Protein for detecting the presence of peptidoglycan, muropeptides or bacterial cells, sensor and its use.

Abstract

The macromolecule peptidoglycan exists only in bacteria. Fragments of peptidoglycan and / or muropeptides are continuously released during bacterial growth, and their presence in sterile tissues and fluids is indicative of a bacterial infection. The main peptidoglycan hydrolase of various species of The genus Staphylococcus is the ATL protein, a bifunctional autolysin with two independent catalytic domains, N-acetylmuramoyl-L-alanine amidase, an endo-β-N-acetylglucosaminodase. Modifications to the primary sequence of the AM protein led to the present invention. The development of a molecule that binds to Peptidoglycan molecules are an advantageous method for detecting bacteria. Proteins or polypeptides that bind specifically to muropeptides and / or fragments of peptidoglycan can be used to detect the presence of bacteria in various configurations. This invention includes nucleic acid molecules whose nucleotide sequence encodes the aforementioned recombinant polypeptides or proteins, vectors and host cells, and applications for their use.

Description

DESCRIPTION PROTEIN TO DETECT THE PRESENCE OF PEPTIDOGLYCAN, MUROPETIDES OR BACTERIAL CELLS, SENSOR AND USE THEREOF Technical Mastery of the Invention

[0001] This invention relates to the recognition of peptidoglycan molecules by recombinant proteins. This invention relates to recombinant or synthesized proteins or polypeptides with the activity of binding, for example, to muropeptide units of peptidoglycan molecules. This invention includes nucleic acid molecules containing a nucleotide sequence encoding the aforementioned recombinant polypeptides or proteins, vectors and host cells, and applications for their use. Background of the Invention

[0002] The peptidoglycan molecule is ubiquitous and present only in bacteria, being the main component of the bacterial cell wall. Peptidoglycan and peptidoglycan fragments called muropeptides are continuously released during bacterial growth, and their presence is indicative of the presence of bacteria. The presence of free muropeptides in tissues and body fluids is indicative of a bacterial infection.

[0003] The peptidoglycan polymer is composed of repeating monomeric units composed of a disaccharide and a peptide composed of 2-5 residues. The peptide of a disaccharide can be cross-linked with another peptide of an adjacent chain, thus achieving a complex three-dimensional structure (1).

[0004] The disaccharide backbone consists of two alternating amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), linked by β-1,4 glycosidic bonds. The peptide is linked to the MurNAc saccharide, and the number of amino acid residues, their composition, and their type of cross-linking vary among bacterial species. For example, in Staphylococcus (S.) aureus, the pentapeptide is composed of L-Ala-D-iso-Gyn-L-Lys-D-Ala-DAla. Cross-linking occurs through a pentaglycine bridge that links the L-Lys of one stem peptide to the fourth D-alanine of a neighboring pentapeptide, and is a characteristic of the peptidoglycan of S. auieus. In Escherichia (E.) coli, the peptide contains L-Ala-D-iso-Glumeso-DAP-D-Ala-D-Ala, and different chains are directly linked by a peptide bond between m-DAP and the fourth D-Ala. The first three amino acids of the peptide may vary in different bacterial species. The amino acid attached to MurNAc is usually L-Ala, but in some cases it may be replaced by Gly or L-Ser. The second residue is usually D-Glu. The acarboxylic group of D-Glu may be free or substituted, being amidated in many organisms, as is the case with S. aureus, or replaced by Gly, as is the case with Micrococcus (M.) luteus. The greatest variation occurs in the third residue, where meso-diaminopimelic acid (m-DAP) is the most commonly found residue in most Gram-negative bacteria and also in numerous other microorganisms. L-Lys is another residue commonly found in the third position, but other residues, mainly meso- or L-diamino acids, may also occur. The fourth and fifth residues are almost always D-Ala-D-Ala.Glycopeptide-resistant enterococcal species have a modified stem peptide containing D-Ala-D-Lactate, which reduces antibiotic affinity.

[0006] Crosslinking between different glycan chains differs among bacterial species and is responsible for the lattice-like structure of peptidoglycan. This reaction results in the removal of the fifth residue of the peptide, and sometimes also the fourth. Crosslinking may occur between the fourth residue of one chain and the second of the other, or between the third and fourth residues of adjacent chains. Some species also contain 3-3 crosslinks in conjunction with 4-3 bridges, and these bridges account for the majority of crosslinks in β-lactam-resistant mycobacteria and enterococci. The most commonly found peptidoglycan contains 4-3 crosslinks, which can be direct or through a bridge between peptides, as occurs in S. aureus, which has a pentaglycine bridge.

[0007] Other bacteria have different peptidoglycan structures (1). For example, M. luteus has the following peptide: L-Ala-D-Glu-(α-Gly)-L-Lys-D-Ala-D-Ala, the second amino acid (D-Glu) is replaced in its α-carboxylic group by a Gly and the crosslink is the 4-3 interchain linkage, made with a peptide identical to the peptide (L-Ala-D-Glu-(α-Gly)-L-Lys-D-Ala). S. epidermidis has a peptide identical to S. aureus and a 4-3 crosslink, made with 5 amino acids mostly Gly but some substituted by L-Ser (one, two, or three, most often in the third residue, but also in the first, sometimes in the fourth) or L-Ala (most commonly in the first) (2, 3). Streptococcus (S.) agalactiae has a peptide identical to S. aureus and a 4-3 chain cross-link; the linking peptide of the two chains is composed of 2 amino acids: L-Ala-L-Ala or L-Ser-L-Ala. S. pyogenes has a peptide identical to S.aureus and a 4-3 cross-link; and a linker peptide composed of 2 / 3 L-Ala residues. Enterococcus (E.) faecalis has a peptide identical to S. aureus and a 4-3 cross-link; a linker peptide composed of 2 L-Ala residues (4). The peptidoglycan structure is very heterogeneous due to the cleavage of the stem peptides from pentapeptides to tetra-, tri-, and dipeptides during maturation, the presence of modifications such as O-acetylation, amidation, and N-deacetylation, and the action of hydrolases from S. aureus, other bacteria, or the host organism, which recognize peptidoglycan and shape its structure. Due to their specificity, peptidoglycan hydrolases bind specifically to their substrate, peptidoglycan, and then act on it, hydrolyzing different bonds in the peptidoglycan structure. This hydrolysis leads to the release of peptidoglycan subunits into the external environment. Furthermore, some bacteria actively secrete peptidoglycan fragments.Thus, the presence of these molecules is indicative of the presence of bacteria.

[0008] The major peptidoglycan hydrolase of S. aureus and other species of the genus Staphylococcus is the Atl protein (5). In S. aureus, Atl is a 13.8 kDa bifunctional autolysin with independent catalytic domains, an N-acetylmuramoyl-L-alanine amidase (AM) domain and an endo-β-N-acetylglucosaminidase (GL) domain, and three repeat domains (Rl, R2, R3) (Fig. 1). The Atl protein is secreted and extracellularly divided into AM-R1-R2 and R3-GL. The repeats are necessary for the attachment of the AM and GL domains to the bacterial cell wall, making them surface exposed. These repeat domains are also extremely promiscuous in their binding, as they can bind not only to peptidoglycan, but also to lipoteichoic acids, fibronectin, vitronectin, and DNA (6-9).

[0009] The AM catalytic domain recognizes and cleaves peptidoglycan and muropeptide molecules (10,11), and homologs exist in most bacterial species, indicating that this family of proteins recognizes and cleaves different peptidoglycan molecules, making it an ideal candidate for peptidoglycan sensing. On the other hand, although the GL catalytic domain is also capable of cleaving peptidoglycan, its association with peptidoglycan from intact S. aureus cells is inefficient and has not been studied extensively (6,9).

[0010] The AM domain binds to peptidoglycan molecules and cleaves the bond between N-acetylmuramic acid and L-Ala of the peptide in the peptidoglycan polymer. The structures of the AM catalytic domain from S. epidermidis and S. aureus have been solved, showing that these domains have a globular α / β fold, in which seven α helices surround a central six-stranded β sheet (10, 11). General Description of the Invention

[0011] The present invention relates to a sequence for detecting the presence of peptidoglycan, muropeptides or bacterial cells, and its use. Furthermore, the present invention also relates to a sensor and its use.

[0012] The development of a molecule that binds to peptidoglycan molecules is an advantageous method for detecting bacteria. Proteins or polypeptides that specifically bind to muropeptides and / or peptidoglycan fragments can be used as a tool to detect the presence of bacteria in various settings. Furthermore, the developed molecule can be specific in the type of peptidoglycan to which it binds or broader in the sense that it is capable of binding to different types of peptidoglycan.

[0013] For the specific detection of peptidoglycan, only the catalytic domain of AM was used in the present invention, in order to guarantee the maximum breadth of binding capacity to peptidoglycan and to minimize non-specific binding interactions with other molecules, particularly those that are part of the human extracellular matrix, such as fibronectin and vitronectin, and also DNA.

