HK1128305B - Mutant type ii beta- lactam acylases - Google Patents

Description

Mutant type II beta-lactam acylases

The present invention relates to mutant type II beta-lactam acylases (acylases), polypeptides encoding such enzymes as well as microorganisms transformed with such polypeptides and methods for producing such mutant type II beta-lactams. The present invention also relates to a process for producing a deacylated β -lactam compound of interest using the mutant type II β -lactam acylases of the present invention.

Beta-lactam antibiotics constitute the most important group of antibiotic-resistant compounds, the clinical use of which has a long history. Among the groups, penicillin and cephalosporin are highlighted. Penicillins are naturally produced by a variety of filamentous fungi, such as Penicillium (e.g., p. chrysogenum). Cephalosporins are naturally produced by a variety of microorganisms, such as Acremonium (e.g., A. chrysogenum) and Streptomyces (e.g., Streptomyces clavuligerus).

As a result of classical strain improvement techniques, the levels of antibiotic production in p.chrysogenum and a.chrysogenum have also increased significantly over the past decades. With the increasing understanding of the biosynthetic pathways for penicillin and cephalosporin production and the advent of recombinant DNA technology, new tools have been available to improve the production strains.

Many enzymes involved in β -lactam biosynthesis have been identified and their corresponding genes have been cloned, e.g., Ingolia and Queener, Med Res Rev (1989) 9: 245-264 (biosynthetic pathway and enzymes) and Aharonowitz, Cohen, and Martin, Ann Rev Microbiol (1992) 46: 461-495 (Gene clone).

The first two steps in the biosynthesis of penicillin in chrysogenum are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L- α -aminoadipic acid) (a), L-cysteine (C) and L-valine (V) to the tripeptide LLD-ACV, followed by the cyclisation of the tripeptide to the form of isopenicillin N. The compounds contain a typical beta-lactam structure. The third step involves replacing the hydrophilic side chain of L-5-amino-5-carboxypentanoic acid with a hydrophobic side chain by the action of an Acyltransferase (AT).

In EP-A-0448180 it is described that AT-mediated enzymatic exchange reactions take place in the intracellular organelle, the microbody. A considerable amount of deacetoxycephalosporin C (DAOC) can be formed by non-precursor (non-precursory) P.chrysogenum transformants expressing deacetoxycephalosporin C synthase (EC 1.14.20.1-DAOCS, also referred to herein as expandase), a phenomenon which suggests the presence of significant amounts of penicillin N, the natural substrate for the expandase, in P.chrysogenum (Alvi et al, J Antibiol (1995) 48: 338-. However, the D- α -amino-adipoyl side chain of DAOC cannot be easily removed.

Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins, such as cephalexin (cephalexin), are produced from penicillin by multiple chemical transformations. Another reason is that only cephalosporins with a D-alpha-amino-adipoyl side chain can be fermented at present. The most important starting material in this respect, cephalosporin C, is readily soluble in water at any pH, thus suggesting that a long and wasteful separation process is to be carried out using cumbersome and expensive column technology. Cephalosporin C obtained in this way must also be converted into a therapeutic cephalosporin by a number of chemical and enzymatic transformations.

The current industry preference for processes for the preparation of 7-amino-desacetoxycephalosporanic acid (7-ADCA) involves complex chemical steps, ring expansion and derivatization of penicillin G. One necessary chemical step in the production of 7-ADCA involves the ring expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure (see, e.g., US 4,003,894). This complex chemical processing is both expensive and environmentally hazardous. It is therefore highly desirable to replace such chemical processes with enzymatic reactions, such as enzymatic catalysis, preferably performed during fermentation. The key to replacing the chemical ring-expanding process by a biological process is the central enzyme in the cephalosporin biosynthesis pathway, expandase. Expandases from the bacterium streptomyces clavuligerus (s. clavuligerus) were found to be able to perform penicillin ring expansion in some cases. When introduced into p.chrysogenum, it converts the penicillin ring structure to a cephalosporin ring structure, such as Cantwell et al, Proc R Soc long B (1992) 248: 283-289. Whereas the expandase enzyme catalyses the expansion of the 5-membered thiazolidine ring of penicillin N to the 6-membered dihydrothiazine ring of DAOC, it would of course be a logical candidate to replace the ring expansion step in chemical processes. Unfortunately, this enzyme acts on the penicillin N intermediate of the cephalosporin biosynthesis pathway, but not on the readily available inexpensive penicillins produced by p. Penicillin N is not commercially available and even when it is expanded, its D- α -amino-adipoyl side chain cannot be easily removed by penicillin acylase.

Expandases have been reported to expand penicillins with specific side chains to the corresponding 7-ADCA derivatives. This feature of expandases has been used in the art disclosed in WO93/05158, WO95/04148 and WO 95/04149. In these publications, the traditional chemical transformation of penicillin G to 7-ADCA in vitro is replaced by in vivo transformation of certain 6-aminopenicillanic acid (6-APA) derivatives in recombinant Penicillium chrysogenum strains transformed with an expandase gene. More specifically, WO93/05158 teaches the in vivo use of expandases in p.chrysogenum in combination with an adipyl side chain (also known as adipyl) as a reservoir, which is a substrate for the acyltransferase in p.chrysogenum. This resulted in the formation of adipoyl-6-APA, which was transformed by an expandase introduced into the P.chrysogenum strain, producing adipoyl-7-ADCA, which was excreted by the fungal cells into the surrounding medium.

In a subsequent step, the side chain of the corresponding 7-ADCA derivative may be cleaved off chemically or enzymatically by means of a acylase, thereby producing 7-ADCA and the corresponding side chain. Various types of microorganisms have been mentioned in the literature as being useful as acylase-producing strains for deacylating β -lactam derivatives obtained by fermentation. Examples of such acylase-producing microorganisms are certain strains of the species Escherichia coli, Kluyvera citrophila, Proteus rettgeri, Pseudomonas sp, Alcaligenes farealis, Bacillus megaterium, Bacillus sphaericus and Arthrobacter viscosus.

