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AssigneeNone recorded
InventorsWolf-Dieter Deckwer, Rolf-Joachim Mueller, Joop van den Heuvel, Ilona Kleeberg, Ute Widow
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The invention relates to DNA sequences coding for enzymes that cleave or hydrolyse ester groups, to the production of those enzymes by genetically recombinant microorganisms and to the use of those enzymes and microorganisms for the degradation of polymers containing ester groups, especially aliphatic-aromatic polyesters.
A DNA sequence that comprises a DNA sequence selected from among:
A DNA sequence that comprises a DNA sequence selected from among:
A DNA sequence that comprises a DNA sequence selected from among:
An amino acid sequence that comprises an amino acid sequence selected from among
An amino acid sequence that comprises an amino acid sequence selected from among
An amino acid sequence that comprises an amino acid sequence selected from among
An amino acid sequence that comprises an amino acid sequence selected from among
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The invention relates to DNA sequences coding for enzymes that cleave or hydrolyse ester groups (hereinafter also EGCEs or EGCE-1 and EGCE-2), to the production of those enzymes by genetically recombinant microorganisms and to the use of those enzymes and microorganisms for the degradation of polymers containing ester groups.
Polymers and macromolecular materials that can be biologically degraded in a controlled manner are becoming increasingly important. A number of such products is already available on the market. Amongst those new types of product, polymers containing ester groups (for example polyesters, polyester urethanes, polyester amides) occupy a central role. Examples of biodegradable polyester-based plastics are poly(β-hydroxybuty-rate-co-β-hydroxyvalerate), poly(ε-caprolactone) and poly(buty-lenesuccinate).
Since, owing to their molecular size, polymers are unable to pass through the outer membrane of microbe cells, the first, and generally the rate-determining, step of degradation is molecular weight reduction (depolymerisation) by extracellular enzymes. Polyesters are potentially bio-degradable because the ester bonds fundamentally represent points of attack for such extracellular hydrolysing enzymes.
Working-up of the Chromosomal DNA of Thermomonospora fusca
1 g of mycelium is thawed and homogenised in 5 ml of TE (10 mM Tris-HCl, 1 m EDTA pH 8.0). 10 mg of lysozyme are added and the solution is mixed for 30 seconds. Incubation is then carried out at 37° C. for from 30 to 60 minutes. 1.2 ml of 0.5 M EDTA and 13 μl of Pronase (20 mg/ml) are then added and the whole is mixed and incubated for a further 5 minutes. After the addition of 0.7% sodium dodecyl sulphate (0.43 ml of a 10% strength SDS stock solution), the whole is mixed and incubated at 37° C. for from 1 to 2 hours. Incubation can be brought to an end when the lysate becomes “clear”. The extract is purified by means of phenol extraction with 10 ml of Rotiphenol/TE, pH 7.5 (Roth, Art. 0038.2). The clear supernatant is then removed with a Pasteur pipette and transferred to a new vessel. The procedure is repeated once, whereupon extraction is carried out twice with 10 ml of chloroform each time. The protein-free supernatant is treated for from 30 to 60 minutes at 37° C. (or overnight at 4° C.) with 4 μl/ml of a stock solution RNAse having a concentration of 10 mg/ml. The volume is made up to 5 ml with TE, and 500 μl of 3 M NaCl and 3.5 ml of isopropanol are added. The DNA precipitation is carried out on ice or over-night at 4° C. The DNA pellet is centrifuged at 4000 g for 10 minutes and is then washed with 70% ethanol. After drying in air, the DNA can be taken up in 500 μl of TE and stored at −20° C. Up to 1 mg of DNA is obtained from 1 g of frozen mycelium.
8 Nested PCR of the chromosomal DNA with degenerate oligonucleotides Reaction A 0.5 μg T.-f.-DNA x μl 10× PCR-buffer 5 μl 2 mM dNTPs 5 μl Primer 1F 100 pmol/μl 1 μl Primer 2R 100 pmol/μl 1 μl Taq-polymerase (2-5 U) 0.5 μl MilliQ-water y μl final volume 50 μl PCR programme: denaturing for 5 min at 95° C., 30 cycles of 1 minute at 95° C., 1 minute at 55° C. and 2 minutes at 72° C. Extension 5 min at 72° C. After end of programme, store at 4° C. Reaction B Product of Reaction A 10.0 μl 10× PCR-buffer 4.0 μl 2 mM dNTPs 5.0 μl Primer 3F 100 pmol/μl 1.0 μl Primer 4R 100 pmol/μl 1.0 μl Taq-polymerase (2-5 U) 0.5 μl MilliQ-water 28.5 μl final volume 50.0 μl