DE102008023447A1 - New polypeptide having an amino acid position against the amino acid sequence, useful as human- or veterinary medicine and as disinfectant, and in food and cosmetics - Google Patents

Abstract

Polypeptide having at least one amino acid position against the amino acid sequence according to SEQ ID NO: 1 (which is not defined) modified amino acid sequence, is new. Independent claims are included for: (1) a nucleotide sequence encoding the polypeptide; (2) a vector comprising the nucleotide sequence; and (3) a host cell comprising the nucleotide sequence or the vector.

Description

The The present invention relates to polypeptides having at least one an amino acid position relative to the amino acid sequence according to SEQ ID NO: 1 changed amino acid sequence. The present invention further relates to the polypeptide encoding Nucleotide sequences, vectors comprising the nucleotide sequences and Host cells for expression of the polypeptides. Furthermore, the present invention the use of the polypeptide human or Veterinary substance, in food, in cosmetics, as a disinfectant or in the environmental field.

Listeria are widespread human and animal pathogenic bacteria from the Food sector, which cause listeriosis as a disease. Often, foods such as fish, meat and dairy are with Listeria burdened. The genus Listeria includes 6 different Species with 16 different serotypes. In detail these are L. monocytogenes with serotypes 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7; L. innocua with the serotypes 3, 6a, 6b, 4ab, U / S; L. ivanovii with serotype 5; L. seeligeri with the Serotypes 1 / 2a, 1 / 2b, 1 / 2c, 4b, 4c, 4d, 6b; L. welshimeri with the Serotypes 1 / 2a, 4c, 6a, 6b, U / S and L. grayi with the serotype Grayi. The two species L. monocytogenes and L. ivanovii are considered pathogenic. A third species, L. seeligeri, is considered to be apathetic, however, a case is known in which L. seeligeri in a human Meningitis caused. The remaining species are considered nonpathogenic. Approximately 90% of listeriosis are on L. monocytogenes serovar 1 / 2a, 1 / 2b and 4b (Wing EJ & Gregory SH, 2002, Listeria monocytogenes: Clinical and Experimental Update, J Infect Diseases 185 (Suppl 1): S18-S24).

Although listeriosis is a rare disease, it is very serious because of the severity of the disease and the high mortality rate. Although only a small proportion of foodborne illnesses are caused by Listeria (about 1% in the US), nearly 30% of the annually fatal diseases caused by foodborne pathogens are attributed to this germ. Affected are especially immunosuppressed persons, eg. As elderly people, diabetics, people with cancer and / or AIDS. Pregnant women and unborn children account for approximately 25% of listeriosis patients. Because of their ability to cross the blood-brain barrier or the placental barrier, Listeria can cause meningitis, encephalitis, fatalities, and stillbirths ( Wing EJ & Gregory SH, 2002, Listeria monocytogenes: Clinical and Experimental Update, J Infect Diseases 185 (Suppl 1): S18-S24 ; Doyle ME, 2001, Virulence Characteristics of Listeria monocytogenes, Food Research Institute, October 2001 ).

Listeria are very well adapted to the survival in the environment in food production. They are tolerant Light acids and are able to be at relative high salt concentrations and at temperatures of 1 ° C to increase to 45 ° C. The main source of infection are foods, especially those that are before consumption are not heat treated, such. Many dairy products, smoked fish, Meat products and increasingly also finished products ("Ready-to-eat" products, especially meat products). Contamination with listeria often occurs in food processing (removal from the cooking containers, slicing, garnishing, packaging, etc.) instead of. For food produced with the help of starter cultures and not heat-treated (eg raw milk cheese, salami), Contamination can also be caused by the starter cultures or the Raw materials themselves or even during maturation and storage respectively. While in the US a zero tolerance for L. monocytogenes in ready-to-eat foods is in many European countries or even Canada for certain foods up to contamination with Listeria 100 KbE (colony forming units) / g food allowed. In any case, however, the food must be on Listerienkontamination be examined. Many of these foods, eg. Seafood, Smoked salmon, dairy products or raw food ready-made products have only a low durability. That's how it often happens to costly product recalls when using these products after delivery a Listeria contamination or contamination over the permitted limit has been established.

Out For this reason there is great interest, both procedures for the detection as well as the decontamination of Listeria provide. Furthermore, there are applications of antimicrobial Substances important, on the one hand to the growth of Listeria Prevent as well as kill already existing Listeria.

EP0781349 describes inter alia the listeriaphage lysine from the phage A511, Ply511, which can be used for the above-mentioned applications. Ply511 is well suited to a variety of Listeria serovars due to its broad host range, but it has a relatively low stability, which is precisely what prevents it from being used in food. Turner et al. ( 2007, Syst. And appl. Microbiol., 30, 58-67 ) point to proteolysis problems in the expression of Ply511 in lactic acid bacteria for potential use in foods and suggest that for a similar use the stability of Ply511 should be increased. However, Turner gives no hints or solutions discloses how the Ply511 can be made more stable.

Therefore The present invention is based on the object, more stable To provide endolysins Ply511.

The Task is defined by the in the claims Object solved.

The The following figures serve to explain the invention.

1 shows the amino acid sequence of the endolysin Ply511. The number of the first amino acid in each row is given at the left margin. The amino acids that make up the EAD are shown in italics and underlined. The amino acids that make up the CBD1 are shown in italics, those of the CBD2 are underlined. K260 is also the last amino acid from CBD1 and the first from CBD2. The domain linker sequences between BAD and CBD1 (amino acids 175 to 203) and between CBD1 and CBD2 (amino acids 245 to 282) are in bold.

2 shows the amino acid sequence of the endolysin Ply511. The number of the first amino acid in each row is given at the left margin. The amino acids that form the BAD are shown in italics and underlined. The amino acids that make up the CBD1 are shown in italics, those of the CBD2 are underlined. K260 is also the last amino acid from CBD1 and the first from CBD2. The bold-labeled amino acids represent cleavage sites for proteases and were determined by N-terminal sequencing of the resulting endolysin fragments.

3 shows the result of polypeptide separation in an SDS-polyacrylamide gel after protease degradation during storage of Ply511. Lane 1 shows a molecular weight standard, lane 2 native full length Ply511, lane 3 Ply511 after storage. The band marked "1" is the full-length Ply511, band "2" represents a degrading band. KDa means kilodalton.

4 shows the result of polypeptide separation in an SDS-polyacrylamide gel after trypsin digestion in comparison between Wt-Ply511 and mutants. 2A shows a comparison between the double mutant Ply511-T241S-T242S and Wt-Ply511. 2 B shows the digestion of the two mutants Ply511-S245A and Ply511-D222A-S245A. The numbers on the left are molecular weight measures in kilodaltons. In the left lane (M) in each case a molecular weight standard is plotted. The kinetics of trypsin digestion are given in minutes above the gels above the respective lanes. The line on the right side marks the position of the unenlarged full length protein.

5 shows the result of polypeptide separation in an SDS-polyacrylamide gel after chymotrypsin digestion in comparison between Wt-Ply511 and mutants. Wt-Ply511 and the mutants Ply511-D222A-S245A and Ply511-K246Q-K248Q were digested with chymotrypsin for 1 min, 2 min or 5 min and then applied to an SDS gel. "K" denotes a control sample without addition of chymotrypsin. The numbers on the left are molecular weight measures in kilodaltons. The line on the right side marks the position of the full-length protein.

