ZA200500794B - Novel lipases and uses thereof - Google Patents

Description

/

NOVEL LIPASES AND USES THERE OF

The invention relaxtes to newly identified polynucleotide sequence comprising a gene that encodes a novel lipolytic enzyme from Aspergillus niger. The invention features the full length nucleotide sequence of the novel egene, the cDNA sequenece comprising the full length coding sequence of the novel lipolytic enzyme as well as the amino acid sequence of the full-length lipolytic enzyme and functional equivalerts thereof. The invention al-so relates to methods of using thnese enzymes in industrial processes and methods eof diagnosing fungal infections. Also included in the inventi on are cells transformed wit a polynucleotide according to the invention and cells whereain a lipolytic enzyme according to the invention is genetically modified to enhance its . activity and/or level of expression.

Background of the invention

Baked products ssuch as bread are prepared from a dough which is usually mde from the basic ingredientss (wheat) flour, water and optionally =salt. Depending on the balked products, other ingrediemts added may be sugars, flavours etceteras. For leavemned products, primarily baker™s yeast is used next to chemical leeavening systems such azs a combination of an acid (generating compound) and bicarbona-te.

In order to improsve the handling properties of the domugh and/or the final properties of the baked products the=re is a continuous effort to develop processing aids with improving properties. Processing aiids are defined herein as compourmds that improve the hanclling properties of the dough aand/or the final properties of the baked products. Dough propemrties that may be improved comprise machineability, gas retaining capability, reduced stickin-ess, eiasticity, extensibility, m oldability etcetera. Properties of thes baked products that mawy be improved comprise loaf volume, crust crispiness, crumb texture and softness, fla—vour relative staleness and sielf life. These dough and/or baked product improving processsing ’ aids can be divided into two groups: chemical additives a, enzymes (also referred t-o as baking enzymes). :

Yeast, enzyme=s and chemical additives are generally added separately t© the dough. Yeast may be added as a liquid suspension, im a compressed form oer as actiwe dry (ADY) or instant dry ye ast (IDY). The difference between these yeast formulations is the water- and yeast dry matter content. Liquid yeast has a yeast dry matter content of less than 25% (w/v). Crean yeast is a particular form of liquid yeaast and has a dry matter content between 17 and 23% (w/v). Compressed yeast has a yeast dry matter comtent between 25-35% (wiv) while the dry yeast formulations have a yeast dry matter coratent between 92-88% (W/V).

Enzymes may be added in a dry, e.g. granulated form or in liguid form. The cheamical additives are in most casses added in powder form. Also, processing aid cormpositions which are tailored to specific baking applications, may be composed of a declicated mixture of chemical additivees and enzyme.

The preparation of a dough from the ingredients and processing =aids described above is well known in the art and comprises mixing of said ingredients awd processing aids and one or more moulding and fewmentation steps.

The preparation of baked products from such doughs Is also well kmown in the art ans=d may comprise molding and shaping and further fermentation of the dough followed by ba king at required temperatures and Ioaking times.

Chemical additives with imp roving properties comprise oxidising amgents such as as corbic acid, bromate and azodicaarbonate, reducing agents such as L-cysteine and glutathione, emulsifiers acting as dough conditioners such as diacetyl tamtaric esters of meono/diglycerides (DATEM), sodium stearoyl lactylate (SSL) or calcium steearoyl lactylate (C=SL), or acting as crumb softeners ssuch as glycerol monostearate (GMS) etceteras, fatty m-aterials such as triglycerides (fat) or- lecithin and others.

As a result of a consumer-d riven need to replace the chemical ad-ditives by more natural products, several baking enzymes have been developed with doug h and/or baked pr-oduct improving properties and which are used in all possible combinatiorss depending on thme specific baking application con ditions.” Suitable enzymes include starch. degrading ewzymes, arabinoxylan- and other hemicellulose degrading enzymes, cellulose degrading emnzymes, oxidizing enzymes, fatty nrmaterial splitting enzymes, protein degraading, modifying o-¥ crosslinking enzymes. Sr .

Starch degrading enzymes are for instance endo-acting enzymess such as alpha- a mylase, maltogenic amylase, puliu fanase or other debranching enzymes and exo-acting e=nzymes that cleave off glucose (armyloglucosidase), maltose (beta-amylase), maltotriose, mnaltotetracse and higher oligosaccharides.

Arabinoxylan- anc other hemicellulose degrading enzymes are for instance= xylanases, pentosanases, tmemicellulase, arabinofuranosidasee, glucanase and others. :

Cellulose degrading enzymes are for instance cellulase, cellobiohydrolase anc] beta-glucosidase. = Oxidizing enzymems are for instance glucose oxidase, hexose oxidase, pyranosee oxidase, sulfhydryl oxidase , lipoxygenase, laccase, polyphen=ol oxidases and others.

Fatty material spliting enzymes are for instanc e lipolytic enzymes such aus triacylglycerol lipases, phosspholipases (such as As, Az, B, C =and D) and galactolipases.

Protein degradingg, modifying or crosslinking enzym-es are for instance endo-actirmg 1€@ proteases (serine proteas es, metalloproteases, aspartyl preoteasss, thiol proteases), exeo- acting peptidases that cle=ave off one amino acid, or dipepticie, tripeptide etceteras from the

N-terminal (aminopeptidasses) or C-terminal (carboxypepticiases) ends of the polypepticle chain, asparagines Of glutamine deamidating enzymes such as deamidase amd peptidoglutaminase or cromsslinking enzymes such as transg Wtaminase. y & Baking enzyme-s may conviently be produced in microorganisms. Microbmial baking enzymes are available from a variety of sources Bacillus spec. are a comm on source of bacterial enzymes, whereas fungal enzyme-s are commonly produced in

Aspergillus spec.