[0014] In the present invention, specific amino acid residues of AM were considered, either specifically for interacting with peptidoglycan, a peptidoglycan-derived molecule, or a bacterial cell such as a Staphylococcus cell. These amino acids are important not only for catalysis, but also for the binding and binding specificity of this interaction. Residues His-67, His-172, and Asp-186 directly coordinate a zinc ion, necessary for catalysis, while amino acid residues Glu-126 and His-184 are positioned to participate in catalysis. In addition, other residues are important for binding to the peptidoglycan ligand during catalysis.The MurNAc portion of the peptidoglycan exhibits fewer direct interactions, and the main binding sites between the protein and peptidoglycan may be maintained by the peptide, which binds in an extended conformation to a recessed concavity formed by the anterior region of the central six-stranded β-sheet. The tetrapeptide backbone anchors this concavity through direct contact or hydrogen bonds between water molecules and the amino acids, while the peptide side chains are also involved in binding, accounting for binding specificity. Furthermore, the D-iso-Gln side chain is a key binding site for AM, hydrogen bonding with Thr-182 and His-172 and interacting with residues Asp-183 and His-184. The entire side chain of the third amino acid, L-Lys, lies parallel to the indole of Trp-112 and interacts with Asn-119, and these interactions are likely responsible for the binding specificity of AM to L-Lys-type peptidoglycan.

[0015] Modifications of the primary sequence of the AM protein led to the present invention. By modifying the AM protein, an instrument was constructed to identify peptidoglycan molecules, and thus bacteria. The modified recombinant AM protein (rAM) is capable of binding to different types of peptidoglycan and muropeptides, without hydrolytic activity.

[0016] While there are inventions that provide descriptions of mechanisms for binding bacteria directly through cell wall-anchored components for bacterial detection, this invention relates to the binding of free peptidoglycan fragments and thus can use binding to these molecules as a surrogate marker for the presence of bacteria. This is particularly useful if the bacteria are immobilized or in small numbers, as is the case in biofilms or on food products.

[0017] The present invention relates to an isolated sequence or recombinant sequence or synthetic sequence comprising at least 90% identity with: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEC ) ID NO: : 4 , SEQ ID NO: 5, SEQ ID NO:6 , SEQ ID NO:7, SEQ IE 1 NO:8, SEQ ID NO:9, í 5EQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, 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:2 0, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:2 8, SEQ ID NO:29, SEQ ID NO:3 0, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:3 3, SEQ ID NO:3 4, SEQ ID NO:3 5, SEQ ID NO:3 6, SEQ ID NO:37, SEQ ID NO:3 8, SEQ ID NO:3 9, SEQ ID NO:4 0, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:4 3, SEQ ID NO: 44, SEQ ID NO:4 5, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:4 8, SEQ ID NO:49, SEQ ID NO:5 0, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 , SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:6 0, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:6 3, SEQ ID NO: 64, SEQ ID NO:6 5, SEQ ID NO:6 6, SEQ ID NO:67, SEQ ID NO:6 8, SEQ ID NO:6 9, SEQ ID NO:7 0, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:7 3, SEQ ID NO:7 4, SEQ ID NO:7 5, SEQ ID NO:7 6, SEQ ID NO:77, SEQ ID NO:7 8, SEQ ID NO:7 9, SEQ ID NO:8 0, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 , SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:9 0, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:9 3, SEQ ID NO:94, SEQ ID NO:9 5, SEQ ID NO:9 6, SEQ ID NO:97, SEQ ID NO:9 8, SEQ ID NO: 99 , SEQ ID NO:100 , SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO: 103 , SEC ) ID NO : 104 , 8 5EQ ID NO:105, SEQ ID NO: 106 , SEQ ID NO: 107, SEQ ID, NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 ou SEQ ID NO:130, onde a referida sequência se liga a peptidoglicano e / ou um muropéptido e / ou uma célula bacteriana.

[0018] Numa concretização, a referida sequência compreende pelo menos 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ou 99% de identidade com: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID SEQ ID NO:7, SEQ ID NO:8, NO:12, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:37, SEQ ID SEQ ID NO:9, NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:38, SEQ ID SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, NO:46, NO:51, NO:56, NO:61, NO: 66 , NO:71, NO:7 6, NO:81, NO:86, NO:91, NO: 96 , ID NO:1 SEQ ID NO:110, SEQ ID NO:119, SEQ ID NO:128, SEQ ID NO:42, SEQ ID NO:43, SEQ SEQ ID NO:47, SEQ ID NO:48, SEQ SEQ ID NO:52, SEQ ID NO:53, SEQ SEQ ID NO:57, SEQ ID NO:58, SEQ SEQ ID NO:62, SEQ ID NO:63, SEQ SEQ ID NO:67, SEQ ID NO:68, SEQ SEQ ID NO:72, SEQ ID NO:73, SEQ SEQ ID NO:77, SEQ ID NO:78, SEQ SEQ ID NO:82, SEQ ID NO:83, SEQ SEQ ID NO:87, SEQ ID NO:88, SEQ SEQ ID NO:92, SEQ ID NO:93, SEQ SEQ ID NO:97, SEQ ID NO:98, SEÇ 01, SEQ ID NO:102, SEQ ID NO:103 NO: 106, SEQ ID NO: 107, SEQ ID SEQ ID NO: 111, SEQ ID NO: 112, NO: 115, SEQ ID NO: 116, SEQ ID SEQ ID NO: 120, SEQ ID NO: 121, NO: 124, SEQ ID NO: 125, SEQ ID SEQ ID NO:129 ou SEQ ID NO:130.ID NO:44, SEQ ID NO:45, SEQ ID ID NO:49, SEQ ID NO:50, SEQ ID ID NO:54, SEQ ID NO:55, SEQ ID ID NO:59, SEQ ID NO:60, SEQ ID ID NO:64, SEQ ID NO:65, SEQ ID ID NO:69, SEQ ID NO:70, SEQ ID ID NO:74, SEQ ID NO:75, SEQ ID ID NO:79, SEQ ID NO:80, SEQ ID ID NO:84, SEQ ID NO:85, SEQ ID ID NO:89, SEQ ID NO:90, SEQ ID ID NO:94, SEQ ID NO:95, SEQ ID 9 ID NO:99, SEQ ID NO:100, SEQ , SEQ ID NO:104, SEQ ID NO:105, NO: 108, SEQ ID NO: 109, SEQ ID SEQ ID NO: 113, SEQ ID NO: 114, NO: 117, SEQ ID NO: 118, SEQ ID SEQ ID NO: 122, SEQ ID NO: 123, NO: 126, SEQ ID NO: 127, SEQ ID

[0019] Numa concretização i, a referida : sequência é SEQ ID NO:1, ! SEQ. ID NO: 2, SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO : 5 , SEQ ID NO : 6 , SEQ ID NO: 7 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO :13, 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, SEQ ID NO: 22 , SEQ ID NO :23, SEQ ID NO :24, SEQ ID NO: 25, SEQ ID NO : 26 , SEQ ID NO: 27 , SEQ ID NO :28, SEQ ID NO :29, SEQ ID NO: 30, SEQ ID NO : 31, SEQ ID NO: 32 , SEQ ID NO :33, SEQ ID NO :34, SEQ ID NO: 35, SEQ ID NO : 36 , SEQ ID NO: 37 , SEQ ID NO :38, SEQ ID NO :39, SEQ ID NO: 40, SEQ ID NO : 41, SEQ ID NO: 42 , SEQ ID NO :43, SEQ ID NO :44, SEQ ID NO: 45, SEQ ID NO : 46 , SEQ ID NO: 47 , SEQ ID NO :48, SEQ ID NO :49, SEQ ID NO: 50, SEQ ID NO : 51, SEQ ID NO: 52 , SEQ ID NO :53, SEQ ID NO :54, SEQ ID NO: 55, SEQ ID NO : 56 , SEQ ID NO: 57 , SEQ ID NO :58, SEQ ID NO : 59 , SEQ ID NO: 60, SEQ ID NO : 61, SEQ ID NO: 62 , SEQ ID NO :63, SEQ ID NO :64, SEQ ID NO: 65, SEQ ID NO : 66 , SEQ ID NO: 67 , SEQ ID NO :68, SEQ ID NO :69, SEQ ID NO: 70, SEQ ID NO : 71,SEQ ID NO: 72 , SEQ ID NO :73, SEQ ID NO :74, SEQ ID NO: 75, SEQ ID NO : 76 , SEQ ID NO: 77 , SEQ ID NO :78, SEQ ID NO :79, SEQ ID NO: 80, SEQ ID NO : 81, SEQ ID NO: 82 , SEQ ID NO :83, SEQ ID NO :84, SEQ ID NO: 85, SEQ ID NO : 86 , SEQ ID NO: 87 , SEQ ID NO :88, SEQ ID NO : 89 , SEQ ID NO: 90, SEQ ID NO : 91, SEQ ID NO: 92 , SEQ ID NO :93, SEQ ID NO :94, SEQ ID NO: 95, SEQ ID NO : 96 , SEQ ID, NO :97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:129 ou SEQ ID NO:130, de preferência em que a sequência é SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ D NO: 21, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 49 ou SEQ ID NO:56 ou de preferência em que a sequência é SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO : 2 8 , SEQ ID NO: 29, SEQ ID NO : 3 0 , SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 , SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 , SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 6 0, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO: 77, SEQ ID NO: 7 8, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 , SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO 104, 105, 106, SEQ ID NO 107, SEQ ID NO 108, 109, 110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 ou SEQ ID NO:130.