6 shows a graphical representation of the evaluation of a liquid lysis test in which listeria cells are lysed by the addition of endolysin. To heat-inactivated cells of Listeria strain 996 (Serovar 1 / 2b) was Wt-Ply511 (o) or the mutant Ply511-K246Q-K248Q (✦) in an amount of 0.1 ug, 0.3 ug or 0.7 ug added (the left-to-right curves correspond to a decreasing protein level) and the decrease in optical density at 600 nm (OD ₆₀₀ ) as a function of time. t means time; [s] means seconds.

7 shows a graphical representation of the evaluation of a liquid lysis test in which listeria cells are lysed by the addition of endolysin. To heat-inactivated cells of Listeria strain 776 (Serovar 4b) Wt-Ply511 (o) or a Ply511 mutant according to the invention (✦) was added and the decrease in optical density at 600 nm (OD ₆₀₀ ) was monitored as a function of time. 7A Wt-Ply511 (o) and mutants Ply511-G249A (✦) at a concentration of 10 μg / ml. 7B Wt-Ply511 (o) and mutant Ply511-Δ195-262 (✦) at a concentration of 0.3 μg / ml. t means time; [s] means seconds.

) und Ply511-D222A (∎) wurden im Photometer aufgeheizt und die Zunahme der Proteinaggregation (entspricht einer Zunahme der Absorption (A) bei einer Wellenlänge von 360 nm) in Abhängigkeit von der Temperatur (T) in Grad Celsius verfolgt. 8th shows a graphical representation of the evaluation of a thermal stability test of Wt-Ply511 and various mutants. Wt-Ply511 (o) and the mutants Ply511-G249A (✦), Ply511-S245A (Δ), Ply511-D222A-S245A (

) and Ply511-D222A (∎) were heated in the photometer and the increase in the Protein aggregation (corresponds to an increase in absorbance (A) at a wavelength of 360 nm) as a function of the temperature (T) followed in degrees Celsius.

9 shows the result of polypeptide separation in an SDS-polyacrylamide gel after protease degradation in an E. coli crude digest. Wt-Ply511 and the mutants Ply511-L243I-L244I, Ply511-Δ195-262 and Ply511-D222A were expressed in E. coli and incubated for different periods of time at 25 ° C in the E. coli crude digest. The positions of the bands for the non-digested protein are indicated on the right margin (1: Ply511 full-length protein, 2: truncated Ply511-Δ195-262). The numbers on the left are molecular weight measures in kilodaltons. The numbers at the bottom indicate the incubation times in days.

10 shows the result of polypeptide separation in an SDS-polyacrylamide gel after protease degradation in an E. coli crude digest. Wt-Ply511 and the mutants Ply511-S245A, Ply511-K246Q-K248Q and Ply511-S245A-K246Q-K248Q were expressed in E. coli and incubated for different periods at 25 ° C in the E. coli crude digest. The positions of the bands for the undigested protein (-1) and two prominent degradation fragments (-2, -3) are given on the right margin. The numbers on the left are molecular weight measures in kilodaltons.

11 shows the result of polypeptide separation in an SDS-polyacrylamide gel after protease degradation in an E. coli crude digest. Wt-Ply511 and the mutants Ply511-K275A ( 11A ), Ply511-K267Q-K268Q and Ply511-K285Q-K289Q ( 11B ) were expressed in E. coli and incubated for different periods at 25 ° C in E. coli crude digestion. The positions of the bands for the undigested protein (-1) and two prominent degradation fragments (-2, -3) are given on the right margin. The numbers on the left are molecular weight measures in kilodaltons. An oval marks the site where the mutant Ply511-K267Q-K268Q lacks the approximately 28 to 30 kDa degradation intermediate.

Of the Term "protease" as used herein an enzyme capable of peptide binding of proteins and / or To split peptides hydrolytically. The term includes both peptidases, the single amino acids from the amino or carboxy end cleave off, as well as proteinases, inside a protein or cleave polypeptide.

Of the Term "wild type" or "Wt" like used herein refers to the amino acid sequence of the endolysin Ply511 from the phage A511 as shown in SEQ ID NO: 1. Of the Term also denotes the nucleic acid sequence encoding for the amino acid sequence according to SEQ ID NO: 1. The nucleic acid sequence isolated from phage A511 encoding the endolysin Ply511 is listed in SEQ ID NO: 2. The term also includes the nucleic acid sequence one that is different for different amino acids codons as listed in SEQ ID NO: 2, but due to the degenerate code the same amino acid sequence coded.

Of the Term "mutation" as used herein a change in the starting amino acid sequence. It can be single or multiple, directly to each other following or unaltered amino acids broken amino acids removed (deletion), added (Insertion or addition) or replaced by others (substitution) become. The term also includes a combination of single mentioned changes. The term concludes also the N- or C-terminal fusion of a protein or peptide tag one.

Of the Term "modification" as used herein be used synonymously for "mutation" but also includes chemical changes the amino acids such. B. biotinylation, acetylation, chemical change of the amino, SH or carboxyl groups

Of the Term "deletion" as used herein Removing 1, 2 or more amino acids from each Starting sequence. The following are the removed amino acids indicated after the symbol "Δ". For example means "Δ195-262" that the Amino acids from position 195 to including position 262 of the starting sequence were.

Of the Term "insertion" or "addition" like used here means adding 1, 2 or more amino acids to the respective starting sequence.

The term "substitution" as used herein means the replacement of an amino acid present at a particular position by another. In the following, the substitutions are listed as follows: After the exchanged amino acid in the one-letter code, the position of the exchanged amino acid is indicated and then in the one-letter code the new amino acid introduced instead. For example, Y4A means that the amino acid tyrosine at position 4 through the amino acid alanine was exchanged.

The term "domain" or "protein domain" as used herein refers to a portion of an amino acid sequence that has either a particular functional and / or structural property. Domains can often be predicted on the basis of amino acid sequence homologies by means of appropriate computer programs, comparing the amino acid sequences in freely available databases with known domains; z. B. Conserved Domgin Database (CDD) at the NCBI ( Marchler-Bauer et al., 2005, Nucleic Acids Res. 33, D192-6 ), Pfam ( Finn et al., 2006, Nucleic Acids Research 34, D247-D251 ) or SMART ( Schultz et al., 1998, Proc. Natl. Acad. Sci. USA 95, 5857-5864, Letunic et al., 2006, Nucleic Acids Res 34, D257-D260 ).

The term "domain linker" as used herein refers to an amino acid sequence that has the function of linking individual protein domains together. Domain linkers usually do not form any or little regular secondary structure elements, such as α-helix or β-sheet, and can adopt different conformations in their respective structural context. The state of the art describes both properties of linker sequences and methods of finding them ( George & Heringa, 2003, Protein Engineering, 15, 871-879, Bae et al., 2005, Bioinformatics, 21, 2264-2270 The endolysin Ply511 in its wild-type form has a length of 341 amino acids. It has three functional domains, each with homology to other known endolysins. The N-terminal amino acids of positions 12 to 166 represent the enzymatically active domain (FAD) with the function of an N-acetylmuramoyl-L-alanine amidase belonging to the group of amidases 2. The cell-binding domain (CBD) of Ply511 is divided into two parts. A first CBD (CBD1) comprising amino acid positions 198 to 260 is similar to CBD of endolysin Ply118 from Listeriaphagen A118. A further C-terminal CBD (CBD2) comprising amino acid positions 260 to 341 bears similarities to the CBD of endolysin from Bacillus phage Φ105. The individual domains are connected to a domain linker. Domain linkers are found between FAD and CBD1 in the region of amino acids 175 to 203 and between the two CBDs in the region of amino acids 245 to 282.

