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    • 专利摘要:
    The present invention relates to pharmaceuticals and modified beta-lactamases. specifically, the invention relates to novel recombinant beta-lactamases and pharmaceutical compositions comprising the beta-lactamases. likewise, the present invention relates to methods for modifying a beta-lactamase, producing the beta-lactamase and treating or preventing adverse effects induced by the beta-lactam antibiotic. further, the present invention relates to beta-lactamase for use as a medicine and the use of beta-lactamase in the manufacture of a medicine to treat or prevent adverse effects induced by beta-lactam antibiotics. still further, the invention relates to a polynucleotide and a host cell that comprises the polynucleotide.
    公开号:BR112012030029B1
    申请号:R112012030029-6
    申请日:2011-05-17
    公开日:2021-08-17
    发明作者:Pertti Koski；Ulla Airaksinen；Katja Välimäki
    申请人:Prevabr Llc；
    IPC主号:
    专利说明:

    field of invention
    The present invention relates to pharmaceuticals and modified beta-lactamases. Specifically, the invention relates to novel recombinant beta-lactamases and pharmaceutical compositions comprising the beta-lactamases.
    Likewise, the present invention relates to methods for modifying a beta-lactamase, producing the beta-lactamase and treating or preventing adverse effects induced by the beta-lactam antibiotic. Furthermore, the present invention relates to beta-lactamase for use as a medicine and the use of beta-lactamase in the manufacture of a medicine to treat or prevent adverse effects induced by beta-lactam antibiotics.
    Still further, the invention relates to a polynucleotide and a host cell that comprises the polynucleotide. Background of the invention
    Beta-lactam antibiotics are characterized by a beta-lactam ring in their molecular structures. The integrity of the beta-lactam ring is essential for biological activity, which results in the inactivation of a set of transpeptidases that catalyze the end-link reactions of peptidoglycan synthesis. Members of beta-lactam antibiotics include penicillins, cephalosporins, clavams (or oxapenams), cephamycins, and carbapenems.
    Beta-lactamases are bacterial defensive enzymes that hydrolyze beta-lactam antibiotics. The production of beta-lactamases is a predominant mechanism for conferring resistance to beta-lactams in Gram-negative bacteria. Beta-lactamases very efficiently catalyze the irreversible hydrolysis of the amide bond of the beta-lactam ring, resulting in biologically inactive product(s).
    Due to the diversity of enzymatic characteristics of different beta-lactamase types, several classification systems have been proposed for their classification. Classifications are based on two main approaches, which are functional and molecular classifications.
    The functional classification scheme for beta-lactamases proposed by Bush et al., (1995, Antimicrob. Agents Chemother. 39:1211-1233) defines four beta-lactamase groups, which are based on their substrate and inhibitor profiles . Group 1 consists of cephalosporinases that are not well inhibited by clavulanic acid. Group 2 consists of broad-spectrum penicillinases, cephalosporinases, and beta-lactamases, which are generally inhibited by active site-targeted beta-lactamase inhibitors. Group 3 consists of metallo-beta-lactamases which hydrolyze penicillins, cephalosporins and carbapenems and which are barely weakly inhibited by almost all beta-lactam-containing molecules. Group 4 consists of penicillinases that are not well inhibited by clavulanic acid. Subgroups were also defined according to the rates of carbenicillin or cloxacillin (oxacillin) hydrolysis by group 2 penicillinases.
    The most widely used classification is the Ambler classification, which divides beta-lactamases into four classes (A, B, C, D) and is based on their amino acid sequences (Ambler 1980, Philos Trans R Soc Lond B Biol Sci 289:321-331). Classes A, C and D bring together evolutionarily distinct groups of serine beta-lactamase enzymes, and class B the zinc-dependent beta-lactamase enzymes (inhibited by EDTA) (Ambler RP et al, 1991 , Biochem J. 276:269- 270). Classes A, C, and D belong to serine beta-lactamases, in which beta-lactam hydrolysis is mediated by serine at an active site. Serine beta-lactamases are related to DD peptidases (D-alanyl-D-alanine carboxypeptidase), the target enzyme of beta-lactams. The mechanism by which serine beta-lactamases hydrolyze beta-lactam antibiotics is believed to follow a three-step pathway, including a non-covalent Henri-Michaelis complex, an acyl-covalent enzyme intermediate, and deacylation (Matagne et al. , 1998, Biochem J 330:581-598). The acylation mechanism is considered to be a common mechanism for all serine beta-lactamase groups, whereas, based on theoretical calculations, the substrate deacylation mechanisms of class A, C and D serine beta-lactamase appear to be different from each other. Deacylation mechanisms have group-specific and common elementary processes (Hata M et al., 2006, Biol Pharm Bull. 29: 2151-2159).
    Serine beta-lactamases from Bacillus spp. and the TEM-1, SHV-1 and CTX-M families were primarily classified as class A beta-lactamases and as penicillinases that have good hydrolyzing capacity, for example, penicillin and ampicillin. Class A beta-lactamases were first identified in penicillin-resistant St. aureus in the 1940s. A plasmid-derived penicillin-resistance gene, TEM-1, was discovered in E. coli 20 years later. Subsequently, it was also shown that serine beta-lactamases develop the ability to hydrolyze most cephalosporins and, in addition, specialize in the hydrolysis of a specific subset of cephalosporins. Most of these extended spectrum beta-lactamases (ESBL) are derived from the enzymes TEM-1, TEM-2 or SHV-1. Recently, there are increasing numbers of reports describing the vast emergence of CTX-M enzymes, a new group of class A ESBLs. Today, CTX-M enzymes are the most frequently observed ESBLs and are subclassified into the big five main families. CTX-M enzymes have a wide range of substrates, including penicillin and first-, second-, and third-generation cephalosporins (Bonnet, R. 2004. Antimicrob Agents Chemother. 48:1-14).
    Although the sequence similarity between class A beta-lactamases (TEM, SHV, CTX-M, Bacillus spp.) is moderate, the crystal structures of all serine beta-lactamases show a particularly high similarity (Matagne et al., 1998, Biochem J 330:581-598; Tranier S. et al., 2000, J Biol Chem, 275: 28075-28082; Santillana E. et al., 2007, Proc Natl Acad Sci. USA, 104: 5354-5359 ). Enzymes are composed of two domains. One domain consists of a five-strand beta sheet packed against three alpha helices, while the second domain, an alpha domain, is composed of eight alpha helices. The active site pocket is the part of the interface between these two domains and is limited by the omega loop. The omega loop is a conserved structural element of all class A beta-lactamases and is essentially involved in the catalytic reaction (Figure 1).
    Several conserved peptide sequences (elements) related to catalysis or substrate recognition have been identified in class A beta-lactamases. The first conserved element 70-Ser-XX-Lys-73 (Ambler classification) includes the active serine residue at site 70 alpha-helix2 and lysine at position 73. The second conserved element is an SXN loop in an alpha domain (at positions between 130 and 132 according to Ambler's classification), where it forms one side of a catalytic cavity. The third conserved element (at positions between 234 and 236 according to Ambler's classification) is in the innermost strand of the beta3 sheet and forms the other side of the catalytic cavity. The third conserved element is usually KTG. However, in some exceptional cases, lysine (K) may be replaced by histidine (H) or arginine (R), and in several beta-lactamases, threonine (T) may be replaced by serine (S) (Matagne et al. ., 1998. Biochem J 330:581-598).