Baking enzyme s may be used in a manifold of baked goods. The term "bated =0 goods” is herein definead as to comprise bread products such as tin bread, loavess of bread, French bread ams well as rolls, cakes, pies, muffins, yeast raised and cake doughnuts and the like.

In the above porocesses, it is advantageous to use baking enzymes that are obtained by recombinarmt DNA techniques. Such recom binant enzymes have a nunmber of advantages over thesir traditionally purified counterpawrts. Recombinant enzymes rmay be produced at a low ceost price, high yield, free from contaminating agents like bactzeria or viruses but also free from bacterial toxins or contamimating other enzyme activities .

Co ‘ it is an object of the invention to provide nove=l polynucleotides encoding rovel lipolytic enzymes with improved properties. A further object is to provide naturally~ and recombinantly produced lipolytic ‘enzymes as well ass recombinant strains producing these. Also fusion potwypeptides are part of the inventieon as well as methods of maaking and using the polynucleotides and polypeptides according to the invention. 1t is also an object of the invention to provide novel lipolytic enzymes, which solve at least one of the above-mentioned probtems or fo provide novel lipolytic enzymess, which have one or more improved properties if used in dough and/or baked products, selected from the group of increased strength of the dough, increased elasticity” of the dough, increased stability of the dough, reduced stickiness of the dough, improved extensibility of the dough, improved machineability of the dough, increased volume ©f the baked product, improved crumb structure of the baked product, improved softness of the baked product, improved flavour of the baked product, improved anti- staling of the baked product, improved colour of the baked product, improved crust of the baked product or which have a broad substrate specificity.

Summary of the invention

The invention provides for novel polynucleotides encoding novel lipolytic enzymes. More in particular, the invention provides for polynucleotides having a nucleotide sequence that hybridises preferably under highly stringent conditions to a sequence selected from the group consisting of SEQIDNO: 1,2,4,5,7, 8, 10, 11, 13, 14, 16, 17,19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and. 38. Consequently, the invention provides nucleic acids that are more thar 40% such as about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, ©8% or 99% homologous to the sequences selected from the group consisting of

SEQ ID NO: 1,2, 4,5, 7,8,10, 11,13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35_ 37 and 38.

In a more preferred embodiment the invention provides for such an isolated polynuacleotide obtainable from a filamentous fungus, in particular Aspergillus niger is preferxed.

In one embodiment, the invention provides for an isolated polynucleotide . comprising a nucleic acid sequence encoding a polypeptide with an amino acid sequence selected from. the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, © 27, 30, 33, 36 and 39 or functional equivalents thereof. ) In a further preferred embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide selected from the group consisting of SEQ ID NO: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33,36 and 39 or feunctional equivalents thereof.

In a preferred embodiment &he Invention provides a lipolytic enzyme gene selected from the group consisting of SEQ ID NO: 1, 4,7, 10, 13, 16, "19, 22, 25, 28, 31,

S4and 37. In another aspect the invermtion provides a polynucleotide, preferably a cDNA encoding an Aspergillus niger lipolytic enzyme whose amino acid secguence is selected rom the group consisting of SEQ ID MNO: 3, 6, 9, 12, 15, 18, 21, 24, =27, 30, 33, 36 and =39 or variants or fragments of that polypeptide. Ina preferred embodinTent the cDNA has a sequence selected from the group consisting of SEQ ID NO: 2, 5,8, 11,14,17,20,23, 26, 29, 32, 35 and 38 or functional eqluivalents thereof.

In an even further preferred embodiment, the Inventio n provides for a polynucleotide comprising the coding sequence of the polynucleotides according to the invention, preferred is the polynucleotide sequence selected from the group consisting of

SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35 and 38.

The invention also relates to vectors comprising a polynucleotide sequence according to the invention and prinreers, probes and fragments that may be used to amplify or detect the DNA according to the invention. in a further preferred errnbodiment, a vector is provided wherein the polynucleotide sequence according two the invention is functionally lirmked with regulatory sequences suitable for expression of the encoded amino acid seq uence in a suitable host cell, such as Aspergillus niger or Aspergillus oryzae. The invention also provides methods for preparing polynucleotides and vectors according to the imvention.

The invention also relates to recombinantly produced hosst cells that contain heterologous or homologous polynucleotides according to the invention. in another embodiment, the invention provides recombinart host cells wherein the expression of a lipolytic enzymes according to the invention is si gnificantly increased or wherein the activity of the lipolytic= enzyme is increased. "In another embodiment the invention provides for a recombinantly produced host cell that contains heterologowus or homologous polynucleoti de according to-the = © 30 invention and wherein the cell is capable of producing a functional lipolytic enzyme according to the invention, preferably a cell capable of over-ex pressing the lipolytic enzyme according to the inventiom, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention.

In yet another aspect of the invention, a purified polypeptides is provided. The polypeptides according to the inwention include: the polypeptides encoded by the polynucleotides according to the inwention. Especially preferred is a polypeptide selected from the group consisting of SEQ 1 DNO: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalents thereof .

Accordingly, in one aspect the present invention provides a lipolytic enzyme composition containing as an active ingredient an enzyme selectead from the group consisting of SEQ ID NO: 3, 6, 9, ~12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalents thereof.

In another aspect, the irmvention provides a method of making baked goods wherein there is Incorporated into the dough used for making the bmaked goods one or more enzymes selected from the group consisting of SEQ ID NO: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalents thereof.