at recombinant expression of Wt-Ply511 in E. coli out that in addition to the full-length protein also a number arising from different fragments. That is true for yourself Purified Ply511 too if stored for a long time. This loss of stability goes hand in hand with a loss the activity, so that each large amounts of Protein must be used to obtain adequate To reach activity. To stabilize the Wt-Ply511 endolysin, it was investigated if there are specific areas within the Ply511 which are particularly sensitive to proteases. The resulting fragments of Ply511 were analyzed by SDS-polyacrylamide gel electrophoresis separated according to their size and then defined protein bands eluted from the gels. By means of an N-terminal Sequencing of the polypeptide bands were the protease cleavage sites certainly. In addition to those in the E. coli lysate or in the purified Protein occurring fragments in which it is not known which Protease responsible for the degradation were also Protease digests with commercially available proteases (eg, chymotrypsin, subtilisin, trypsin, pepsin, staphylococcus Peptidase I, proteinase K) are known in which is what amino acids they prefer to cut. It turned out that the protease interfaces are not uniform within the endolysin but certain areas were particularly sensitive. The proteins became common in the area of the N-terminus that lies before the start of the EEAS, cut. Several interfaces were also within the EAD and CBD1 and found in the linker between CBD1 and CBD2. numerous Protease cleavage sites exist within the amino acid sequence LLSKIK comprising amino acid positions 243 to 248 and at the C-terminal end of the CBD1 positions the present The invention thus relates to polypeptides which are opposite to the naturally occurring endolysin Ply511 with the Amino acid sequence according to SEQ ID NO: 1 have an altered amino acid sequence. Further The present invention relates to the invention Polypeptides which additionally have modifications. The The present invention further relates to encoding the nucleotide sequences for the polypeptides of the invention. The polypeptides of the invention have the lytic Activity of Wt-Ply511 endolysin on, with activity higher, equal or lower, but not complete is lost. The activity is known to those skilled in the art Assays measured, e.g. B. the plate lysis test or the liquid lysis test.

The changes in the amino acid sequence can deletions, insertions or additions, substitutions or combinations thereof.

The in the amino acid sequence according to SEQ ID NO: 1 of the naturally occurring Ply511 introduced Deletions should preferably have the amino acid sequence so shorten that protease interfaces are removed, without losing the protein's activity.

The Deletions can be one or more amino acids affect. If several amino acids are deleted, then the deleted amino acids follow each other directly. Furthermore, individual deleted amino acids or areas with multiple deleted amino acids one or more non-deleted amino acids from each other be separated. In the starting sequence of Ply511 according to SEQ ID NO: 1 can therefore insert one or more deletions become.

Preferably become deletions in the range of amino acid positions from 186 to 341 of the amino acid sequence according to SEQ ID NO: 1, in particular the amino acid positions 186 to 341, 195 to 255, 195 to 262, 238 to 341, 241 to 341, 267 to 341 and 270 to 341 of the amino acid sequence according to SEQ ID NO: 1 introduced. Particularly preferred are deletions in the amino acid sequence according to SEQ ID NO: 1 C-terminal of position 237, more preferably C-terminal Position 266, where the deleted area is the specified position until the end of the protein, ie until amino acid position 341. Also preferred are deletions which are only part of the C-terminus especially deletions of amino acids 195-262 and 195-255 according to the amino acid sequence SEQ ID NO: 1. These deletion polypeptides are complete soluble expressible and show both in the plate lysis test as well as in the liquid lysis test opposite the Wt-Ply511 one increased activity.

Further the proteins were more stable against protease degradation.

Surprisingly showed that N-terminal deletions in the range of amino acid positions of 1 to 11, especially from 2 to 9 not only the solubility significantly reduce the protein but also the activity the protein was completely lost.

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 996: Listeria monocytogenes (1/2b)
• 1095: Listeria monocytogenes (1/2a)
• 1147: Listeria innocua (6b)
• Löslichkeit: + wie Wt-Ply511, – schlechter als Wt-Ply511
• –/+: kaum Lyseaktivität nachweisbar
• +: Zelllyse deutlich schlechter als beim Wt
• ++: Zelllyse geringfügig schlechter als beim Wt
• +++: Zelllyse vergleichbar dem Wt
• ++++: Zelllyse besser als beim Wt
• 6 × H: N-terminaler His-Tag mit 6 Histidinen

By way of example, preferred polypeptides according to the invention are summarized in Table 1. Table 1 Lysis of bacterial strain mutation solubility 776 996 1095 1147 6xH-Ply511-Δ186-341 ₊ ++ ++ ++ ++ Ply511-Δ270-341 + ++ +++ ++ +++ Ply511-Δ267-341 + +++ ++ ++ +++ Ply511-Δ241-341 + - / + - / + - / + - / + Ply511-Δ238-341 + - / + - / + - / + - / + Ply511-Δ195-262 + ++++ +++ ++ ++++ Ply511-Δ195-255 + ++++ +++ ++ ++++

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 996: Listeria monocytogenes (1 / 2b)
• 1095: Listeria monocytogenes (1 / 2a)
• 1147: Listeria innocua (6b)
• Solubility: + like Wt-Ply511, - worse than Wt-Ply511
• - / +: hardly detectable lysis activity
• +: Cell lysis significantly worse than Wt
• ++: Cell lysis slightly worse than Wt
• +++: cell lysis comparable to Wt
• ++++: cell lysis better than the Wt
• 6 × H: N-terminal His tag with 6 histidines

The in the amino acid sequence according to SEQ ID NO: 1 of the naturally occurring Ply511 introduced Substitutions should preferably be the amino acid sequence change so that protease interfaces are removed, without, however, losing the activity of the protein.

The Substitutions can be one or more amino acids affect. If several amino acids are substituted, so The substituted amino acids can be immediate follow one another. Furthermore, individual substituted Amino acids or regions with multiple substituted amino acids by one or more unsubstituted amino acids be separated from each other. In the starting sequence of Ply511 according to SEQ ID NO: 1 can therefore insert one or more substitutions become.

preferred Substitutions to remove protease cleavage sites are Y4A and T5P. Further preferred substitutions at amino acid deposition 4 are G, T, S, C, I, V, E, Q, D, N, R and K. Further preferred Substitutions of the E7 amino acid to any other Amino acid residues. Especially preferred are the substitutions E7A and E7Q. Also preferred are Ply511 mutants having a Combination of substitutions at the N-terminus and further substitutions, especially Ply511-Y4A-E7Q, Ply-511-T5P-E7A, Ply511-T5P-E7A-Δ195-262, Ply511-T5P-E7A-K246A, Ply511-Y4A-E7Q-K246A, Ply511-Y4A-E7Q-K246H and Ply511-Y4A-E7Q-Δ195-262.