According to the literature, several types of acylases can be considered, based on their molecular structure and substrate specificity (Vandamm E.J.Penicillin acylases and beta-lactames "In:" Microbial Enzymes and bioconversion "E.H.Rose (Ed.), Economic microbiology 5(1980) 467. 552, Acad. Press, New York).

The type I acylase is specific for penicillin V. These enzymes are composed of four identical subunits, each having a molecular weight of 35 kDa. The complete nucleotide sequence of a cloned gene from Bacillus sphaericus has been reported (Ollson A. appl. environm. Microb. (1976), 203).

Type II acylases all share the following common molecular structure: these enzymes are heterodimers of small alpha-subunits (20-25kDa) and large beta-subunits (60-65 kDa). Type II acylases can be further divided into two groups in terms of substrate specificity.

Type IIA acylases are very specific for penicillin G and are therefore commonly referred to as penicillin acylases. Generally, they are not very specific for motifs adjacent to the amide nitrogen atom (this may be a cephamycin core (cephem) group, a penem (penem) group, an amino acid, etc.), but are substrate specific for the acyl motif of the substrate. The acyl moiety must be very hydrophobic, and is preferably benzyl or (short) alkyl. Examples of substrates which are not hydrolysed by acylases of type IIA are those having a dicarboxylic acid as the acyl moiety: succinyl, glutaryl, adipyl and aminoadipyl, the side chain of CefC. Examples of acylases of type IIA are the enzymes from Escherichia coli, Kluyvera citrophila, Proteus rettgeri and Alcaligenes faecalis.

It has been reported that acylases of type IIB hydrolyze cephalosporins (including desacetoxy derivatives) and even CefC having succinyl, glutaryl, adipyl and α -ketoadipyl as acyl motifs to a very limited extent. The group of type IIB acylases can also be subdivided into two groups based on amino acid sequence homology. These subgroups are defined herein as SY77 and SE83 groups, which are named for acylases from Pseudomonas SY77 and Pseudomonas SE83-acyII, respectively.

Matsuda et al(J.Bacteriol(1985)，1631222 the gene encoding SY 77-acylase has been cloned and sequenced, which shows that the enzyme is active on glutaryl-7-ACA, but much less active on succinyl-7-ACA and adipoyl-7-ACA. The three-dimensional structure of SY 77-precursor is known (j. biol. chem. (2002),277，2823)。

thereafter, Matsuda et al (J.Bacteriol (1987),1695815 and J.Bacteriol. (1987),1695821) the gene encoding the SE83-acyII acylase was cloned and sequenced, which showed that the enzyme was active against glutaryl-7-ACA, adipoyl-7-ACA, succinyl-7-ACA and CEFC (cephalosporin C) (descending order). All studies related to SE83 focused on the ability of the enzyme to hydrolyze 70ACA derivatives, in particular the CEFC.

It has been shown in WO91/16435 that the amino acid homology between SY77 and SE83-acyII is very low: the alpha-subunit and beta-subunit of the acylase are about 25% and 28%, respectively.

WO9512680 discloses another group of SE83 acylases from Brevundimonas diminuta, which is designated N176, which is approximately 94% homologous to SE83-acyII and tested for its CEFC-acylase activity. The third member of group SE83 is V22 from Brevundimonas diminuta V22. The amino acid sequences of these three acylases are disclosed by Aramori et al in Journal of Fermentation and Bioengineering 72, 232-243 (1991). Table 1 shows the full-length sequence identity matrix for the amino acid sequences of various type IIB acylases of the SE83 group.

TABLE 1

Type-IIB acylase	SE83acyii	N176	V22
				SE83acyii	100	94	93
N176	94	100	98
				V22	93	98	100

There have been several attempts to increase the CEFC-acylase activity of several existing acylases: WO2005014821 discloses mutants of SE 83-acylase and EP-A-1553175 discloses mutants of N176-acylase, all for increasing CEFC-acylase activity. None of the references mentioned are concerned with improving the deacylation of other acylated beta-lactam compounds of interest, such as adipoyl-7-ADCA. Thus, there remains a strong need for acylases having improved deacylation activity towards adipoyl-7-ADCA and which can be advantageously used in processes for the production of 7-ADCA from adipoyl-7 ADCA (e.g., produced by fermentation of a transformed Penicillium strain).

FIG. 1: multiple alignments of the amino acid sequences of beta-lactam acylases-type II SE83-acyii from Pseudomonas SE83 (SEQ ID No.1), N176 from Brevundimonas diminuta N-176 (SEQ ID No.2) and V22 from Brevundimonas diminuta V22(SEQ ID No. 3).

FIG. 2: conversion of adipoyl-7-ADCA by immobilized acylase at pH 8.8 and 30 ℃ (fig. 2a) and at pH 9.5 and 40 ℃ (fig. 2 b). Peudomonas SE83ACYIi wild type immobilized acylase (solid line) and mutant L161T immobilized acylase (dotted line). The rate (ml KOH/min, on the Y-axis) is plotted as a function of percent conversion (%, on the X-axis).

In a first aspect, the present invention provides a mutant type II β -lactam acylase which is a variant of a model polypeptide having type II β -lactam acylase activity, wherein the mutant β -lactam acylase has an in vitro β -lactam acylase activity towards adipoyl-7-ADCA which is improved by at least 1.5-fold compared to the model polypeptide having β -lactam acylase activity. The determination of in vitro beta-lactam acylase activity for adipoyl-7-ADCA is described in detail in the materials and methods section. More preferably, the mutant type II β -lactam acylase has an in vitro β -lactam acylase activity towards adipoyl-7-ADCA which is increased at least 2 fold, more preferably at least 2.5 fold, more preferably at least 3 fold, more preferably at least 4 fold, more preferably at least 5 fold, more preferably at least 6 fold, more preferably at least 7 fold, more preferably at least 8 fold, more preferably at least 9 fold, more preferably at least 10 fold, more preferably at least 11 fold.

