Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-02-11
2001-06-12
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S183000, C435S325000, C435S320100, C536S023200, C536S023100
Reexamination Certificate
active
06245524
ABSTRACT:
The present invention relates to an enzyme useful in the synthesis of penicillins from intermediates involved in penicillin biosynthesis. The present invention also relates to processes for the preparation of the enzyme and DNA coding for the enzyme.
The biochemical pathway for Penicillin G is disclosed in the literature (Queener (1990) Antimicrobial Agents and Chemotherapy 34(6), 943-948; Martin (1992) J. Industrial Microbiol 9, 73-90; Luengo (1995) J. Antibiotics 48 (11), 1195-1212). The pathway has been the subject of considerable study with a view to increasing the yield (titre) in fermentation processes.
Phenylacetate (PAA) and Phenoxyacetate (POA) are activated to the corresponding CoA thioesters in
Penicillium chrysogenum
by a ligase enzyme (e.g. PAA-CoA ligase). These thioesters are then used for the biosynthesis of Penicillin G in the case of PAA and penicillin V in the case of POA. PAA-CoA ligase is therefore thought to be essential in the biosynthesis of these commercially important therapeutic antibiotics.
An enzyme from
Pseudomonas putida
having PAA-CoA ligase activity has been isolated (J. Biol. Chem. 267(12), 7084-7090 (1990) in a purification procedure involving ammonium sulphate precipitation and potassium chloride elution from a DEAE-Sephacel column. The enzyme has a molecular weight of 48 kDa +/−1 kD, a pH optimum of 8.2 and is involved in PAA catabolism.
Attempts to assay an enzyme having PAA-CoA ligase activity from
P. chrysogenum
by the hydroxymate method (a colorimetric assay detecting phenylacetyl-hydroxamate or phenoxyacetyl-hydroxamate at 540 nm) have been reported by Kogekar & Deshpande (1983) Ind. J. Biochem. Biophys 20, 208-212 and by Brunner & Rohr (1975) Methods Enzymol 43, 476-481; however other workers (Martinez-Blanco et al (1992) J. Biol. Chem. 26(8), 5474-5481) are of the view that the protein had not been purified or the activity characterised in detail. Moreover the latter authors failed to find the enzyme by the reported procedure.
WO 96/10085 (Gist Brocades, published Apr. 4, 1996) reviews these and other attempts at isolating a PAA CoA ligase which operates in the penicillin pathway. In WO 96/10085 an acyl-CoA enzyme synthetase is described as being obtained from a strain of
Penicillium chrysogenum
B 10 which is held by PanLabs (USA). Among the specific properties attributed to the enzyme are the following: molecular weight about 50 kDa (as determined by gel filtration), pH optimum pH 8 to 8.5 (low activity at pH 7 or below), temperature optimum at 40° C., pI higher than 7.25. Importantly the enzyme can be purified by ammonium sulphate precipitation. It has a fairly wide specificity (ie. it is able to catalyze the formation of phenoxyacetyl-coenzyme A, phenylacetyl-coenzyme A, adipyl-coenzyme A and hexanoyl-coenzyme A from Mg 2+, ATP, CoASH, and phenoxyacetic acid, phenylacetic acid, adipic acid or hexanoic acid respectively but does not show any significant activity towards acetic acid. Also it is said that the enzyme is stabilised by reducing agents. A high concentration of ammonium sulphate or glycerol also stabilises the enzyme. No indications of purity are given for the enzyme activity obtained by the methods disclosed and no sequence or N-terminal sequence is given for the enzyme and the corresponding DNA is not characterised or its sequence given.
In spite of all these efforts little is known about the authentic PAA-CoA ligase enzyme that operates in Penicillium sp. in vivo . It had been speculated that the enzyme responsible may be involved in primary metabolism (Smith et al (1990) Biotechnology 8, 39-41). Martinez-Blanco et al (1992) ibid selected one possible candidate enzyme, acetyl CoA synthetase, and purified it from
P. chrysogenum
on the basis of acetyl CoA synthetase activity. They were able to show that in addition to forming the CoA derivative of acetate, acetyl CoA synthetase was able to activate several fatty acids (C2 -C8) and some aromatic molecules (including PAA) in vitro.