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 1147: Listeria innocua (6b)
• Löslichkeit: + wie Wt-Ply511, – schlechter als Wt-Ply511 keine Lyseaktivität nachweisbar
• +: Zelllyse deutlich schlechter als beim Wt
• ++: Zelllyse geringfügig schlechter als beim Wt
• +++: Zelllyse vergleichbar dem Wt
• ++++: Zelllyse besser als beim Wt

By way of example preferred polypeptides according to the invention are summarized in Table 2. Table 2 Lysis of bacterial strain mutation solubility 776 1147 Ply511-Y4a + +++ +++ Ply511-T5P + +++ +++ Ply511-E7a + +++ +++ Ply511-E7Q + +++ ++++ Ply511-Y4a-E7Q - nd ++ Ply511-Y4a-E7Q-K246A - nd +++ Ply511-Y4a-E7Q-Δ195-262 - nd ++ Ply511-T5P-E7a - (50%). +++ +++ Ply511-T5P-E7a-Δ195-262 - (50%) +++ +++ Ply511-T5P-E7AP5A7A246 + ++ ++ Ply511-T5P-E7a-K246H + + +++ Ply511-MVKYTVENKI (1-10) MASKKTNANK - (50%) - - Ply511-MVKYTVENKI (1-10) MASGGG - (30%) - -

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 1147: Listeria innocua (6b)
• Solubility: + as Wt-Ply511, - no lysing activity detectable worse than Wt-Ply511
• +: Cell lysis significantly worse than Wt
• ++: Cell lysis slightly worse than Wt
• +++: cell lysis comparable to Wt
• ++++: cell lysis better than the Wt

It has been shown that the mutants Ply511-Y4A, Ply511-T5P, Ply511-E7A are equivalent in activity to Wt-Ply511, whereas Ply511-E7Q has even higher activity. Of the multiple mutants, Ply511-T5P-E7A-Δ195-262 and Ply511-T5P-E7A P5 A7 A246 prove to be advantageous, while the other multiple mutants listed in Table 2, while still having enzyme activity, have a lower solubility than Wt-Ply511. Substituting the first 10 amino acids of Ply511 (MVKYTVENKI) for the amino acids MVKYTVENKI (mutant Ply511-MVKYTVENKI (1-10) MASKKTNANK) or the sequence MASGGG (mutant Ply511-MVKYTVENKI (1-10) MASGGG) results in insoluble proteins , which have no activity at all, so that the importance of the N-terminus is underlined again.

Prefers are also substitutions within the range of BAD in the range from amino acid position 12 to 166, especially substitutions of acidic amino acid residues and aromatic amino acid residues. Preferred are substitutions at the amino acid positions 24, 43, 83 and 99 selected from the group G, T, S, C, I, V, E, Q, D, N, R and K.

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 996: Listeria monocytogenes (1/2b)
• 1095: Listeria monocytogenes (1/2a)
• 1147: Listeria innocua (6b)
• Löslichkeit: + wie Wt-Ply511, – schlechter als Wt-Ply511
• n. d.: Versuch nicht durchgeführt
• Keine Zelllyseaktivität
• +: Zelllyse deutlich schlechter als beim Wt
• ++: Zelllyse geringfügig schlechter als beim Wt
• +++: Zelllyse vergleichbar dem Wt
• ++++: Zelllyse besser als beim Wt

Preferred substitutions are summarized in Table 3. Table 3 Lysis of bacterial strain mutation solubility 776 996 1095 1147 Ply511-F24I + ++++ +++ +++ ++++ Ply511-E40A + + ++ + + Ply511-E40Q + + + + + Ply511-Y43A + +++ +++ ++ +++ Ply511-Y43S + +++ +++ ++ +++ Ply511-E40Q-Y43S + - + - + Ply511-F24I-Δ195-262 + ++++ ++++ +++ ++++ Ply511-E40A-Δ195-262 + ++ ++ + + Ply511-E40Q-Δ195-262 + ++ ++ + + Ply511-Y43A-Δ195-262 + +++ ++++ ++ +++ Ply511-Y43S-Δ195-262 + +++ +++ ++ +++ Ply511-E40Q-Y43S-Δ195-262 + - + + + Ply511-Y83I + nd nd nd ++++ Ply511-E89A + nd nd nd - Ply511-E89Q + nd nd nd - Ply511-F99A - nd nd nd +++

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 996: Listeria monocytogenes (1 / 2b)
• 1095: Listeria monocytogenes (1 / 2a)
• 1147: Listeria innocua (6b)
• Solubility: + like Wt-Ply511, - worse than Wt-Ply511
• nd: try not done
• No cell lysis activity
• +: Cell lysis significantly worse than Wt
• ++: Cell lysis slightly worse than Wt
• +++: cell lysis comparable to Wt
• ++++: cell lysis better than the Wt

It shows that substitutions at positions 24, 43 and 83 while substitutions to the Positions 40 and in particular 89 the activity negative influence. The mutants Ply511-Y43A, Ply511-Y43S are in their Activity equaled the Wt-Ply511, while the mutants Ply511-F24I and Ply511-Y83I are even more active than the Wt are. This also applies to combinations of these substitutions, especially in connection with the deletion Δ195-262. The mutant Ply511-F24I-Δ195-262 proved to be especially advantageous. The mutant Ply511-F99A remains after removal Although the protease interface receives the activity, however, the solubility of the protein is increasing from the Wt.

When Mutation that increases the protease stability can they are still used, if a little less Solubility is accepted. Negative on the enzyme activity Substitutions affect positions 40 and 89, in particular the mutants Ply511-E40A, Ply511-E40Q, Ply511-E89A, Ply511-E89Q have little or no activity. this applies even for such combinations of mutations in which the incoming muzzations have a positive effect on the function and stability of Ply511, e.g. Ply511-E40Q-Y43S, Ply511-E40A-Δ195-262, Ply511-E40Q-Δ195-262 and Ply511-E40Q-Y43S-Δ195-262.

Prefers Furthermore, substitutions within the CBD1 are in the range of amino acid positions 198 to 260, in particular substitutions of the aromatic amino acid residues at positions 208, 218 and 228. Preferred substitutions amino acid positions 208, 218 and 228 are A, V, I, L and M. Also preferred is the substitution at position 222 of D to A.

• 1147: Listeria innocua (6b)
• Löslichkeit: + wie Wt-Ply511, – schlechter als Wt-Ply511
• +: Zelllyse deutlich schlechter als beim Wt
• +++: Zelllyse vergleichbar dem Wt

Preferred substitutions are summarized in Table 4. Table 4 mutation solubility Lysis of bacterial strain 1147 Ply511-Y218V + +++ Ply511-D222A + +++ Ply511-Y228I + +++ Ply511-Y233I + + Ply511-Y233M + +

• 1147: Listeria innocua (6b)
• Solubility: + like Wt-Ply511, - worse than Wt-Ply511
• +: Cell lysis significantly worse than Wt
• +++: cell lysis comparable to Wt

While for substitutions at positions 218 and 228, in particular at the mutants Ply511-Y218V and Ply511-Y228I the activity is maintained and the stability increases, goes with substitutions at position 233, in particular at the mutants Ply511-Y233I and Ply511-Y233M significantly slowed down the activity and There is even a greater degradation in E. coli lysate than with the original amino acid Y. In the substitution D222A are expression rate, solubility and activity comparable to the Wt-Ply511, however, showed Surprisingly, a significantly increased Thermostability of the mutant Ply511-D222A the wild type and increased protease stability in trypsin digestion and in E. coli digestion.

Prefers are also substitutions in the range of amino acid positions from 243 to 248, which have the amino acid sequence LLSKIK as well as at the amino acid positions N-terminal or C-terminal following this sequence, especially the area of the amino acids 240 to 249. Here, single as well as multiple mutations be introduced.