In the context of the present invention, an "altered or mutant type II β -lactam acylase" refers to any enzyme having acylase activity which is not obtained from a natural source and which differs in its amino acid sequence from the complete amino acid sequence of a natural type II β -lactam acylase.

The present invention also provides a mutant type II beta-lactam acylase which is a variant of a model polypeptide having type II beta-lactam acylase activity, wherein the mutant type II beta-lactam acylase is modified at least at an amino acid position selected from the group consisting of positions 161, 270, 296, 442 and 589 or the group consisting of positions 10, 29, 274, 280, 314, 514, 645, 694, 706 and 726 or the group consisting of positions 10, 29, 161, 270, 274, 280, 296, 314, 442, 514, 589, 645, 694, 706 and 726 or the group consisting of positions 10, 29, 270, 274, 280, 442, 514, 589, 645, 694 and 726, wherein the amino acid position numbering of the amino acid sequence of SE83-acyII acylase of Pseudomonas is used (SEQ ID NO: 1).

More preferably, the present invention also provides a mutant type II β -lactam acylase which is a variant of a model polypeptide having type II β -lactam acylase activity, wherein the mutant type II β -lactam acylase has an in vitro β -lactam acylase activity towards adipoyl-7-ADCA which is increased at least 1.5-fold, more preferably at least 2-fold, more preferably at least 2.5-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, more preferably at least 10-fold, more preferably at least 11-fold compared to the model polypeptide having β -lactam acylase activity, and wherein the mutant type II β -lactam acylase is at least at a position selected from the group consisting of position 161, position b acylase, and wherein the mutant type II β -lactam acylase is a variant of a model polypeptide having type II β -lactam acylase, 270. The amino acid positions of the group consisting of positions 296, 442 and 589 or of positions 10, 29, 274, 280, 314, 514, 645, 694, 706 and 726 or of the group consisting of positions 10, 29, 161, 270, 274, 280, 296, 314, 442, 514, 589, 645, 694, 706 and 726 or of the group consisting of positions 10, 29, 270, 274, 280, 442, 514, 589, 645, 694 and 726 are modified, using the amino acid position numbering of the amino acid sequence of SE83-acyII acylase from Pseudomonas (SEQ ID NO: 1).

The present invention preferably provides mutant type II beta-lactam acylases which are variants of a model polypeptide having type II beta-lactam acylase activity, wherein the mutant type II beta-lactam acylase is modified at least at position 10 or at least at position 29, at least at position 161 or at least at position 270 or at least at position 274 or at least at position 280 or at least at position 296 or at least at position 314 or at least at position 442 or at least at position 514 or at least at position 589 or at least at position 645 or at least at position 694 or at least at position 706 or at least at position 726, wherein the amino acid position numbering of the amino acid sequence of SE83-acyII acylase from Pseudomonas (SEQ ID NO: 1) is used. In one embodiment of the present invention, there is provided a mutant type II β -lactam acylase having a single modification at position 161 or 296.

The present invention also provides a mutant type II beta-lactam acylase which is a variant of a model polypeptide having type II beta-lactam acylase activity, wherein the mutant type II beta-lactam acylase is at least a combination at position 161+270 or at least a combination at position 161+296 or at least a combination at position 161+442 or at least a combination at position 161+589 or at least a combination at position 270+296 or at least a combination at position 270+442 or at least a combination at position 270+589 or at least a combination at position 296+442 or at least a combination at position 296+589 or at least a combination at position 161+270+296 or at least a combination at position 161+270+442 or at least a combination at position 161+270+589 or at least a combination at position 161+296+589 or at least a combination at position 161+442 +296+589 or at least a combination at position 161+296+589 +296 or at least a combination at position 161+589 Or at least in the combination of positions 296+442+589 or at least in any combination of 4 positions selected from the group consisting of 161, 270, 296, 442 and 589 or at least in the combination of positions 161, 270, 296, 442 and 589, and wherein said mutant type II beta-lactam acylase may have modifications at amino acid positions other than these positions or all the possible combinations thereof mentioned before, wherein the amino acid position numbering of the amino acid sequence of SE83-acyII acylase from Pseudomonas (SEQ ID NO: 1) is used.

The model polypeptide having type II β -lactam acylase activity used herein is selected from the group consisting of: having type II β -lactam acylase activity and preferably having the amino acid sequence of SEQ ID NO: 1 (i.e. SE83-acyII acylase of the Pseudomonas species SE 83) or a nucleic acid sequence having the amino acid sequence of SEQ ID NO: 2 (i.e., N176 acylase of Pseudomonas species N176) or a polypeptide having the amino acid sequence of SEQ ID NO: 3 (i.e., the V22 acylase of Brevundimonas diminuta V22) and a polypeptide having an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence having a percent identity to SEQ ID NO: 2, or an amino acid sequence having a percent identity to SEQ ID NO: 3 and having type II β -lactam acylase activity, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%. As a model polypeptide having type II β -lactam acylase activity for use in the present invention, more preferred is a polypeptide having type II β -lactam acylase activity and having the sequence of seq id NO: 1 or a polypeptide having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having SEQ id no: 3. As model polypeptide having type II beta-lactam acylase activity, the most preferred is SE83-acyII acylase from Pseudomonas (SEQ ID NO: 1).

