Background of the invention Penicillins and cephalosporins are the most widely used antibacterial agents. Both are secondary metabolites which are industrially produced by filamentous fungi like Penicillium chrysogenum and Acremonium chrysogenum, respectively, in several enzymatic steps (Miller and Ignolia (1989), Mol. Microbiol. 3: 689-695).

The main steps in the biosynthetic pathways leading to penicillins and cephalosporins have been elucidated in the past 30 years. The pathways share two enzymatic steps. In the first step a tripeptide is formed from a-aminoadipic acid, cysteine and valine. The enzyme which is responsible for this step is b-(L-a-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS). In the second step the ACV is cyclised by the action of isopenicillin N synthase (IPNS). The reaction product is isopenicillin N (IPN), a compound which contains the typical ß-lactam ring structure and which possesses antibacterial activity. The biosynthesis of penicillin involves an unique third and last step in which the a-aminoadipic acid side- chain of IPN is exchanged for a hydrophobic side-chain. The hydrophobic side-chains commonly used in industrial production are phenylacetic acid and phenoxyacetic acid, yielding penicillin G and penicillin V, respectively. The side-chain exchange has been proposed to be a reaction catalysed by a single enzyme referred to as acyltransferase (AT). Cephalosporins are formed from IPN in a number of steps, including epimerisation of IPN to penicillin N, ring expansion and hydroxylation.

One of the precursor amino acids, the toxic amino acid L-cysteine, is present in a relatively low concentration

inside the cell (Jrgensen (1993), Ph. D. Thesis, Technical University of Denmark, Lyngby, Metabolic fluxes in P. chrysogenum). Biosynthesis of L-cysteine in fungi may occur via two different pathways, the transsulfuration and/or the direct sulfhydrylation pathway. L-Cysteine, synthesised via the transsulfuration pathway, is formed by cleavage of L- cystathionine derived from the intermediate L-homocysteine, which is formed from L-methionine or from O-acetyl-L- homoserine. In the direct sulfhydrylation pathway acetylation of L-serine yields 0-acetyl-L-serine which, in the presence of sulphide, is converted to L-cysteine by action of the enzyme O-acetyl-L-serine sulfhydrylase.

Reducing the energy costs of production and/or increasing the internal pool of the amino acid precursors may improve the yield of ß-lactam compounds. In a theoretical study it has been demonstrated that the yield of penicillin on glucose would be substantially higher when L-cysteine is synthesised via the direct sulfhydrylation pathway as compared with the biosynthesis via the transsulfuration pathway (Hersbach et al. (1984), in: Biotechnology of industrial antibiotics, E. J. Vandamme (ed.), Marcel Dekker, NY, pp. 45-140; Jorgensen et al. (1995), Biotechnol.

Bioengineer. 46: 117-131).

In the ß-lactam producing organisms Aspergillus nidulans and Acremonium chrysogenum both pathways exist, but the direct sulfhydrylation pathway is the main route in A. nidulans, contrary to A. chrysogenum where the transsulfuration pathway dominates (Pieniazek et al. (1973), Biochim. Biophys. Acta 297: 37-47; Treichler et al. (1979), in: Genetics of industrial microorganisms, Sebek and Laskin (eds.), AMS, Washington, pp. 97-104).

Up to now, only the presence of the transsulfuration pathway has been reported in Penicillium chrysogenum.

Moreover, Treichler et al. (1979) and Döbeli and Nuesch (1980, Antimicrob. Agents Chemother. 18: 111-117) claimed that no activity of O-acetyl-L-serine sulfhydrylase could be detected in P. chrysogenum. Furthermore, mutants disturbed in the transsulfuration pathway were unable to grow on inorganic

sulphate (Dobeli and Nüesch, 1980), the main source of sulphate during industrial fermentations.

The present invention now surprisingly shows that P. chrysogenum also contains an enzyme associated with the direct sulfhydrylation pathway.

Summary of the invention The present invention discloses a novel enzyme obtainable from P. chrysogenum which is an 0-acetyl-L-serine sulfhydrylase and the use of said enzyme to improve a ß- lactam production process.

According to the present invention, it has been shown that said O-acetyl-L-serine sulfhydrylase is able to catalyze the formation of L-cysteine from O-acetylserine via the direct sulhydrylation pathway.