Preferred substitutions are summarized in Table 5. Table 5 Lysis of bacterial strain mutation solubility 776 996 1095 1147 Ply511-K246A + ++ +++ +++ +++ Ply511-K246H - (70%) ++ +++ +++ +++ Ply511-I247P - (80%) + + + + Ply511-L243I-L244I + nd nd nd ++ Ply511-L243I-L244I-K246A + nd nd nd +++ Ply511-L243I-L244I-K246A-K248A - nd nd nd + Ply511-D222A-L243I-L244I-K246A-K248A + nd nd nd + Ply511-K248A + nd nd nd +++ Ply511-K246Q + nd nd nd +++ Ply511-K248Q + nd nd nd +++ Ply511-K246Q-K248Q + +++ +++ +++ +++ Ply511-L243I-L244I-K246Q + nd nd nd + Ply511-L243I-L244I-K248Q + nd nd nd + Ply511-L243I-L244I-K246Q-K248Q + nd nd nd + Ply511-D222A-L243I-L244I-K246Q-K248Q + nd nd nd + Ply511-D222A-K246Q-K248Q + nd nd nd +++ Ply511-T241S-T242S + nd nd nd +++ Ply511-D222A-T241S-T242S + nd nd nd +++ Ply511-T241S-T242S-K246Q-K248Q + nd nd nd ++++ Ply511-N240Q + nd nd nd ++ Ply511-D222A-N240Q + nd nd nd ++ Ply511-K246Q-K248Q-N240Q + nd nd nd ++ Ply511-S245A + nd nd nd +++ Ply511-D222A-S245A + nd nd nd +++ Ply511-S245A-K246Q-K248Q + nd nd nd ++ Ply511-G249A + nd nd nd ++ Ply511-D222A-G249A + nd nd nd ++ Ply511-K246Q-K248Q-G249A + nd nd nd + Ply511-D222A-K246Q-K248Q-G249A + nd nd nd + Ply511-N240A + nd nd nd + Ply511-T241A + nd nd nd +++ Ply511-T242A + nd nd nd +++ Ply511-L243A + nd nd nd +

• 776: Listeria monocytogenes Scott A (Serovar 4b)
• 996: Listeria monocytogenes (1 / 2b)
• 1095: Listeria monocytogenes (1 / 2a)
• 1147: Listeria innocua (6b)
• Solubility: + like Wt-Ply511, - worse as Wt-Ply511
• n. d .: attempt not performed
• +: Cell lysis significantly worse than Wt
• ++: cell lysis a little worse than at Wt
• +++: cell lysis comparable to Wt
• ++++: cell lysis better than the Wt

A Range of substitutions are suitable, the protease stability to improve and yet the enzyme activity of Wt-Ply511 to obtain. In particular, these are the mutants Ply511-K246A, Ply511-K246H, Ply511-S245A, Ply511-T241A, Ply511-T242A and the double mutant Ply511-T241S-T242S. Also in combination with other mutations these substitutions show positive effects. The double mutation T241S-T242S works also in combination with the mutations D222A and K246Q-K248Q slightly stabilizing and positive for the activity. The Mutation K246A is suitable for the negative effect of the double mutation L243I-L244I mitigate, so the activity in the triple mutant Ply511-L243I-L244I-K246A again the level of Wt protein reached. The mutation S245A is already taken in itself suitable, the stability of Ply511 in E. coli crude digestion increase and have a positive effect on the destabilizing Double mutation K246Q-K248Q effect.

The Double mutant Ply511-D222A-S245A exhibits even at a longer Incubation period in trypsin digestion and in E. coli digestion the Wt protein a significantly higher protease stability on. Nevertheless, the mutant Ply511-S245A is in the thermal stability test destabilized against the wild type and only the Combination of the mutations D222A and S245A leads to the thermostability test to a protein with the same stability as Wt-Ply511. For the mutants Ply511-K248A, Ply511-K246Q and Ply511-K248Q as well the double mutant Ply511-K246Q-K248Q remains the enzyme activity and protein solubility, protease stability however, is reduced by these mutations.

It also affect several substitutions in this sequence area negative for enzyme activity and / or protease stability out. These are in particular the mutations Ply511-I247P, Ply511-G249A, Ply511-N240Q, Ply511-N240A and Ply511-L243A and the double mutation Ply511-L243I-L244I. Although the mutant Ply511-G249A shows a reduced thermostability and protease stability during purification, however, the enzyme activity is both in the plate lysis test as well as in the liquid lysis test only slightly above the Wild type decreased. The double mutation Ply511-L243I-L244I also has negative effects in all combinations with other mutations on protease stability and enzyme activity out. This also applies to combinations with the mutations N240Q and G249A.

• 1147: Listeria innocua (6b)
• Löslichkeit: + wie Wt-Ply511, – schlechter als Wt-Ply511
• +++: Zelllyse vergleichbar dem Wt
• ++: Zelllyse schlechter als beim Wt

Also preferred are substitutions within CBD2 in the range of amino acid positions 260 to 341. Further preferred are substitutions in the mutant Ply511-Δ195-262 at amino acid position 278. Further preferred substitutions are summarized in Table 6. Table 6 Lysis of bacterial strain mutation solubility 1147 Ply511_W278I + +++ Ply511-K46A-W278I + +++ Ply511-Δ195-262-W278I + ++ Ply511-I247P-W278I + - Ply511-K267Q-K268M + +++ Ply511-K275A + +++ Ply511-K285Q + +++ Ply511-K289Q + +++ Ply511-K285Q-K289Q + +++

• 1147: Listeria innocua (6b)
• Solubility: + like Wt-Ply511, - worse than Wt-Ply511
• +++: cell lysis comparable to Wt
• ++: Cell lysis worse than Wt

at the mutant Ply511-W278I shows that although the enzyme activity retained at the level of the Wt protein, but the protease sensitivity in E. coli digestion is significantly worse. this applies also for a combination of the mutation with further mutations. The mutations Ply511-K267Q-K268M, Ply511-K275A, Ply511-K285Q, Ply511-K289Q, in particular the double mutant Ply511-K267Q-K268M prove to be suitable for the protease stability of the Ply511 to increase endolysins, at the same time a the Wt protein comparable enzyme activity is maintained. Especially the mutations at positions K267 and K268 against others Amino acids except R, especially in the mutant Ply511-K267Q-K268M prove to be suitable, protease-sensitive areas within to stabilize the CBD2.

mutations which increase the protease stability of Ply511, are also suitable for the stability of fragments of the endolysin Ply511 such as the EAD, the CBD1, CBD2 or a combination from CBD1 and CBD2 increase. The amino acid region, which includes the BAD is here 1 to 166, for the CBD1 the range 198 to 260 and for the CBD2 260 to 341 accordingly the sequence according to SEQ ID NO: 1. The entire CBD thus covers the range starting from amino acid 160. The domains can also be shortened even further N- or C-terminal as long as they continue to have activity. For Areas that comprise the BAD is the lysis of Listeria cells (Plate lysis test or liquid lysis test), for areas, which only comprise the CBD, this is no longer the lysis of Listeria cells, but only their binding, since the CBD no amidase activity having. All the mutations described above, which are positive affect the protease stability and within the are also suitable, to stabilize corresponding fragments of the endolysin Ply511.

modifications such as N- or C-terminal tags or chemical modifications of individual Amino acids can be added to the preparation of the protein (eg, his-tag or strep-tag for easier Cleaning), to improve its application (eg Strep-Tag, Avi-Tag, JS tag or chemical biotinylation for immobilization on surfaces, having streptavidin or avidin) or solubility or increase stability (eg, PEGylation).

Preferably the invention further relates to the nucleic acid molecules, encoding the described invention modified polypeptides. Furthermore, the present invention relates Vectors comprising the Nukeinsäuremoleküle invention and suitable host cells for expression of the invention Polypeptides.

The modified Ply511 endolysins according to the invention all have a lysing activity, which also the naturally occurring Ply511 has. In addition, the modifications described above have positive effects that are advantageous for a commercial application of endolysins. These positive effects may be increased protease stability, thermostability or stability to chemical denaturants. In addition, stabilization can lead to a higher expression rate, solubility or longer storage life. The positive effect can also be expressed in an increased activity.

