EP0876489A2 - Acides nucleiques codant des polypeptides presentant l'activite de la lipase d'absidia - Google Patents

Acides nucleiques codant des polypeptides presentant l'activite de la lipase d'absidia

Info

Publication number
EP0876489A2
EP0876489A2 EP97904764A EP97904764A EP0876489A2 EP 0876489 A2 EP0876489 A2 EP 0876489A2 EP 97904764 A EP97904764 A EP 97904764A EP 97904764 A EP97904764 A EP 97904764A EP 0876489 A2 EP0876489 A2 EP 0876489A2
Authority
EP
European Patent Office
Prior art keywords
acid sequence
nucleic acid
seq
set forth
absidia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97904764A
Other languages
German (de)
English (en)
Inventor
Randy M. Berka
Karuppan C. Boominathan
Thomas Sandal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Novozymes Inc
Original Assignee
Novo Nordisk AS
Novo Nordisk Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS, Novo Nordisk Biotech Inc filed Critical Novo Nordisk AS
Publication of EP0876489A2 publication Critical patent/EP0876489A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • nucleic acid sequence endogenous to an Absidia strain which is capable of hybridizing under medium stringency conditions with (i) the nucleic acid sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (ii) any of their complementary strands;
  • nucleic acid sequence which is capable of hybridizing under medium stringency conditions with (l) the nucleic acid sequence set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or (ii) any of their complementary strands;
  • Absidia griseola var. iguchii PCR product Both PCR products appear to be approximately 870 bp in size.
  • the lipolytic enzymes of the present invention may have activity towards triglycerides (lipase activity, E C 3 1 1.3), e g , 1,3 -positionally specific lipase activity
  • the present invention relates to isolated nucleic acid sequences encoding polypeptides with lipase activity which are capable of hybridizing under high, medium, or low stringency conditions with an oligonucleotide probe which hybridizes under the same conditions with the nucleic acid sequence set forth in SEQ ID NO.l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO 7, or SEQ LD NO:9, its complementary strand or a subsequence thereof (J.
  • the present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences capable of directing the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokmase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3 -phosphogly cerate kinase gene.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokmase gene
  • ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes
  • Saccharomyces cerevisiae 3 -phosphogly cerate kinase gene Other useful promoters for yeast host cells are descnbed by Romanos et
  • Aspergillus oryzae TAKA amylase Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-hke protease.
  • the control sequence may also be a signal peptide coding region, which codes for an ammo acid sequence hnked to the ammo terminus of the polypeptide which can direct the expressed polypeptide mto the cell's secretory pathway.
  • the 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked m translation reading frame with the segment of the coding region which encodes the secreted polypeptide
  • the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide
  • the foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the lipase relative to the natural signal peptide coding region normally associated with the coding sequence
  • a processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlm and Ogrydziak, 1994, Yeast 10: 67-79; Fuller et al , 1989, Proceedings of the National Academy of Sciences USA 86: 1434-1438; Julius et ai, 1984, Cell 37 1075-1089; Julius et ai, 1983, Cell 32: 839-852).
  • the host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
  • a mammalian cell such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
  • Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
  • yeast host cell is a Saccharomyces carlsbergensis
  • Example 2 PCR Amplification of Absidia griseola NN000987 and Absidia griseola var. iguchii NN000591 Lipase Gene Segments Based on the ammo acid sequences of the Absidia griseola NN000987 lipase and the Absidia griseola var.
  • coli Y1090ZL cells and the lipase genes were subsequently excised from the ⁇ ZipLox vector as pZL 1 -derivatives (D'Alessio et al , 1992, Focus® 14: 76).
  • the recombinant DNA segments were inserted within the phagemid pZLl portion of the vector, and the phagemid harboring the cloned insert was recovered in the autonomously replicating pZLl using in vivo excision by infection of E coli DHlOBzip cells (Life Technologies, Gaithersburg, MD)
  • the lipase clones isolated in this manner were prepared for DNA sequence analysis using a Wizard 373 DNA pu ⁇ fication kit (Promega, Madison, WI)
  • DNA sequencing of the lipase clones descnbed in Example 4 was performed with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the pnmer walking technique with dye-terminator chemistry (Giesecke et al , 1992, Journal of Virol Methods 38 47-60) Oligonucleotide sequencing pn ers were synthesized on an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions
  • Absidia lipases share extensive amino acid sequence homology between each other with 87-96% identity, and limited homology to Rhizomucor miehei lipase (SEQ ID NO 15) with 53-55% identity and Humicola lanuginosa lipase (SEQ ID NO 16) with 22-24% identity as shown in Table 3 and Figure 8 Table 3 Amino acid sequence similarity among lipases from Absidia species, R miehei, and H lanuginosa
  • the plasmid also contained the Aspergillus nidulans amdS gene as a selectable marker for fungal transformations
  • the following primers were used for PCR amplification: Forward Primer: 5'-CCCATTTAAATATGCGTTTTTATTCAGTAGTATCAT-3' (SEQ ID NO: 17)
  • the coding region of the A. sporophora-variabihs lipase was amplified using the original genomic clone as the template for Pwo polymerase (Boehringer-Mannheim Biochemicals, Indainapohs, IN) in a PCR reaction which contained the following: 61 ml sterile water, 10 ml of diluted template DNA (ca. 5 ng/ml), 1 ml pnmer 1 (ca. 30 pmol. dATGATGCATTCTCATTTTGTAGTCTTATTG, SEQ ID NO:19), 1 ml primer 2 (ca.
  • transformants After a 48 hour incubation at 30 °C, 80 of 84 transformants showed distinct clea ⁇ ng zones on the tributynn agar plates indicating production of extracellular lipase activity. Ten of these transformants were further tested in shake flask cultures of MY50 medium containing 50 g of maltodextrin, 2 g of MgS0 4 -7H 2 0, 10 g of KH 2 P0 4J 2 g of citric acid, 2 g of K 2 S0 4 , 0.5 ml of trace metals solution, 10 g of yeast extract, 2 g of urea pH 6.0 per liter.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Detergent Compositions (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention se rapporte à des séquences d'acides nucléiques isolées codant des polypeptides présentant l'activité de la lipase d'Absidia. L'invention se rapporte également à des constructions d'acides nucléiques, à des vecteurs et à des cellules hôtes comprenant les séquences d'acides nucléiques, ainsi que des procédés de production des polypeptides. L'invention se rapporte enfin à des compositions comprenant ces polypeptides.
EP97904764A 1996-01-24 1997-01-21 Acides nucleiques codant des polypeptides presentant l'activite de la lipase d'absidia Withdrawn EP0876489A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US1361696P 1996-01-24 1996-01-24
US13616P 1996-01-24
PCT/US1997/000598 WO1997027276A2 (fr) 1996-01-24 1997-01-21 Acides nucleiques codant des polypeptides presentant l'activite de la lipase d'absidia