The novel OAS sulfhydrylase can be used in connection with improvement of the biosynthesis of various -lactam antibiotics.

Also contemplated is a DNA fragment comprising a DNA sequence encoding said novel enzyme exhibiting O-acetyl-L- serine sulfhydrylase activity, and an expression cassette comprising said DNA sequence.

It is a further object of the invention to provide a vector or transformation vehicle comprising said expression cassette.

Also contemplated according to the invention is a microbial cell comprising said expression cassette or said vector or transformation vehicle.

Detailed description of the invention The present invention discloses an enzyme obtainable from P. chrysogenum which is an O-acetyl-L-serine sulfhydrylase (OAS sulfhydrylase). The present invention for the first time shows that P. chrysogenum contains this enzyme activity associated with the direct sulfhydrylation pathway.

The OAS sulfhydrylase of the invention is able to convert 0- acetyl-L-serine (OAS) and sulphide into cysteine and acetate.

The present invention thereby provides the possibility to increase the synthesis of L-cysteine via the direct sulfhydrylation pathway and, consequently, to improve the yield of ß-lactam compounds, especially penicillins or cephalosporins, on glucose.

The OAS sulfhydrylase of the invention is purified from P. chrysogenum biomass using conventional protein purification techniques. For instance, the enzyme is purified from cell-free extracts of P. chrysogenum by precipitation with ammonium sulphate and by elution from columns packed with various gels (e. g. Sephadex G-75@ and DEAE-Sepharose CL- 6B@). The OAS sulfhydrylase enzyme activity is associated with a protein which in an isolated form comprises two polypeptides, being approximately 59 and 68 kDa in size, respectively, as measured under denaturing conditions. The enzyme has been found to be specific for O-acetyl-L-serine, no O-acetyl-L-homoserine could be used as a substrate.

In the context of the present invention, OAS sulfhydrylase enzymes include mature proteins or precursor forms thereof as well as functional fragments thereof which essentially have the activity of the full-length polypeptide.

Further contemplated according to the invention are homologues of OAS sulfhydrylase. Such homologues comprise enzymes exhibiting OAS sulfhydrylase activity with an amino acid sequence exhibiting a degree of identity of at least between 50% and 70%, preferably between 70% and 80%, more preferably up to 100%, with the amino acid sequence of the OAS sulfhydrylase from P. chrysogenum.

The degree of identity may be determined by conventional methods (see for instance Altshul et al. (1986), Bull. Math. Bio. 48: 603-616 and Henikoff and Henikoff (1992), Proc. Natl. Acad. Sci. USA 89: 100915-10919). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and"blosum 62"scoring matrix of Henikoff and Henikoff (supra).

Alternatively, a homologue of the OAS sulfhydrylase according to the invention may be an OAS sulfhydrylase encoded by a nucleotide sequence hybridizing with an

oligonucleotide probe prepared on the basis of the nucleotide sequence of said enzyme exhibiting OAS sulfhydrylase activity as obtained from P. chrysogenum.

Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using standard detection procedures (e. g. polymerase chain reaction (PCR) technology, Southern blotting).

Homologues of the present polypeptide may have one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions which do not adversely affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain (see in general Ford et al.

(1991), Protein Expression and Purification 2: 95-107).

Examples of conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).

It will be apparent to persons skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active enzyme. Amino acids essential to the activity of the OAS sulfhydrylase of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells (1989), Science 244: 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e. g. OAS sulfhydrylase activity) to identify amino

acid residues that are critical to the activity or the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labelling (see, for example, de Vos et al. (1992), Science 255: 306-312, Smith et al.

(1992), J. Mol. Biol. 224: 899-904 and Wlodaver et al. (1992), FEBS Lett. 309: 59-64).

The homologue may be an allelic variant, i. e. an alternative form of a gene that arises through mutation, or an altered enzyme encoded by the mutated gene, but having substantially the same activity as the OAS sulfhydrylase of the invention. Hence mutations can be silent (no change in the encoded enzyme) or may encode OAS sulfhydrylases having altered amino acid sequence.

Further homologues of the present OAS sulfhydrylase are those which are immunologically cross-reactive with antibodies raised against the OAS sulfhydrylase obtainable from P. chrysogenum.