The present invention preferably provides a mutant of a model type II β -lactam acylase selected from the group consisting of a mutant having the amino acid sequence of SEQ ID NO: 1 and an acylase having the amino acid sequence of SEQ ID NO: 2 and an acylase having the amino acid sequence of SEQ ID NO: 3, and an acylase having an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence having a percent identity to SEQ ID NO: 2, or an amino acid sequence having a percent identity to SEQ ID NO: 3 and having type II β -lactam acylase activity, and said mutant has a modification at least at position 10 or at least at position 29, at least at position 161 or at least at position 270 or at least at position 274 or at least at position 280 or at least at position 296 or at least at position 314 or at least at position 442 or at least at position 514 or at least at position 589 or at least at position 645 or at least at position 694 or at least at position 706 or at least at position 726. In one embodiment, the present invention provides a mutant type II β -lactam acylase having a single modification at position 161 or 296, wherein the amino acid position numbering of the amino acid sequence of SE83-acyII acylase from Pseudomonas (SEQ ID NO: 1) is used.

The modification at an amino acid position may comprise a substitution of an additional amino acid selected from the group of the naturally occurring 20L-amino acids, see table 1. Alternatively, the modification at an amino acid position may comprise a deletion of an amino acid at that position. Furthermore, the modification at the amino acid position may comprise substitution of one or more amino acids on the C-terminal side or the N-terminal side of the amino acid.

TABLE 1

Amino acids	Three letter code	Single letter code
			Alanine	Ala	A
Arginine	Arg	R
			Asparagine	Asn	N
Aspartic acid	Asp	D
			Cysteine	Cys	C
Glutamic acid	Glu	E
			Glutamine	Gln	Q
Glycine	Gly	G
			Histidine	His	H
Isoleucine	Ile	I
			Leucine	Leu	L
Lysine	Lys	K
			Methionine	Met	M
Phenylalanine	Phe	F
			Proline	Pro	P
Serine	Ser	S
			Threonine	Thr	T
Tryptophan	Trp	W
			Tyrosine	Tyr	Y
Valine	Val	V

The mutant type II β -lactam acylase of the present invention is preferably selected from the group consisting of those having SEQ ID NO: 1 and an acylase having the amino acid sequence of SEQ ID NO: 2 and an acylase having the amino acid sequence of SEQ ID NO: 3 may carry one or more of the following modifications:

glutamic acid (SEQ ID NO: 1) or alanine (SEQ ID NO: 2 and SEQ ID NO: 3) at position 10 is substituted with a positively charged amino acid residue (e.g., lysine or arginine) or a small amino acid residue having a conformational preference for α -helix formation (e.g., alanine), preferably with lysine;

serine at position 29 is substituted with an amino acid having an aromatic (like) side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine) or an amino acid having a larger non-charged polar side chain or a positively charged side chain (e.g., asparagine, glutamine, arginine, and lysine), preferably with asparagine or phenylalanine;

the leucine at position 161 is substituted by smaller and more polar amino acids (e.g. threonine, serine, glycine and cysteine) or amino acids with positive charges around pH 9 (e.g. arginine and lysine), preferably by serine or threonine or glycine, most preferably by threonine;

histidine at position 274 is substituted with an amino acid residue which contains a carbon, oxygen or sulfur atom at least at position γ of the side chain and is smaller in size than histidine (e.g., leucine, isoleucine, cysteine, threonine, serine, asparagine, valine and proline), preferably with leucine, isoleucine, cysteine or threonine;

arginine at position 280 is substituted by amino acid residues that are positively charged by negative charges (e.g. aspartic acid and glutamic acid) or by amino acid residues having unbranched and uncharged polar side chains (e.g. glutamine, asparagine and serine), preferably by glutamine and asparagine, most preferably by glutamine;

the histidine at position 296 is substituted by a charged or polar amino acid or an amino acid residue capable of replacing a hydrogen bond existing in the model acylase bonded to the N-delta or N-epsilon atom of the histidine residue, for example, by asparagine and glutamine, preferably by glutamine;

isoleucine at position 314 is substituted with a smaller amino acid residue having a β -branch (e.g., valine) or an amino acid residue having a medium-sized polar side chain (e.g., glutamine, asparagine, serine, and threonine), preferably with valine or glutamine;

the glutamic acid at position 442 is substituted with an amino acid residue having no hydrophobic side chain or a small hydrophobic side chain (e.g., glycine, alanine, valine, and isoleucine), preferably with glycine;

proline at position 514 is substituted with amino acid residues having more polar and/or more flexible side chains (which can bring about additional hydrogen bonds), such as glutamine, asparagine, threonine, serine, cysteine, aspartic acid and glutamic acid, preferably with glutamine;

replacing the arginine at position 589 with an amino acid residue capable of retaining a positive charge under certain circumstances (e.g., histidine and lysine) or with an amino acid residue having an aromatic side chain capable of forming a hydrogen bond (e.g., tyrosine and tryptophan) or with an amino acid residue capable of replacing a hydrogen bond existing in the model acylase bonded to the N- δ or N-epsilon atom of the histidine residue (e.g., asparagine and glutamine), preferably with histidine;

alanine at position 645 is substituted with small amino acid residues with increased preference for β -chain formation (e.g., threonine, valine, serine, cysteine, and leucine), preferably with threonine;

the asparagine at position 694 is substituted with an amino acid residue having a side chain smaller than asparagine (e.g., alanine, threonine, serine, cysteine, valine, and glycine), preferably with threonine;

the tyrosine at position 706 is substituted with an amino acid residue having no side chain or a side chain smaller than leucine (e.g., glycine, alanine, valine, serine, cysteine, threonine, and proline), preferably with glycine;

valine at position 726 is substituted with an amino acid residue having a larger hydrophobic side chain (e.g., isoleucine, leucine, and methionine), and preferably with isoleucine.

