EP0034470A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Genetically engineered microorganisms for massive production of amylolytic enzymes and process for preparing same
Assignee: Unilever Bestfoods North America
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Filing: 1981-02-12
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Granted: —
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Expires: 2001-02-12
Abstract
Abstract There is claimed, as a new composition of matter, recombinant DNA containing an amylase coding gene, prepared by the in vitro process of cleaving DNA derived from a bacterial donor microorganism and combining the resulting DNA fragments with a vector, which has been similarly cleaved, characterized in that said vector comprises a plasmid or the DNA of a derivative of phage lambda. Also claimed are genetically engineered microorganisms containing the novel DNA, processes for preparing the novel DNA, and novel amylase enzymes, their production from the DNA and their use in hydrolysing starch. The donor microorganism is preferably a Bacillus or a Klebsiella strain. The vector may be a phage or a plasmid. The host microorganism is preferably E.coli or B.subtilis. The amylase enzyme produced is preferably an alpha-amyLase, a beta-amylase or a pullulanase.
Inventors
- 👤 Charles Antoine Colson
- 👤 Pierre Emile Cornelis
- 👤 Colette Simone Digneffe
- 👤 Raoul G.P. Walon
- 👤 Corrine Walon
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 20:04
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2414; C12N9/2417; C12N9/2425; C12N9/2451; C12N9/2457; C12Y; C12Y302/00; C12Y302/01; C12Y302/01041; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/832; Y10S435/839; Y10S435/848; Y10S435/849
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
59 words
As a new composition of matter, recombinant DNA containing an amylase coding gene, prepared by the in vitro process of cleaving DNA derived from a bacterial donor microorganism and combining the resulting DNA fragments with a vector, which has been similarly cleaved, characterized in that said vector comprises a plasmid or the DNA of a derivative of phage lambda.
Claim 2
Dependent Claim
59 words
As a new composition of matter, recombinant DNA containing an amylase coding gene, prepared by the in vitro process of cleaving DNA derived from a bacterial donor microorganism and combining the resulting DNA fragments with a vector, which has been similarly cleaved, characterized in that said vector comprises a plasmid or the DNA of a derivative of phage lambda.
Claim 3
Dependent Claim
17 words
The recombinant DNA of claim 1 characterized in that the bacterial donor microorganism comprises a Bacillus strain.
Claim 4
Dependent Claim
27 words
The recombinant DNA of claim 1 characterized in that the bacterial donor microorganism is a Bacillus megaterium, a Bacillus coagulans, a Bacillus cereus or a Klebsiella pneumcniae.
Claim 5
Dependent Claim
38 words
The recombinant DNA of claims 1 or 3 characterized in that the bacterial donor microorganism is Bacillus megaterium NCIB No. 1 1568, Bacillus coagulans NCIB No. 11571, Bacillus cereus ATCC No. 31102, or Klebsiella pneumoniae ATCC No. 15050.
Claim 6
Dependent Claim
23 words
The recombinant DNA of any of the preceding claims characterized in that the vector comprises plasmid pBR 322, pACYC 184, or pC 194.
Claim 7
Dependent Claim
25 words
The recombinant DNA of any of claims 1-4 characterized in that the vector comprises phage lambda NM 590, lambda NM 781, or lambda NM 989.
Claim 8
Dependent Claim
62 words
The recombinant DNA of any of claims 1-4 or 6 characterized in that it comprises phages lambda NM 590 Amy 1, NCIB No. 11569; lambda NM 781 alpha Amy 1, NCIB No. 11572; lambda NM 781 beta Amy 1, NCIB No. 11574; lambda NM 781 Pul 1, NCIB No. 11593; lambda NM 781 Pul 2, NCIB No. 11604; variants or mutants thereof.
Claim 9
Dependent Claim
23 words
A genetically engineered microorganism containing recombinant DNA, characterized in that said recombinant DNA comprises the recombinant DNA of any of the preceding claims.
Claim 10
Dependent Claim
32 words
The microorganism of claim 8 characterized in that it is capable of producing, under suitable conditions of cultivation, amylase in a quantity substantially greater than that produced by the original donor microorganism.
Claim 11
Dependent Claim
15 words
The microorganism of claims 8 or 9 characterized in that it comprises E.coli or B.subtilis.
Claim 12
Dependent Claim
83 words
The microorganism of claims 8, 9 or 10 characterized in that it comprises E.coli CL 7001 (pCP 1), NCIB No.11570; E.coli CL 7002 (pCP 2), NCIB No.11573; E.coli CL 7003 (lambda alpha Amy 1), NCIB No. 11586; E.coli CL 7004 (pCP 3), NCIB No. 11602; E.coli CL 7005 (lambda beta Amy 1), NCIB No. 11603; E.coli CL 7006 (pCP 4), NCIB No. 11605; E.coli CL 7007 (lambda Pul 2), NCIB No.11606; B.subtilis CL 8001 (pCH 1), NCIB No. 11629; variants or mutants thereof.
Claim 13
Dependent Claim
66 words
The process of preparing the recombinant DNA of claim 1 comprising cleaving and bonding the DNA's of a bacterial donor microorganism capable of producing amylase and a vector, introducing them into a suitable host and screening and selecting the resultant clones on the basis of the presence of an amylase coding gene, characterized in that the vector comprises the DNA of a derivative of phage lambda.
Claim 14
Dependent Claim
65 words
The process of claim 12 including the additional step of extracting and cleaving the DNA of a selected clone, subcloning into another vectur, and again screening and selecting on the basis of the presence of an amylase coding gene, the other vector comprising a plasmid or the DNA of another derivative of phage lambda; said subcloning optionally being followed by one or more subsequent subclonings.
