleasure-or-nutritious preparation or cosmetic preparation with reduced acrylamide content, preferably, wherein the preparation is selected from the group consisting of fried or deep-fried potato products, roasted grains or products containing them, corn products, coffee products such as solid or liquid coffee extracts and green beans, chicory extracts, grain coffee products, coffee substitutes, snacks, wheat products, cosmetics such as baked goods and pastries such as biscuits, cookies, rusks, cereal bars, scones, ice cream cones, waffles, crumpets, gingerbread, crispbread, and bread substitutes, pasta, rice, fish products, meat products, grains, beer, nuts, complementary foods for children and infants, hair care products, personal care products, hair care products, and facial care products, and the method consists of or includes the following steps. (I)(a) A step of providing a product obtained by a process according to the present invention (as described herein, preferably as described herein as preferred or particularly advantageous), (b) A step of obtaining a preparation that provides pleasure or nourishment, or a cosmetic preparation, by further processing of the product and / or adding one or more additional ingredients. Or, (II)(i) The step of providing the enzyme of the present invention (as described herein, preferably as described herein as preferred or particularly advantageous), (ii) A preparation containing acrylamide that provides pleasure or nutrition, or a cosmetic preparation, comprising the step of adding the enzyme of the present invention of step (i), (iii) Incubating the preparation obtained from step (ii) at a temperature preferably in the range of 40°C to 80°C, more preferably in the range of 45°C to 75°C, for at least 20 minutes. (iv) Optionally, the incubated preparation resulting from step (iii) is heated to a temperature of at least 90°C and maintained at a temperature above 90°C for at least 15 minutes to cause enzyme inactivation; optionally, the incubated preparation is cooled to obtain a preparation with a lower acrylamide content than the preparation provided in step (ii). In one embodiment of the method according to the present invention, the semi-treated product with reduced acrylamide content is further treated to obtain a final product to obtain a preparation that provides pleasure or nutrition or a cosmetic preparation. In a further embodiment, the semi-treated product is treated with the enzyme according to the present invention to obtain a preparation with reduced acrylamide content. This preparation may then be heated above 90°C to inactivate the enzyme. In yet another embodiment, the inactivated enzyme may then be removed from the preparation by methods commonly used by those skilled in the art, such as filtration, absorption or adsorption.

[0087] In a further embodiment, the present invention relates to the use of an enzyme according to the present invention for the decomposition of acrylamide and / or for preparations or cosmetic preparations that provide pleasure or nutrition, wherein the acrylamide content is reduced to preferably <2000 μg / kg, preferably <850 μg / kg, and particularly preferably <500 μg / kg, based on the total weight of the preparation. In a further embodiment, the acrylamide content may be reduced by 60%, preferably 65%, particularly preferably 70%, even more preferably 75%, particularly preferably 80%, even more preferably 85%, particularly preferably 90%, even more preferably 95%, and very particularly preferably 100%, compared to a preparation in which the enzyme according to the present invention is not used.

[0088] Further aspects of the present invention relate to preparations for pleasure or nutrition or cosmetic preparations (preferably those described above) prepared or prepareable by the methods of the present invention, each having an acrylamide content of <2000 μg / kg, preferably <850 μg / kg, and particularly preferably <500 μg / kg based on the total weight of the preparation (and / or the acrylamide content is reduced as described above, preferably as described above).

[0089] The present invention is described in more detail below using selected non-limiting embodiments. [Examples]

[0090] 1. Development of enzymes to break down acrylamide in coffee: 1.1 Preparation of Enzyme Preparations Genes encoding amidase and its variants are cloned into the expression plasmid pLE1A17(Novagen). Subsequently, E. coli BL21(DE3) cells are transformed with these plasmids.

[0091] Cells are cultured at 37°C in ZYM505 medium (Non-Patent Literature 11) supplemented with kanamycin (50 mg / l), and enzyme expression is induced with a final concentration of 0.1 mM IPTG when the logarithmic growth stage is reached. After induction, the cells are cultured further at 30°C for approximately 20 hours.