[0020] Numí a. concretização, 0 X, Xi OU X2 de SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO : 4 , SEQ ID NO : 5 , SEQ ID NO: 6 , SEQ ID NO : 7 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, 2 ;eq ID 10:11, SEQ ID NO:12, SEQ ID NO:13, 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, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:2 8, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:3 3, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:3 6, SEQ ID NO:37, SEQ ID NO:3 8, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:4 3, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:4 8, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 , SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:6 3, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:6 8, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:7 3, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:7 6, SEQ ID NO:77, SEQ ID NO:7 8, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 , SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:9 3, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:9 8, SEQ ID NO:99, SEQ ID NO:100, SEÇ 9 II NO:101, SEQ ID NO:102, SEQ ID NO: 103, SEÇ ) ID NO:104, SEQ ID NO : 105 , SEQ ID NO: 10( SEQ ID NO:107, SEQ ID NO:108 , SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112 , SEQ ID NO: 113, SEQ ID NO:] -14 , SEÇ D ID NO : 115 c SEQ ID NO: 116, SEQ ID NO:117 , SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121 , SEQ ID NO: 122, SEQ ID NO: 123, SEÇ D ID NO : 124 , £ 5EQ ID NO:125, SEQ ID NO:126 , SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO 129 ou SEQ ID NO:130 é selecionado de A, C, D, E , F, G, Η, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; and X, X i, or X2 are selected independently of each other.

[0021] In one embodiment, X, Xi, or X2 are A or R.

[0022] In an embodiment, H67 may be replaced by A, Y, N, I, M, L, V, W or F.

[0023] In an embodiment, H172 may be replaced by A, Y, N, I, M, L, V, W or F.

[0024] In an embodiment, H184 may be replaced by A, Y, N, I, M, L, V, W or F.

[0025] In an embodiment, V, M, L, F, I, Y, or W.

[0026] In an embodiment, W, F, Y, or H.

[0027] In an embodiment, H, M, I, V, Y, F, L, or W.

[0028] In an embodiment, V, M, L, F, I, Y, or W.

[0029] In an embodiment, S, G, K, L, N, D, or P.

[0030] In an embodiment, Q, K, L, G, W, Y, I, or F. D186 can be replaced by A, T182 can be replaced by A, N119 can be replaced by A, D183 can be replaced by A, W112 can be replaced by A, E126 can be replaced by A, And, N, R, S, D, S, And, N, F, Y, I, D,

[0031] In one embodiment, the bacterial cell may be a Staphylococcus cell.

[0032] In one embodiment, the sequence may be for use in a method of in vivo diagnosis of a bacterial infection, preferably where the bacterial infection is a Staphylococcus bacterial infection.

[0033] The present invention also relates to an isolated DNA sequence encoding any of the sequences disclosed herein or to a vector or expression cassette or host cell or phage comprising any of the sequences disclosed herein.

[0034] Furthermore, this invention also relates to a sensor comprising any of the sequences disclosed herein, preferably comprising any of the sequences selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, 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:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO: 110, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO: 119, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 116, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO: 104, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 113, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 and / or SEQ ID NO: 130. The advantage of this embodiment is that immobilizing one or more of these sequences to the substrate surface through a cysteine ​​allows the sequence to be covalently linked to the substrate surface, contributing to the sensor's lifetime. The sensor's lifetime depends on its ability to retain the sequence or sequences immobilized on its surface.

[0035] In one embodiment, the sensor now disclosed may include any of the sequences disclosed herein, where such sequence or sequences are immobilized on the surface of the substrate via the biotin / streptavidin pair, preferably immobilized by adsorption or covalently.

[0036] In a preferred embodiment, the now disclosed sensor further comprises: a substrate and a liquid crystal, wherein the substrate has a sequence or plurality of different sequences immobilized on its surface; wherein the sequence or plurality of different sequences is capable of binding to peptidoglycan and / or a muropeptide and / or a bacterial cell of a sample; and wherein the liquid crystal can adopt a first liquid crystal orientation or a second liquid crystal orientation such that the liquid crystal changes from the first liquid crystal orientation to the second liquid crystal orientation when the liquid crystal interacts with a sample having peptidoglycan and / or muropeptide and / or bacterial cell bound to the sequence or plurality of different sequences such that a light signal is generated.

[0037] In one embodiment, the sensor comprises two substrates, a first substrate and a second substrate, such that the surface of the first substrate having the immobilized sequence or plurality of sequences faces the surface of the second substrate having the immobilized sequence or plurality of immobilized sequences, preferably wherein the sequence or plurality of different sequences immobilized on the surface of the first substrate is identical to the sequence or plurality of different sequences immobilized on the surface of the second substrate; or wherein the sequence or plurality of different sequences immobilized on the surface of the first substrate is different from the sequence or plurality of different sequences immobilized on the surface of the second substrate.

[0038] In one embodiment, the liquid crystal is in a nematic phase.

[0039] In an embodiment, the first liquid crystal molecular orientation is homogeneous perpendicular or parallel to the substrate and the second liquid crystal molecular orientation is heterogeneous.

[0040] In one embodiment, the liquid crystal is between the two substrates.

[0041] In one embodiment, the liquid crystal is selected from 4-cyano-41-pentylbiphenyl, N-(4-methoxybenzylidene)-4-butylaniline; 4-cyano-4'-n-heptyl-biphenyl; 4-cyano-41-n-oxyoctyl-biphenyl; 4-cyano4'-n-pentyl-p-triphenyl; and / or a mixture consisting of 51% 4-cyano4'-n-pentyl-biphenyl, 25% 4-cyano-41-n-heptyl-biphenyl, 16% 4cyano-4'-n-oxyoctyl-biphenyl (8OCB), and 8% 4-cyano-4'-n-pentyltriphenyl (5CT). Cyano-41-n-pentyl-biphenyl (5CB) has CAS number: 40817-08-1, 4-cyano-4-n-heptyl-biphenyl (7CB) has CAS number: 41122-71-8, 4-cyano-4'-n-oxyoctyl-biphenyl (8OCB) has CAS number: 52364-73-5 and 4-cyano-411-n-pentyl-triphenyl (5CT) has CAS number: 54211-46-0.

[0042] In one embodiment, the substrate is selected from: glass substrate, silicon substrate, polydimethyloxane substrate, acrylic substrate, and other transparent substrates.

[0043] In one embodiment, the sensor further comprises a sample input and a sample output.

[0044] The present invention also relates to a method of detecting peptidoglycan and / or muropeptides and / or bacterial cells in a sterile fluid or water sample comprising the following steps: Use of the sensor disclosed herein with a sterile fluid or a water sample; subjecting said sensor to polarized light and determining a light signal generated by changes from a first liquid crystal orientation to a second liquid crystal orientation when the liquid crystal interacts with a sample having peptidoglycan and / or bacterial cells bound to a different sequence or plurality of sequences as disclosed herein.

[0045] The present invention also relates to a method of detecting peptidoglycan and / or muropeptides and / or bacterial cells in a sterile fluid sample, the sensor having a detection limit that is less than 102CFUs / ml.

[0046] Furthermore, this invention also relates to the use of a sequence as disclosed herein as a bacterial detector, preferably as a water bacteria detector.

[0047] While there are inventions that provide descriptions of mechanisms for binding bacteria directly through cell wall-anchored components for bacterial detection, this invention relates to the binding of free peptidoglycan fragments and thus can use binding to these molecules as a surrogate marker for the presence of bacteria. This is particularly useful if bacteria are immobilized or in low numbers, as is the case in biofilms or on food products. Brief description of the figures

[0048] For a better understanding of the invention, attached are figures representing preferred embodiments of the invention which, however, are not intended to limit the scope of protection of the present invention.

[0049] Figure 1 - Structural organization of the S. aureus Atl protein. The Atl precursor protein (~138 kDa) is exported from the cytoplasm through an N-terminal signal sequence (black and white mosaic) and undergoes proteolytic processing in two cleavage events to generate two extracellular hydrolases, with ~51 kDa and ~63 kDa, that recognize peptidoglycan. The mature AM domain comprises the RI and R2 repeat regions, while the GL domain is associated with the R3 repeat region. The PP propeptide of unknown function is removed during proteolytic processing. The AM catalytic domain of the rAM protein encompasses the region between amino acids Ala-1 and Thr-226, represented in the figure.

[0050] Figure 2 - Analysis of purified recombinant rAM protein by SDS-PAGE. ET: Total extract, F: fraction of proteins not bound to the column, L: Washing, E: Elution. Molecular mass of protein standards indicated on the right, in kDa.

[0051] Figure 3 - Recombinant AM proteins were added to heat-inactivated S. aureus cells and hydrolysis was followed by measuring the optical density at a wavelength of 600 nanometers for 2 hours. The addition of a cysteine ​​residue at the C-terminus did not alter the hydrolytic activity of the protein. The amino acid substitutions H67A or D186A abolished virtually all of the amidase hydrolytic activity of the recombinant proteins, while the amino acid substitution N119A led to an approximately 30% decrease in the hydrolytic activity of the recombinant AM protein. A double amino acid substitution H67A-N119A also led to the abolition of the hydrolytic activity of the rAM protein.