A further aspect of the present invention relates to nucleotide sequences encoding the OAS sulfhydrylase proteins according to the invention.

A DNA fragment comprising a nucleotide sequence encoding the OAS sulfhydrylase according to the invention may suitably be of genomic or cDNA origin.

Said DNA fragment may for instance be obtained by preparing a genomic or cDNA library, screening said library for clones comprising DNA sequences coding for all or part of the polypeptide of the invention by hybridization using synthetic oligonucleotide probes, selecting hybridizing clones and identifying clones containing the DNA fragment encoding the. OAS sulfhydrylase according to the invention, in accordance with standard techniques (cf. Sambrook et al.

(1989), Molecular Cloning: A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

Suitable synthetic oligonucleotide probes are obtained by preparing degenerate oligonucleotide sequences from amino acid sequences, for instance from amino acid sequences as obtained from the protein as purified from P. chrysogenum or

from conserved amino acid boxes as present within homologous proteins from various other organisms.

Partial amino acid sequences can be obtained from the N-terminus of the full-length or mature protein and/or from the N-termini of internal fragments. In the event that the enzyme preparation is not substantially homogeneous, or in the event that a peptide mixture has been prepared by chemical or enzymatical fragmentation of the purified enzyme, partial amino acid sequences can be directly determined from protein or peptide bands separated by electrophoresis of a protein or peptide preparation on a denaturing SDS gel (Matsudaira (1990), Methods Enzymol. 182: 602-613).

The DNA fragment comprising the genomic or cDNA sequence encoding the OAS sulfhydrylase of the invention may also be prepared by PCR, using the oligonucleotide sequences as described hereinabove as primers, for instance using the method as described in US 4,683,202 or Saiki et al. (1988), Science 239: 487-491).

In addition, a DNA fragment comprising a cDNA encoding the OAS sulfhydrylase of the invention may be obtained by complementing a specific mutant strain containing a defective OAS sulfhydrylase gene with a cDNA library prepared from an organism of interest.

In a preferred embodiment of the invention, the DNA fragment comprising the DNA sequence encoding the OAS sulfhydrylase of the invention is obtainable from a filamentous fungus belonging to genus of Aspergillus, Penicillium or Acremonium, preferably from a strain belonging to the species of P. chrysogenum, A. chrysogenum, or A. nidulans, more preferably from a strain of P. chrysogenum.

In a further aspect, the present invention relates to an expression cassette comprising a genomic DNA or cDNA sequence encoding said enzyme according to the invention exhibiting OAS sulfhydrylase activity.

In the expression cassette, the DNA sequence encoding' the polypeptide of the invention is operably linked to additional segments required for transcription of the DNA.

The term,"operably linked"indicates that the segments are arranged so that they function in concert for their intended

purposes, e. g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.

The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

An overview of fungal promoters can be found in, for instance, Applied Molecular Genetics of filamentous fungi (Kinghorn, Turner (eds.) (1992), Blackie, Glasgow, UK).

Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al. (1985), EMBO J. 4: 2093-2099) or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral a-amylase, A. niger acid stable a-amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, P. chrysogenum ACV synthetase, P. chrysogenum isopenicillin N synthase, P. chrysogenum acyltransferase, P. chrysogenum phosphoglycerate kinase, P. chrysogenum gene Y. Preferred are the A. niger glucoamylase or P. chrysogenum promoters.

It is often advantageous to use identical or similar promoters to regulate two or more of the biosynthetic genes in order to obtain a synchronized production of the intermediates involved in the ß-lactam antibiotic synthesis.

If the production of intermediates is not synchronized an accumulation of intermediates (bottle neck) might arise.

Consequently the production of -lactam antibiotic may be retained.

In an embodiment of the invention the promoter of said OAS sulfhydrylase gene is replaced by the promoter from another gene involved in the biosynthesis of 0-lactames.

The DNA fragment encoding the OAS sulfhydrylase of the invention may also, if necessary, be operably connected to a' suitable terminator.

The expression cassette comprising the DNA sequence encoding said enzyme exhibiting OAS sulfhydrylase activity may be incorporated in a recombinant vector or transformation

vehicle. The vector into which the expression cassette of the invention is inserted may be any vector which may conveni- ently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i. e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e. g. a plasmid.

Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.

The recombinant vector may also comprise a selectable marker, e. g. a gene the product of which complements a defect in the host cell, or one which confers resistance to a drug.

Examples of the latter are phleomycin or hygromycin. For filamentous fungi, additional selectable markers include amdS, pyrG, argB, niaD, facA, and sC (Applied Molecular Genetics of Filamentous fungi (ibid.), Biotechnology of Filamentous fungi, Finkelstein, Ball (eds.) (1992), Butterworth-Heinemann, Boston).

It is also possible to introduce the expression cassette comprising the DNA sequence encoding the OAS sulfhydrylase of the invention into a host cell on a DNA fragment separate from the vector comprising a selectable marker, by a so-called co-transformation process.

The present invention also relates to hosts being transformed with an expression cassette comprising the DNA sequence encoding the OAS sulfhydrylase of the invention or with a vector or transformation vehicle comprising said expression cassette. Preferred hosts are bacteria belonging to the genus Streptomyces, Nocardia or Escherichia, or filamentous fungi belonging to the genus Aspergillus, Acremonium or Penicillium.

According to the invention, several options exist for the improvement of the fermentative production of a-lactam compounds.

For instance, such improved processes are provided if the production of -lactam antibiotics of interest cakes

place in the presence of increased OAS sulfhydrylase activity.

According to the invention, such an improved process for the production of a P-lactam antibiotic comprises fermentation of a microorganism capable of producing said/3- lactam antibiotic and, optionally, recovery of said ß-lactam antibiotic, wherein said fermentation takes place in the presence of an increased OAS sulfhydrylase activity, said increase being obtained by modifying said microorganism and/or modifying said fermentation conditions, in comparison to the OAS sulfhydrylase activity present when fermentation occurs with the original microorganism and/or under the original fermentation conditions.

An increased OAS sulfhydrylase activity is defined as an enhanced conversion of O-acetyl-L-serine towards cysteine, in comparison to the unmodified original microorganism and/or the original fermentation conditions. Said original microorganism, capable of producing ß-lactams, lacks or only has a relatively low OAS sulfhydrylase activity and/or said original fermentation conditions do not give any or result in only a relatively low OAS sulfhydrylase expression.

The increased expression of OAS sulfhydrylase activity may be accomplished by any suitable way.

As an example, according to an embodiment of the invention, the OAS sulfhydrylase activity may be increased by modulation of the physical conditions of the fermentation process, such as temperature and pH. Another possibility is subjecting the microorganism to compounds or agents leading to an increased expression of OAS sulfhydrylase. The nature of said compound or agent depends on e. g. the promoter used for initiating the expression of the OAS sulfhydrylase.

Further, by interfering with the cellular control mechanisms controlling the OAS sulfhydrylase expression, increased expression of OAS sulfhydrylase activity can be achieved.

In an embodiment of the invention said OAS sulfhydrylase activity or increased OAS sulfhydrylase activity is obtained by modifying said microorganism.

This modification can be done by well known procedures, such as introducing at least one copy of an

expression cassette or a recombinant vector, comprising a DNA sequence encoding said OAS sulfhydrylase activity, into said original microorganism to be fermented. The introduction of said expression cassette or recombinant vector into a host cell may the performed according to, for instance, Applied Molecular Genetics of Filamentous fungi (supra) or Biotechnology of Filamentous fungi (supra).

It is also possible to modulate the cellular localization of the OAS sulfhydrylase. For instance, it is possible to target the OAS sulfhydrylase to the cytosol, the cytosol being the site where the first steps of a-lactam biosynthesis occur. In addition, other enzymes of the direct sulfhydrylation pathway may also be targeted to the cytosol.

It is also possible that the OAS sulfhydrylase activity or increased OAS sulfhydrylase activity is obtained by random mutagenesis of said microorganism.

Furthermore, a modification leading to increased OAS sulfhydrylase activity may be obtained by amino acid substitutions, deletions or additions of the OAS sulfhydrylase enzyme.

The DNA sequence encoding the OAS sulfhydrylase of the invention may thereby be derived from a species which is similar to or different from the microorganism in which it is to be introduced.