Fusion proteins comprising a polypeptide according to thes invention are also within the scope of the invention— The invention also provides metzhods of making the "polypeptides according to the invesntion.

The invention also relates to the use of the lipolytic enzyeme according to the invention in any industrial processs as described herein.

Detailecd description of the invention

A lipolytic enzyme is defined herein as an enzyme exhibiting at least one and preferably two or three or four oT more of the following lipolytic ac=tivities: triacylglycerol lipase, phospholipase A;, pho=spholipase Az phospholipase £8, phospholipase C, phospholipase D, lysophospholipaase and galactolipase.

Polynucleotides

The present invention provides polynucleotides encodi ng lipolytic enzymes =30 having an amino acid sequence selected. from the group consisting of SEQ ID NO: 3, 6, 9; 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalents thereof. The sequences of the seven genes encoding the lipolytic enzymess NBE028, NBE029,

NBE030, NBE031, NBE032, NE3E033, NBE034, NBE036, NBEO=38, NBE039, NBE043,

NBE045 and NBE042 respectively were determined by sequermicing genomic clones

_. WOneesesn PC~T/EP2003/009145 obta ined from Aspergillus niger. Thee invention provides polynucleotiide sequences com prising the genes encoding the lipolytic enzymes NBEO028, NBES029, NBEO030,

NBE=031, NBE032, NBEO33, NBEO034 , NBE036, NBE038, NBE039, NBsE043, NBE045 and NBEO42 as well as thelr complete cDNA sequences and their coding sequences (Table 1). Accordingly, the invention welates to isolated polynucleotides comprising the nucleotide sequences selected from tie group consisting of SEQIDNO=1,2,4,5,7,8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38 or functional equivalents thereof.

Table. — weer ® | " —weeor |__| * [| ®

More in particular, the invention relates to an isolatexd polynucleotide hwybridisable under stringent conditions, preferably under highly stringent conditions, to a a peolynucleotide selected from the greoup consisting of SEQ ID NO: 1,2,4,5,7,8,10, 11, 1.3, 14, 16, 17,19, 20, 22, 23, 25, 246, 28, 29, 31, 32, 34, 35, 37 and 383. Advantageously, ~ g-uch polynucleotides may be obtained from filamentous fungi, In particular from ~ mspergillus niger. More specifically, the invention relates to an isolated polynucleotide tmaving a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 4,

5,7,8,10 , 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38.

The invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide having an =mino acid sequences selected frorm the group corsisting of SEQ ID NO: 3, 6, 9, 12, 45, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalents thereof. ’

A\s used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein, e.g. an Aspe»rgillus niger lipolytic enzyme. A genee may include c=oding sequences, non-coding seque=nces, introns and regulatory sequences.

Moreover, a gene refers to an isolated nucleic acid molecule as defined herein.

A nucleic acid molecule of the present invention, such as a nucleic acid molecule= having the nucleotide sequence sele=cted from the group consisting of SEQID

NO: 1, 24, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38 or a functional equivalent thereof, can be isolated using standard mo lecular 156 biology teechniques and the sequence information provided herein. For example, usssing all or portio-n of the nucleic acid sequence selected from the group consisting of SEQ 1D

NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19. 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and =38 as a hybridization probe, nucleic acid molecules according to the in=vention can be i=solated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J... Fritsh, E. F,, and Maniatis, T. Molecular Cloning: A Labworatory

Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory” Press,

Cold Spering Harbor, NY, 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of the nucleic acid secyuence selected from the group consisting of SEQ ID NO: 1, 2, 4,5 7,8, 10,11, o5 13, 14, 16, 17,19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information. contained in the nucleic acid sequence selecte«d from the group consisting of SEQ IDNO0:1,2,4,5,7,8, 10, 11, 13, 14, 16, 17, 19, 20, 22,23, 25,26, 28, 29, 31, 32, 34, 35, 37 and 38. -

A nucleic acid of the invention can be amplified using cDNA, maRNA or alterna-tively, genomic DNA, as a template and appropriate oligonucleotide . primers according to standard PCR amplification te chniques. The nucleic acid so ampl ified can be clored into an appropriate vector and chaaracterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to of Mhybridisable to nucleotide sequences according fo the invention can be prepared by starmdard synthetic techniques, e. g., using an automa.ted DNA synthesizer.

In one prefereed embodiment, an isolated nucleic acicd molecule of the invention comprises the nucleo®tide sequence shown in SEQ ID NO: 2 The sequence of SEQ ID

NO: 2 corresponds to the coding region of the Aspergillus nigesr gene provided in SEQ ID

NO: 1. This cDNA comprises the sequence encoding the Aspergillus niger NBE028 polypeptide as showrm in SEQ ID NO: 3. in a second preferred embodiment, an Isolated nucleic acid molecule of the invention comprises &he nucleotide sequence shown in SEQ ID NO: 5. The sequence of

SEQ ID NO: 5 corres ponds to the coding region of the Asperagillus niger gene provided in

SEQ ID NO: 4. Thiss cDNA comprises the seguence enceading the Aspergillus niger

NBE029 polypeptide as shown in SEQ ID NO: 6. in a third poreferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEC» ID NO: 8. The sequence of

SEQ ID NO: 8 corresponds to the coding region of the Aspergillus niger gene provided in

SEQ ID NO: 7. This cDNA comprises the sequence encoding the Aspergillus niger

NBEO030 polypeptide as shown in SEQ ID NO: S. in a fourth prefered embodiment, an isolated n ucleic acid molecule of the invention comprises the nucleotide sequence shown in SE-Q ID NO: 11. The sequence of SEQ ID NO: 14 comesponds to the coding region of the Aspergillus niger gene provided in SEQ ID NO: 10. This cDNA comprises the sequence encoding the

Aspergillus niger NE3EO31 polypeptide as shown in SEQ ID NO:12.