[0092] Cells were collected by centrifugation and digested with lysis buffer (50 mM potassium phosphate (pH 7.2), 2 mM magnesium chloride, 0.5 mg / mL lysozyme, 0.02 U / μL nucleanese). Digestion was performed mechanically by multiple freeze-thaw cycles in liquid nitrogen or by sonication. After centrifugation and separation of insoluble components, a crude extract containing soluble enzymes was obtained.

[0093] 1.2 Measurement of Enzyme Activity Standard measurements of amidase activity track the release of ammonia from acrylamide at pH 5.5 and 40°C. One amidase unit corresponds to the release of 1 μmol of ammonia per minute from 25 mM acrylamide in 50 mM sodium acetate buffer (pH 5.5) at 40°C. Quantitative determination of the released ammonia is performed, for example, using the Megazymes Rapid Ammonia Kit. The activity, expressed in U / mL, refers to the mL of crude extract with an optical density (measured at 600 nm) of 100.

[0094] Activity is similarly measured at other pH values ​​(pH 5.0) and temperatures (50 / 60°C). Parameters that changed compared to standard activity are shown individually.

[0095] 1.3 Measurement of enzyme stability 1.3.1 T at various pH values m Measurement of Values To record the temperature stability of amidase, T mMeasure the 50% value. For this purpose, the crude extract containing the enzyme is incubated in 50 mM sodium acetate buffer (pH 4.5 to pH 5.5) at various temperatures ranging from 25°C to 85°C for 15 minutes. Then, the crude extract is incubated on ice for 15 minutes, centrifuged, and the supernatant is used to perform activity measurements under standard conditions as described in Section 1.2. The activity value of the untreated sample is set to 100%, and all other values ​​are normalized to this. m The 50 value corresponds to the temperature at which the enzyme is still 50% active.

[0096] 1.3.2 Measurement of stability at various pH values ​​and temperatures In studies on the long-term stability of enzymes, crude extracts are incubated for an extended period at a specific pH (e.g., in the range of pH 4.5–5.5) and a specific temperature (e.g., in the range of 50–75°C). Periodic sampling is performed over 24 hours, during which time the samples are mixed with one volume equivalent of 100 mM NaAc buffer (pH 5.5) and immediately frozen in liquid nitrogen. After thawing, the samples are centrifuged, and the supernatant is used to perform activity measurements under standard conditions as described in Section 1.2. Percent residual activity is calculated by comparing the activity value measured after incubation with the activity of the untreated sample at time 0, with the activity value of the untreated sample set as 100%, and the activity value measured after incubation is compared to this to show the percentage residual activity.

[0097] 1.4 Examination of enzyme variants Based on the wild-type sequence of SEQ ID NO: 2, single mutants were generated, and amino acid substitution selections were combined from these mutants.

[0098] [Table 1]

[0099] If data is not shown in the table, or if the term nd ("not measured") is used, the corresponding enzyme variant was not recorded.

[0100] Similar to activity at high temperatures and acidic pH, stability at temperatures above 50°C can be achieved with the enzyme of the present invention.

[0101] Further investigations have revealed that the enzymes according to the present invention, particularly those described as preferred herein, are especially suitable for the purposes and requirements described herein, especially as introductions.

[0102] 1.5 Investigation of various amino acid substitutions and their effects on enzyme stability and activity. Using the enzyme of Sequence ID No. 11 as a template, single mutants were generated based on it. Each amino acid substitution exhibits different effects on the enzyme's activity and stability. For this characterization, the mutants were selected based on two criteria: stability and activity. Each property is strongly dependent on the substituted amino acid and its position. In particular, these studies resulted in enzymes particularly suitable for the present invention (as described herein).