    Beta-lactamase-mediated resistance to beta-lactams is widespread among pathogenic and commensal microbiota, because of the abundant use of beta-lactams in recent decades. In fact, antibiotic resistance is a well-known clinical problem in humans and veterinary medicine, and hundreds of different beta-lactamases derived from gram-positive and gram-negative bacteria have been purified and characterized in the scientific literature. Due to the fact that the use of antimicrobials has not reduced and, in addition, antimicrobial resistance has become part of everyday life, new approaches are invariably and urgently needed to solve these medical problems.
    The intestinal microbiota of humans is a complex bacterial community that plays an important role in human health, for example, by stimulating the immune response system, aiding in food digestion and preventing the overgrowth of potentially pathogenic bacteria. Antimicrobial agents, eg beta-lactams are known to have an effect on the normal microbiota. The effectiveness of antimicrobial agents in promoting changes in the normal intestinal microbiota is associated with several factors, including drug dosage, route of administration and the pharmacokinetics/dynamics and properties of antibiotics (Sullivan A. et al., 2001, Lancet 1 : 101-114). Although the gut microbiota has a tendency to return to normal after the end of antibiotic treatment, long-term persistence of selected resistant commensal bacteria has been reported (Sjolund M. et al., 2003, Ann Intern Med. 139:483-487 ). Such persistence and the exchange of antibiotic resistance genes make the commensal microbiota a putative reservoir of antibiotic resistance genes.
    Certain parenterally-administered beta-lactams such as ampicillin, ceftriaxone, cefoperazone, and piperacillin are eliminated in part through biliary excretion in the proximal part of the small intestine (duodenum). Residual unabsorbed beta-lactams in the intestinal tract can have an undesirable effect on the ecological balance of the normal intestinal microbiota, resulting in antibiotic-associated diarrhea, overgrowth of pathogenic bacteria such as vancomycin-resistant enterococci (VRE), gram-negative producing bacilli of extended-spectrum beta-lactamase (ESBL), Clostridium difficile and fungi, and the selection of antibiotic-resistant strains among the normal intestinal microbiota and potentially pathogenic bacteria.
    The therapeutic purpose of beta-lactamases is to inactivate unabsorbed antibiotics in the gastrointestinal tract (GIT), thus maintaining a normal intestinal microbiota and preventing their overgrowth with potentially pathogenic microorganisms (WO 93/13795).
    There are at least three requirements for beta-lactamase drug products, which are suitable for GIT-targeted therapy. The first requirement is to preserve the enzyme activity under the conditions prevailing in the GIT. Resistance against proteolytic breakdown by various proteases secreted by various glands in the GIT is a quintessential precondition for the feasibility of beta-lactamase therapy. Another important consideration is the range of pH values prevailing in the different compartments of the small intestine. These pH values typically range between 5 (duodenum) and 7.5 (ileum). Therefore, in order to qualify as candidates for the intended therapeutic purpose, a beta-lactamase needs to have high enzymatic activity in the pH range of 5 to 7.5.
    The second requirement of a beta-lactamase or a product thereof is to efficiently hydrolyze the beta-lactam. The concentration of a beta-lactam antibiotic in the chyme of the small intestine during an antibiotic treatment episode is primarily related to the elimination of the particular beta-lactam through biliary excretion. An adequate beta-lactamase must have kinetic parameters that allow it to effectively hydrolyze lower GIT beta-lactam concentrations below the levels that cause changes in the gut microbiota. The ideal set of kinetic parameters consists of a numerically low value for the Km of the Michaelis constant, combined with a numerically high value for the maximum reaction rate Vmax. A high Vmax value is needed to provide a sufficient degree of breaking capacity, while a low KM value is needed to ensure beta-lactam degrading activity at low substrate concentrations.
    The third requirement of a beta-lactamase or a product thereof is to tolerate conditions, such as relatively high temperatures, in the manufacture of pharmaceutical compositions. Furthermore, in the production process, the mixing dispersion of the aqueous excipients and the drug requires a high degree of solubility at a suitable pH.
    An enzyme therapy, called Ipsat P1A, is being developed to prevent the adverse effects of e-lactam antibiotics within the intestine. The Ipsat P1A delivery system is designed to inactivate parenterally administered beta-lactams of the penicillin group (eg, penicillin, amoxicillin, ampicillin and piperacillin) with or without beta-lactamase inhibitors (eg, tazobactam, sulbactam, acid clavulanic) excreted through the biliary system (WO 2008065247; Tarkkanen, AM et al., 2009, Antimicrob Agents Chemother. 53:2455-2462). The P1A enzyme is a recombinant form of the small 749/C beta-lactamase exo from Bacillus licheniformis (WO 2008065247) which belongs to class A and is grouped into subgroup 2a in the functional classification. Beta-lactamase from B. licheniformis and its P1A derivative are considered to be penicillinases that have a high hydrolytic capacity to degrade, for example, penicillin, ampicillin, amoxicillin or piperacillin (Table 1) and they are generally inhibited by beta-lactamase inhibitors targeted to an active site, such as clavulanic acid, sulbactam, or tazobactam (Bush K. et al., 1995, Antimicrob Agents Chemother 39: 1211-1233).
    However, the P1A enzyme has only a very limited ability to inactivate beta-lactam antibiotics that belong to the cephalosporin or carbapenem group. Since the beta-lactamases used have weak activity against cephalosporins, they cannot be applied in conjunction with parenteral cephalosporin therapy for the inactivation of unabsorbed beta-lactam in the small bowel tract.
    Therefore, new beta-lactamases or P1A derivatives with extended substrate profile, for example, as seen in metallo-beta-lactamases, are indispensable.
    The present invention provides new genetically adapted derivatives of beta-lactamases P1A and, in addition, new methods for modifying and producing the beta-lactamases. Brief description of the invention
    The new recombinant beta-lactamase P1A derivatives of the invention meet the three aforementioned requirements for suitable beta-lactamases (ie, they have the ability to preserve enzymatic activity, hydrolyze beta-lactams efficiently and tolerate conditions in the manufacture of pharmaceutical compositions ) and, in addition, they have extended substrate profiles. The beta-lactamases of the invention can also be used in conjunction with parenteral cephalosporin therapy to inactivate the beta-lactam eliminated by bile in the small bowel tract.
    The present invention highlights preclinical and preliminary studies of a new pharmaceutical protein Ipsat P3A (a substituted D276N derivative of P1A) and features a single drug substance dose.
    The present invention allows for fast and efficient methods to modify beta-lactamases and to produce them. Furthermore, by the present invention, more effective and specific treatments are made available.
    The enzymes of the invention are suitable for large-scale manufacture for a drug substance to treat or prevent adverse effects induced by various groups of beta-lactam antibiotics.
    The object of the present invention is to provide new beta-lactamases, especially beta-lactamases from B. licheniformis, and to provide products, methods and uses related to beta-lactamases. Tools for further developments in the pharmaceutical industries are also presented by the invention.
    The present invention relates to a beta-lactamase comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 and having a hydrophilic amino acid residue at a position of SEQ ID NO: 1 corresponding at position 276, according to Ambler's classification, or a variant or fragment thereof.
    The invention also relates to a pharmaceutical composition comprising the beta-lactamase of the invention.
    The invention also relates to a method of modifying a beta-lactamase which comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1, wherein an amino acid of the beta-lactamase at one position of SEQ ID NO: 1 corresponding to position 276 according to the Ambler classification is substituted with a hydrophilic amino acid.
    Furthermore, the invention relates to a method of producing beta-lactamase, the method comprising the following steps: i) providing a gene encoding the beta-lactamase of the invention; ii) transforming a host cell with the gene; iii) obtaining a host cell that produces the beta-lactamase; iv) recovering beta-lactamase.