[0052] Figure 4 - Recombinant AM proteins were added to heat-inactivated cells of different bacterial species and hydrolysis was followed by measuring the optical density at a wavelength of 600 nanometers for 2 hours. The average values ​​(n=3) of the percentage of hydrolysis of the heat-inactivated cells of the different bacterial species after 2 hours of incubation are shown.

[0053] Figure 5 - The purified proteins were incubated with heat-inactivated cells from different microorganisms. After incubation, the cells were separated by centrifugation and the amount of protein bound to the cells was determined by Western blot with anti-AM antibody. The addition of the CIS amino acid did not alter the binding capacity of the rAM protein. There are no visible differences in the binding of the recombinant proteins to the different types of cell walls.

[0054] Figure 6 - Antibody-based assay for determining protein binding on the glass surface. The glasses were immobilized with 0.1 mg / ml of rAM-Cis protein, a control without immobilized protein was also used.

[0055] Figure 7 - Visualization of molecular alignment of liquid crystals using a polarized light microscope. (A) Glass slides with non-aligned immobilized protein. (B) Rubbed glass slides with aligned immobilized protein.

[0056] Figure 8 - Glass slides were submerged in heat-inactivated S. auieus cells and the decrease in optical density at a wavelength of 600 nanometers was followed for 100 minutes.

[0057] Figure 9 - Visualization of silanized glass slides without (A) and with (B) immobilized rAM-Cis protein by AFM (Atomic Force Microscopy).

[0058] Figure 10 - Binding of heat-inactivated cells to protein immobilized on the glass surface was assessed by MFA. (A) Glass slides with immobilized rAM-Cis protein. (B) Silanized glass slides without immobilized protein.

[0059] Figure 11 - The binding of purified peptidoglycan from S. aureus to protein immobilized on the glass surface was evaluated by MFA. (A) Glass slides with immobilized rAM-Cis protein. (B) Silanized glass slides without immobilized protein.

[0060] Figure 12 - The binding of S. aureus peptidoglycan, hydrolyzed with mutanolysin, to the protein immobilized on the glass surface was evaluated by MFA. (A) Glass slides with immobilized rAM-Cis protein. (B) Silanized glass slides without immobilized protein.

[0061] Figure 13 - Visualization of the molecular alignment of the liquid crystal using a polarized light microscope. (A) Glass slides with the space between them filled with liquid crystal, with aligned immobilized protein. (B) Glass slides with the space between them filled with liquid crystal, with aligned immobilized protein after the presence of heat-inactivated S. aureus cells. Detailed description of the invention

[0062] In the context of the present invention, the term rAM refers to proteinaceous material, including single or multiple proteins, and extends to those proteins with the amino acid sequence described herein and identified as SEQ ID NO 1-20. In addition, proteins that exhibit substantially equivalent or altered activities are also contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or they may be accidental, such as those obtained through mutations in hosts that are producers of amidase AM. In addition, the term amidase is intended to include within its scope proteins specifically cited herein, as well as all analogs that are substantially homologous and allelic variations.

[0063] In the context of the present invention, the term altered includes amino acid substitutions introduced into the protein or polypeptide sequence, for example, through peptide synthesis or site-directed mutagenesis.

[0064] The part of the protein responsible for peptidoglycan-binding activity can be used to produce recombinant or synthetic proteins or polypeptides that bind to peptidoglycan with or without hydrolytic activity. The peptidoglycan-binding proteins of the present invention are useful for the detection of peptidoglycan molecules and / or fragments derived from the same and / or a bacterial cell.

[0065] In the present invention, the rAM protein was altered to be as small as possible without compromising its secondary structure, which is important for binding and hydrolytic activity. The altered proteins and polypeptides need only be large enough to allow binding to peptidoglycan fragments and peptidoglycan. The rAM protein was designed to have the amino acid sequence of the AM catalytic domain of the Atl protein (Uniprot SEQ ID Q5HH31) from amino acid Ala-199 to Thr-424 (Fig. 1). Amino acid numbering for rAM starts at Ala-1 through Thr-226.

[0066] The rAM protein or derived protein / polypeptide was designed to include substitutions at different amino acid residues, through peptide synthesis or site-directed mutagenesis. Certain amino acids were substituted for others in the rAM protein structure without appreciable loss of peptidoglycan-binding ability. These amino acids belong to different categories, being catalytic and / or important for binding specificity. Residues His67, His-172, Asp-186, Glu-126, and His-184 are catalytic, while residues Thr-182, His-172, Asp-183, Asn-119, and Trp-112 are responsible for binding specificity. The catalytic residues were substituted to remove hydrolytic activity. Removing the catalytic activity of rAM is important because the molecule to which rAM binds, peptidoglycan or fragments thereof, must not be degraded during the detection process.On the other hand, the residues responsible for the interaction with the amino acids D-iso-Gln and L-Lys were substituted to allow the protein to bind to different types of peptidoglycan, different from the peptidoglycan of S. auieus, i.e., without D-iso-Gln or L-Lys.

[0067] The invention includes any AM protein with any of the described amino acid substitutions, whether a single substitution or multiple substitutions. Any naturally occurring AM variants or substitutions not disclosed in this invention are also encompassed by the present invention. Examples

[0068] The following examples are provided by way of illustration and are not intended to limit the claims. Cloning

[0069] The 226 amino acid rAM protein spanning from amino acid Ala-199 to Thr-424 of the amidase domain of At1 was generated. The rAM nucleotide sequence was amplified by polymerase chain reaction (PCR) using oligonucleotides pexpl (5'CCAGGGCTTCAGCAACCAAGATCAG-3') and pexp5 (5'CCAGTCGACTTAGGTAGGTTGTAGTSGTAGCATTG-3'). The oligonucleotides included flanking sequences with BamHI and SalI restriction sites for cloning purposes. The pexp5 oligonucleotide also included a termination codon. The amplification product was generated from genomic DNA of S. auieus strain COL, amplified for 30 cycles at an annealing temperature of 55°C, using a high-fidelity enzyme (Thermo Fisher Scientific).The resulting DNA fragment was purified using the Zymo DNA clean and concentrator kit (Zymo Research), digested with BamHI and SalI restriction enzymes (Thermo Fisher Scientific), and ligated into the pET28a(+) expression vector also digested with BamHI and SalI restriction enzymes using T4 DNA ligase (Thermo Fisher Scientific). The resulting ligation reaction was used to transform the appropriate host, E. coli DH5, and the plasmid pET28a-rAM was generated. The pET28a(+) plasmid contains an N-terminal 6x-His tag to facilitate protein purification, and the rAM protein has a total of 260 amino acids: the N-terminal 6x-His tag followed by a spacer (MGSSHHHHHHSSSGSGSHMASMTGGQQMGRGS) and the 226 amino acids of the AM catalytic domain.By expressing the target protein in fusion with an affinity tag, such as a 6x-His, the chimeric protein can be effectively and selectively recovered from the cell extract by passage through a column containing an immobilized ligand.

[0070] To construct proteins with substitutions at different residues, a site-directed mutagenesis protocol was used. Plasmid pET28a-rAM was amplified using mutagenesis oligonucleotides that included the point mutation. For example, to construct plasmid pET28a-rAM-H67A, oligonucleotides pH67Afw (5'GGTCGTCCTGAAGGTATCGTAGTTgCAGATAGCTAATGTCGTTCGACG-3') and pH67Arv (5'CGTCGAACGATCATCATTAGCTCTTATCTAACACACACACACACTSCACTTCAGGACGACC-3') were used in a PCR reaction, amplifying the entire plasmid for 30 cycles at an annealing temperature of 80°C, using a high-fidelity polymerase enzyme (Thermo Fisher Scientific). After amplification, the initial plasmid was hydrolyzed by the action of the restriction enzyme Dpnl, and the appropriate host, E. coli DH5, was transformed with the amplified plasmid. Protein overexpression

[0071] For protein expression, E. coli BL21(DE3) expression host cells were transformed with the generated plasmids. All proteins were expressed using an autoinduction method (6,12). To induce protein expression, E. coli BL21(DE3) strains carrying the appropriate plasmids were grown aerobically in lysogeny broth liquid culture medium supplemented with 1 mM MgSO4, 0.5% glycerol, 0.05% glucose, 0.2% β-lactose monohydrate, 50 mM Na2HPC>4, 50 mM KH2PO4, 25 mM (NH4)2SO4, and 100 μ9 / π1 kanamycin for 18 h at 37 °C. Cells were harvested by centrifugation at 8421 g for 10 min at room temperature, the supernatant was discarded, and the cell pellets were kept at -20 °C. Protein purification