In a fifth preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEEQ ID NO: 14. The sequence of SEQ ID NO: 1-4 corresponds to the coding region o-f the Aspergillus niger gene provided in SEQ ID NO: 13. This cDNA comprises the sequence encoding the

Aspergillus niger N BEO32 polypeptide as shown in SEQ ID NO: 15.

In a sixtta preferred embodiment, an isolated mucleic acid molecule of the invention comprises the nucleotide sequence shown in SEEQ ID NO: 17. The sequence of SEQ ID NO: 87 corresponds to the coding region of the Aspergillus niger gene provided in SEQ ID NO: 16. This CDNA. comprises the sequence encoding the

Aspergillus niger NIBE033 palypeptide as shown in SEQ ID NO: 18.

In a seventh preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 20. The sequence of SEQ ID NO: 20 corresponds to the coding region of tine Aspergillus niger gene provided in SEQ ID NO: 19. This cDNA comprises thes sequence encoding the

Aspergillus niger NBEO34 polypeptide as shown in SEQ ID NO: 21.

In a eight preferred embodiment, an isolated nucleic acid molecule of the invention comprises thes nucleotide sequence shown in SEQ ID NO: 23. The sequence of SEQ ID NO: 23 corresponds to the coding region of the Aspergillus niger gene provided in SEQ ID INO: 22. This cDNA comprises the sequence encoding the

Aspergillus niger NBEO34 polypeptide as shown in SEQID N OQ: 24. : in a nineth preferred embodiment, an isolated nuacleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 26. The sequence of SEQ ID NO: 26 comnesponds to the coding region of the Aspergillus niger gene provided in SEQ ID NO: 25. This cDNA comprises thee sequence encoding the

Aspergillus niger NBEO 34 polypeptide as shown in SEQ ID NO: 27,

In a tenth preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 29. The sequence of SEQ ID NO: 29 corresponds to the coding region of the Aspergillus niger gene provided in SEQ ID NO: 28. This cDNA comprises tine sequence encoding the

Aspergillus niger NBEO34 polypeptide as shown in SEQ ID MNO: 30.

In a eleventh preferred embodiment, an isolated mnucleic acid molecule of the invention comprises the nucleotide sequence shown in SE=Q ID NO: 32. The sequence of SEQ ID NO: 32 corresponds to the coding region of the Aspergillus niger gene provided in SEQ ID NO: 31. This cDNA comprises the sequence encoding the

Aspergillus niger NBE©34 polypeptide as shown in SEQ ID NO: 33.

In a twelvth preferred embodiment, an isolated mucleic acid molecule of the invention comprises tke nucleotide sequence shown in SEQ ID NO: 35. The sequence of SEQ ID NO: 35 corresponds to the coding region of the Aspergillus niger gene provided in SEQ ID NO: 34 This cDNA comprises he sequence encoding the

Aspergillus niger NBEE034 polypeptide as shown in SEQ ID NO: 36. . in a thirteenth preferred embodiment, an isolatedl nucleic acid molecule of the “invention comprises the nucleotide sequence shown in SEZQ iD NO: 38. The sequence of SEQ ID NO: 38 corresponds to the coding region oof the Aspergillus niger gene

~ provided in SEQ ID N#O: 37. This cDNA comprises tthe sequence encoding the

Aspergillus niger NBEO34 polypeptide as shown in SEQ ID NO: 39.

In another prefered embodiment, an isolated mucleic acid molecule of thhe invention comprises a nucleic acid molecule which is a complement of the nucleotiede sequence selected from the group consisting of SEQ IDN O: 1, 2,4,5,7, 8,10, 11, R3, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, =®5, 37 and 38 ora functioral equivalent of these nucleotide sequences. A nucleic acid moleculs, which is complementary to another nucleotide sequence, is one that is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleot ide sequence thereby formirmga stable duplex.

One aspect of the invention pertains to isolated nucleic acid molecules t-hat encode a polypeptide of the invention or a functional equivalent thereof such ass a biologically active fragment or domain, as well as nuclei-c acid molecules sufficient for use as hybridisation pro bes to identify nucleic acid molecwules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. An “isolated polynucleotide” or sisolaated nucleic acid” is a DNA or RNA that is not immediamtely contiguous with both of" the coding sequences with whicsh it is immediately contigu-ous (one on the 5’ end and one on the 3’ end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embod@ ment, an isolated nucleic sacid includes some or all of the 5 non-coding (e.g.. P romotor) sequences that are immediately contiguouss to the coding sequence. The term therefore includes, for example, a recombinart DNA that is incorporated into & vector, into an autonomomusly replicating plasmid or \itus, or into the genomic DNA of a prokaryote or eukaryotes, or which exists as a sepparate molecule (e.g., a cDNA or a genomic DNA fragrment produced by PCR owr restriction endonuclease treatment) independent of Other sequences. It also inclLades a recombinant DNA that is part of a hybrid gene encodinzg an additional polypeptide that is substantially free of cell ular material, viral materiaml, or culture medium (whe=n. produced by recombinant [DNA techniques), or chermical : 30 precursors or other chemicals (when chemically synth-esized). Moreover, an “iso lated nucleic acid fragment™ is a nucleic acid fragment that is not naturally occurring as a fragment and would nowt be found in the natural state. : ~

As used hereain, the terms “oolynucleotide” or “nucleic acid molecule™ are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide amnalogs. The nucleic acid molecule can be single-stranded or double-stranded, but oreferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g. inosine or phosphorothioate nucleotides). Such oligomucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

Another embodiment of the inventior provides an isolated nucleic acid molecule whic h is antisense to a nucleic acid molecule according to the invention. M\so included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.