    Furthermore, the invention relates to a method of treating or preventing beta-lactam antibiotic-induced adverse effects in the gastrointestinal tract by administering the beta-lactamase of the invention simultaneously or sequentially with a beta-lactam antibiotic to a subject.
    Still further, the present invention relates to beta-lactamase for use as a medicine.
    Still further, the present invention relates to a use of beta-lactamase in the manufacture of a medicament for the treatment or prevention of adverse effects induced by beta-lactam antibiotics in the gastrointestinal tract.
    Still further, the invention relates to a polynucleotide, which comprises a sequence of any one of SEQ ID NOs: 2 or 4, or a degenerate thereof, or it encodes the beta-lactamase of the invention. The invention also relates to a host cell which comprises the polynucleotide. Brief description of the figures
    Figure 1 shows the 3D structure of beta-lactamase from Bacillus licheniformis (exo small form of PenP). The conserved amino acid residues and side chain residues of R-244 and D-278 are marked. The diagram was generated using the MolSof-Browser program.
    Figure 2 shows the nucleotide and deduced amino acid sequences of the beta-lactamase gene D276N from Bacillus licheniformis (P1A derivative). The amino acid sequence corresponds to the sequence of SEQ ID NO: 3, where Xaa is asparagine (Asn). The nucleotide sequence corresponds to the sequence of SEQ ID NO: 4, wherein the nucleotide triplet nnn is aat. The open reading frame encodes a 299 amino acid polypeptide that has a 31 amino acid long (underlined) signal sequence from the amyQ gene derived from the secretion vector pKTH141 (WO 2008/065247). The predicted signal peptidase cleavage site is after alanine (A) at position -1. The HindIII cloning site encoding an NH2-QAS extension is expressed in bold. The mutant mature D276N enzyme starts from glutamine (Q) at a +1 position. Thus, the mature mutant beta-lactamase D276N comprises 268 amino acid residues, including the NH2-QAS extension encoded by HindIII. A single amino acid substitution from aspartic acid (D) to asparagine (N) is located at position 280 (expressed as a bold character) corresponding to position 276 in the Ambler classification system and corresponding to amino acid position 249 in the SEQ ID sequence NO: 3.
    The NH2-terminal sequence of the purified D276N mutant enzyme was determined by automated Edman degradation in a protein sequencer. The analysis demonstrated that the mutant enzyme D276N lacks the pentapeptide NH2-QASKT at its deduced amino terminus similarly to the parent p1A enzyme (WO 2008/065247). The major fraction of the purified mutant D276N enzyme, which was used in examples 4 and 6 of this application, starts from glutamic acid at position +6 and is composed of 263 amino acid residues with a molecular mass of 29,272.
    Figure 3 shows the nucleotide and deduced amino acid sequences of the substituted beta-lactamase gene D276N of P1A derived from Bacillus licheniformis. The amino acid sequence corresponds to the sequence of SEQ ID NO: 3, where Xaa is arginine (Arg). The nucleotide sequence corresponds to the sequence of SEQ ID NO: 4, wherein the nucleotide triplet nnn is cgc.
    Figure 4 shows the effect of orally administered enteric-coated beta-lactamase D276N (P3A) pellets on ceftriaxone concentrations in the jejunal chyme of beagle dogs (n = 5) after intravenous administration of ceftriaxone (30 mg of ceftriaxone per kg of body weight) (closed squares). Beta-lactamase pellets were received 10 minutes before ceftriaxone injection. Closed diamonds represent the jejunal ceftriaxone concentrations achieved after a single dose of ceftriaxone (i.v.) without beta-lactamase treatment. Detailed description of the invention
    Beta-lactamases have been used to inactivate unabsorbed beta-lactams in the gastrointestinal tract to prevent beta-lactam-induced adverse effects, including alterations in the normal intestinal microbiota and the overgrowth of beta-lactam-resistant bacteria (WO 9313795, WO 2008065247, WO 2007147945. The present invention now provides a modified beta-lactamase from Bacillus licheniformis, which exhibits a surprising altered substrate profile.
    As used herein, a beta-lactamase refers to an enzyme that hydrolyzes beta-lactams. Hydrolysis of the amide bond of the beta-lactam ring renders antimicrobial agents biologically inactive. As used in this document, class A beta-lactamases (Ambler's classification) refer to serine beta-lactamases, in which beta-lactam hydrolysis is mediated by serine at the active site, usually at amino acid position 70 in alpha -helix2. Class A beta-lactamases include, but are not limited to, Len-1, SHV-1, TEM-1, PSE-3/PSE-3, ROB-1, Bacillus cereus as 5/B type 1, 569/ H type 1 and 569/H type 3, Bacillus anthrasis sp, Bacillus licheniformis such as PenP, Bacillus weihenstephanensis, Bacillus clausii, Staphylococcus aureus, PC1, Sme-1, NmcA, and beta-lactamases type IMI, PER, VEB, GES, KPC , CME and CTX-M. Sequence identity of peptides and polynucleotides
    The amino acid sequences of the beta-lactamase mutant of the present invention (D276X, P1A derivative) are set forth as SEQ ID NO: 1 and SEQ ID NO: 3. The corresponding nucleotide sequences are set forth as SEQ ID NO: 2 and SEQ ID NO: 4. SEQ ID NO: 1 establishes the amino acid sequence that participates in the formation of the beta-lactamase secondary structure. SEQ ID NO: 3 sets forth the full length amino acid sequence of the protein, including the 31 amino acid long signal sequence.
    A beta-lactamase of the invention may comprise an amino acid sequence having at least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63. 64, 65, 66, 67 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 99.5, 99.8, 99.9 or 100% identity with SEQ ID NO: 1 or 3.
    According to a specific embodiment of the invention, the peptide is at least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9 or 100% identity with SEQ ID NO: 1 or 3.
    In a preferred embodiment of the invention, the beta-lactamase of the invention comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1. In another preferred embodiment of the invention, the beta-lactamase has at least 60 % sequence identity with SEQ ID NO: 1 or 3.
    In one embodiment of the invention, the beta-lactamase comprising an amino acid sequence having any above-mentioned sequence identity with SEQ ID NO: 1 has a hydrophilic amino acid selected from a group consisting of arginine (R), histidine ( H), lysine (K), asparagine (N), glutamine (Q), serine (S) and threonine (T) at a position of SEQ ID NO: 1 corresponding to position 276 according to the Ambler classification.
    In a preferred embodiment of the invention, the peptide has the sequence shown in SEQ ID NO: 1 or 3. In an embodiment of the invention, the beta-lactamase has the sequence as shown in SEQ ID NO: 1 or 3, wherein one residue of hydrophilic amino acid at the position corresponding to position 276 according to the Ambler classification (marked as Xaa in SEQ ID NO: 1 or 3) is an arginine (R, Arg). In another embodiment of the invention, the beta-lactamase has the sequence as shown in SEQ ID NO: 1 or 3, wherein a hydrophilic amino acid residue at the position corresponding to position 276 according to the Ambler classification (marked as Xaa in SEQ ID NO: 1 or 3) is an asparagine (N, Asn).
    The identity of any sequence with the sequence of this invention refers to the identity of the entire sequence of the present invention. Sequence identity can be determined by any conventional bioinformatics method, for example using BLAST (Basic Local Alignment Search Tools) or FASTA (FAST-AII).