[0072] Cell pellets were resuspended in lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8), supplemented with benzonase nuclease (2.5 U / ml, Novagen) and EDTA-free protease inhibitors (Thermo Fisher Scientific). Cells kept on ice were mechanically lysed using a sonicator (Ultrasonic processor UP200S, Hielscher Ultrasonics) at an amplitude of 80%, 0.5 cycles in pulse mode, for 10 cycles of 1 minute with pauses. After cell lysis, proteins were present in the soluble fraction. Extracts were obtained by centrifugation at 8421 g for 60 minutes at 42°C. Purification was performed using Ni-NTA columns (Thermo Fisher Scientific) under native conditions. The supernatant was loaded onto the pre-packed column with lysis buffer. The column was washed with lysis buffer and wash buffer (50 mM Na2HPC>4, 30 mM imidazole, 300 mM NaCl, pH 8.0).To recover the protein, elution buffer (50 mM NazHPCU, 300 mM imidazole, 300 mM NaCl, pH 8.0) was used. The most concentrated elution fractions were buffer exchanged using PD10 desalting columns (GE Healthcare Life Sciences) to 10 mM potassium phosphate buffer, 10% glycerol, pH 8. Protein concentrations were determined using a Nanodrop ND-1000 Spectrophotometer using the theoretical molar extinction coefficient and the molecular mass of the protein. Protein purity was assessed by polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2). The rAM protein has a molecular mass of 29 kDa. Hydrolytic activity

[0073] Since the AM protein is a peptidoglycan hydrolase, the hydrolytic activity of the proteins was tested using heat-inactivated S. aureus cells as a substrate. Heat-inactivated S. aureus cells were prepared from cultures with an optical density at a wavelength of 600 nm (ODeoo) of 0.7, harvested by centrifugation at 8421 g for 10 minutes at 4°C. The cells were washed twice with distilled water before heat inactivation by autoclaving at 121°C for 15 minutes. The cells were washed twice more in distilled water and maintained in 0.05% NaN3.

[0074] The hydrolytic activity of the purified proteins was tested by the decrease in the optical density at a wavelength of 600 nm (ODeoo) of the heat-inactivated cells, over a period of 2 hours at 37°C, with shaking. Approximately 2 x 108 of heat-inactivated cells were resuspended in 50 mM potassium phosphate pH 7.85, and subsequently each purified protein was added to was added at a concentration of 10 pg / ml. The optical density at a wavelength of 600 nm was monitored over time and a decrease in this value was observed when the proteins had hydrolytic activity, while proteins with amino acid substitutions that rendered the protein hydrolytically inactive showed the same optical density value at 600 nm throughout the assay (Fig. 3).

[0075] The same assays were performed using heat-inactivated cells of other relevant microorganisms, such as, but not limited to, Micrococcus luteus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Escherichia coli, allowing to infer the specificity of the hydrolytic activity for different types of bacterial cells (Fig. 4). Peptidoglycan Binding

[0076] To analyze the binding capacity of the purified proteins to the peptidoglycan structures of different types of bacterial cells, a binding assay was designed. The different proteins (1 μρ) were incubated with approximately 2 x 108 heat-inactivated S. auieus cells in 50 mM potassium phosphate buffer, pH 7.85, for 10 minutes at 4°C, with shaking. At the end of the incubation period, the proteins that did not bind to the bacterial cells were separated from the proteins bound to the cell wall by centrifugation at 20,000 g for 10 minutes. At this stage, the proteins that did not bind to the bacterial cells were present in the supernatant, while the proteins that bound to the bacterial cell wall were in the bacterial cell pellet. The bacterial cell pellet was resuspended in Laemmli buffer (Bio-rad). Laemmli buffer was added to the supernatant, and both fractions were analyzed by SDS-PAGE.Separated proteins were transferred to a nitrocellulose membrane (Hibond-ECL, GE Healthcare Life Sciences) in a Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-rad) with transfer buffer (0.192 M glycine, 0.025 M Tris, 10% ethanol, pH 8.3). Blocking of nonspecific binding sites on the membrane was performed using PBST buffer [PBS 1X - Tween 20 0.5% (w / v)] with 5% (w / v) nonfat dry milk, and washing was performed with PBST buffer. ECL Western Blot Detection Reagents (Perkin Elmer) and ECL IgG (rabbit) secondary antibody conjugated with horseradish peroxidase (Perkin Elmer) were used according to the manufacturers' instructions to detect the primary anti-AM antibody (rabbit polyclonal antibody, 1:10000) at a dilution of 1:20000. Detection was performed in a dark room using autoradiography film (GE Healthcare Life Sciences).Analysis of both fractions by Western Blot allows us to infer the binding preferences of the different recombinant proteins in relation to the cell wall.

[0077] The same assay was performed using heat-inactivated cells from other relevant microorganisms, such as, but not limited to, M. luteus, S. epidermidis, S. agalactiae, S. pyogenes, E. faecalis, E. coli, allowing to infer the binding specificity of recombinant AM proteins to different types of bacterial cells (Fig. 5). Example 2 Muropeptide detection with a liquid crystal-based biosensor

[0078] A liquid crystal biosensor was developed using microfluidics (MF) for the detection of muropeptides through molecular interactions with the rAM protein.

[0079] The basic configuration of the system is an MF cell composed of a chemically activated surface on which the rAM protein is immobilized. The activated surface is capable of inducing the homeotropic alignment of the nematic liquid crystal (LC). In contact with the analysis sample, the protein immobilized on the surface binds to muropeptides, if present. The sensor cell will then be filled with nematic LC and the visualization of the test result will be performed using two crossed polarizers to detect the interaction of the incident light with the LC molecules. Two situations can occur, corresponding to a negative or positive biological sample: • No analyte (muropeptide) binds to the sensor; all CL molecules remain perpendicular to the surface as a result of the induced homeotropic alignment. Incident light will be filtered by polarizer 1 and, during the optical path through the cell, will remain polarized in the direction of polarizer 1. This polarization will not undergo rotation due to the molecular orientation of the liquid crystal. Therefore, when it reaches polarizer 2 (crossed with respect to polarizer 1), the light is extinguished. The sensor color remains black, indicating that no muropeptides were detected - Negative result. • analyte molecules (muropeptides) bind to the sensor surface, causing the homeotropic alignment of the CL to be distorted by the formed complexes (rAM protein-muropeptides). The CL molecules near the active surface will no longer be uniformly aligned along a preferred direction. The incident light will be filtered by polarizer 1 and, during the optical path through the cell, will undergo polarization rotations due to the optical anisotropy of the misaligned CL molecules. Some of the light will be able to emerge at the top of the cell after passing through polarizer 2. The sensor color will change to colors other than black, indicating the presence of muropeptides - Positive Result. Example 3 Construction of the biosensor prototype

[0080] The recombinant rAM protein was immobilized on the surface of glass slides. The glass slides were cut into 6 mm wide pieces and cleaned with piranha solution (75% H2SO4, 25% H2O2) before being silanized with a solution of 2% (v / v) (3-Aminopropyl)triethoxysilane 99% (APTES, Sigma-Aldrich) in toluene for 16 hours in a fume hood. Glass slides were functionalized with 0.5 mg / ml sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo SMCC, Thermo Fisher Scientific) in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) with 10 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) for 2 hours at room temperature with slow shaking. The recombinant rAM protein to be immobilized was reduced with 5 mM tris(2carboxyethyl)phosphine (TCEP, Sigma-Aldrich) for 30 minutes at room temperature with slow shaking.The reduced protein solution was added to functionalized glass slides and incubated for 4 hours at room temperature, with slow agitation, to promote protein binding to the glass slides. Evaluation of protein immobilization

[0081] To test protein immobilization to the glass slide surface, an antibody-based assay was developed using the 6x His-tag tail present at the N-terminal end of the protein as a probe. The silanized glass with immobilized protein was blocked with 1% (w / v) bovine serum albumin (BSA) in PBS-T (PBS, 0.5% (v / v) Tween 20), washed three times for 15 minutes with PBS-T, and incubated with a monoclonal antibody specific for 6x-His tags, conjugated with horseradish peroxidase (Thermo Scientific) at a dilution of 1:3000 in PBS-T with 1% BSA. After a one-hour incubation period and three 15-minute washes in PBS-T, the glass slide was incubated in a 2% solution of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Thermo Scientific, colorimetric substrate for horseradish peroxidase). After 15, 30, and 60 minutes, absorbance at a wavelength of 405 nm was measured with a microplate reader.After reaction with peroxidase, ABTS produces a green compound, indicating the presence of the protein on the glass surface (Fig. 6). A control assay was performed using a glass slide without immobilized protein. Protein alignment and visualization

[0082] To test whether the protein immobilized on the surface of the glass slide could be aligned, a rubbing procedure was used. The proteins immobilized on the glass surface were aligned with the aid of a soft cloth, and the glass was rubbed in the same direction approximately 30 times, to subsequently induce the alignment of the liquid crystal to be added to the biosensor.

[0083] To build a prototype biosensor, two glass slides with immobilized protein were glued using epoxy resin with 4 drops at the corners, and, after drying, a drop of liquid crystal 4-cyano-4'-pentylbiphenyl (5CB) was introduced between the glass slides by capillary action. As a control, glass slides with unaligned proteins were also used. The alignment of the liquid crystal was evaluated with a polarized light microscope, showing that friction can induce protein alignment in the glass slide matrix (Fig. 7). Binding of immobilized proteins to the peptidoglycan substrate

[0084] Proteins immobilized on the glass surface lost their hydrolytic activity, as verified by hydrolytic tests performed by following the optical density at a wavelength of 600 nm (DOeoo) over time (Fig. 8).