A highly preferred embodiment of the present invention is a mutant of a model type II β -lactam acylase selected from the group consisting of a mutant having the amino acid sequence of SEQ ID NO: 1 and an acylase having the amino acid sequence of SEQ ID NO: 2 and an acylase having the amino acid sequence of SEQ ID NO: 3, and an acylase having an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence having a percent identity to SEQ ID NO: 2, or an amino acid sequence having a percent identity to SEQ ID NO: 3 and having type II β -lactam acylase activity, and said mutant carrying one of the following modifications: H296Q, L161G, L161S or L161T.

A more highly preferred embodiment of the present invention is a mutant of a model type II β -lactam acylase selected from the group consisting of a mutant having the amino acid sequence of SEQ ID NO: 1 and an acylase having the amino acid sequence of SEQ ID NO: 2 and an acylase having the amino acid sequence of SEQ ID NO: 3, and an acylase having an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence having a percent identity to SEQ ID NO: 2, or an amino acid sequence having a percent identity to SEQ ID NO: 3 and having a type II β -lactam acylase activity, and said mutant carries a mutation at a position. Most preferred are the following mutant acylases with modifications at the following combinations of 2 positions: [161+10], [161+29] or [161+694] or [161+726] or [161+274] or [161+706] or [161+442] or [161+589] or [161+314], or at a combination of 3 positions: [161+29+274], [161+29+706], [161+29+514], [161+274+589], or [161+274+706], or at a combination of the following 4 positions: [161+29+274+726], [161+274+280+314], or at a combination of the following 5 positions: [161+29+274+314+694], [161+274+280+514+726], or a modification at a combination of the following 6 positions: [161+29+280+314+645+726].

The invention preferably provides mutants of the SE83-acyII acylase of Pseudomonas (SEQ ID NO: 1) having at least the combination of positions L161+ M270 or at least the combination of positions L161+ H296 or at least the combination of positions L161+ E442 or at least the combination of positions L161+ R589 or at least the combination of positions M270+ H296 or at least the combination of positions M270+ E442 or at least the combination of positions M270+ R589 or at least the combination of positions H296+ E442 or at least the combination of positions H296+ R589 or at least the combination of positions E442+ R589 or at least the combination of positions L161+ M270+ H296 or at least the combination of positions L161+ M270+ E442 or at least the combination of positions L161+ M270+ R589 or at least the combination of positions L161+ M296 + R296 or at least the combination of positions L161+ M296 + E442 or at least the combination of positions L161+ M296 + E589 or at least the combination of positions L161+ E442 or at least the combination of positions L161+ E296 or at least the combination of positions L161+ R589 or at least the combination of positions L296 or at least the combination of positions L161+ E296 and L296 or at A modification at any combination of the 4 positions from the group consisting of L161, M270, H296, E442 and R589 or at least at a combination of positions L161, M270, H296, E442 and R589, and wherein said mutant type II β -lactam acylase may have modifications at amino acid positions other than these positions or all possible combinations thereof as described above. A highly preferred embodiment of the present invention is the Pseudomonas SE83-AcyII mutant of tables 2-5 in the examples.

In a second aspect, the present invention provides a polynucleotide encoding a mutant type II β -lactam acylase of the present invention. The present invention also provides polynucleotides encoding the alpha-subunit of the mutant type II beta-lactam acylase and polynucleotides encoding the beta-subunit of the mutant type II beta-lactam acylase. WO2005/014821 is disclosed on pages 8 and 9: the gene encoding the acylase of SE 83-group encodes a polypeptide consisting of an alpha-subunit, a spacer peptide and a beta-subunit (in this order). After undergoing transcription and translation in a host cell, the acylase derived from Pseudomonas sp.SE83 is produced in the form of an inactive single-chain polypeptide of about 84kDa in size. Then, SEQ ID NO: 1 between the amino acids at positions 230 and 231 and at positions 239 and 240, which resulted in the removal of the spacer peptide consisting of 9 amino acids and separation into a 25kDa alpha-subunit and a 58kDa beta-subunit. One α -subunit and one β -subunit are combined by hydrophobic interaction to form a heterodimer with acylase activity of about 83 kDa. As is well known to the skilled worker, the first codon (ATG) coding for the N-terminal methionine is necessary for translation initiation during protein synthesis in prokaryotes. Methionine is removed post-translationally.

The polynucleotide encoding a mutant type II β -lactam acylase or an α -subunit or β -subunit according to the invention may be any polynucleotide encoding a suitable amino acid sequence according to the invention. Alternatively, the polynucleotide of the invention may comprise a coding sequence wherein the codon usage for the various amino acids is derived from the codon usage in Pseudomonas. For example, the codon usage may be altered to suit the codon usage of a particular host cell that will be or has been transformed with a DNA fragment encoding an altered type II β -lactam acylase.

In a third aspect, the present invention provides an expression vector or cassette comprising a polynucleotide of the invention as defined hereinbefore.

In a fourth aspect, the invention provides a transformed host cell transformed with a polynucleotide of the invention or an expression vector or cassette of the invention. The transformed host cell may be used to produce the mutant type II β -lactam acylase of the present invention.

The host cells for producing the mutant type II β -lactam acylase of the present invention are preferably those known in the art to be capable of extracellular or intracellular high efficiency production of proteins or enzymes, e.g., microorganisms such as fungi, yeasts and bacteria. Examples of preferred host cells include, but are not limited to, the following genera: aspergillus (e.g. a. niger, a. oryzae), Penicillium (e.g. p. emersonii, p. chrysogenum), Saccharomyces (e.g. s. cerivisiae), Kluyveromyces (e.g. k. lactis), Bacillus (e.g. b. subtilis, b. licheniformis, b. amyloliquefaciens), Escherichia (e.g. e.coli), Streptomyces (e.g. s. clavuligerus), Pseudomonas.

In a fifth aspect, the present invention provides a process for producing a mutant type II β -lactam acylase of the invention, said process comprising culturing a transformed host cell under conditions conducive to the production of the mutant expandase, and optionally recovering the mutant expandase.