A increased protease stability is already important in the recombinant production of the protein. This is how larger ones can be Amounts of Ply511 due to the onset of production Protease degradation is extremely time-consuming produce. You could get larger quantities added to protease inhibitors, but this is expensive and a Variety of additives in the endolysin preparation would bring with it. Furthermore, one could mined Full-length protein protein with sophisticated chromatographic techniques which is difficult because part of the fragments of the decomposition, which are only a few kilodaltons smaller than the full-length protein, so that the Abbaufragmente cleaning technically similar Have properties like the native protein. An increased Protease stability is also important in storage of the isolated Ply511. Desired is the protease stability even with the use of Ply511 in foods containing a variety of Contain proteases. An improved protease stability increases the duration of the effectiveness of the added inventive modified Ply511.

A increased thermal stability also proves as advantageous. In food technology often become higher Temperatures used such. B. in the cheese or yogurt production. Ply511 endolysin can only be used antimicrobially for the lysis of listeria be used if it is still active at appropriate temperatures is. An increased thermal stability proves also in the recombinant production of the invention Polypeptides as advantageous. Proteins that are poorly soluble or are unstable, often at lower Temperatures (eg 25 ° C or 30 ° C) expressed so that the expression product is soluble at all. Expression at higher temperatures (eg 37 ° C) However, it offers economic benefits because protein production goes faster at these temperatures and higher Cell densities are achieved so that more protein is produced can.

proteins, the increased thermal stability, protease stability or also stability to chemical dentining agents usually show up during storage over stable for a longer period of time. This proves both for the manufacturer as well as for the user as cost-effective, as it holds larger quantities can. High solubility of the protein is important to doing so the endolysin are produced efficiently and inexpensively can. Insoluble protein is usually denatured and no longer has its native conformation and therefore also not his full activity anymore. Is the expression product insoluble, you can try to refold it, so that it regains its native form and activity. However, this is technically complex, expensive and many Proteins in the yield of native protein that is obtained not very efficient, so prefers well-soluble proteins are expressed. Higher activity brings economic Benefits, as used in the application correspondingly less enzyme must become.

The The present invention further relates to the use of the invention Proteins as human and veterinary substance, in food, in cosmetics, as a disinfectant.

The following examples illustrate the invention and are not to be construed as limiting. Unless otherwise stated, standard molecular biological methods were used, such. B. from Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York , described.

Example 1. Assay for Expression and Solubility

To be examined E. coli clones containing plasmids for Ply511 endolysins were incubated in 1 ml LB cultures at 30 ° C with shaking until turbidity was evident. The induction of the cultures except the negative control was done with 1 mM IPTG. After incubation for 3-4 h at 30 ° C, the cells were harvested in the bench centrifuge (13,000 rpm for 10 min at 4 ° C). For expression assays, the pellet was boiled in 100 μl of 1X SDS sample buffer (95 ° C for 5 min) and analyzed on SDS gels. For solubility testing, the pellet was resuspended in cell disruption buffer (25mM Tris, 250mM NaCl, pH 7.5) and disrupted via an ultrasound probe (20s). After separation of the insoluble proteins by centrifugation (13000 rpm for 10 min at 4 ° C) aliquots of supernatant (soluble protein) and pellet (insoluble protein) were added to sample buffer and boiled (5 min at 95 ° C). In both cases, the samples were analyzed by SDS gels electrophoresis and subsequent Coomassie staining of the gels.

Example 2. Purification of the modified Endolysins Ply511 and the naturally occurring Ply511.

Purification of Ply511 proteins was from cells from induced E. coli cultures (30 ° C, 1 mM IPTG). The cell pellets were digested in the loading buffer A1 (25mM Tris, 250mM NaCl, 1mM MgCl ₂ , pH 8.0) with a high pressure homogenizer (Microfluidizer). After centrifugation, the supernatant was prepurified on a Streamline Direct HST column (cation exchanger, GE Healthcare). To this was washed 10 volumes of buffer A1 and 10 volumes of buffer A2 (25 mM borate, 250 mM NaCl, pH 9.0), then eluted with buffer B1 (25 mM borate, 500 mM NaCl, pH 10.0). A second purification step was carried out over phenylsepharose HP. Buffer B4 (25 mM NaBorate, 1.1 M ammonium sulfate, pH 8.0) was added, eluting with buffer A5 (25 mM NaBorate pH 8.0) with the Ply511 derivatives in the flow-through fractions. For removal of salts, dialysis was then carried out with buffer changes twice in a period of about 18 h against 40 mM Tris, 100 mM NaCl, pH 8.0 at 4 ° C.

Example 3. Analysis of degradants after Storage of Ply511

purified Ply511 was stored for 2 days at 4 ° C in storage buffer (20 mM Tris, 500 mM NaCl, pH 8.0) and then in comparison analyzed for freshly purified Ply511 on SDS gels. It showed itself, that during storage a prominent Abbaubande of about 26 kDa size and next smaller ones Degraded, so the protein degraded by a protease was stored stable and not for long periods could be. Protein preparations with protease degradation point a lower activity than the full-length protein.

Example 4. Determination of protease-sensitive Areas within the Ply511 sequence.

Around Find out which areas of the endolysin Ply511 especially Protease digestion experiments were carried out with different commercially available proteases (eg, chymotrypsin, Trypsin, pepsin, subtilisin, staphylococcal peptidase I, proteinase K, thermolysin). Protease incubation of the Ply511 each carried out at room temperature or at 37 ° C for different time intervals (minutes to several hours) in different buffers specified by the manufacturers. The resulting protease fragments were separated on SDS gels. The resulting protein bands were blotted on PVDF (polyvinylidene fluoride) membranes, good separable bands were excised and N-terminally sequenced. In addition to the commercially available proteases were also the resulting fragments from the E. coli digestion sequenced. As often fragments with very similar Size developed, but not all fragments were sequenced and also other proteases with different Specificities exist, it can be assumed that in addition to the indicated positions also nearby amino acids lie in protease-sensitive areas.

Example 5. Plate lysis test for examination of activity

to Preparation of lysis plates were heat-inactivated cells (20 min at 80 ° C) from L. monocytogenes Scott A (Serovar 4b, Strain No. 776), L. monocytogenes (Serovar 1 / 2b, strain No. 996 or Serovar 1 / 2a, strain no. 1095) or Listeria innocua (serovar 6b, Strain No. 1147) or other Listeria strains in LB-TopAgar poured, so that a dense, turbid cell lawn was created. If the lysis activity of transformed E. coli clones should be tested, was included in LB-TopAgar IPTG and ampicillin. To test for lysis activity were subsequently either with plasmids for modified Ply511 variants transformed E. coli clones, cell disruptions induced Spotted E. coli clones or purified protein solutions (about 5 μl solution, in E. coli cloning Impföse from single colony) and plates at 30 ° C Incubated overnight. If the Ply511 endolysins have lysing activity had formed around the spots where protein spotted Lysies were made out as holes in the otherwise dense bacterial lawns were recognizable. The size The lysis court is a measure of the activity the proteins. The activity is given in comparison to the activity of the Wt protein. All activity data from the given tables were using the plate lysis test determined and the symbols (+++, ++, +, +/-) from the size the lysis farms compared to the wild-type Ply511 determined.