[0103] [Table 2]

[0104] 1.6 Investigation of various combinations of mutations, and their effects on the activity and stability of enzyme variants. Several single mutants were generated starting from SEQ ID NO: 11, and amino acid substitution selections were combined within the recombinant bank. Subsequently, appropriate mutant selections were screened for stability and activity.

[0105] [Table 3]

[0106] 1.7 Saturation mutation at position 453 We examined all amino acid substitutions at position 453 in the mutant of SEQ ID NO: 16. A mutant with increased activity was obtained, which is a result of improved soluble expression in E. coli. Enzyme stability was maintained.

[0107] [Table 4]

[0108] 2. Manufacturing of an exemplary product according to the present invention: 2.1 Process for preparing acrylamide-reduced coffee products 2.1.1 Production of coffee extract Brazilian Arabica coffee beans were roasted to a color value of 110 according to the Neuhaus Neotec Colortest II. Acrylamide is formed by roasting the coffee beans at temperatures between 145°C and 230°C. The roasted coffee was ground to level 13 in a VT6 coffee grinder (Mahlkoenig). The ground coffee was first poured into the percolator of the extraction unit, and then filled with 70L of water at 85°C. The mixture was allowed to swell for 1 hour. Then, extraction was started at 85°C. 100L of water was passed through the percolator to the collection container at 85°C and under an overpressure of 6 bar. The hot water dissolves the soluble components of the coffee, including acrylamide.

[0109] 2.1.2 Enzymatic treatment of coffee extract Following extraction, the obtained coffee extract is heated to 70°C in a collection container and maintained at this temperature. Then, the enzyme according to the present invention, for example, the enzyme of SEQ ID NO: 22, is added. The enzyme is added in an amount that results in an enzyme concentration of 1000 U / L.

[0110] The enzyme is added to the extract, stirred, and incubated at 70°C for 30 minutes. After incubation, the extract is heated to 95°C for 15 minutes to inactivate the amidase. The extract is then cooled and prepared for freeze-drying.

[0111] Different reduction rates of acrylamide are achieved depending on the pH of the coffee extract. Acrylamide concentrations below the detection limit can also be achieved depending on the amount of enzyme used. However, the objective of the present invention was to provide an enzyme suitable for use in coffee matrices from an economic standpoint. For example, using 1000 U / L of enzyme at pH 4.8 achieved a 60% reduction in acrylamide. Using 1000 U / L of enzyme at pH 5.3 achieved a 90% reduction in acrylamide. Therefore, acrylamide-reduced products with acrylamide content of only 554 and 129 μg / kg, respectively, were obtained.

[0112] 2.1.3 Further processing of coffee extract after enzyme treatment After extraction and enzymatic treatment, the coffee extract is concentrated using freeze-concentration or evaporation. Since the extract obtained after extraction has a solid content of approximately 2-6% by weight, concentration is an intermediate step to increase the solid content in the extract. For fluidized bed drying, at least 20% by weight of solids is required. For freeze-drying, a higher solid content is advantageous, but not absolutely necessary.

[0113] Subsequently, the concentrated extract is dried by freeze-drying or fluidized bed drying to obtain an acrylamide-reduced final product (dried dissolved coffee) with a solid content of typically about 96% by weight.

[0114] 2.2 Process for preparing acrylamide-reduced coffee substitute products As a coffee substitute, chicory can be used, for example. The roots of the chicory plant are used for this purpose. They are dried, ground, roasted like coffee at a temperature between 150 and 200°C, and then ground. Further processing to obtain a soluble acrylamide-reduced extract is carried out in the same manner as described for coffee in Example 2.1.2.

[0115] Dried soluble chicory extract can be used as a final product or as a compounding additive for, for example, grain coffee or coffee mix products.