In a sixth aspect, the present invention provides a process for producing a deacylated β -lactam compound of interest, said process comprising the step of deacylating an acylated precursor of a β -lactam compound of interest using a mutant type II β -lactam acylase of the present invention. The deacylated β -lactam compound of interest may be a derivative of a naturally occurring penicillin or cephalosporin, e.g. 6-APA, 7-ACA, 7-ADCA, 7-ADAC, 7-amino-3-carbamoyloxymethyl-3-cephamycin nucleus-4-carboxylic acid (e.g. WO2004/106347) etc. Preferably, the deacylated β -lactam compound of interest is 7-ADCA or 7-ACA, most preferably 7-ADCA. The acylated precursor of the β -lactam compound of interest may have an acyl group belonging to the group consisting of dicarboxylic acids. Preferred acyl groups are succinyl, glutaryl, adipyl, alpha-ketoadipyl and aminoadipyl. More preferred are adipyl and aminoadipyl, and most preferred is adipyl. Preferred acylated precursors of the β -lactam compounds of interest are adipyl-7-ADCA, adipyl-7-ACA, aminoadipyl-7-ADCA and aminoadipyl-7-ACA, the latter known as CEFC; most preferred is adipoyl-7-ADCA.

The process of the present invention for producing a deacylated β -lactam compound of interest may be carried out in a batch mode, wherein the mutant type II β -lactam acylase is used in dissolved state in a solution comprising an acylated precursor of the β -lactam compound of interest.

More preferably, the mutant type II β -lactam acylase is used as immobilized form. This has the advantage that after the deacylation reaction the mutant type II β -lactam acylase can be recovered and reused for further deacylation reactions. In this way, the cost of using mutant type II β -lactam acylases can be significantly reduced, thereby increasing the economic attractiveness of deacylation. The conditions for the deacylation reaction and the immobilization of the enzyme are known in the art (e.g., Kallenberg, A.I.et. Adv.Synth. Catal. (2005), 347, 905. sub.926).

In a seventh aspect, the present invention relates to the use of a mutant type II β -lactam acylase of the invention in a process for producing a deacylated β -lactam compound of interest, said process comprising the step of deacylating an acylated precursor of the β -lactam compound of interest. The deacylated β -lactam compound of interest may be a derivative of a naturally occurring penicillin or cephalosporin, e.g. 6-APA, 7-ACA, 7-ADCA, 7-ADAC, 7-amino-3-carbamoyloxymethyl-3-cephamycin core-4-carboxylic acid, etc. Preferably, the deacylated β -lactam compound of interest is 7-ADCA or 7-ACA, most preferably 7-ADCA. The acylated precursor of the β -lactam compound of interest may have an acyl group belonging to the group consisting of dicarboxylic acids. Preferred acyl groups are succinyl, glutaryl, adipyl, alpha-ketoadipyl and aminoadipyl. More preferred are adipyl and aminoadipyl, and most preferred is adipyl. Most preferred acylated precursors of the β -lactam compounds of interest are adipyl-7-ADCA, adipyl-7-ACA, adipyl-7-amino-3-carbamoyloxymethyl-3-cephamycin core-4-carboxylic acid, α -ketoadipyl-7-ADCA, α -ketoadipyl-7-ACA, aminoadipyl-7-ADCA and aminoadipyl-7-ACA, the latter known as CEFC. The process of the present invention for producing a deacylated β -lactam compound of interest may be carried out in a batch mode, wherein the mutant type II β -lactam acylase is used in dissolved state in a solution comprising an acylated precursor of the β -lactam compound of interest. More preferably, the mutant type II β -lactam acylase is used in immobilized form.

Materials and methods

Preparation of acylase

Coli Top 10 cells (Invitrogen) were transformed with plasmids having the wt or mutant genes. Cells were seeded in 100ml flasks using 20ml of 2XTY medium (containing 50. mu.g/ml Zeocin) at 37 ℃ and 280 rpm. After 24 hours, 50. mu.l of the culture was inoculated at 1: 1000 into a flask with 100ml of 2XTY medium, 50. mu.g/ml Zeocin and 0.05% arabinose and cultured at 25 ℃ and 280 rpm. The culture was centrifuged and frozen at-20 ℃. To prepare cell-free extracts, the pellet was resuspended in extraction buffer (50mM Tris/HCl, 0.1mg/ml DNASel, 2mg/ml Lysozyme, 10mM DTT (dithiothreitol), 5mM MgSO4) and incubated. After 30 minutes, the extract was centrifuged and the supernatant containing the acylase activity was used for the activity measurement.

The acylase content was determined using SDS-PAGE gel electrophoresis and analytical HPLC size exclusion chromatography (performed on a TSK3000SWx1 column with 0.1M phosphate buffer (pH 7.0) as eluent). Chromatographic conditions applied: the flow rate was 1.0 ml/min and the detection was at 280 nm. By comparing the area of the observed peaks of the acylase, the protein content of different samples can be compared. Use 154350 (M)^-1.cm^-1) The molar extinction coefficient of (c) was calculated from OD280 for the amount of acylase protein. In the case of other peaks in the HPLC chromatogram, the E280 value of the sample is added to the effect of the other peaksTo correct for.