Example 6. Liquid Lysis Test for Investigation of the activity

For each liquid lysis batch, 1 ml of heat inactivated cells (20 min at 80 ° C) from a bacterial culture incubated to an OD ₆₀₀ of 1.0 +/- 0.1 were incubated by L. monocytogenes Scott A (Serovar 4b), L. monocytogenes (Serovar 1 / 2b or 1 / 2a) or Listeria innocua (Serovar 6b) or other Listeria strains in PEST (20 mM sodium phosphate, 120 mM sodium chloride, 0.5% Tween, pH 8.0) used in cuvettes. After adding endolysin (protein concentrations between 0.1 μg / ml and 10 μg / ml), the decrease in OD _{600 was} measured as a function of time at 30 ° C. The respective cell suspension served as a control without addition of the endolysin. The activity was calculated as the decrease in absorbance at 600 nm per minute (ΔA / min) based on the amount of protein in μmol (ΔA μmol / min). The activities of the modified endolysins were measured in each case in comparison to Wt-Ply511. In a comparative lysis test with Wt-Ply511 and Ply511-K246Q-K248Q with Listeri strain 996 (Serovar 1 / 2b), 0.1 μg, 0.3 μg or 0.7 μg endolysin were added in each case. As the amount of protein increased, listeria lysis became faster, but both Wt-Ply511 and the mutant Ply511-K246Q-K248Q showed approximately the same lysis activity at all three protein concentrations. In further liquid lysis tests, comparative lysis data between Wt-Ply511 and mutants with the Listeri strain 776 (Serovar 4b) were determined. Wt-Ply511 and the mutant Ply511-G249A (10 μg / ml) also showed a very similar lysis activity, whereas the mutant Ply511-Δ195-262 shows even faster lysis compared to Wt-Ply511 (concentration 0.3 μg / ml) ,

Example 7. Protease digestion in E. coli Information on the test of protease stability

induced 1 ml cultures of E. coli were incubated after 3-4 h harvested at 30 ° C (13 000 rpm, 10 min, 4 ° C). After that the pellet was resuspended in cell disruption buffer (25 mM Tris, 250 mM NaCl, pH 7.5) and via ultrasound probe open minded (20 s). After separation of insoluble constituents and non-disrupted cells by centrifugation (4 ° C, 13000 rpm, 10 min), the cell disruption supernatant was at room temperature or incubated at 37 ° C. Sampling took place in each case At the beginning of the incubation period (t = 0), then at room temperature 24 h (t = 1, 2 or 3 days), for samples incubated at 37 ° C within 24 hours (eg t = 1, 16, 21 h). After boiling in SDS sample buffer (5 min, 95 ° C) became the batches analyzed on SDS gels. While in wild-type Ply511 at 25 ° C a significant protein degradation by E. coli own proteases took place and after 2 days hardly more full length protein available was mutant Ply511-D222A and mutant Ply511-Δ195-262 the degradation is significantly delayed, so after 2 days of incubation still full-length protein was present and the 2nd Abbaubande With smaller molecular weight within this time did not occur. The double mutant Ply511-L243I-L244I, however, was clear destabilized, so that already after 2 days of incubation at 25 ° C only protein with the molecular weight of the smaller Abbaubande (smaller 25 kDa molecular weight) was present. Another protease digestion in E. coli was incubated for up to 3 days at 25 ° C. While in the wild-type protein the band of the full-length protein (Bande 1) is decreasing and a degradation band (band 2) is increasing in the mutant Ply511-S245A significantly more of the full-length protein available. The double mutation K246Q-K248Q destabilizes the protein, however, so that after 3 days virtually no full-length protein more is present, but a degradative with a shorter molecular weight (Volume 3) is added. In combination with the double mutation K246Q-K248Q The mutation S245A in turn stabilizes, so that in the Triple mutant Ply511-S235A-K246Q-K248Q by the end of the Experiment full length protein is present and at the same time Abbaubande 3 is less populated. It turned out that within the CBD2 a trypsin-sensitive interface was present, leading to a Abbaufragment with about 28 to 30 kDa size led. Various mutations were used to try this protease cleavage site to find and stabilize. Wt-Ply511 and the mutants Ply511-K275A, Ply511-K267Q-K268M and Ply511-K285Q-K289Q were for 1, 16 or 21 hours at 37 ° C in E. coli Incubated and showed all protease stabilities, which were at least equal to the Wt-Ply511. It showed However, that along with the potential trypsin interface at the mutant Ply511-K267Q-K268M also has a general protease interface was removed because this mutant, the degradation intermediate of about 28-30 kDa size (band - 2) no longer occurred.

Example 8: Trypsin Digest for Testing Protease stability.

Wt-Ply511 (SEQ ID NO: 1) and the mutants tested (Ply511-T241S-T242S, Ply511-S245A, and Ply511-D222A-S245A) were purified as described in Example 2. Before protease digestion, they were dialysed 2 times for a total of ca. 18 h against 25 mM Na phosphate, 100 mM NaCl, pH 8.0. The dialysis buffer was also used for trypsin digestion. In each case 30 μg of endolysin were used in 150 μl sample volume. 2.5 μl of a trypsin stock solution (1 mg / ml in 25 mM Na phosphate, 100 mM NaCl, pH 8.0) was used in the digestion and incubated for 1 min, 2 min, 5 min, 13 min, 25 min and Digested for 35 min at room temperature. Samples were taken at the indicated times, SDS sample buffer added, and then all samples were analyzed on a 12% SDS gel. The control was a sample which was not incubated with trypsin. While the double mutant Ply511-T241S-T242S is degraded with similar kinetics as the Wt protein, the two mutants Ply511-S245A and in particular Ply511-D222A-S245A are stabilized against protease degradation. The kinetics of the degradation takes place much slower. There are mainly 2 defined degradation bands with a molecular weight of about 29 kDa and 26 kDa. Note that the Pro tesa stability to trypsin in the described mutants has increased, although no amino acids have been exchanged which are direct interfaces for trypsin (lysine and arginine). This means that the mutations described generally stabilize the protein against proteases, and not only in a narrower sense, that certain cleavage sites are removed from certain, sequentially defined proteases.

Example 9. Chymotrypsin Digest to Test on protease stability.

Wt-Ply511 and the mutants Ply511-D222A-S245A and Ply511-K246Q-K248Q were purified as described in Example 2. Before the protease digestion they were twice 2 times for a total of about 18 h against 25 mM Na phosphate, Dialyzed 100 mM NaCl, pH 8.0. The dialysis buffer was also used for used the Trypsinverdau. 24 μg Ply511 were used with each 3 μg chymotrypsin for 1 min, 2 min or 5 min incubated at room temperature in 150 ul sample volume, too mixed with SDS sample buffer at the indicated times and then analyzed on 12% SDS gels. As a control served a sample, which was not incubated with chymotrypsin. While the Double mutant Ply511-K246Q-K248Q a little faster than that Wt protein is degraded, the mutant Ply511-D222A-S245A is against Protease degradation stabilized significantly.

Example 10. Thermal stability test for protein aggregation

For the thermal stability test was 100 μg of the respective Proteins in 25 mM Na-phosphate, 100 mM NaCl, pH 8.0 in a stirrable Quartz cuvette (volume 1 ml) used. The increase in optical density (light scattering caused by aggregation of the protein occurs) while heating from 20 to 90 ° C (Heating rate 1 ° C / min) was in the photometer at one wavelength followed by 360 nm. In an exemplary experiment Wt-Ply511 and the mutants Ply511-G249A, Ply511-S245A, Ply511-D222A-S245A and Ply511-D222A in the photometer heated and the increase in protein aggregation tracked as a function of the temperature. It appears, that Wt-Ply511 aggregates at about 65 ° C while the destabilizing mutations G249A and S245A in the mutants Ply511-G249A and Ply-S245A already aggregate at something 60 ° C lead. The mutant Ply511-D222A, on the other hand is significantly more thermostable and aggregates only at about 75 ° C. Combining the mutations S245A and D222A, the double mutant exhibits Ply511-D222A-S245A has a thermostability opposite to that Wt is slightly increased, so that the effects of single mutations behave additively.