    The present invention also relates to any variants or fragments of the novel beta-lactamases. As used herein, a beta-lactamase fragment or variant refers to any part or variant that has a biological function, that is, that is enzymatically active. A variant refers to a peptide that has minor changes in the peptide sequence, for example, mutations, minor deletions or insertions. Fragments and variants must include the hydrophilic amino acid in the position corresponding to position 276 according to the Ambler classification. The hydrophilic amino acid is typically different from aspartic acid (D).
    There are several short forms of beta-lactamases, which are obtainable from SEQ ID NO: 3 and which are secreted outside the cell. These are called exoforms. Exoforms are the result of the hydrolytic activity of proteases in the cell wall or in the culture medium. D276X, D276N, D276R, the mutant form, the P1A or P3A derivative as used herein encompasses any active beta-lactamase fragment and/or variant of SEQ ID NO: 3 or variant which comprises the explicitly determined amino acid sequence ( SEQ ID NO: 1). Especially, the beta-lactamase of the invention is an NH2-truncated form, meaning that it has been truncated at the amino terminus. In addition to NH2-truncation, it can include one or more amino acid deletions, substitutions and/or insertions, as long as it has beta-lactamase activity. Said modifications can be naturally occurring variations or mutants, or artificial modifications introduced, for example, by gene technology.
    Differently aminoterminally truncated exoforms were found in the B. licheniformis growth medium. Such exoforms are also encompassed in this document. Matagne et al. described several extensions of microheterogeneity in the extracellular forms produced by the natural host B. licheniformis 749/C (Matagne A. et al., 1991. Biochem J. 273:503-510). The following five different secreted exoforms with different N-terminal amino acid residues have been identified: SQPAEKNEKTEMKDD KALNMNGK EKTEMKDD KALNMNGK KTEMKDD KALNMNGK EMKDD KALXMNGK MKDD KALNMNGK
    Initial amino acid residues are shown in bold. C-terminal amino acid residues are indicated on the right. The exoform starting from serine (S) is called the "large secreted form" of beta-lactamase from B. licheniformis, and the one starting from lysine (K) is called the "small secreted form".
    The first alpha-helix (ai-helix) starts from aspartic acid (D) (shown in italics) and the end of the last alpha-helix (ai-helix) ends at asparagine (N) (shown in italics). According to an embodiment of the invention, the beta-lactamase comprises at least amino acids i-258 of SEQ ID NO: i or amino acids 7-264 of SEQ ID NO: 3, which form part of the secondary structure of the protein (Knox JR et al., 99i. J. Mol Biol. 220: 435-455).
    According to another embodiment of the invention, one or more of said amino acids i-258 of SEQ ID NO:i or amino acids 7-264 of SEQ ID NO:3 have been deleted or replaced.
    According to yet another embodiment of the invention, the amino terminus of the beta-lactamase begins with NH2-KTEMKDD (amino acids 4-10 of SEQ ID NO: 3). This exoform called ES-betaL may not yet have up to 2i contiguous residues, as described by Gebhard et al. (Gebhard L.G. et al., 2006, J. Mol. Biol. 2i: 358(i)280-288). According to another embodiment of the invention, the amino terminus begins with glutamic acid (E) of SEQ ID NO:3 and it especially begins with NH2-EMKDD (amino acids 6-10 of SEQ ID NO:3), or alternatively it begins with NH2-MKDD (amino acids 7-10 of SEQ ID NO:3 or amino acids 1-4 of SEQ ID NO:i).
    The variable region in the amino-terminal sequence of beta-lactamase does not have a rigid structure that represents the constancy of enzymatic parameters of various forms of beta-lactamase.
    The last four amino acids at the carboxy terminus of beta-lactamase, MNGK-COOH (amino acids 265-268 of SEQ ID NO: 3), are not part of the secondary structure and therefore can also be eliminated without losing activity. In another embodiment, up to nine C-terminal amino acids can be eliminated. The C-truncated forms of the protein were described by Santos et al. (Santos J. et al., 2004. Biochemistry 43:1715-1723).
    All the different forms of beta-lactamase defined above are encompassed by the present invention, along with other forms of protein with beta-lactamase activity.
    A polynucleotide of the invention can comprise or have a sequence of any one of SEQ ID NO: 2 or 4, or a degenerate thereof. A polynucleotide that is a degenerate of a sequence shown in any one of SEQ ID NOs: 2 or 4 refers to a polynucleotide that has one or more different nucleotides compared to SEQ ID NOs: 2 or 4, but which encodes the same amino acid. Preferably, the nucleotide triplet nnn of SEQ ID NO: 2 or 4 encodes a hydrophilic amino acid, more preferably N or R. A "polynucleotide", as used herein, is a sequence of nucleotides, such as a DNA or RNA sequence, and it can be a single-stranded or double-stranded polynucleic acid. The term polynucleotide encompasses cDNA, mRNA and genomic DNA.
    According to a specific embodiment of the invention, the polynucleotide is at least 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8 or 99.9% identity to any of the nucleotide sequences of SEQ ID NOs: 2 or 4, or fragments thereof.
    In a specific embodiment of the invention, the polynucleotide has a sequence shown in either sequence SEQ ID NO: 2 or 4. Amino acids at position 276 (Ambler) of class A beta-lactamases
    Asparagine (Asn, N), at amino acid position 276, is present in a wide variety of class A beta-lactamases. The function of Asn276 has been intensively studied in TEM and SHV beta-lactamases, where n276 forms hydrogen bonds with the guanidium group of arginine (Arg, R) 244 and therefore limits the mobility of the Arg244 side chain.
    Asparagine (Asn, N) substitutions in either TEM enzymes have been recognized as a major contributor to resistance to serine beta-lactamase inhibitors such as clavulanic acid, sulbactam or tazobactam. N276D (Asp) substitution variants of the beta-lactamase TEM-1 are present in the inhibitor-resistant beta-lactamases (IRT enzymes such as TEM-35 and TEM-36). An N276D variant is more resistant to clavulanic acid and tazobactam than the wild-type TEM-1 enzyme, but concomitantly, the catalytic efficiencies (kcat/Km) of the N276D variant for various penicillins are less than 50% of those for the wild-type enzyme HAS-1. The catalytic efficiencies of the N276D variant to cephalosporins are reduced compared to those of wild-type TEM-1 (Saves I et al., 1995, J Biol Chem. 270:18240-18245).
    Similar to TEM-1, the N276D substitution in beta-lactamase SHV-1 or SHV-5 increases resistance to serine beta-lactamase inhibitors, but reduces their efficiencies in most beta-lactams (Giakkoupi P. et al. , 1999, J Antimicrobiol Chemother, 43: 23-29). Furthermore, the N276D substitution in the SHV-1 or SHV-5 enzymes moderately improves their ability to degrade the “fourth generation” cephalosporins cefpiroma and cefepime.
    In beta-lactamase type SHV OHIO-1, an N276G (Gly) mutant has been shown to be highly resistant to clavulanic acid, whereas an N276G mutant derived from TEM-1 has only moderate resistance to clavulanic acid (Bono-mo RA et al., 1995, Biochim Biophys Acta. 1247:121-125).