Purification of

Cell pellets from 100ml cultures were resuspended in 1ml 20mM Tris pH 8. After sonication (Soniprep 150I-BU03) on ice at an amplitude of 10 μ for 9X 10 seconds, the cell suspension was centrifuged in a microfuge tube at 14000rpm and 4 ℃ for 5 minutes. After the supernatant was adjusted to pH 5.3-5.4 with 0.1M HCl, it was centrifuged to remove the precipitate. Subsequently, the supernatant was titrated back to pH8 with NaOH. Approximately 100. mu.l of the suspension was applied to a 1ml MonoQ column equilibrated with 20mM Tris pH8 containing 10% NaCl. Buffer A (20mM Tris pH8) and buffer B (20mM Tris pH8+1M NaCl) were mixed during elution as follows: 0-1 min, 10% B/90% A; 1-5 minutes, 20% B/80% A; 5-9 minutes, 40% B/60% A; 9-12 minutes, 60% B/40% A; 12-15 min, 100% B. The peak fractions containing the acylase activity were collected and applied to a gel filtration column TSKGel3000SWx1, which had been equilibrated with 100mM sodium phosphate buffer pH 7. The peak fractions were collected and stored for later use.

Reagent

adipoyl-7-ADCA can be prepared from adipic acid and 7-ADCA by enzymatic synthesis as described in WO 9848037. Furthermore, adipoyl-7-ADCA can be prepared by chemical synthesis as described in Shibuya et al, Agric.biol.chem., 1981, 45(7), 1561-1567, but starting from adipic anhydride (instead of glutaric anhydride).

An 8% (w/v) stock of adipoyl-7-ADCA substrate was prepared in a suitable buffer and adjusted to the desired pH with 4N NaOH.

The pigment reagent was freshly prepared by dissolving 200mg of 4- (dimethylamino) -benzaldehyde (p-DMBA) in 100ml of citric acid (315.5 g of citric acid monohydrate dissolved in 1 liter of ethanol).

The activity measurements were carried out in 0.2M CHES buffer (2- (N-cyclohexylamino) ethanesulfonic acid) at a pH in the range from pH 8.0 to pH 10.0, the buffer being adjusted to the desired pH with 4N HCl or 4N NaOH, if necessary.

Measurement of acylase Activity

Mu.l of a suitable buffer are mixed with 200. mu.l of a stock of substrate in the corresponding buffer and 20. mu.l of enzyme solution and incubated for 20 minutes at the desired temperature, usually at room temperature, if not indicated otherwise. The reaction was stopped by adding 600. mu.l of pigment reagent. After 10 minutes at room temperature, the absorbance was measured at 415 nm. Blank measurements were made by adding a pigment solution to the test system prior to the addition of the enzyme. The acylase activity was calculated as the increase in optical density per minute (OD) (δ OD/min). To calculate the absolute activity, a 7-ADCA calibration line in the range of 0.1 to 1g 7-ADCA per liter was used.

K_MAssay and pH profile.

Performing K according to said test_MAnd (4) measuring. However, adipoyl-7-ADCA concentrations varied between 0.5 and 4% (w/v) adipoyl-7-ADCA.

Examples

Example 1 acylase Activity of SE83ACYii mutant

The acylase activity of the mutants was measured at pH 8.5 and pH 9.5 using adipoyl-7-ADCA as substrate. The results are shown in Table 2.

Table 2: relative activities of wild-type and mutant acylases to adipoyl-7-ADCA, the activity of wild-type at pH 8.5 was set to 1. The activity at pH 9.5 relative to the wild-type activity is in parentheses. The assay was performed at room temperature. The initial rate was measured in the presence of 4% (w/w) adipoyl-7-ADCA.

At pH 8.5 and pH 9.5, the activity of the mutant was significantly higher than the wild-type acylase. In addition, when the activity at pH 9.5 was compared with that at pH 8.5, it was clearly seen that the activity of the mutant acylase at pH 9.5 was relatively higher than that of the wild type. The pH activity curves of the mutants shifted towards higher pH, which makes these mutants particularly suitable for use at elevated pH. This is particularly important because the conversion yield will increase at higher pH, since the thermodynamic equilibrium is further towards completion of the hydrolysis reaction.

Since the concentration of adipoyl-7-ADCA will increase during the conversion of 7-ADCA to 7-ADCA and adipic acid, product inhibition may reduce the enhancing effect of the mutant as measured at the initial rate conditions. Thus, the activity of the wild type and mutant acylases was measured in the presence of 1.5% (w/v) adipic acid. Table 3 shows that under these conditions, the activity of the mutant was also significantly higher than the wild type at pH 8.5 and pH 9.5. The pH activity curves of these mutants did not migrate to higher pH.

The acylase activities of the wild-type and mutant acylases were determined using adipoyl-7-ADCA as substrate in the presence of 1.5% (w/v) adipic acid at pH 8.6, 9.1 and 9.5. The results are shown in Table 4.

Table 4 shows that the activity of the mutant was significantly higher than the wild-type acylase at pH 8.6, 9.1 and 9.5. When the activity at pH 9.1 was increased compared to that at pH 8.6, it was clearly seen that the activity of the mutant acylase at pH 9.1 was increased more significantly than that of the wild type. The pH activity curves of the mutants shifted towards higher pH, which makes these mutants particularly suitable for use at elevated pH. When pH 9.5 was compared to pH 8.6, the activity of most mutant acylases increased more at pH 9.5 than at pH 8.5 compared to the wild type.

Table 3: relative activities of wild-type and mutant acylases to adipoyl-7-ADCA, the activity of wild-type at pH 8.5 was set to 1. The activity at pH 9.5 relative to the wild-type activity is in parentheses. The assay was performed at room temperature. The starting substrate concentration was measured in the presence of 4% (w/w) adipoyl-7-ADCA. The initial activity was measured in the presence of 1.5% (w/v) adipic acid.

Table 4: relative activities of wild-type and mutant acylases to adipoyl-7-ADCA, the activity of wild-type at pH 8.6 was set to 1. In parentheses are the activities relative to the wild type activity at pH 9.1 and pH 9.5, respectively. The assay was performed at room temperature. The starting substrate concentration was measured in the presence of 2% (w/w) adipoyl-7-ADCA. The initial activity was measured in the presence of 1.5% (w/v) adipic acid.