According to the invention, the above mentioned recipient or host microorganism preferably is a strain belonging to the genus Penicillium chrysogenum, Penicillium notatum, Acremonium chrysogenum, Aspergillus nidulans, Nocardia lactamdurans or Streptomyces clavuligerus.

In an embodiment of the invention, an increased OAS sulfhydrylase activity is used in combination with other modifications in biosynthetic routes leading to the production of 0-lactames.

In a preferred embodiment of the invention, an increased OAS sulfhydrylase activity is used in combination with other modifications in the direct sulfhydrylation pathway. Other modifications in the direct sulfhydrylation pathway include an increase in serine-acetyl transferase activity and/or a removal of product inhibition (by e. g.

cysteine) of serine-acetyl transferase and/or OAS sulfhydrylase. It is also possible to combine an increased OAS sulfhydrylase activity with additional modifications in the transsulfuration pathway, e. g. a disruption of one or more genes encoding enzymes of the transsulfuration pathway.

In an embodiment of the invention the expression of said OAS sulfhydrylase activity is synchronized to the expression of other genes belonging to the ß-lactam biosynthetic pathway. Said genes may e. g. be the pcbAB, pcbC and/or penDE genes.

The above mentioned ß-lactam compound preferably is a penicillin, a cephalosporin or a cephamycin, more preferably a penicillin or a cephalosporin.

In each situation of an increased amount of OAS sulfhydrylase activity, an increased production of the amino acid precursor cysteine would result. An increased production of cysteine subsequently would result in an increased production of the tripeptide ACV, in turn resulting in a significantly elevated yield of a ß-lactam compound of interest. Due to formation of less byproduct (s), the recovery and purification of ß-lactam compounds is facilitated.

All the above mentioned advantages make the processes of producing industrial important ß-lactam antibiotics more efficient.

Example 1 Cysteine production from O-acetyl-L-serine and sodium sulphide using cell-free extracts from P. chrysogenum Media and organisms Rice containing spores of P. chrysogenum strain Wis54- 1255 (ATCC 28089), or any other suitable P. chrysogenum strain, was prepared as described by Lein (Lein (1986), in: Overproduction of microbial metabolites, Vanek and Hostalek (eds.) Buttersworth, Boston, pp. 105-139). Conidiospores were' inoculated at 105-106 conidia/ml in a production medium containing (g/1): glucose. H2O, 5; lactose. H20,80; (NH2) 2CO, 4.5; (NH4) 1. 1; Na2SO4,2.9; KH2PO4,5.2; K2HPO4.3H2O, 4.8; trace elements solution (citric acid. H2O, 150; FeSO4.7H, O, 15;

MgSO,. 7H2O, 150; H3BO3,0.0075; CuS04.5H2O, 0.24; CoSO,. 7H2O, ZnS04.7H2O, 1.5; MnS04. H20,2.28; CaCl2. 2H2O, O. 99), 10 (moi/1) looi potassium phenoxyacetate solution, pH 7,75 (ml/1). (pH before sterilization 6.3).

The culture was incubated at 25 OC in an orbital shaker at 280 rpm for 96 hours. At the end of the fermentation, the medium fluid was removed by filtration and the mycelium was washed with cold 0.9% NaCl. Afterwards, the filter with mycelium was frozen in liquid nitrogen and freeze-dried.

Preparation of cell-free extract Freeze-dried mycelium was mortared and resuspended at 50 mg/ml in 0.2 M KH2PO4, pH 7.2. Mycelia were disrupted by stirring the suspensions for 30 min at 300-500 rpm. The resulting supernatant was used as cell-free extract.

O-Acetyl-L-serine sulfhydrylase assay Formation of L-cysteine was measured according to Gaitonde (Gaitonde (1967) Biochem. J. 104: 627-633). The reaction mixture contained 0.2 moles of DTT, 0.04 moles of pyridoxal 5'-phosphate, 2.5 moles of 0-acetyl-L-serine and 40 Al of cell-free extract. After the 0-acetyl-L-serine sulfhydrylase reaction was initiated by adding 10 yl of Na2S in a 0.5 M KH2PO4-buffer (pH 6.8), the final volume of the reaction mixture was 100 Ul with a KH2PO4 concentration of 0.15 M at pH 7.2. The reaction mixture was incubated at 25 °C for 15 minutes and then stopped by adding 200 Fl of Gaitonde's reagent (solution of 250 mg ninhydrin in a mixture of 4 ml concentrated HCl and 16 ml glacial acetic acid). The test tubes were placed on a boiling water bath for 5 minutes and cooled on an ice-bath. Subsequently, 400 gel of 96% ethanol was added to the test tubes and a pink coloured complex formed, which was quantified spectrophotometrically at 560 nm. The absorbance at 560 nm was shown to be proportional with the cysteine concentration, and thus the reaction velocity of OAS sulfhydrylase could be determined.