    In the CTX-M family of enzymes, arginine (Arg, R) is typically present at position 276 (Bonnet R., 2004,
    Antimicrob Agents Chemother, 48: 1-14) and Arg276 mutations affect the extent of enzyme activity. Relative hydrolysis rates of CTX-M enzymes against cefotaxime are moderately reduced by Arg276 substitution. Furthermore, the CTX-M mutant enzymes Arg276Trp, Arg276Cys, Arg276Ser and Arg276Gly do not affect the level of beta-lactamase inhibitor resistance (Bonnet R., 2004, Antimicrob Agents Chemother, 48: 1-14; Perez-Llarena FJ et al. al., 2008, J
    Antimicrobiol Chemother, 61:792-797). Table 1. Amino acid residues located at position 276 (Ambler's classification) among class A beta-lactamases (Matagne A et al., 1998, Biochem J 330: 581-598; Tranier S. et al., 2000, J Biol Chem, 275: 28075-28082)


    Now, in the present invention, beta-lactamases comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 (PenP derivative from Bacillus licheniformis, i.e. P1A derivative) and having a residue of hydrophilic amino acid at a position of SEQ ID NO: 1 corresponding to position 276, according to Ambler's classification, exhibit an extended beta-lactam as well as improved catalytic effects on beta-lactams.
    Previously, the role of amino acid substitutions of aspartic acid (D) at position 276 in resistance to serine beta-lactamase inhibitors or in the catalytic properties for various beta-lactams has not been studied among beta-lactamases from Bacillus spp., specifically beta- lactamase from B. licheniformis.
    As used herein, amino acid residue 276 according to the Ambler classification corresponds to amino acid position 243 of SEQ ID NO: 1 and amino acid position 249 of SEQ ID NO: 3.
    Typically, the beta-lactamases of the present invention have a hydrophilic amino acid in a position corresponding to position 276 of the Ambler classification, different from aspartic acid (D). Amino acids are classified based on the chemical and/or structural properties of their side chains. The amino acid classification groups include hydrophilic amino acids, which are divided into the following groups: polar and positively charged hydrophilic amino acids; hydrophilic polar and neutrally charged amino acids; polar and negatively charged hydrophilic amino acids; aromatic, polar and positively charged hydrophilic amino acids. As used herein, "hydrophilic amino acid" includes all of the aforementioned groups of hydrophilic amino acids, that is, it refers to hydrophilic and/or polar and positively charged amino acids, hydrophilic polar and neutrally charged amino acids; hydrophilic and/or polar and negatively charged amino acids aromatic, polar, positively charged hydrophilic amino acids (http://www.biomed.curtin.edu.aU/biochem/tutorials/AAs/AA.html) "A polar, positively charged hydrophilic amino acid" refers to arginine (R ) or lysine (K). "A polar, neutral hydrophilic amino acid" refers to asparagine (N), glutamine (Q), serine (S) or threonine (T). "A polar and negatively charged hydrophilic amino acid" refers to aspartate (D) or glutamate (E). "An aromatic, polar and positively charged hydrophilic amino acid" refers to histidine (H).
    In one embodiment of the invention, the hydrophilic amino acid is a neutral or positively charged hydrophilic amino acid selected from the group consisting of arginine (R), histidine (H), lysine (K), asparagine (N), glutamine (Q), serine (S ) and threonine (T) at a position of Seq ID NO: 1, corresponding to position 276 according to Ambler's classification.
    In a preferred embodiment of the invention, the hydrophilic amino acid of beta-lactamase at a position of SEQ ID NO: 1 corresponding to position 276 according to the Ambler classification is selected from polar and positively charged hydrophilic amino acids from the group consisting of arginine (R ), histidine (H) and lysine (K). More preferably, the amino acid at position of SEQ ID NO: 1 corresponding to position 276 according to the Ambler classification is arginine.
    In another preferred embodiment of the invention, the hydrophilic amino acid is selected from the polar and neutral hydrophilic charge amino acids from the group consisting of asparagine (N), glutamine (Q), serine (S) and threonine (T). More preferably, the amino acid at position of SEQ ID NO: 1 corresponding to position 276 is asparagine.
    In another preferred embodiment of the invention, the hydrophilic amino acid at position SEQ ID NO: 1 corresponding to position 276 is located on an alpha helix. An alpha helix is a portion of the protein's secondary structure, resembling a coiled conformation. Alpha-helices may be of particular importance in the DNA binding moieties (e.g., helix-turn-helix, leucine zipper and zinc finger moieties). In a preferred embodiment of the invention, amino acid residue 276 is located in the final alpha-helix 11 (Figure 1). This alpha-helix11 is not conserved among class A beta-lactamases. Specific characteristics of class A beta-lactamases
    A specific feature of class A beta-lactamases is a guanidinium group of Arg278. CTX-M enzymes have Arg278, Arg244 or Arg220, which are found in equivalent positions in the three-dimensional structures. It is shown that arginine at position 220 or 244 is essential for the catalytic properties of TEM-1 (Leu220 and Arg244) and beta-lactamase from Streptococcus albus G (Arg220 and Asn244). A basic guanidinium group of arginine 244 or arginine 220 is proposed to contribute to the beta-lactam binding or inactivating chemistry of "suicidal" inhibitors such as clavulanic acid (Matagne et al., 1998, Biochem J. 330:582 -598; Perez-Llarena et al., 2008, J Antimicrobiol Chemother, 61: 792-797). In B. licheniformis PenP, the Arg-244 residue forms a salt bond with aspartic acid 276 (Herzberg, O. 1991, J Mol Biol. 217: 701-719; Knox, JR, and PC Moews, 1991, J Mol Biol. 220: 435-555).
    In a preferred embodiment of the invention, the beta-lactamase further comprises at least one amino acid selected from the group consisting of Leu220 and Arg244, according to the Ambler classification, which corresponds to Leu189 and Arg212, respectively, of SEQ ID NO: 1. Beta-lactamase from Bacillus licheniformis (PenP, P1A)
    The beta-lactamase of the invention originates from the 749/C strain of Bacillus licheniformis. Beta-lactamase from B. licheniformis 749/C (PenP; penicillin starch-beta-lactam hydrolase, EC3.5.2.6) belongs to a subgroup 2a in the functional classification of class A beta-lactamases (Bush K. et al. al., 1995, Antimicrob Agents Chemother 39: 1211-1233 ). Beta-lactamase from B. licheniformis can be considered as a penicillinase, which has a high hydrolytic capacity to degrade, for example, penicillin, ampicillin, amoxicillin or piperacillin and is generally inhibited by beta-lactamase inhibitors directed to active sites, such as clavulanic acid, sulbactam or tazobactam (Bush K. et al., 1995, Antimicrob Agents Chemother. 39: 1211-1233).
    Beta-lactamase from Bacillus licheniformis 749/C is expressed as a preprotein of 307 amino acid residues. After translocation and removal of its signal sequence 26 amino acid residues in length, it becomes a membrane-anchored lipoprotein, in which the amino-terminal cysteine (C27) forms a thioether bond with a diacylglyceride. Beta-lactamase from B. licheniformis is also found as the secreted (extracellular) forms, which are proteolytic products of the lipoprotein form (Izui K. et al., 1980, Biochemistry 19: 1882-1886; Matagne A. et al., 1991, Biochem J, 273: 503-510). The beta-lactamase gene region of Bacillus Licheniformis 749/C encoding the small secreted form (exo small form; P1A) of amino acid residues 43-307 was chosen as a DNA fragment for adaptation of the host vector production system of the Bacillus subtilis (WO 2008065247). Occupation
    Beta-lactamases hydrolyze beta-lactam antibiotics that comprise a beta-lactam ring such as penicillins, cephalosporins, clavams (or oxapenams), cephamycins and carbapenems. In a preferred embodiment of the invention, beta-lactamase hydrolyzes penicillins and/or cephalosporins. "Penicillins" refers to several natural or semi-synthetic variants of penicillin, which is originally derived from Penicillium. Penicillins include, but are not limited to, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, hetacillin, oxacillin, mezlocillin, penicillin G, penicillin V and piperacillin.