Example 11. Thermal stability test to protein activity

Around to test for a potentially higher thermal stability on the protein activity, were different Ply511 variants (protein concentration 0.3 mg / ml) in each case 20 min at elevated temperature in buffer with 40 mM Tris, 100 mM NaCl, pH 8.0 and then the residual activity in the liquid lysis test (see Example 6). It was the decrease in absorbance at 600 nm per minute (ΔA / min) in the beginning of the lysis curve as a measure of the Activity taken. Wt-Ply511 and the mutant Ply511-G249A (concentration 3 μg / ml each) were added to PEST for 20 min Incubated at 50 ° C while controls at 4 ° C were stored. After this time, the residual activity became measured in the liquid lysis test at room temperature. It showed itself, that Wt-Ply511 under these conditions 98% of the activity while the mutant Ply511-G249A retained only 15% residual activity had.

Example 12. Stability versus chemical denaturants.

Proteins in their native form have characteristic fluorescence emission spectra. Denaturation in chemical denaturants such as guanidinium chloride or urea changes both the position of the emission maximum and the level of the fluorescence signal. At the wavelength at which the largest signal change occurs between native and denatured protein, the protein fluorescence is measured as a function of the addition of denaturing agent in order to arrive at a statement about the stability of a protein. The higher (in M denaturants) the center of the denaturation transition of a protein, the more stable the protein. For the modified Ply511 endolysins, the stability is compared with that of the Wt protein. GdmCl stock solutions are prepared in water between 0 and 8 M in 0.5 M steps and the denaturant concentration subsequently checked in the refractometer. A protein stock solution is prepared at 100 μg / ml in 4X concentrated PBS buffer (PBS: 20 mM sodium phosphate, 120 mM sodium chloride, pH 8.0). Both GdmCl stock solutions and the PBS buffer are sterile filtered. Each 0.75 ml of the stock protein solution is mixed with 2.25 ml of the various GdmCl stock solutions and the samples are incubated at 25 ° C. For fluorescence measurement, 0.75 ml each are taken from the samples and the fluorescence signal is measured. This will be for each measurement Point control values for the buffer are subtracted with the corresponding GdmCl concentration without addition of protein. The measurement is repeated after about 7 days in order to check whether an equilibrium state for the protein denaturation has already set in and if necessary to repeat it again later. The corrected fluorescence values are then plotted against denaturant concentrations and the denaturant center determined.

Example 13. Cell Binding Test for non-enzymatically active variants of Ply511, in particular fragments, contain the CBDs.

Ply511 CBD Fragments are tagged with N- or C-terminal tags such as His-tag or Strep tag fused and heterologously expressed in E. coli. For fluorescence detection The bound CBDs may still be between day and Ply511 CBD sequence GFP be fused as a marker. The proteins are using of the tag via affinity chromatography according to data the manufacturer cleaned. 50 μl of a preculture of L. monocytogenes Scott A (Serovar 4b), L. monocytogenes (Serovar 1 / 2b or 1 / 2a) or Listeria innocua (Serovar 6b) or others Listeria strains are treated with about 2 μg of the purified Protein and incubated for 10 min at room temperature. After addition of 1 ml of PEST (10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20, pH 8.0), the cells are centrifuged off, 2 × in 0.5 ml and resuspended in 50 ml PEST. The binding of Ply511 CBDs Listeria cells are monitored under the fluorescence microscope. It is also possible for Ply511 CBD fusions with Strep-Tag To use magnetic particles that streptavidin on the surface or avidin. Strep-tag Ply511-CBDs bound to bacteria in this case, will be with the corresponding Magnetbeads incubated and then the complexes of Listeria cells and Ply511CBD separated from the sample using a magnetic separator. The detection of the Listeria is then carried out using conventional methods (eg PCR, microbial detection techniques).

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• EP 0781349 [0006]

Cited non-patent literature

• - Wing EJ & Gregory SH, 2002, Listeria monocytogenes: Clinical and Experimental Update, J Infect Disease 185 (Suppl 1): S18-S24 [0003]
• - Doyle ME, 2001, Virulence Characteristics of Listeria monocytogenes, Food Research Institute, October 2001 [0003]
• - 2007, Syst. And appl. Microbiol., 30, 58-67 [0006]
• Marchler-Bauer et al., 2005, Nucleic Acids Res. 33, D192-6 [0028]
• -. Finn et al, 2006, Nucleic Acids Research 34, D247-D251 [0028]
• Schultz et al., 1998, Proc. Natl. Acad. Sci. USA 95, 5857-5864, Letunic et al., 2006, Nucleic Acids Res 34, D257-D260 [0028]
• - George & Heringa, 2003, Protein Engineering, 15, 871-879, Bae et al., 2005, Bioinformatics, 21, 2264-2270 [0029]
• Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [0065]

Claims (14)

A polypeptide having one at least one amino acid position opposite to the amino acid sequence according to SEQ ID NO: 1 altered amino acid sequence. The polypeptide of claim 1, wherein the altered Amino acid sequence is a deletion, addition and / or substitution is. A polypeptide according to claim 1 or 2, wherein the altered Amino acid sequence on immediately consecutive amino acids or at amino acid positions by one or several unaltered amino acids are separated. Polypeptide according to one of claims 1 to 3 with deletions in the range of amino acid positions of 186 to 341 of the amino acid sequence according to SEQ ID NO: 1. A polypeptide according to claim 4 having deletions of amino acid positions 186 to 341, 195 to 255, 195 to 262, 238 to 341, 241 to 341, 267 to 341 and 270 to 341 of the amino acid sequence according to SEQ ID NO: 1. Polypeptide according to one of claims 1 to 3 with one or more substitutions at the amino acid positions 4, 5, 7, 24, 43, 46, 83, 99, 208, 218, 222, 228, 246, 249, 267, 268, 275, 278, 285 or 289, or in the range of amino acid positions from 12 to 166, 198 to 260, 240 to 249, 260 to 341. The polypeptide of claim 6, wherein the substitution at amino acid positions 4, 24, 43, 83 and 99 G, T, S, C, I, V, E, Q, D, N, R or K, at amino acid position 249A and at amino acid positions 208, 218 and 228A, V, I, L or M is. The polypeptide of claim 6, wherein the substitution at amino acid position 4A, at amino acid position 5P, at amino acid position 7A or Q, at amino acid deposition 24I, at amino acid position 43A or S, at amino acid deposition 83I, at amino acid position 99A, at amino acid deposition 222A, at amino acid position 246A or H, at amino acid position 245A, at amino acid position 241A, at amino acid deposition 242A, at amino acid position 248A or Q, at amino acid deposition 246Q, at amino acid position 275A, at amino acid deposition 278I, at amino acid position 285Q, at amino acid position 289Q, at amino acid position 267 and 268 is not R. Polypeptide according to one of claims 6 to 8, wherein the polypeptide substitutions at the amino acid position Y4A and E7Q, or T5P and E7A, or T5, E7A and K246A, or Y4A, E7Q and K246A, or Y4A, E7Q and K246H, or D222A and S245A, or T241S and T242S, or T241S, T242S and D222A, or T241S, T242S, K246Q and K248Q, or L243I, L244I and K246A, or S245A, K246Q and K248Q, or K246Q and K248Q, or K267Q and K268M, or K46A and W278I. Polypeptide according to one of claims 1 to 3, wherein the polypeptide deletions and substitutions at the amino acid positions T5P, E7A and Δ195-262, or Y4A, E7Q and Δ195-262, or F24I and Δ195-262, or W278I and Δ195-262 having. Nucleotide sequence encoding a polypeptide according to one of claims 1 to 10. A vector comprising a nucleotide sequence according to claim 11th A host cell comprising a nucleotide sequence Claim 11 or a vector according to claim 12. Use of a polypeptide according to any one of the claims 1 to 10 as a human or veterinary substance, in food, in cosmetics, as a disinfectant.