Sequencing emors

The sequence information as provided herein should not be so narrowly consstrued as to require inclusion of erroneously identified bases. The speci. fic sequences discelosed herein can be readily used to issolate the complete gene fror filamentous fungi, in particular Aspergillus niger whichs in tum can easily be subjected to further seq uence analyses thereby identifying sequ encing errors.

Unless otherwise indicated, all nucleotide sequences determined Eby sequencing a DNA molecule herein were determined tising an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules detesmined herein wenwre predicted by translation of a DNA seq uence determined as above. T herefore, as is known in the art for any DNA sequence cletermined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleo®ide sequences determined by automation are typically at least about 90% identical, m~ore typically at least about 95% to at least about 99.9% identical to the actual nucleoticde sequence of thes sequenced DNA molecule. The actual sequence can be more precissely determined by other approaches including manual DNLA sequencing methods well krown in the art.

As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a de=termined nucleotide sequence will bes completely different from t-he amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the poirt of such an insertion or deletion.

The per=son skilled in the art is capable of id entifying such erroneously identified bases and knowss how to correct for such errors.

A nuclesic acid molecule according to the invention may comprise only a pOortion or a fragment of™ the nucleic acid sequence selected from the group consisting of SEQID

NO: 1,2 4,5, 7,8,10,11,13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 3-4, 35, 37 and 38, for example a fragment which can [oe used as a probe or primer— or a fragment encoding a portion of protein accordirag to the invention. The nucleotide sequence determined from the cloning of the lipolytic enzyme gene and cDNA allowws for the generation eof probes and primers designed for use in identifying and/or clonings other lipolytic enzyme family members, as well as lipolytic enzyme homologues from other species. The probe/primer typically comprises =substantially purified oligonuc eotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably abowut 22 or 25, more preferably about =30, 35, 40, 45, 50, 55, 60, 65,0'T 75 or more consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 4,5,7,8,10, 11, 13, 14, 16, 17, 19, 20, 22, 23, =25, 26, 28, 29, 31, 32, 34,35, 37 and 38orofa functional equivalent thereof.

Probess based on the nucleotide sequernces provided herein can be Lised to detect transcripts or genomic sequences encoding the same or homologous protesins for instance in other organisms. In preferred embod iments, the probe further comprises a label group afttached thereto, e.g., the label grou p can be a radioisotope, a fluosrescent compound, ar enzyme, or an enzyme cofactor. S uch probes can also be used ass part of a diagnostic test kit for identifying cells that express a lipolytic enzyme protein. -30 entity & homeology ~~ ’ The terms “homology” or “percent identity” are used interchangeably * herein. For time purpose of this invention, it is defined here that in order to determmine the percent iden-tity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optinnal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for ogotimal alignment with a second amino or nucleic acid sequence). The amino acid resiclues or nucleotides at corresponding amino acid positions or nucleotide positions are tien compared. When a position In the first sequence is occupied by the same amino aci d residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequenczes is a function of the number of identical positions shared by the sequences (i.e., %0o identity = number of identical positionsitotal numer of positions (i.e. overlappimng positions) X 100). ~40 Preferably, the two sequences are the same length.

The skilled person wiill be aware of the fact that seweral different computer programmes are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of perceant identity between two sequences can be accomplished using a mathematical algorithm. In a preferred 45 embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at hitp:/fiwww.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8,6, or4 and a length weight of 1, 2, =3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yiel d slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms. in yet another em bodiment, the percent identity Bostween two nucleotide sequences is determined using the GAP program in the GCG software package (available at hittp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a tength weight of 1, 2, 3, 4, 5, or 6 - In another embodiment, the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 411-17 (1989) which has been incorporated into the ALIGN program (version 2.0) (available atv http://vega.igh.cnrs fribin/align-guess.cgi) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nudleic acid and protein sequences of the presert invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such se arches can be mperformed using the BLASTN and EBLASTX programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 21 5:403—10. BLAST nucleotide searches can be performed with the

BLASTN program, score = 100, word length = 12 to obtain nucleokide sequences homologous to PLPO3 nucleic acid rnolecules of the invention. BLAST p rotein searches can be performed with the BLAST>< program, score = 50, word lengtkr = 3 to obtain amino acid sequences homologouss to PLP03 protein molecules of th e invention. To obtain gapped alignments for comparison purposes, Gapped BLAST camn be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing

BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See http:/Awww.ncti.nim.nih.gov.

Hybridisation

As used herein, the tem "hybridizing" is intended to descritoe conditions for hybridization and washing under vvhich nucleotide sequences at leasst about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95% hormologous to each other typically remain hybridized to each other.

A preferred, non-limitings example of such hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 1x SSC, 0.1 % SDS at 50°C, preferably at 55°C, pereferably at 60°C and even more preferably at 65°C.

Highly stringent conditiors include, for example, hybridizin g at 68°C in 5x . SSC/5x Denhardt's solution / 1.0%% SDS and washing in 0.2x $SC/0. 1% SDS at room temperature. Alternatively, washing may be performed at 42°C.

The skilled artisan will kreow which conditions to apply for staringent and highly stringent hybridisation conditions . Additional guidance regarding s uch conditions is 30. readily available in the art, for example, in Sambrook et al., 1989, Maslecular Cloning, A

Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel est al. (eds.), 1995,

Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.)