[0085] To determine whether the immobilized proteins were present on the glass surface and capable of binding to the substrate, although without hydrolytic activity, the atomic force microscopy (AFM) technique was used.

[0086] The appearance of glass slides visualized by MFA was different whether or not they had immobilized protein, suggesting that protein can be visualized on the glass surface using this technique (Fig. 9).

[0087] To determine whether the protein was able to bind to its substrate, glass slides were submerged in an aqueous solution of heat-inactivated cells of S. auieus strain NCTC 8325-4 at a concentration of ~2x108 cells / ml for 30 minutes, washed twice with water, and visualized by MFA (Fig. 10A). Glass slides without immobilized protein were subjected to the same treatment and visualized by MFA as a control (Fig. 10B). The technique revealed that heat-inactivated cells bound to the glass slides with immobilized rAM-Cis protein, but did not bind to the control silanized glass slides.

[0088] To test smaller cell wall fragments, ~500 μg purified peptidoglycan from S. auieus (Fig. 11) and ~500 μg peptidoglycan resulting from hydrolysis of purified peptidoglycan with mutanolysin (Sigma Aldrich) were also used as substrate in the same protein binding assay (Fig. 12). The results were similar to those obtained with heat-inactivated cells, showing binding of both the purified peptidoglycan and the peptidoglycan fragments to the glass slide only when the rAM-Cis protein was immobilized on the glass slide.

[0089] Collectively, these results showed that, although catalytically inactive, the rAM-Cis protein immobilized to the surface of the glass slide was still capable of binding to its substrate, whether heat-inactivated cells or purified peptidoglycan or peptidoglycan fragments. Visualization of binding of immobilized proteins to heat-inactivated cells by polarized optical microscopy

[0090] To evaluate whether protein binding to the substrate destabilizes the 5CB liquid crystal molecules, two glass slides with immobilized protein were rubbed to align the proteins and subsequently submerged in an aqueous solution of heat-inactivated cells of S. aureus strain NCTC 8325-4 at a concentration of ~2 x 108 cells / ml, as previously described. Subsequently, the two glass slides were glued by placing drops of epoxy resin on the 4 corners, and, after drying, a drop of 5CB liquid crystal was introduced between the glass slides by capillary action. A control without heat-inactivated cells was also performed, and the alignment of the liquid crystal was evaluated with a polarized light microscope (Fig. 13). Visualization of the glass slides using the polarized light microscope showed that the presence of heat-inactivated S. aureus cells disorients the liquid crystals, resulting in a colored image. SEQUENCE LISTING SEQ ID NO: 1 Main sequence > rAM protein without amino acid substitutions ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 2 > rAM protein with H67X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 3 > rAM protein with H172X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTXYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 4 > rAM protein with H184X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDXADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 5 > rAM protein with D186X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHAXPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 6 > rAM protein with T182X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGXDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 7 > rAM protein with N119X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 8 > rAM protein with D183X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 9 > rAM protein with W112X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 10 > rAM protein with E126X substitution ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVXIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 11 > rAM protein without amino acid substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 12 > rAM protein with H67X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 13 > rAM protein with H172X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTXYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 14 > rAM protein with H184X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDXADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 15 > rAM protein with D186X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHAXPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 16 > rAM protein with T182X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGXDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 17 > rAM protein with N119X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 18 > rAM protein with D183X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 19 > rAM protein with W112X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 20 > rAM protein with E126X substitution plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVXIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 21 > rAM protein with H67X T182X1 substitutions ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 22 > rAM protein with substitutions H67X D183X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXiHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 23 > rAM protein with substitutions H67X W112X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 24 > rAM protein with substitutions H67X N119X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 25 > rAM protein with substitutions T182X D186X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGXDHAXiPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 26 > rAM protein with substitutions D183X DI86X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXHAXiPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 27 > rAM protein with substitutions W112X DI86X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHAXiPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 28 > rAM protein with substitutions N119X DI86X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHAXiPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 29 > rAM protein with substitutions D183X H184X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXXiADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 30 > rAM protein with substitutions W112X H184X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDXiADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 31 > rAM protein with substitutions N119X H184X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDXiADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 32 > rAM protein with substitutions W112X T182X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 33 > rAM protein with substitutions N119X T182X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGXiDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 34 > rAM protein with substitutions W112X H172X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTXiYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 35 > rAM protein with substitutions N119X H172X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTXiYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 36 > rAM protein with substitutions W112X D183X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXiHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 37 > rAM protein with substitutions N119X D183X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 38 > rAM protein with substitutions E126X T182X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVXIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGXiDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 39 > rAM protein with substitutions E126X D183X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVXIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXiHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 40 > rAM protein with substitutions W112X E126X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 41 > rAM protein with substitutions N119X E126X1 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 42 > rAM protein with substitutions H67X T182X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTWTHYAVSKYLGGX1DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 43 > rAM protein with substitutions H67X D183X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 44 > rAM protein with substitutions H67X W112X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 45 > rAM protein with substitutions H67X N119X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 46 > rAM protein with substitutions T182X D186X plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGXDHAXiPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 47 > rAM protein with substitutions D183X D186X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXHAX1PHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 48 > rAM protein with substitutions W112X DI86X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHAX1PHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 49 > rAM protein with substitutions N119X DI86X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHAXiPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 50 > rAM protein with substitutions D183X H184X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXXiADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 51 > rAM protein with W112X H184X1 substitutions plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDXiADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 52 > rAM protein with substitutions N119X H184X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX1ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 53 > rAM protein with W112X T182X1 substitutions plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 54 > rAM protein with substitutions N119X T182X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 55 > rAM protein with substitutions W112X H172X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTXiYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 56 > rAM protein with substitutions N119X H172X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTXiYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 57 > rAM protein with substitutions W112X D183X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 58 > rAM protein with substitutions N119X D183X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 59 > rAM protein with substitutions E126X T182X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVXIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 60 > rAM protein with substitutions E126X D183X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVXIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTXiHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 61 > rAM protein with substitutions W112X E126X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 62 > rAM protein with substitutions N119X E126X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVX1IVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 63 > rAM protein with substitutions H67X W112X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 64 > rAM protein with substitutions H67X N119X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2HADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 65 > rAM protein with substitutions H67X W112X1 T182X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 66 > rAM protein with substitutions H67X N119X1 T182X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 67 > rAM protein with substitutions H67X W112X1 H172X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSX1GVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 68 > rAM protein with substitutions H67X N119X1 H172X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 69 > rAM protein with substitutions H67X W112X1 D183X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 70 > rAM protein with substitutions H67X N119X1 D183X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 71 > rAM protein with substitutions W112X H184X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX1AX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 72 > rAM protein with substitutions N119X H184X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX1AX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 73 > rAM protein with substitutions W112X T182X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 74 > rAM protein with substitutions N119X T182X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 75 > rAM protein with substitutions W112X H172X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 76 > rAM protein with substitutions N119X H172X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 77 > rAM protein with substitutions W112X D183X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 78 > rAM protein with substitutions N119X D183X1 DI86X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 79 > rAM protein with substitutions W112X T182X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 80 > rAM protein with substitutions N119X T182X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 81 > rAM protein with substitutions W112X H172X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 82 > rAM protein with substitutions N119X H172X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 83 > rAM protein with substitutions W112X D183X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTWTHYAVSKYLGGTX1X2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 84 > rAM protein with substitutions N119X D183X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1X2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 85 > rAM protein with substitutions W112X H172X1 T182X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGX2DHADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 86 > rAM protein with substitutions N119X H172X1 T182X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGX2DHADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 87 > rAM protein with substitutions W112X H172X1 D183X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTX2HADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 88 > rAM protein with substitutions N119X H172X1 D183X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTX2HADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 89 > rAM protein with substitutions W112X E126X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVX1IVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 90 > rAM protein with substitutions N119X E126X1 H184X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVX1IVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 91 > rAM protein with substitutions W112X E126X1 T182X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 92 > rAM protein with substitutions N119X E126X1 T182X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 93 > rAM protein with substitutions W112X E126X1 H172X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 94 > rAM protein with substitutions N119X E126X1 H172X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 95 > rAM protein with substitutions W112X E126X1 D183X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 96 > rAM protein with substitutions N119X E126X1 D183X2 ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTT SEQ ID NO: 97 > rAM protein with substitutions H67X W112X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 98 > rAM protein with substitutions H67X N119X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2HADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 99 > rAM protein with substitutions H67X W112X1 TI82X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 100 > rAM protein with substitutions H67X N119X1 T182X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGX1PRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 101 > rAM protein with substitutions H67X W112X1 H172X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 102 > rAM protein with H67X N119X1 H172X2 substitutions more cistern and C-terminal ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXiPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 103 > rAM protein with H67X W112X1 D183X2 substitutions cistern and C-terminal ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXiGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 104 > rAM protein with substitutions H67X N119X1 D183X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWXD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGX1PRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 105 > rAM protein with substitutions W112X H184X1 DI86X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX1AX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 106 > rAM protein with substitutions N119X H184X1 DI86X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX1AX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 107 > rAM protein with substitutions W112X T182X1 DI86X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 108 > rAM protein with substitutions N119X T182X1 DI86X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 109 > rAM protein with substitutions W112X H172X1 DI86X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 110 > rAM protein with substitutions N119X H172X1 DI86X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDHAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 111 > rAM protein with substitutions W112X D183X1 DI86X2 plus cisterna at the C-terminus ASAQPRSAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1HAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 112 > Proteína rAM com substituições N119X D183X1 DI86X2 mais cisterna no C-terminal ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYARNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGGNGTVWTHYAVSKYLGGTX1HAX2PHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 113 > Proteína rAM com substituições W112X T182X1 H184X2 mais cisterna no C-terminal ASAQPRSAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 114 > rAM protein with substitutions N119X T182X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX1DX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 115 > rAM protein with substitutions W112X H172X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 116 > rAM protein with substitutions N119X H172X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 117 > rAM protein with substitutions W112X D183X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX1X2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 118 > rAM protein with substitutions N119X D183X1 H184X1 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTWTHYAVSKYLGGTX1X2ADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 119 > rAM protein with substitutions W112X H172X1 T182X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGX2DHADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 120 > rAM protein with substitutions N119X H172X1 T182X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGX2DHADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 121 > rAM protein with substitutions W112X H172X1 D183X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTX2HADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 122 > rAM protein with substitutions N119X H172X1 D183X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVEIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX1YAVSKYLGGTX2HADPHGYLRSHNYSYDQLYD LINEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 123 > rAM protein with substitutions W112X E126X1 H184X2 plus cisterna at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVX1IVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 124 > rAM protein with substitutions N119X E126X1 H184X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDX2ADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 125 > rAM protein with substitutions W112X E126X1 T182X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 126 > rAM protein with substitutions N119X E126X1 T182X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGX2DHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 127 > rAM protein with substitutions W112X E126X1 H172X2 plus cysteine ​​at C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 128 > rAM protein with substitutions N119X E126X1 H172X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVX1IVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTX2YAVSKYLGGTDHADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 129 > rAM protein with substitutions W112X E126X1 D183X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSXGVGAVGNPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC SEQ ID NO: 130 > rAM protein with substitutions N119X E126X1 D183X2 plus cysteine ​​at the C-terminus ASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHD TANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGXPRFINVXiIVHTHDYASF ARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTX2HADPHGYLRSHNYSYDQLYDL INEKYLIKMGKVAPWGTQSTTTC References 1. Schleifer KH, Kandler O. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36 : 407 -77 . 2. Loza-Correa M, Aiala JA, Perelman I, Hubbard K, Kalab M, Li QL, Taha M, de Pedro MA, Ramirez-Arcos S. 2019. The peptidoglycan matrix and biofilm of Staphylococcus epidermidis undergo structural changes when exposed to human platelets. PLoS One 14:e0211132. 3. Tipper DJ, Berman MF. 1969. Structures of the cell wall peptidoglycans of Staphylococcus epidermidis Texas 26 and Staphylococcus aureus Copenhagen. I. Chain length and average sequence of cross-bridged peptides. Biochemistry 8: 2183–92. 4. lang H, Singh M, Kim SJ, Schaefer J. 2017. Characterization of the tertiary structure of Enterococcus faecalis peptidoglycan. Biochim Biophis Ata Biomembr 1859:2171–2180. 5. Oshida T, Sugai M, Komatsuzawa H, Hong IM, Suginaka H, ​​Tomasz A. 1995. A Staphylococcus aureus autolysin domain having an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc Natl Acad Sci UA 92: 285–9. 6. Grilo IR, Ludovice AM, Tomasz A, de Lencastre H, Sobral RG. 2014. The glucosaminidase domain of Atl - the major Staphylococcus aureus autolysin - has DNA-binding activity. Microbiology 3: 247–56. 7. Biswas R, Voggu L, Simon UK, Hentschel P, Thumm G, Gõtz F. 2006. Activity of major staphylococcal autolysis Atl. FEMS Microbiol Lett 259: 260–8. 8. Zoll S, Schlag M, Shkumatov AV, Rautenberg M, Svergun Dl, Gõtz F, Stehle T. 2012. Ligand-binding properties and conformational dynamics of autolysis repeat domains in staphylococcal cell wall recognition. J Bacteriol 194: 3789–802. 9. Heilmann C, Hussain M, Peters G, Gõtz F. 1997. Evidence for autolysis-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 24:1013-24. 10. Búttner FM, Zoll S, Nega M, Gõtz F, Stehle T. 2014. Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage. J Biol Chem 289:11083–94. 11. Zoll S, Pâtzold B, Schlag M, Gõtz F, Kalbacher H, Stehle T. 2010. Structural basis of cell wall cleavage by staphylococcal autolysis. PLoS Pathog 6:el000807 12. Studier FW. 2005. Protein production by autoinduction in high-density shaken cultures. Protein Expr Purif 41:207–34.