    In cephalosporins, the beta-lactam ring is fused to a six-membered dihydrothiazine ring rather than the five-membered thiazolidine ring found in penicillins. Based on their biological activities, cephalosporins are divided into six generations, but some cephalosporins have not been grouped into a specific generation. In a specific embodiment of the invention, beta-lactamase has improved catalytic efficiency in cephalosporins compared to wild-type beta-lactamases. According to the present invention, beta-lactamase from Bacillus licheniformis, in which the aspartic acid (Asp, D) at position 276, numbered according to the classification of
    Ambler, which is replaced by a hydrophilic amino acid residue such as asparagine (N) or arginine (R), has a prolonged activity to beta-lactam antibiotics such as cephalosporins.
    In one embodiment of the invention, the cephalosporins are selected from the group consisting of cefoperazone, ceftriaxone and cefazolin.
    As used herein, the catalytic efficiency of beta-lactamases refers to the ability to hydrolyze the beta-lactam antibiotics. Improved catalytic efficiency can be measured by any conventional methods in vitro, ex vivo or in vivo of any biological sample or an individual. . Methods of production and modification of beta-lactamases
    The beta-lactamase of the invention can be produced by modifying the enzyme with any conventional method of genetic engineering. Methods such as rational design, random mutagenesis, DNA shuffling (random recombination), phage display, whole genome shuffling, heteroduplex, random chimeragenesis in the assembly of transient templates of engineered oligonucleotides, mutagenic and unidirectional reassembly, exon shuffling, block shuffling Y-link based, non-homologous recombination, and rational combination design with directed evolution can be used in production. In addition, mutant enzymes can be obtained by using site-directed mutagenesis and splicing by overlap extension techniques.
    In one embodiment of the invention, a method of modifying a beta-lactamase comprises a step of modifying a beta-lactamase which comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 by replacing a amino acid at a position of SEQ ID NO: 1 corresponding to position 276 according to the Ambler classification as a hydrophilic amino acid. The hydrophilic amino acid can be any hydrophilic amino acid, for example, selected from the group consisting of arginine (R), histidine (H), lysine (K), asparagine (N), glutamine (Q), serine (S) and threonine (T ).
    In one embodiment of the invention, a non-hydrophilic amino acid is substituted with a hydrophilic amino acid at a position of SEQ ID NO: 1 corresponding to position 276 according to the Ambler classification.
    The beta-lactamase of the invention can also be produced, for example, by synthetic methods, for example, peptide synthesis or by recombinant production in a host cell.
    In a preferred embodiment of the invention, the enzyme is recombinant. As used herein, "recombinant" genetic material refers to material that is typically a combination of genetic material, for example, strands of DNA of various origins, and that was produced by combining or inserting the sequences. The polynucleotide of the invention can, for example, be inserted under the control of any endogenous or exogenous regulators, such as promoters. Recombinant protein is derived from recombinant DNA.
    At least one polynucleotide or polynucleotide fragment of interest can be isolated from a cell or produced synthetically. This polynucleotide or polynucleotide fragment can be transformed into a host cell. A suitable host cell for producing any peptide of the invention can be any eukaryotic or prokaryotic cell, preferably bacteria, more preferably a strain of Bacillus spp. such as Bacillus subtilis, Bacillus licheniformis, Bacillus pumilis or Bacillus amyloliquefaciens.
    As used herein, "transformation" refers to a genetic alteration of a cell by foreign genetic material, preferably DNA, resulting in the expression of that genetic material. Foreign genetic material can be introduced as such or as incorporated into any other genetic material such as vectors, plasmids, etc. Any genetic engineering method or any molecular cloning methods can be used to transform a host cell with the polynucleotide of the invention. There are several methods of introducing foreign material into a eukaryotic cell. Materials such as polymers (eg DEAE-dextran or polyethylenimine), liposomes and nanoparticles (eg gold) have been used as vehicles for transformation. Genetic material can also be introduced into cells using, for example, viruses or vectors as vehicles. Other methods for introducing foreign material into a cell include, but are not limited to, nucleofection, electroporation, conjugation, transfection, sonoporation, heat shock, and magnetofection.
    After a host cell has produced the peptide of the invention under appropriate conditions, the peptide can, for example, be purified from the cell or a secreted form of the peptide can be recovered, for example, from the culture medium. In a preferred embodiment of the invention, beta-lactamase is secreted. Pharmaceutical composition
    The pharmaceutical composition of the invention comprises the beta-lactamase of the invention. The composition may comprise only one beta-lactamase or more, such as at least two, three, four, etc. different beta-lactamases.
    The pharmaceutical compositions of the invention may also comprise any other active ingredients other than the beta-lactamases of the invention.
    The pharmaceutical compositions can be used, for example, in solid, semi-solid or liquid form, such as in the form of tablets, pellets, capsules, solutions, emulsions or suspensions. Preferably, the composition is for oral administration or enteral applications.
    In addition to at least one beta-lactamase of the invention or polynucleotides or host cells comprising the polynucleotides of the invention, the pharmaceutical composition may comprise carrier(s), adjuvant(s), excipient(s), excipient(s) adjuvant(s) , antiseptic(s), stabilizing agent(s), binding agent(s), filler(s), lubricating agent(s), suspending agent(s), plasticizer, dyes, builders of film, sugar, alcohols, lubricating agents and thinning agents and/or components normally found in corresponding products.
    The product or pharmaceutical composition of the invention comprises the beta-lactamases in an amount sufficient to produce the desired effect.
    The pharmaceutical products or compositions can be manufactured by any conventional processes known in the art. Beta-lactamases can be added to any pharmaceutical product or mixed with any agents during any preparation step. The beta-lactamase of the invention can also be produced, for example, by expressing the beta-lactamase gene under appropriate conditions in a pharmaceutical product or in the target tissue, after the pharmaceutical product has been degraded.
    In a preferred embodiment of the invention, the beta-lactamase(s) and the beta-lactam antibiotic are administered together as an enteric coated pellet to a subject. Water-based coating forms appear to be the most favorable materials for hydrophilic P1A protein coating processes. Aqueous polymers commonly used to achieve enteric properties and also usable in the present invention are polymethacrylates, such as Eudragit®, cellulose-based polymers, for example, cellulose ethers, for example, Duodcell®, or cellulose esters, for example , Aquateric®, or polyvinyl acetate copolymers, for example, Opadry®.
    The beta-lactamase of the invention or a pharmaceutical composition of the invention can be administered to a subject simultaneously or sequentially with a beta-lactam antibiotic. In one embodiment of the invention, the beta-lactamase or pharmaceutical composition is administered before a beta-lactam antibiotic, for example, 5 to 30 minutes before a beta-lactam antibiotic. The beta-lactamase and a beta-lactam antibiotic/antibiotics may be in the same or different formulations. Adverse effects of beta-lactams and treatments
    Adverse effects, ie, adverse drug reactions to beta-lactam antibiotics may include, but are not limited to, diarrhea, nausea, rash, urticaria, superinfection, fever, vomiting, erythema, dermatitis, angioedema, and pseudomembranous colitis.
    In a preferred embodiment of the invention, the adverse effects to be treated or prevented occur in the gastrointestinal tract (GIT). As used in this document, gastrointestinal tract refers to the digestive structures that extend from the mouth to the anus. The gastrointestinal tract comprises the mouth, esophagus, stomach, duodenum, jejunum, ileum, small intestine, colon, cecum, rectum and anus.