[0116] 3. Measurement of acrylamide content and reduction of acrylamide: The reduction in acrylamide is measured by extracting a sample of specially roasted coffee with hot water. The extract is then divided into two portions, and only the first portion is treated with amidase and incubated. The second portion is treated similarly, except for the addition of amidase, and serves as the reference sample. At the end of the incubation period, the reaction is stopped by heating once to a temperature that safely denatures the enzyme. Acrylamide analysis of both extracts is performed using LC-MS / MS according to DIN EN ISO18862. The reduction rate is calculated from the acrylamide content in the treated sample and the reference sample.

[0117] 4. Sensory evaluation: In comparative sensory tests of various coffee extracts and coffee substitutes conducted by a trained sensory panel, no perceptible sensory differences were observed between untreated and treated extracts.

Claims

1. An enzyme for reducing the amount of acrylamide in a preparation, comprising or consisting of the amino acid consensus sequence according to Sequence ID No. 1, The aforementioned amino acid sequence is not the sequence shown in Sequence ID No. 2, The enzyme comprises, or consists of, an amino acid sequence with respect to a sequence selected from the group consisting of sequences from Sequence ID No. 3 to Sequence ID No. 41, having at least 95%, 96%, 97%, 98%, or 99% sequence identity.

2. The 33rd position of the aforementioned amino acid sequence is arginine, tyrosine, histidine, or phenylalanine, and / or The 41st position is tyrosine, and / or The 68th position is asparagine, and / or The 74th position is tyrosine, and / or The 94th position is isoleucine, and / or The 175th position is alanine, and / or The 201st position is phenylalanine, and / or The 217th position is arginine, and / or The 221st position is glycine, and / or The 225th position is threonine, and / or The 229th position is cysteine, and / or The 317th position is isoleucine, and / or The 328th position is arginine, and / or The 424th position is valine, and / or The 445th position is arginine or serine, and / or The 448th position is histidine, and / or The 453rd position is aspartic acid, cysteine, asparagine, glutamine, glutamic acid, lysine, arginine, or serine, and / or The 454th position is asparagine, and / or The 457th position is glycine, and / or The enzyme according to claim 1, wherein the 507th position is proline.

3. The 33rd position in the aforementioned amino acid sequence is arginine or tyrosine, and / or The 68th position is asparagine, and / or The 74th position is tyrosine, and / or The 201st position is phenylalanine, and / or The 225th position is threonine, and / or The 424th position is valine, and / or The 445th position is alanine, and / or The 448th position is histidine, and / or The enzyme according to claim 1 or 2, wherein the 453rd position is aspartic acid or cysteine.

4. The enzyme according to any one of claims 1 to 3, comprising, or consisting of, an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a sequence selected from the group consisting of the sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22.

5. The enzyme according to any one of claims 1 to 4, comprising, or consisting of, an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a sequence selected from the group consisting of the sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22.

6. The enzyme according to any one of claims 1 to 5, wherein the enzyme is amidase.

7. The enzyme according to any one of claims 1 to 6, which exhibits catalytic activity up to a temperature of 50°C or higher, preferably at least 60°C, more preferably at least 70°C, and even more preferably at least 80°C.

8. The enzyme according to any one of claims 1 to 7, which has catalytic activity in the range of pH 4 to pH 7, preferably in the range of pH 4 to pH 6.5, and more preferably in the range of pH 4.5 to pH 5.

5.

9. The enzyme according to any one of claims 1 to 8, which exhibits catalytic activity up to a temperature of 50°C or higher, preferably at least 60°C, more preferably at least 70°C, and even more preferably at least 80°C, in the range of pH 4 to pH 7, preferably in the range of pH 4 to pH 6.5, more preferably in the range of pH 4.5 to pH 5.

5.