Claims

1. Sequência isolada ou sequência recombinante ou sequência sintética que contém pelo menos 9 0% de identidade com: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:2 0, SEQ ID NO:2 9, SEQ ID NO:3 8, SEQ ID NO:47, SEQ ID NO:56, SEQ ID NO:6 5, SEQ ID NO:7 4, SEQ ID NO:8 3, SEQ ID NO:9 2, SEQ ID NO:16, SEQ ID NO:2 5, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:7 0, SEQ ID NO:7 9, SEQ ID NO:8 8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:3 0, SEQ ID NO:39, SEQ ID NO:4 8, SEQ ID NO:57, SEQ ID NO:6 6, SEQ ID NO:7 5, SEQ ID NO:84, SEQ ID NO:9 3, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:3 5, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:6 2, SEQ ID NO:71, SEQ ID NO:8 0, SEQ ID NO:89, SEQ ID NO:13, SEQ ID NO:2 2 , SEQ ID NO:31, SEQ ID NO:4 0 , SEQ ID NO:49, SEQ ID NO:5 8 , SEQ ID NO:67, SEQ ID NO:7 6 , SEQ ID NO:8 5 , SEQ ID NO:9 4 , SEQ ID NO:18, SEQ ID NO:27 , SEQ ID NO:36,SEQ ID NO:4 5, SEQ ID NO:54, SEQ ID NO:6 3, SEQ ID NO:7 2, SEQ ID NO:81, SEQ ID NO:9 0, SEQ ID NO:14, SEQ ID NO:2 3, SEQ ID NO:3 2, SEQ ID NO:41, SEQ ID NO:5 0, SEQ ID NO:59, SEQ ID NO:6 8, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:9 5, SEQ ID NO:19, SEQ ID NO:2 8, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:5 5, SEQ ID NO:6 4, SEQ ID NO:7 3, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO:15, SEQ ID NO:2 4, SEQ ID NO:3 3, SEQ ID NO:42, SEQ ID NO:51, SEQ ID NO:6 0, SEQ ID NO:6 9, SEQ ID NO:7 8, SEQ ID NO:87, SEQ ID NO:9 6, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:10 5, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:10 8, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO: 118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125 SEQ ID NO:126 SEQ ID NO:127, SEQ ID NO:128,SEQ ID NO:129 or SEQ ID NO:130, wherein said sequence binds a peptidoglycan and / or a muropeptide and / or a bacterial cell., 2. Sequência de acordo com a reivindicação anterior, contendo pelo menos 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ou 99% de identidade com: SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO:5 , SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, 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: 2 0, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:2 3, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 , SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 3 8, SEQ ID NO: 39 , SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 , SEQ ID NO: 49, SEQ ID NO:5 0, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 , SEQ ID NO: 58,SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 , SEQ ID NO:67, SEQ ID NO:6 8, SEQ ID NO: 69, SEQ ID NO : 7 0 , SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 , SEQ ID NO: 76, SEQ ID NO:7 7, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 , SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 , SEQ ID NO:94, SEQ ID NO:9 5, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 10 0, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119, SEQ ID NO: 12 0, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO: 124,SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:128, SEQ ID NO:129 OU SEQ ID NO:

130. Sequência de acordo com qualquer uma das reivindicações anteriores, em que a sequência é SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:8: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, 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, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 2 8, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO :42, SEQ ID NO:51, SEQ ID NO:6 0, SEQ ID NO:6 9, SEQ ID NO:7 8, SEQ ID NO:87, NO:3 8, SEQ ID NO:47, SEQ ID NO:56, SEQ ID NO:6 5, SEQ ID NO:7 4, SEQ ID NO:8 3, SEQ ID SEQ ID NO:4 3, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:7 0, SEQ ID NO:7 9, SEQ ID NO:8 8, NO:3 9,SEQ ID NO:4 8, SEQ ID NO:57, SEQ ID NO:66, SEQ ID NO:7 5, SEQ ID NO:84, SEQ ID SEQ ID NO:44, SEQ ID NO:5 3, SEQ ID NO:6 2 , SEQ ID NO:71, SEQ ID NO:8 0 , SEQ ID NO:89, NO:4 0, SEQ ID NO:49, SEQ ID NO:58, SEQ ID NO:67, SEQ ID NO:7 6, SEQ ID NO:8 5, SEQ ID SEQ ID NO:4 5, SEQ ID NO:54, SEQ ID NO:6 3, SEQ ID NO:7 2, SEQ ID NO:81, SEQ ID NO:9 0, NO:41, SEQ ID NO:5 0, SEQ ID NO:59, SEQ ID NO:6 8, SEQ ID NO:7 7, SEQ ID NO:86, SEQ ID SEQ ID NO:46, SEQ ID NO:5 5, SEQ ID NO:6 4, SEQ ID NO:7 3, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO: 92, SEQ ID NO: 93 SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQID NO: 121, SEQ ID NO: 122,SEQ ID NO: 123, SEQ ID NO: 124, SEQID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 ou SEQ ID NO:130 de preferência em que a sequência é SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:14: 45, SEQ ID NO: 49 ou SEQ ID NO:56 ou de preferência em que a sequência é SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:3 2, SEQ ID NO:41, SEQ ID NO:5 0, SEQ ID NO:59, SEQ ID NO:6 8, SEQ ID NO:2 8, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:5 5, SEQ ID NO:6 4, SEQ ID NO:7 3, SEQ ID NO:3 3, SEQ ID NO:42, SEQ ID NO:51, SEQ ID NO:6 0, SEQ ID NO:6 9, SEQ ID NO:29, SEQ ID NO:3 8, SEQ ID NO:47, SEQ ID NO:56, SEQ ID NO:6 5, SEQ ID NO:7 4, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO : 6 3 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:7 7, SEQ ID NO:7 8, SEQ ID NO:7 9 , SEQ ID NO:8 0, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:8 5, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:8 8 , SEQ ID NO:89, SEQ ID NO:9 0, SEQ ID NO:91, SEQ ID NO:9 2, SEQ ID NO:9 3, SEQ ID NO:9 4, SEQ ID NO:9 5, SEQ ID NO:9 6, SEQ ID NO:97, SEQ ID NO:9 8, SEQ ID NO:9 9, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO 104, 105, 106, SEQ ID NO 107, SEQ ID NO 108, 109, 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 12 0, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128,SEQ ID NO:129 ou SEQ ID NO:130., 4 . Sequência de acordo com qualquer uma das reivindicações anteriores, onde X, Xi ou X2 da SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:2 3, SEQ ID NO:32, SEQ ID NO:41, SEQ ID NO:5 0, SEQ ID NO:59, SEQ ID NO:6 8, SEQ ID NO:7 7, SEQ ID NO:86, SEQ ID NO:9 5, NO:19, SEQ ID NO:2 8, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:6 4, SEQ ID NO:7 3, SEQ ID NO:82, SEQ ID NO:91, SEQ ID SEQ ID NO:24, SEQ ID NO:3 3, SEQ ID NO:42, SEQ ID NO:51, SEQ ID NO:6 0, SEQ ID NO:6 9, SEQ ID NO:7 8, SEQ ID NO:87, SEQ ID NO:9 6, NO:2 0, SEQ ID NO:29, SEQ ID NO:3 8, SEQ ID NO:47, SEQ ID NO:56, SEQ ID NO:6 5, SEQ ID NO:7 4, SEQ ID NO:83, SEQ ID NO:9 2, SEQ ID SEQ ID NO:2 5 , SEQ ID NO:34, SEQ ID NO:4 3 , SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:7 0 , SEQ ID NO:7 9 , SEQ ID NO:8 8 , SEQ ID NO:9 7 , NO:21,SEQ ID NO:3 0, SEQ ID NO:3 9, SEQ ID NO:4 8, SEQ ID NO:57, SEQ ID NO:6 6, SEQ ID NO:7 5, SEQ ID NO:84, SEQ ID NO:9 3, SEQ ID SEQ ID NO:26, SEQ ID NO:3 5, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:6 2, SEQ ID NO:71, SEQ ID NO:8 0, SEQ ID NO:89, SEQ ID NO:9 8, NO:22, SEQ ID NO:31, SEQ ID NO:4 0, SEQ ID NO:49, SEQ ID NO:5 8, SEQ ID NO:6 7, SEQ ID NO:7 6, SEQ ID NO:85, SEQ ID NO:9 4, SEQ ID SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:4 5, SEQ ID NO:54, SEQ ID NO:6 3, SEQ ID NO:7 2, SEQ ID NO:81, SEQ ID NO:9 0, SEQ ID NO:9 9, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO :116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQID NO:128,SEQ ID NO:129 or SEQ ID NO:130 is selected from A, C, D, E, F, G, Η, IK, L, Μ, N, P, Q, R, S, T, V, W or Y; and X, Xi or X2 are selected independently of each other., 5. The sequence according to any one of the preceding claims, wherein X, Xi or X2 are A or R.

6. Sequence according to any one of the preceding claims, wherein the bacterial cell is a Staphylococcus cell.

7. A sequence according to any one of the preceding claims for use in a method of in vivo diagnosis of a bacterial infection, preferably where the bacterial infection is a Staphylococcus bacterial infection.

8. Isolated DNA sequence encoding a sequence according to any one of the preceding claims 1-7.

9. A vector or expression cassette or host cell or phage comprising a sequence according to any one of the preceding claims 1-7.

10. Sensor comprising a sequence according to any one of the preceding claims 1-7.

11. Sensor de acordo com a reivindicação anterior que inclui uma sequência selecionada a partir do SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:61, NO:18, SEQ ID NO:4 8, SEQ ID NO:57, SEQ ID SEQ ID NO:44, SEQ ID NO:5 3, SEQ ID NO:6 2, NO:19, SEQ ID NO:49, SEQ ID NO:5 8, SEQ ID SEQ ID NO:20, SEQ ID NO:42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 6 0, SEQ ID NO: 97, SEQ ID NO : 9 8 , SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQID NO: 124,SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 and / or SEQ ID NO: 130., 12. The sensor of any one of the preceding claims 10-11, further comprising: a substrate and a liquid crystal, wherein the substrate has a sequence or plurality of sequences immobilized on its surface; wherein the sequence or plurality of different sequences is capable of binding to peptidoglycan and / or muropeptides and / or bacterial cells in a sample; and wherein the liquid crystal has a first liquid crystal orientation and a second liquid crystal orientation such that the liquid crystal changes from the first liquid crystal orientation to the second liquid crystal orientation when the liquid crystal interacts with a sample having peptidoglycan and / or muropeptides and / or bacterial cells bound to the sequence or plurality of different sequences such that a light signal is generated.

13. The sensor according to the preceding claim, wherein the sensor comprises two substrates, a first substrate and a second substrate, such that the surface of the first substrate with the immobilized sequence or plurality of immobilized sequences is exposed to the surface of the second substrate with the immobilized sequence or plurality of immobilized sequences.

14. Sensor according to any one of the preceding claims 11-13, wherein the sequence or plurality of different sequences immobilized to the surface of the first substrate is identical to the sequence or plurality of different sequences immobilized to the surface of the second substrate; or wherein the sequence or plurality of different sequences immobilized to the surface of the first substrate is different from the sequence or plurality of different sequences immobilized to the surface of the second substrate.

15. Sensor according to any one of the preceding claims 12-14, wherein the liquid crystal is in a nematic phase.

16. Sensor according to any one of the preceding claims 12-15, wherein the first liquid crystal molecular orientation is homogeneous perpendicular or parallel to the substrate and the second liquid crystal molecular orientation is heterogeneous.

17. Sensor according to any one of the preceding claims 13-16, wherein the liquid crystal is between the two substrates.

18. The sensor according to the preceding claim, wherein the liquid crystal is selected from 4-cyano-41-pentylbiphenyl, N-(4-methoxybenzylidene)-4-butylaniline; 4-cyano-4'-n-heptyl-biphenyl; 4-cyano-4'-n-oxyoctyl-biphenyl; 4-cyano-41-n-pentyl-triphenyl; and / or a mixture consisting of 51% 4-cyano-4'-n-pentyl-pentyl-biphenyl, 25% 4-cyano-4'-n-heptyl-biphenyl, 16% 4-cyano-41-n-oxyoctyl-biphenyl (8OCB) and 8% 4-cyano-4'-n-pentyltriphenyl (5CT).

19. Sensor according to any one of the preceding claims 12-18, wherein the substrate is selected from: glass substrate, silicon substrate, polydimethylsiloxane substrate, acrylic substrate and other transparent substrates.

20. The sensor of any one of the preceding claims, further including a sample inlet and a sample outlet.

21. Sensor according to any one of the preceding claims, wherein the sequence or sequences are immobilized on a substrate surface via biotin / streptavidin, preferably covalently or by adsorption.

22. Sensor according to any one of the preceding claims, wherein its detection limit is less than 102 CFUs / ml.

23. A method for detecting peptidoglycan and / or muropeptides and / or a bacterial cell in a sterile fluid or a water sample, comprising the following steps: Using a sensor according to any one of the preceding claims 10-22 in a water sample or in a sterile liquid sample; subjecting said sensor to polarized light and determining a light signal generated by changes from a first liquid crystal orientation to a second liquid crystal orientation when the liquid crystal interacts with a sample having peptidoglycan and / or muropeptides and / or bacterial cells bound to a different sequence or plurality of sequences according to any one of the preceding claims 17.

24. Use of a sequence according to any one of the preceding claims 1-7 as a bacterial detector, preferably as a water bacteria detector.