    The beta-lactamase of the invention or the pharmaceutical composition of the invention can be administered to a subject orally or directly to the gastrointestinal tract. Drug product(s) of the enzyme combinations are intended to inactivate unabsorbed beta-lactam in the GIT or other unwanted body compartments such as the skin or vaginal cavity. The pharmaceutical composition may be an orally administered drug product, a dermatological formulation or a vaginal suppository, and may comprise liquid, immediate, delayed or sustained release dosage formulations.
    In a preferred embodiment of the invention, the beta-lactamase(s) is/are administered orally. In another preferred embodiment of the invention, the beta-lactamase(s) is/are administered directly to a patient's gastro-intestinal tract.
    A treated individual can be a human or an animal such as a pet or farm animal, for example a dog, cat, cow, pig, chicken or horse. In a preferred embodiment of the invention, the individual is a human being.
    The present invention is illustrated by the following examples, which are not intended to be limited in any way. Example 1. Construction of mutant enzymes D276N and D276R
    Bacillus licheniformis beta-lactamase D276N and D276R mutants were constructed by overlap splice extension mutagenesis (SOE) using plasmid pRSI-110 encoding beta-lactamase P1A as a template for initial PCR reactions according to previously published procedures (Horton RM et al., 1989, Gene 77:61-68). Primers were designed to provide two different PCR products with a common sequence region. The fragments were then fused in a subsequent PCR amplification by the aid of overlapping regions. The desired mutations were achieved using mutagenic primers in the initial PCR.
    For the D276N mutant, the mutation was made at the desired position in the wild-type gene, converting a GAT codon to an AAT codon. The primers used in the first PCR amplifications are shown in Table 2. The size of the fragments amplified in the first PCR was 800 nt and 220 nt, which had a long overlap region of 21 nt. Table 2. Oligonucleotide PCR primers. Complementary regions are shaded and modified codons are expressed in bold. The Normal-1 and Reverso-1 10 primers were used in the amplification of the fused fragment in the second PCR.

    In the second PCR reaction (SOE reaction), the two overlapping fragments were fused in a subsequent extension reaction. The inclusion of the outer primers (Normal-1 and Reverso-1) in the extension reaction amplifies the fused product by PCR. The purified SOE product was digested with restriction enzyme HindIII and ligated to the HindIII cleaved secretion vector pKTH141 as described in WO 2008/065247.
    Competent Bacillus subtilis RS303 cells were transformed with a ligation mixture. Positive clones on Luria-kanamycin plates were tested by suspending the bacterial mass from a single colony in nitrocefin solution. Positive clones effectively hydrolyzed the nitrocefin, changing the color of the nitrocefin solution from yellow to red. The hybrid plasmid was purified from cells from a single clone. The correct sequence of the region generated by PCR was verified by DNA sequencing.
    For mutant D276R, the mutation was made at the desired position by converting a GAT codon to a CGC codon. The construction of mutant strain D276R was performed similarly to that of mutant D276N, except that reverse primers D276R and normal D276R were used in the initial PCR (see Table 2). Example 2. Nucleotide sequence of mutant beta-lactamase gene D276N (penP)
    The expression construct was isolated from a positive clone and the insert was subjected to DNA sequencing. The complete nucleotide sequence and deduced amino acid sequence of the mutant beta-lactamase gene D276N revealed that an Asp to Asn substitution occurred correctly at the desired codon (Figure 2). Furthermore, the DNA sequence of the mutant beta-lactamase gene D276N revealed in the frame fusion between the nucleotide sequence encoding a signal sequence of 31 amino acids in length the alpha-amylase of Bacillus amyloliquefaciens, the HindIII cloning site and the complete sequence of the mutant D276N gene. The signal peptidase is predicted to sever the peptide bond between alanine (A) at the -1 position and glutamine (Q) at the +1 position. The mature beta-lactamase D276N has an NH2-terminal extension of an NH2-QAS-tripeptide derived from the HindIII cloning site in the expression construct. Therefore, based on the deduced amino acid sequence, the mature mutant beta-lactamase D276N is comprised of 268 amino acid residues. Example 3. Nucleotide sequence of the mutant beta-lactamase gene D276R (penP)
    To confirm the desired substitution of aspartic acid to arginine at position 276 (Ambler's classification) in the beta-lactamase gene from Bacillus licheniformis, the expression construct was isolated from a positive clone and the nucleotide sequence of the insert was sequenced similarly to example 2. According to the nucleotide sequence obtained, the deduced amino acid sequence contains the desired D276R substitution and the mature mutant D276R enzyme is composed of 268 amino acid residues (Figure 3). Example 4. Biochemical analysis of mutant beta-lactamase D276N (P3A)
    The purity of the enzyme preparation was estimated to be greater than 95 percent by SDS-PAGE analysis (data not shown).
    The kinetic parameters of beta-lactamases from wild-type (P1A) and mutant D276N (P3A) B. licheniformis were determined for the hydrolysis of various types of beta-lactams and are summarized in Table 3. Enzymatic reactions were carried out in phosphate buffer a 20 mM (pH 7), at 30°C, using the appropriate enzyme concentration and various concentrations of penicillin or cephalosporin substrates. The kcat and Km values were obtained using the Hanes linearization method. The main results are described below. (i) Penicillins
    The D276N substitution effect on the hydrolysis of penicillins (ampicillin, amoxicillin or piperacillin) was not drastic with enzymatic efficiencies of percentages from 51 to 80 of those of the wild-type enzyme. Consequently, the Km values of the mutant enzyme D276N for penicillins were reduced to a maximum of two-fold or less. (ii) Cephalosporins
    As expected, in relation to penicillins, wild-type beta-lactamases had poor enzymatic efficiency for several cephalosporins, including the first (cafazolin), second (cefuroxime) and third (ceftriaxone, cefotaxime, ceftadizime, cefoperazone and cefepime) generation cephalosporins ( Table 1). Surprisingly, the enzymatic efficiencies of the mutant enzyme D276N for certain cephalosporins, preferably for cefoperazone and more preferably for ceftriaxone, were essentially improved compared to those obtained with wild-type enzymes. The Km constants for ceftriaxone and cefoperazone were reduced and, concomitantly, the turnover numbers (kcat) for ceftriaxone and cefoperazone were increased compared to those for the wild-type enzyme (P1A). Thus, the substitution of aspartic acid-asparagine at position 276 of the beta-lactamase of Bacillus licheniformis contributes to the extension of the beta-lactam substrate profile in the beta-lactamase of Bacillus licheniformis. Table 3. Kinetic parameters for the hydrolysis of beta-lactam substrates by wild-type (P1A) and D276N mutant enzymes of beta-lactamases from Bacillus licheniformis.
    (1) Relative catalytic efficiency (k /K ) of D276N compared to that of wild-type enzyme (P1A). Example 5. Biochemical characterization of mutant enzyme D276R
    The mutant enzyme D276R was constructed to assess whether
    Asp-276 tolerates substitutions and assess the contribution of the D276R substitution to the extent of beta-lactamase activity observed in the D276N enzyme.
    Crude enzyme samples of D276R and D276N obtained from the culture supernatants were used as test materials. The purity and quantity of enzyme samples were estimated by performing SDS-PAGE analysis. The hydrolysis rate of the mutant enzymes D276R and D276N for various beta-lactams was performed by determining the Vmax values. The results obtained are presented as relative activities (%) compared to those of the D276N enzyme in Table 4.