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' temminal poly(A) tract of mRNAs), or to & complementary stretch of ~T {or U) resides, woulcd not be included In a polynucleotide= of the invention used to sp-ecifically hybridize to & portion of a nucleic acid of the inwention, since such a polynucleotide would hybridize to any nucleic acid molecule c=ontaining a poly (A) stretcln or the complement thereof (e.g., practically any double-staranded cDNA clone). :

Obtaining full length DNA fron other organisms in a typical approach, cDNA libraries coanstructed from other organisms, 6.9. filamentous frungi, in particular from the species Asspergillus can be screened. :

For example, Aspergillus strains czn be screened for hommologous polynucleoticles by Northern blot analysis. Upon detection of transcripts homologous to polynucleoticles according to the invention, cDNA libraries can be constructed —from RNA isolated from the appropriate strain, utilizing stanclard techniques well known teo those of skill in the amt. Alternatively, a total genomic DNA library can be screened usirmg a probe hybridisable fo a polynucleotide according to the imvention.

Hormologous gene sequences can be Isolated, for example, by performing PCR using two deegenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.

The template for the reaction can be cD»NA obtained by reverse trans=scription of mRNA prepeared from strains known or suspecte«d to express a polynucleotides according to the invertion. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new PLP03 nucleic acid sequence, or a functiomal equivalent thereof.

Thee PCR fragment can then be used €o isolate a full length cDNA clone by a variety of k_nown methods. For example, the amplified fragment can be labele=d and used to screen = bacteriophage or cosmid cDNA lib rary. Altematively, the labelead fragment can be use=d to screen a genomic library.

PCR technology can also be used to Eisolate full-length cDNA seqiaences from other organisms. For example, RNA can be #isolated, following. standard procedures, - from an a-ppropriate cellular or tissue source. A reverse transcription reaction can be performed on the RNA using an oligonucleotides primer specific for the most =5' end of the amplified fragment for the priming of first strand synthesis. N ) -

The resulting RNA/DNA hybrid can the=n be "tailed" (e.g., with guani&nes) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C psrimer). Thus, CDNA sequences upstream of the amplified fragment can easily be isolated. For a review of useful cloning strategies, see e.cs.,Sambrook et al., supra; and Ausutoel et al, supra.

Vectors

Another aspect of thes invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a protein according two the invention or a —0 functional equivalent thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it mas been linked. One type of vector is a “plasmid”, wrhich refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segmemts can be ligated into the viral gen ome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and espisomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) a_re integrated into the genome of a host cell upon in troduction into the host cell, and tlnereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such veectors are referred to herein as “expression vectors” . In general, expression vectors of utility in recombinant

DNA techniques are often in the form of plasmids. The terms “plassmid” and “vector” can be used interchangeably herexin as the plasmid is the most cornmonly used form of vector. However, the invention is intended to include such othewr forms of expression vectors, such as viral vectors (e.g., replication defective retrovirusses, adenoviruses and adeno-associated viruses), whi ch serve equivalent functions.

The recombinant expression vectors of the invention commprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes ones or more regulatory sequences, selected on the basis of the host cells to be used foar expression, which is . operatively linked to the nucleic acid sequence to be expressed.. Within a recombinant expression vector, “operatively linked” is intended to mean that th e nucleotide sequence of interest is linked to the ragulatory sequence(s) in a mariner which allows for exp-ression of the nucleotide sequerice (e.g., in an in vitro tra_nscription/transiation system or in a host cell when the vector is introduced into the host cell). The term “recyulatory sequence” is intended to irmcdude promoters, enhancers =and other expression coratol elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goedd el; Gene Expression Tech nology: Methods in

En=zymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences inc 1ude those which direct constitutive or inducible expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide secyuence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the e=xpression vector can de pend on such factors as the choice of the host cell to be trarsformed, the level of exspression of protein desired, etc. The expression vectors of the invention can be int_roduced into host cells to thereby produce proteins or peptidess, encoded by nucleic ac-ids as described herein (e.g. lipolytic enzymes, mutant lipolytic enzymes, fragments thesreof, variants or functional equivalents thereof, fusion proteins, estc.).

The recombinant expression vectors of the invention can be designed for expression of lipolytic enzymes in prokaryotic or eukaryotic cells. Wor example, a protein according to the invention can be expressed in bacterial cells— such as E. coli and

Bexcillus species, insect cells (usirmg baculovirus expression vesctors) yeast cells or m.ammalian cells. Suitable host cells are discussed further in Goe—-ddel, Gene Expression

Technology: Methods in Enzymology 185, Academic Press, S an Diego, CA (1 990).

Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter- regulatory sequences and Ta polymerase.

Expression vectors useful in the present invention finclude chromosomal, e-pisomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, b acteriophage, yeast episome, weast chromosomal elemerts, viruses such as beaculoviruses, papova viruses, vaccinia viruses, adenovirus es, fowl pox viruses, peseudorabies viruses and retroviruses, and vectors derived frormn combinations thereof, ssuch as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. =

The DNA insert should ber operatively linked to an apperopriate promoter, such as the phage lambda PL promoter, the E. coli ac, trp and tac preomoters, the SV40 early zand late promoters and promoters of retroviral LTRs, to name a few. The skilled person will know other suitable promoters. In a specific embodiment, promoters ar=e preferred that are capable of directing a high expression level of lipolytic enzymes in #ilamentous fungi. Such promoters are known in the art. The expression constructs may c=ontain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of thee mature transcripts expressed by the constructs will include a translation initiating #AUG at the beginning and a termination cordon appropriately positioned at the end of the polypeptide to be translated.