OAS sulfhydrylase activity was detected in a cell-free extract of P. chrysogenum up to 0.30 Umol/mg protein/h.

Example 2 Purification of O-acetyl-L-serine sulfhydrylase from P. chrysogenum Cultivation conditions The enzyme can be obtained from any penicillin producing culture of P. chrysogenum. Convenient protocols for purification are fed-batch cultivations of high-producing strains as P. chrysogenum P2 (ATCC 48271), or continuous cultivation carried out as described by Christensen et al.

(Renno et al. (1992) Curr. Genet. 21: 49-54; Christensen et al. (1995) J. Biotechnol. 42: 95-107).

To generate a large amount of biomass for easy purification, continuous culture cells were harvested when steady state was obtained, at a specific growth rate of 0.05 h-1, by withdrawing samples of approximately 700 ml from the bioreactor. Each sample was rapidly filtered, washed with a cold 0.9% NaCl-solution and then transferred to a plastic petri-dish and stored in liquid nitrogen.

O-Acetyl-L-serine sulfhydrylase assay The assay conditions were as descibed in Example 1.

Quantitative determination of the protein content was done by using the method of Bradford (Bradford (1976), Anal. Biochem.

72: 248-254).

Purification procedure Cell-free extract : 68 g (wet weight) of mycelium was homogenized at 3-4 °C in a coffee mill and transferred to a mortar containing liquid nitrogen and 250 ml of 0.1 M Tris buffer (pH 7.2) containing 0.002 M DTT (buffer A). After grinding for 5 minutes, the suspension was centrifuged at 20,000 g for 30 minutes and the supernatant was used for the purification.

Protamine sulphate precipitation: The supernatant was adjusted to 0.07 W (w/v) protamine sulphate by adding 6 ml of 3 % (w/v) protamine sulphate solution. After 20 minutes stirring at 3-4 °C the solution was centrifuged at 20,000 g

for 30 minutes. The supernatant was used for further purification.

Ammonium sulphate precipitation : 62.5 g of ammonium sulphate was slowly added to the supernatant and 40 % saturation was achieved. After 30 minutes stirring at 3-4 °C the solution was centrifuged at 20,000 g for 30 minutes and 36.7 g of ammonium sulphate was slowly added to the supernatant until everything was dissolved in order to achieve 60 o saturation. Again the suspension was centrifuged after stirring at 3-4 °C for 30 minutes. The resulting precipitate was dissolved in 18 ml of buffer A.

Sephadex G-75 gel filtration : This fraction was loaded onto a Sephadex G-75 column (63.4 cm x 5.3 cm2) equilibrated with buffer A containing 0.05 M potassium citrate. The gel filtration was carried out at a flow rate of 20 ml/h, and fractions of 3 mL were collected. SDS-PAGE (12.5 0-.), protein determination, and the 0-acetyl-L-serine sulfhydrylase assay were carried out after elution of the column. Fraction 39 to 46 containing the 0-acetyl-L-serine sulfhydrylase activity were pooled and used for further purification.

DEAE-Sepharose CLUBS chromatography: The pool from the Sephadex G-75@ column was loaded onto a DEAE-Sepharose CL-6B column (12.7 cm x 5 cm2) equilibrated with buffer A.

The column was washed with 120 ml of buffer A and a NaCl- gradient from 0 M to 0.5 M NaCl with a total volume of 500 ml, was pumped through the column. The chromatography was carried out at a flowrate of 60 ml/h and 4 ml-fractions were collected. After elution of the column, 12.5 W SDS-PAGE, protein determination and the O-acetyl-L-serine sulfhydrylase assay were carried out. Fraction 22 and 23 were pooled and used for determination of the kinetics and temperature dependence. The 93 times purified protein showed a specific activity of 15.4 ymol/mg protein/h.