    In general, the catalytic efficiencies of beta-lactamase D276R for both penicillins and cephalosporins are comparable to those of the enzyme D276N. Compared to the D276N enzyme, the D276R enzyme has reduced activity relative to ceftriaxone and improved activity relative to cefoperazone. This study showed that the extended spectrum of beta-lactams can be achieved by replacing a hydrophilic amino acid residue, such as arginine or asparagine, with aspartic acid at position 276 in the beta-lactamase from Bacillus licheniformis. It also indicates that a desired enzyme modification can be achieved by replacing another hydrophilic amino acid residue, such as glutamine (Q), lysine (K), serine (S) or threonine (T) with aspartic acid at position 276. Table 4. Relative activities (%) of mutant enzyme D276R compared to those of mutant enzyme D276N

    Example 6. In vivo study of beta-lactamase D276N
    The ability of the mutant beta-lactamase enzyme D276N from Bacillus licheniformis to inactivate ceftriaxone (ORC), which was excreted into the gastrointestinal tract during parenteral therapy was investigated in a canine model. Laboratory beagles in the study have a nipple valve surgically inserted into the jejunum approximately 170 cm away from the pylorus, allowing samples to be collected for analysis. Bowel surgery did not alter bowel motility. Five beagle dogs were used in each experiment.
    The study was carried out as two sequential treatments: in the first treatment (control experiment without beta-lactamase therapy), a single dose of ceftriaxone (30 mg of ceftriaxone (CRO) per kg of body weight, which corresponds to about 1 gram of ORC in humans) was administered intravenously 20 minutes after the dogs' first feeding. Jejunal samples were collected at various time points over ten hours. Dogs were re-fed five hours and forty minutes after administration of ceftriaxone to induce biliary excretion of ceftriaxone accumulated in the gallbladder.
    Jejunal chyme samples were immediately frozen and stored at -20°C to await analysis. The chyme samples were pretreated with perchloric-citric acid to precipitate interfering substances. Precipitates were removed by centrifugation. A reversed-phase high pressure chromatography method with UV detection was used for the quantification of ceftriaxone in supernatants.
    In the second treatment, the mutant beta-lactamase D276N was provided as enteric coated pellets filled into hard gelatin capsules 10 minutes before the injection of ceftriaxone. Enteric coated dosage forms are common among oral products in the pharmaceutical industry. Enteric coated drug products are designed to bypass the stomach as an intact form and release the contents of the dosage form into the small intestine. The reasons for applying solid enteric formulations are to protect the drug substance from the destructive action of the enzymes or low pH environment of the stomach or to avoid the drug substance-induced drug from the gastric mucosa, nausea or bleeding or to supply the drug substance. drug in undiluted form at a target site in the small intestine. Based on these criteria, enteric coated drug products can be considered as a type of delayed action dosage forms. Eudragit® L 30 D-55 polymethacrylic acid copolymer was used to achieve a pH dependent enteric coating dosage form. A single dose of enteric coated pellets containing about 0.44 mg of beta-lactamase D276N active ingredient per kg of body weight was used in the second treatment.
    The results obtained from both treatments are shown in Figure 4.
    Treatment 1 showed that high concentrations of ceftriaxone were excreted within the small intestinal tract during parenteral ceftriaxone therapy. The highest jejunal concentration (about 1500 micrograms per gram of jejunal chyme) was found 60 minutes after ceftriaxone injection. Increased jejunal ceftriaxone levels were observed after the dogs' second feeding (at a time point of 340 minutes) which indicates the accumulation of food-stimulated ceftriaxone-containing bile excretion in the gallbladder.
    Treatment 2 showed that orally administered mutant beta-lactamase D276N is able to reduce jejunal ceftriaxone levels near the limit of quantification (10 micrograms of ceftriaxone per microgram of jejunal chyme). This finding shows that the mutant beta-lactamase D276N is a potent candidate drug substance for reducing side effects related to the use of parenteral ceftriaxone. Furthermore, based on the high activities for penicillins such as ampicillin, amoxicillin and piperacillin, the mutant enzymes D276N or D276R can be used as an alternative drug substance in the beta-lactamase therapy described in WO 2008065247.
    权利要求:
    Claims (11)
    [0001]
    1. Beta-lactamase consisting of an amino acid sequence of SEQ ID NO: 1, characterized in that the beta-lactamase has an amino acid at position 243, corresponding to position 276 according to the Ambler classification replaced by an amino acid selected from the group that consists of arginine (R), histidine (H), lysine (K), asparagine (N), glutamine (Q), serine (S) and threonine (T) and hydrolyzes penicillins and/or cephalosporins.
    [0002]
    2. Beta-lactamase according to claim 1, characterized in that the amino acid in the position of SEQ ID NO: 1 corresponding to position 276 is asparagine (N).
    [0003]
    3. Beta-lactamase according to claim 1, characterized in that the amino acid in the position of SEQ ID NO: 1 corresponding to position 276 is arginine (R).
    [0004]
    4. Beta-lactamase according to any one of claims 1 to 3, characterized in that the hydrophilic amino acid in the position of SEQ ID NO: 1 corresponding to position 276 is located in an alpha helix.
    [0005]
    5. Beta-lactamase according to any one of claims 1 to 4, characterized in that the beta-lactamase further comprises at least one amino acid selected from the group consisting of Leu220 and Arg244, according to the classification of Ambler, in that Leu refers to leucine and Arg refers to arginine.
    [0006]
    6. Beta-lactamase according to any one of claims 1 to 5, characterized in that the beta-lactamase has improved catalytic efficiency for cephalosporins, compared to the beta-lactamase of SEQ ID NO: 1 with aspartic acid (D) in position corresponding to position 276 of the Ambler classification.
    [0007]
    7. Beta-lactamase according to claim 1, characterized in that the cephalosporins are selected from the group consisting of cefoperazone, ceftriaxone and cefazolin.
    [0008]
    8. Method for producing beta-lactamase as defined in any one of claims 1 to 7, characterized in that the method comprises the following steps: i) providing a gene encoding beta-lactamase as defined in any of the claims 1 to 7; ii) transforming a host cell with the gene; iii) obtaining a host cell that produces beta-lactamase; iv) beta-lactamase recovery, wherein the gene encoding beta-lactamase is a polynucleotide of any one of SEQ ID NO:s 2 or 4 modified to encode a hydrophilic amino acid selected from the group consisting of arginine (R), histidine (H), lysine (K), asparagine (N ), glutamine (Q), serine (S) and threonine (T) at position 276 according to the Ambler classification.
    [0009]
    9. Polynucleotide, characterized in that it consists of the sequence of any one of SEQ ID NO: 2 or 4, modified to encode a hydrophilic amino acid selected from the group consisting of arginine (R), histidine (H), lysine (K), asparagine ( N), glutamine (Q), serine (S) and threonine (T) at position 276 according to the Ambler classification, or encode betalactamase as defined in any one of claims 1 to 7.
    [0010]
    Pharmaceutical composition characterized comprising beta-lactamase as defined in any one of claims 1 to 7.
    [0011]
    11. Beta-lactamase according to any one of claims 1 to 7, characterized in that it is for use as a medicine.
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    法律状态:
    2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2019-12-17| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2020-02-04| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-07-07| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-11-17| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-03-02| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-06-08| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2021-07-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    2021-07-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-08-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/05/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |
    优先权:
    申请号 | 申请日 | 专利标题
    FI20105572A|FI20105572A0|2010-05-24|2010-05-24|Modified beta-lactamase and methods and uses related thereto|
    FI20105572|2010-05-24|
    PCT/FI2011/050450|WO2011148041A1|2010-05-24|2011-05-17|Modified beta-lactamases and methods and uses related thereto|
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