Claim 15
Dependent Claim
36 words
The process of claims 12 or 13 characterized in that the screening is conducted by plating the clones on a starch-containing culture medium and staining said medium with iodine by exposing the plates to iodine vapors.
Claim 16
Dependent Claim
45 words
The process of claims 12 or 13 characterized in that the amylase coding gene comprises a pullulanase- coding gene, and the screening is conducted by plating the clones on a BBL Trypticase medium containing pullulan, whereby positive clones are evidenced by opaque rings surrounding same.
Claim 17
Dependent Claim
23 words
The process of claims 12 or 13 characterized in that the donor microorganism comprises Bacillus megaterium, Bacillus coagulans, Bacillus cereus or Klebsiella pneumoniae.
Claim 18
Dependent Claim
35 words
The process of claims 12 or 13 characterized in that the donor microorganism comprises Bacillus megaterium NCIB No. 11568 Bacillus coagulans NCIB No. 11571, Bacillus cereus ATCC Na.. 31Z02, or Klebsiella pneumoniae ATCC No. 15050.
Claim 19
Dependent Claim
27 words
The process of claims 12 or 13 characterized in that the phage to which the donor DNA is bonded comprises lambda NM 590 or lambda NM 781.
Claim 20
Dependent Claim
30 words
The process of claim 13 wherein the DNA of the selected clones is bonded with a plasmid, characterized in that the plasmid comprises pBR 312, pACYC 184 or pC 194.
Claim 21
Dependent Claim
32 words
The process of claim 13 wherein the DNA of the selected clones is bonded with the DNA of another derivative of phage lambda, characterized in that the phage comprises lambda NM 989.
Claim 22
Dependent Claim
17 words
The process of producing amylase comprising cultivating under amylase-producing conditions, the genetically engineered microorganism of claim 8.
Claim 23
Dependent Claim
28 words
The process of claim 21 wherein the recombinant DNA is present in the form of a lytic phage and the cultivation is performed in an infected bacterial host.
Claim 24
Dependent Claim
36 words
The process of claim 21 wherein the recombinant DNA is present in the form of a phage in a lysogenic E.coli and the cultivation is performed so as to induce the phage multiplication by heat treatment.
Claim 25
Dependent Claim
33 words
The process of claim 21 wherein the recombinant DNA is present in the genetically engineered microorganism in the form of a plasmid, and the cultivation is performed by growth to the stationary phase.
Claim 26
Dependent Claim
36 words
The process of claim 24 wherein the plasmid is a derivative of pBR 322 or pACYC 184 and an amplification is performed by cultivation in the presence of chloramphenico] prior to growth to the stationary phase.
Claim 27
Dependent Claim
17 words
Amylase, characterized in that it is produced by cultivating the genetically engineered-microorganism of any of claims 8-11.
Claim 28
Dependent Claim
28 words
The amylase of claim 26 characterized in that it comprises an alpha-amylase and the recombinant DNA of said microorganism contains an amylase coding gene obtained from Bacillus megaterium.
Claim 29
Dependent Claim
25 words
The amylase of claims 26 or 27 characterized in that the recumbinant DNA of said microorganism comprises phage lambda NM590 Amy 1, NCIB No. 11569.
Claim 30
Dependent Claim
21 words
The amylase of claims 26 or 27 characterized in that said microorganism comprises E.coli CL 7001 (pCP 1), NCIB No. 11570.
Claim 31
Dependent Claim
29 words
The amylase of claim 26 characterized in that it comprises a thermoduric alpha-amylase and the recombinant DNA of said microorganism contains an amylase coding gene obtained from Bacillus coagulans.
Claim 32
Dependent Claim
27 words
The amylase of claims 26 or 30 characterized in that the recombinant DNA of said microorganism comprises phage lambda NM 781 alpha Amy 1, NCIB No. 11572.
Claim 33
Dependent Claim
41 words
The amylase of claims 26 or 30 characterized in that said microorganism comprises E.coli CL 7002 (pCP 2), NCIB No. 11573; E. coli CL 7003 (lambda alpha Amy 1), NCIB No. 11586; or B.subtilis CL 8001 (pCH 1), NCIB No.11629 .
Claim 34
Dependent Claim
28 words
The amylase of claim 26 characterized in that it comprises a beta-amylase and the recombinant DNA of said microorganism contains an amylase coding gene obtained from Bacillus cereus.
Claim 35
Dependent Claim
26 words
The amylase of claims 26 or 33 characterized in that the recombinant DNA of said microorganism comprises, phage lambda NM781 beta Amy 1, NCIB No. 11574.
Claim 36
Dependent Claim
32 words
The amylase of claims 26 or 33 characterized in that said microorganism comprises E.coli CL 7004 (pCP 3), NCIB No. 11602 or E.coli CL 7005 (lambda beta Amy 1), NCIB No. 11603.
Claim 37
Dependent Claim
28 words
The amylase of claim 26 characterized in that it comprises a pullulanase and the recombinant DNA of said microorganism contains an amylase coding gene obtained from Klebsiella pneumoniae.
Claim 38
Dependent Claim
36 words
The amylase of claims 26 or 36 characterized in that the recombinant DNA of said microorganism comprises phage lambda NM 781 Pul 1, NCIB No. 11593, or phage lambda NM 781 Pul 2, NCIB No. 11604.
Claim 39
Dependent Claim
31 words
The amylase of claims 26 or 36, characterized in that said microorganism comprises E.coli CL 7006 (pCP 4), NCIB No. 11605, or E.coli CL 7007 (lambda Pul 2), NCIB No. 11606.
Claim 40
Dependent Claim
22 words
The process of enzymatically hydrolyzing starch, characterized in that the enzyme employed is an amylase of any of claims 26 - 38.
References Cited (Backward Citations)
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