10. The enzyme according to any one of claims 1 to 9, which is a modified amidase derived from Pseudocardia thermophila.

11. A method for decomposing acrylamide, preferably by reducing the amount of acrylamide in the preparation, (i) the step of providing the enzyme according to any one of claims 1 to 10, (ii) Provide a mixture containing acrylamide, preferably a preparation, comprising the step of adding the enzyme of step (i), (iii) The step of incubating the mixture obtained as a result of step (ii) at a temperature preferably in the range of 40°C to 80°C, more preferably in the range of 45°C to 75°C, for at least 20 minutes, (iv) Optionally, the incubated mixture resulting from step (iii) is heated to a temperature of at least 90°C and maintained at a temperature higher than 90°C for at least 15 minutes so that the enzyme is inactivated, and optionally, the mixture is cooled. A method comprising or including the step of obtaining a product with a lower acrylamide content than the mixture or preparation provided in step (ii).

12. The method according to claim 11, wherein in step (ii), a preparation containing acrylamide is provided, wherein the acrylamide contained is preferably a product of the Maillard reaction, the preparation is a preparation that provides pleasure or nutrition, or a cosmetic preparation, or a semi-processed product for the manufacture of such preparations, preferably the preparation is selected from the group consisting of fried or deep-fried potato products, roasted grains or products containing them, corn products, coffee products such as solid or liquid coffee extracts and green beans, chicory extracts, grain coffee products, coffee substitutes, snacks, wheat products, cosmetics such as baked goods and pastries such as biscuits, cookies, rusks, cereal bars, scones, ice cream cones, waffles, crumpets, gingerbread, crispbread and bread substitutes, pasta, rice, fish products, meat products, grains, beer, nuts, complementary foods for children and infants, hair care products, personal care products, hair care products, and facial care products.

13. The method according to any one of claims 11 or 12, wherein the acrylamide content in the obtained product is <2000 μg / kg, preferably <850 μg / kg, and particularly preferably <500 μg / kg, based on the total weight of the product.

14. A method for producing a preparation or cosmetic preparation that provides pleasure or nutrition and has a reduced acrylamide content, wherein the preparation is selected from the group consisting of fried or deep-fried potato products, roasted grains or products containing them, corn products, coffee products such as solid or liquid coffee extracts and green beans, chicory extracts, grain coffee products, coffee substitutes, snacks, wheat products, cosmetics such as baked goods and bread products such as biscuits, cookies, rusks, cereal bars, scones, ice cream cones, waffles, crumpets, gingerbread, crispbread, and bread substitutes, pasta, rice, fish products, meat products, grains, beer, nuts, supplemental foods for children and infants, hair styling products, personal care products, hair care products, and facial care products. A method comprising or including the following steps: (I) (a) the step of providing a product obtained by the method described in any one of claims 11 to 13, (b) A step of further processing the product and / or adding one or more additional ingredients to obtain the preparation or cosmetic preparation that provides pleasure or nourishment, Or, (II) (i ) A step of providing the enzyme according to any one of claims 1 to 10, (ii) A preparation or cosmetic containing acrylamide that provides pleasure or nourishment, comprising the step of adding the enzyme of step (i), (iii) The step of incubating the preparation obtained as a result of step (ii) at a temperature preferably in the range of 40°C to 80°C, more preferably in the range of 45°C to 75°C, for at least 20 minutes, (iv) Optionally, the incubated preparation resulting from step (iii) is heated to a temperature of at least 90°C and maintained at a temperature higher than 90°C for at least 15 minutes so that the enzyme is inactivated, and optionally the preparation is cooled. A step of obtaining a preparation with a lower acrylamide content than the preparation provided in step (ii).

15. Use of the enzyme according to any one of claims 1 to 10 for the production of a preparation or cosmetic preparation for the decomposition of acrylamide, and / or for providing pleasure or nutrition, wherein the acrylamide content is reduced to preferably <2000 μg / kg, preferably <850 μg / kg, and particularly preferably <500 μg / kg, based on the total weight of the preparation.

16. A preparation or cosmetic preparation prepared or that can be prepared by any one of claims 11 to 13, wherein the acrylamide content is <2000 μg / kg, preferably <850 μg / kg, and particularly preferably <500 μg / kg, based on the total weight of the preparation.

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