Vector DNA can be Introduced into prokaryotic or eukaryoti ¢ cells via conventional transformation or transfection “techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of arit-recognized te chniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride «o-percipitation, DEAE-dextr=an-mediated transfection, transduction, infection, lipofection, cationic lipidmediated tramnsfection or electroporation. Suitable methods for transforming or transfecting host cells can be found ira Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2" ed. Cold S=pring Harbor

Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manumals.

For stable transfection of mammalian calls, it is known that, depencding upon the expression vector and transfection techniques used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to =antiblotics) is generally introduced into the host cells along with the gene of intere=st. Preferred selectable markers include those that confer resistance to drugs, such as G418, raygromycin and methatrexate. A nucleic acid encoding a selectable marker is preferably introduced into a host cell on the same vector as that encoding a protein. according to the invention or an be introduced on a separate vector. Cells stably transfeacted with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells d ie).

Expression of proteins in prokaryotes is often carried out in E. coeli with vectors ° containing constitutive or inducible promoters directing the expression of e=ither fusion or ron-fusion proteins. Fusion vectors add a number of amino acids to a preotein encoded " gherein, e.g. to the amino terminus of the recombinant protein. Such #usion vectors " gypically serve three purposes: 1) to increase expression of recombinant: protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification Of the recombinant protein by acting as a ligand im affinity purification. Often, in fausion expression veectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and tine recombinant protein to enable separation of the recombinant proteirm from the fusion mnolety after purification of the fussion protein. Such enzymes, and their cognate recoagnation sequences, include Factor Xa, thrombin and enterokinase.

As i ndicated, the expression vectors will preferably contain selectable ma rkers.

Such markers include dihydrofolate reductase or neomycin resistance for eukarot ic cell culture and t-etracyline or ampicilling resistance for culturing in E. coli and other baacteria.

Representat@ive examples of appropriate host include bacterial cells, such as E. col,

Streptomyce=s and Salmonella typhimurium; fun gal cells, such as yeast; insect cellss such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture» mediums and conditions for the &bove- described host cells are known in the art.

Ameong vectors preferred for use in bacteria are pQE70, pQE60 and FPQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pIENHS8A, pNH16A, p NH18A, pNH46A, available from Stratagene; and ptrc99a, pKK. 223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Strateagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable \wectors will be readi ly apparent to the skilled artisan.

Known bacterial promoters for use in the present invention include E. c=oli lacl and lacZ pr-omoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSV thymidine kinase promoter, the early a=nd late

SV40 promeoters, the promoters of retroviral LTRs, such as those of the Rous s=arcoma virus (“RS\W/"), and metallothionein promoters, such as the mouse metallothi onein- promoter.

Inserting an enhancer sequence into the vector may increase transcripotion of the DNA e=ncoding the polypeptides of the present invention by higher eukamryotes. '30 Enhancers are cis-acting elements of DNA, ussually about from 10 to 300 bp tha tactto increase transcriptional activity of a promotesr in a given host cell-type. Exam ples of } enhancers include the SV40 enhancer, which is located on the late side of the replication - - origin at bgp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late sid e of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into tie lumen of the endoplasmic reticulum, into the perifolasmic space or into the extracellular environment, appropriate secretion signal may be incorporated into the expressed polypeptide. The signals enay be endogenous to the p olypeptide or they may be heterologous signals.

The polypeptid-e may be expressed in a modified form, such as a fusion protein, and may include not omnly secretion signals but also additional heterologous functi-onal regions. Thus, for inst-ance, a region of additional amino acids, particularly chamrged amino acids, may be aclded to the N-terminus of the polwypeptide to improve stabllity and persistence in the hosst cell, during purification or during subsequent handling and storage. Also, peptide rnoieties may be added to the polypeptide to facilitate purification.

Polypeptides according to the imvention

The invention provides an isolated polypeptide having the amino acid sequ=ence selected from the grougp consisting of SEQ ID NO: 3,6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39, an amino acid sequence obtainable by expressing the polynucle otide selected from the group consisting of SEQ ID NO: 1, 2, 4,5,7,8,10, 11,13, 14,16, 17, 19, 20, 22, 23, 25, 26, 28,29, 3, 32, 34, 35, 37 and 38 in an appropriate host. Al so, a peptide or polypeptide= comprising a functional equivaleent of the above polypeptides is comprised within the= present invention. The above polypeptides are collec=tively comprised in the term =*polypeptides according to the invention” :

The terms "peptide” and “oligopeptide” are considered synonymous ( as is commonly recognizecdl) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkagess. The word "polypeptide” is used herein for chains containi ng more, than seven amino acid residues. All oligopept-ide and polypeptide formulas or Sequences herein are writter from left to right and in the direction from amino terminus tos carboxy terminus. The one -letter . code of amino acids used herein is commonly knowan in the art and can be found in

Sambrook, et al. (Moadecular Cloning: A Laboratory Mazanual, 2®.ed. Cold Spring Harbor

Laboratory, Cold Sprirg Harbor Laboratory Press, Colc3 Spring Harbor, NY, 1989) i By “isolated™ polypeptide or protein is intended a polypeptide or porotein removed from its native environment. For example, recombinantly pro-duced

WP 0 2004/018660 PCT/EP-2003/009145 polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

The lipolytic enzyme according to the invention can be recovered =and purified fromm recombinant cell cultures by well-known methods including ammoniurm sulfate or ettnanol precipitation, acid extraction, anion or cation exchange chrormatography, pheosphocellulose chromatography, hydrophobic interaction chromatogragphy, affinity charomatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is ermnployed for pu rification., .