Publications (1)

Publication Number Publication Date
EP0876489A2 true EP0876489A2 (fr) 1998-11-11

Family

ID=21760853

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97904764A Withdrawn EP0876489A2 (fr) 1996-01-24 1997-01-21 Acides nucleiques codant des polypeptides presentant l'activite de la lipase d'absidia

Country Status (5)

Country Link
EP (1) EP0876489A2 (fr)
JP (1) JP3333521B2 (fr)
CN (1) CN1209842A (fr)
AU (1) AU1747797A (fr)
WO (1) WO1997027276A2 (fr)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10507639A (ja) * 1994-10-26 1998-07-28 ノボ ノルディスク アクティーゼルスカブ 酵素界面活性剤組成物
US6936289B2 (en) 1995-06-07 2005-08-30 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
US6406723B1 (en) 1997-04-09 2002-06-18 Danisco A/S Method for preparing flour doughs and products made from such doughs using glycerol oxidase and lipase
EP2302044A1 (fr) 1998-11-27 2011-03-30 Novozymes A/S Variants d'enzyme lipolytique
US7226770B2 (en) 2000-04-28 2007-06-05 Novozymes A/S Lipolytic enzyme variant
ATE426668T1 (de) 2001-01-10 2009-04-15 Novozymes As Variante eines lipolytischen enzyms
WO2002094123A2 (fr) 2001-05-18 2002-11-28 Danisco A/S Procede permettant d'ameliorer la qualite de la pate et du pain
MXPA05007654A (es) 2003-01-17 2005-09-30 Danisco Metodo.
GB0405637D0 (en) 2004-03-12 2004-04-21 Danisco Protein
PT3219806T (pt) 2004-03-25 2020-06-30 Novozymes Inc Métodos para degradar ou converter polissacarídeos da parede celular de plantas
CN102533440B (zh) 2004-07-16 2017-09-19 杜邦营养生物科学有限公司 用酶使油脱胶的方法
US10030215B2 (en) 2014-04-15 2018-07-24 Novozymes A/S Polypeptides with lipase activity and polynucleotides encoding same
CN104928193A (zh) * 2015-06-26 2015-09-23 云南大学 一株犁头霉菌株及其应用
CN114292829A (zh) * 2015-07-06 2022-04-08 诺维信公司 脂肪酶变体以及编码它们的多核苷酸
EP3384019B1 (fr) 2015-12-01 2020-06-24 Novozymes A/S Procédés de production de lipases
CN109337828B (zh) * 2018-11-29 2021-10-12 中国科学院成都生物研究所 一种水稻秸秆发酵处理工艺
EP3994255A1 (fr) 2019-07-02 2022-05-11 Novozymes A/S Variants de lipase et compositions de ceux-ci

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3634195A (en) * 1969-09-08 1972-01-11 Miles Lab Production of lipase
DK88892D0 (da) * 1992-07-06 1992-07-06 Novo Nordisk As Forbindelse

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9727276A3 *

Also Published As

Publication number Publication date
WO1997027276A3 (fr) 1997-10-02
AU1747797A (en) 1997-08-20
JPH11510395A (ja) 1999-09-14
CN1209842A (zh) 1999-03-03
JP3333521B2 (ja) 2002-10-15
WO1997027276A2 (fr) 1997-07-31

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