The acetyl CoA synthetase gene has been sequenced (International Patent WO92/07079, Gouka et al (1993) Appl. Microbiol. Biotechnol. 3, 514-519, Martinez-Blanco et al (1993) Gene 130, 265-270) and has been shown to have homology with other acetyl CoA synthetases from fungi. However mutations in this gene selected by fluoroacetic acid do not appear to alter penicillin production levels (International Patent WO92/07079) suggesting that in vivo another enzyme is actually responsible for activation of PAA.
In the present invention a direct assay for PAA-CoA ligase activity has been developed in conjunction with a specific purification protocol and this has allowed purification of and subsequent cloning of what is believed to be the authentic PAA-CoA ligase. The enzyme isolated by the present invention has a different N-terminal amino acid sequence to the acetyl CoA synthetase mentioned above and possesses a number of different properties (e.g. molecular weight) indicating that a different enzyme has been isolated from all the enzymes attributed with this role to date. The properties characteristic of the enzyme isolated in the present invention include an absolute dependence on CoASH as substrate while the enzyme isolated by Kogekar & Deshpande (1983) ibid was assayed in conditions where CoASH was omitted. This and other differences between the isolated proteins (e.g. pH optima and other characteristics given in the examples below) show that the enzyme in the present invention is different to any of those described in the prior art. It is believed that the present work represents the first isolation of a pure form of the enzyme PAA CoA ligase from Penicillium sp. In particular the presence of an SKI C-terminal peptide is consistent with this enzyme having a real role in penicillin biosynthesis. The enzyme of the present invention differs from that in WO 96/10085 mentioned above in that it has a different molecular weight and the enzyme of the present invention, unlike that of WO 96/10085 is sensitive to ammonium sulphate precipitation and chloride salts.
Accordingly, the present invention provides an enzyme having PAA-CoA ligase activity obtainable from
Penicillium chrysogenum
by culturing, harvesting and sonicating the mycelium, removing cell debris and fractionating the sonicate by anion-exchange chromatography, followed by hydrophobic interaction chromatography, affinity chromatography with substrate elution and gel filtration chromatography wherein the active chromatographic fractions are detected using a PAA and coenzyme A dependent assay.
The enzyme is preferably in purified form, advantageously in substantially pure form.
The enzyme of this invention has an apparent molecular mass of 63 kDa (by SDS PAGE)
Preferably the enzyme includes the sequence of N-terminal amino acids
VFLPPKESGQLDP
In particular, the enzyme comprises the sequence of amino acids in FIG.
1
/ID SEQ 1
In a further aspect of the invention there is provided a method of preparing an enzyme having PAA-CoA ligase activity by culturing Penicillium sp. followed by extraction and purification wherein the active fractions are detected using a PAA and Co-enzyme A dependent assay. In particular the Penicillium sp. is
P. chrysogenum
and the mycelium is treated by sonication, followed by fractionation by anion-exchange, hydrophobic interaction, affinity and gel filtration chromatography so as to provide an approximate 1000 fold increase in purity.
The enzyme can be used in in vitro biotransformations. For example for CoA ester synthesis or for penicillin synthesis when mixed with acyl-CoA: 6-APA acyltransferase. The in vitro biotransformations can be carried out using whole cells, cell free extracts, permeabilised cells or the isolated enzyme from the microorganisms or any of these in immobilised form.
Where the biotransformation is carried out using whole cells, the microorganism may be in the form of a growing culture, resting culture, washed mycelium, immobilised cells or protoplasts.
When cell-free extracts are used these are suitably produced by shear
Gledhill Linden
Greaves Philip Andrew
Griffin John Patrick
Carlson Karen Cochrane
Kerekes Zoltan
Kinzig Charles M.
SmithKline Beecham p.l.c.
Venetianer Stephen
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