Molecular weight All the fractions from the large DEAE-Sepharose CL-6B column containing O-acetyl-L-serine activity showed two bands on a silver stained SDS-PAGE gel. SDS-PAGE was performed

according to Laemmli (Laemmli (1970), Nature 277: 680-685). A horizontal electrophoresis system by LKB Bromma was applied (2117 Multiphor II horizontal electrophoresis unit, 2219 Multiphor II thermostatic circulator). Low molecular weight standards of Bio-Rad were used and detection of proteins was carried out by silver staining the gel just after electrophoresis was completed. The 7.5t SDS-PAGE gel showed two bands with an estimated molecular weight of 59 kDa and 68 kDa, respectively.

Example 3 Biochemical properties of O-acetyl-L-serine sulfhydrylase from P. chrysogenum O-Acetyl-L-serine sulfhydrylase assay The assay conditions were as described in example 1.

Influence of the temperature Temperature dependence of the specific activity of purified O-acetyl-L-serine sulfhydrylase was examined at 19.0 °C, 24.5 °C, 29.0 °C, 36.0 °C, 43.7 °C, and 54.8 °C.

Apparently the activity of 0-acetyl-L-serine sulfhydrylase has a temperature optimum at 45 °C. When the data were plotted according to the Arrhenius equation a straight line appears in the approximate temperature range of 24 °C to 45 °C.

Influence of the pH The influence of pH on the specific activity of 0- acetyl-L-serine sulfhydrylase was examined from cell-free extracts at seven different pH values in the pH range of 5.0 to 7.4. O-Acetyl-L-serine sulfhydrylase exhibited the maximum specific activity at a pH level above pH 7.0, verifying that the purification of 0-acetyl-L-serine sulfhydrylase at pH 7.2 took place in an environment where the enzyme manifests its maximum specific activity. The pH dependence of 0-acetyl-L- serine sulfhydrylase above pH 7.4 was not examined due to the O-to N-acetyl shift, giving N-acetylserine, occurring

rapidly at pH values above 7.6 (Flavin and Slaughter (1965), Biochemistry 4: 1370-1375).

Kinetics The kinetics of the O-acetyl-L-serine sulfhydrylase reaction followed Michaelis-Menten kinetics. By varying the O-acetyl-L-serine concentration at a fixed sulphide concentration, the k value of O-acetyl-L-serine was estimated. By varying the O-acetyl-L-serine concentration at a constant sulphide concentration of 0.01 M, the Vmax was estimated to 6.6 ymoles/mgP/h, and the k value was found to be 1.3 mM. It was not possible to derive a K value with respect to sulphide.

Because of the instability of O-acetyl-L-serine sulfhydrylase, the Vmax value was lower than the reaction rates observed from the different fractions of the DEAE- Sepharose CL-6B column. The kinetic studies were carried out 90 hours after the purification of O-acetyl-L-serine sulfhydrylase was accomplished, and due to the linear decrease of the enzymatic activity versus time, the kinetic data could be adjusted, and hereby Vmax was estimated to 14.9 ymoles/mgP/h.

Example 4 Substrate specificity of the purified O-acetyl-L-serine sulfhydrylase from P. chrysogenum O-Acetyl-L-homoserine sulfhydrylase assay O-Acetyl-L-homoserine was synthesised according to Wiebers and Garner (Wiebers and Garner (1967), J. Biol. Chem.

242: 5644-5649). Examination of O-acetyl-L-homoserine sulfhydrylase was done by an assay similar to the one of O- acetyl-L-serine sulfhydrylase. The only difference in the reaction mixture was the presence of 0-acetyl-L-homoserine instead of O-acetyl-L-serine. Formation of L-homocysteine was terminated by freezing the reaction mixture in liquid nitrogen, and subsequently detecting the L-homocysteine by an HPLC assay originally developed by Fahey and Newton (Fahey

and Newton (1987), Meth. Enzymol. 143: 85-109) and further optimised by Jrgensen (1993).

No homocysteine could be detected after incubation of the purified O-acetyl-L-serine sulfhydrylase with 0-acetyl-L- homoserine. Therefore, it was concluded that the enzyme purified from P. chrysogenum was a genuine 0-acetyl-L-serine sulfhydrylase.