Example 2K by comparison with the SE83ACYIi mutant _M Measurement of indicator substrate affinity

Table 5 shows K relative to wild type measured for various mutants_MThe value is obtained. Michaelis constant K_MRepresents the substrate concentration when the enzyme is operating at 50% of its maximum speed. At less than K_MAt substrate concentrations of (c), the enzyme will be slower, whereas above K_MAt high substrate concentrations, the enzyme will operate faster until the enzyme is fully saturated and operates at maximum speed at high substrate concentrations. At the end of the enzymatic conversion (when the substrate is exhausted), the low K_MIs critical to maintain comparable activity. Relative K in the mutant_MAt values < 1.00, this indicates at lower substrate concentrations (e.g.at conversion)End point), the mutant has an advantage in maintaining a relatively high activity compared to the wild type.

Table 5: in relation to K_MValues represent relative substrate affinity. K was performed using the test described previously_MAnd (4) measuring. The concentration of adipoyl-7-ADCA varied between 0.5 and 4% adipoyl-7-ADCA.

EXAMPLE 3 acylase Activity of immobilized SE83ACYIi mutant

Immobilization was carried out as described in WO97/04086, using gelatin and chitin as gelling agents and glutaraldehyde as crosslinking agent. Performance of the immobilized wild-type acylase and mutant acylase was measured by performing a complete hydrolysis of adipoyl-7-ADCA in a temperature and pH controlled 100ml reactor. Experiments were performed with 3.2% adipoyl-7-ADCA. The immobilized enzyme is provided in such a way that at least 90% conversion is obtained within 120 minutes under the desired conditions. The conversion was carried out at pH 8.8 and 30 ℃ and pH 9.5 and 40 ℃. The same amounts (by weight) of wild type and mutant acylase were used for the transformation. During the reaction, the pH was kept constant by adding 1M KOH solution. The activity of the immobilized acylase is expressed as KOH ml per minute. In fig. 2a and 2b, the rate as a function of conversion (expressed as ml KOH per minute) is shown.

The average of six times was taken at pH 8.8 and 30 ℃. The first 30% of the data converted was not included because the system was not yet fully stable, making the data largely divergent. The average of two times was taken at pH 9.5 and 40 ℃. Therefore, the variation is larger. However, the calculated slope gives a good indication of activity. Figures 2a and 2b show that the activity of the mutant acylase is significantly higher throughout the transformation period.

The stability of the immobilized acylase was determined by measuring the subsequent conversion 20 times for 180 minutes with the same batch of immobilized acylase. The rate between 30 and 50% of the conversion in each incubation was measured. The residual activity of the immobilized acylase is defined herein as the activity at the 20 th incubation relative to the first incubation.

Table 6 summarizes the results. It was observed that the stability of the mutated acylase was significantly improved compared to the wild type, especially under conditions (at high temperature and high pH) which allow a shift of the kinetic equilibrium of the hydrolysis reaction to a more complete conversion.

As a result of this higher hydrolytic activity and higher stability of the mutated acylase, the production capacity per gram of mutated acylase is also considerably increased.

Table 6: residual activity after 20 conversions carried out under the conditions indicated.

Claims (13)

1. A mutant type II beta-lactam acylase which is a variant of a model polypeptide having type II beta-lactam acylase activity, wherein,

said mutant β -lactam acylase having an in vitro β -lactam acylase activity improved by at least 1.5-fold compared to said model polypeptide having β -lactam acylase activity, and,

wherein the mutant type II β -lactam acylase is modified by a mutation selected from L161, L161+ E442, L161+ H589, L161+ E10, L161+ S29 + H274, L161+ I314, L161+ V726, L161+ Y706 + S29, L161+ S29 + P514, L161+ S29 + H274 + I314 + N694, L161+ S29 + R280 + I314 + A645 + V726, L161+ S29 + H274 + V726, L161+ H274 + R280 + I314, L161+ H274 + R589, L161+ H274 + Y760 or L161+ H274 + R514 + V726 to the amino acid sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 of SE83-acyII acylase of Pseudomonas, and

wherein the model polypeptide is SEQ ID NO: SE83-acyII acylase of Pseudomonas as shown in 1.

2. A polynucleotide encoding the mutant type II β -lactam acylase of claim 1.

3. An expression vector or cassette comprising the polynucleotide of claim 2.

4. A host cell transformed with the polynucleotide of claim 2 or the expression vector or cassette of claim 3.

5. A process for producing a mutant type II β -lactam acylase according to claim 1, said process comprising the step of cultivating the host cell according to claim 4 under conditions conducive to the production of the mutant type II β -lactam acylase, and optionally, the step of recovering the polypeptide.

6. A process for producing a deacylated β -lactam compound, said process comprising the step of deacylating an acylated precursor of a β -lactam compound using the mutant type II β -lactam acylase of claim 1.

7. The process according to claim 6, wherein the deacylated β -lactam compound is 6-APA, 7-ACA, 7-ADCA, 7-ADAC or 7-amino-3-carbamoyloxymethyl-3-cephamycin nucleus-4-carboxylic acid.

8. The process as claimed in claim 6 or 7, wherein the acylated precursor of the β -lactam compound has an acyl group with a dicarboxylic acid as acyl moiety.

9. The process according to claim 6 or 7, wherein the mutant type II β -lactam acylase is used in immobilized form.

10. Use of the mutant type II β -lactam acylase according to claim 1 for deacylating an acylated precursor of a β -lactam compound.

11. The use of claim 10, wherein the β -lactam compound is 7-ADCA or 7-ACA.

12. Use according to claim 10 or 11, wherein the acylated precursor of the β -lactam compound has an acyl group with a dicarboxylic acid as acyl moiety.

13. The use of claim 10 or 11, wherein the mutant type II β -lactam acylase is used in immobilized form.