Polypeptides of the present invention include naturally purified products, products of chemical synthetic procesdures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bac=terial, yeast, higher plant, insect and mammalian cslls. Depending upon the host employed in a reccombinant production procedure, the polypeptides of the present inven—tion may be ghwcosylated or may be non-glycosylated. In addition, polypeptides of the irmvention may alsso include an initial modified methionine residue, in some cases as a reasult of host- me=diated processes.

A lipolytic enzyme according to the invention may be advantageomusly used in bamking processes. The amount of enzyme to be added to the dough iss determined emmpirically. It may depend on the quality of the flour used, the degree of Emprovement which is required, the kind of bread or baked goods, the method of preparing the dough, thee proportion of other ingredients etcetera. : Protein fragments :

The invention also features biologically active fragments of the polypeptides according to the invention. ; “ 30 . Biologically active fragments of a polypeptide of the invertion include polypeptides comprising amino acid sequences sufficiently identical to or derived from th a amino acid sequence of the lipolytic enzyme (e.g., the amino acid sequeance selected from the group consisting of SEQ ID INO: 3, 6, 9, 12, 15, 18, 21,24, 27, 30, 33, 36 and

39), which include fewer amino acids than the full lengtkn protein, and exhibit at leeast one biological activity of the corresponding full-length protein. Typically, biological ly active fragments comprise a domain or motif with at least one activity of the correspording full length protein.

A biologically acstive fragment of a protein of tte invention can be a polypeptide which is, for example, 1 0, 25, 50, 100 or more amino- acids in length. Moreower, other biologically active portiosns, in which other regions of the protein are deleted, can be prepared by recombinamnt techniques and evaluated for one or more of the Koiological activities of the native fomrm of a polypeptide of the inver—tion.

The invention also features nucleic acid fracyments which encode time above biologically active fragmeents of the lipolytic enzyme prowtein.

Fusion proteins

The proteins o=f the present invention or furictional equivalents ther—eof, e.g., biologically active portions thereof, can be operativelwy linked to a nondipolyticc enzyme polypeptide (e.g., heterologous amino acid sequencess) to form fusion proteins... As used herein, a lipolytic enzymme "chimeric protein” or "fus ion protein” comprises aa lipolytic enzyme polypeptide operatively linked to a non-iipolytJc enzyme polypeptide. /A\ "lipolytic enzyme polypeptide” reafers to a polypeptide having &an amino acid sequence selected from the group consistirmg of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 3 3, 36 and 39, whereas a "non-lipeoiytic enzyme polypeptide” re=fers to a polypeptide having an amino acid sequence c-orresponding to a protein which is not substantially ho- mologous to the lipolytic enzyme, e.g., a protein which is different from the lipolytic en—=zyme and which is derived from tie same or a different organisrm. Within a lipolytic enzyme fusion protein the lipolytic enzzyme polypeptide can correspond to all or a portion of a lipolytic enzyme protein. In a preferred embodiment, a lipolytics enzyme fusion protein ecomprises at least one biologica lly active fragment of a liposlytic enzyme protein. tm another preferred embodiment a lipolytic enzyme fusion protein comprises at least two biologically active portisons of a lipolytic enzyme protein. Within the fusion protein, the term “operatively linkedi" is intended to indicate that tise lipolytic enzyme polypeptide and the non-lipolytic enzyme polypeptide are fused in-frarme to each other. The ncon-lipolytic enzyme polypeptide cam be fused to the N-terminus ox C-terminus of the lipolytic enzyme polypeptide.

For example, i none embodiment, the fusion protein is a GST-lipolytic erzyme fusion protein in which the lipolytic enzyme sequence iss fused to the C-terminus of the

GST sequence. Such fusion proteins can facilitate the purification of recom’binant lipolytic enzyme(s). in another embodiment, the fusiom protein is a lipolytic ernzyme protein containing a heterologous signal sequence atits N-terminus. In certain hosst cells (e.g., mammalian and “Yeast host cells), expression and/or secretion of lipolytic emnzyme can be increased through use of a hetereologous signal sequence.

In another example, the gp67 secretory sequemce of the baculovirus en—velope protein can be used ass a heterologous signal sequences (Current Protocols in Mo_Jecular

Biology, Ausubel et al_, eds., John Wiley & Sons, 1992). Other examples of euk-aryotic heterologous signal se=quences include the secretory sequences of melittin and Bhuman placental alkaline ph-osphatase (Stratagene; La Jotla, California). In yet &xanother example, useful prokamnytic heterologous signal sequerces include the phoA se cretory signal (Sambrook et a_l., supra) and the protein A secretory signal (Pharmacia Biotech;

Piscataway, New Jersey).

A signal sequeence can be used fo facilitate semcretion and isolation of a protein or polypeptide of the imwvention. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or mcare cleavage events. Such signal peptides contain processiryg sites that allow cleavage Of the signal sequence from the mature proteins as the=y pass through the secretory~ pathway. The signal sequence directs secretion of the protein, such as from a eukanwotic host into which the expression vector is transformed, =and the signal sequence is ssubsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alterratively, the signal sequence can be linked to the protein of interest using a sequences which facilitates purification. such as with a GST domain. “Thus, for instance, the sequence encoding the polypeptide may be fused to a marker- sequence, such as a sequence encoding a peptide, which facilitates purification of -the fused polypeptide. In. certain preferred embodimermts of this aspect of the invention , the marker sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As describexd in Gentz et al, Proc. Natl. Acad.

Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for comnvenient purificaton of the fusior protein. The HA tag is another peeptide useful for purification which corresponds to aan epitope derived of influenza herinaglutinin protein, which has been described by Wilson et al., Cell 37-767 (1984), for instance.

Preferably, a chimeric or fusion protein of the invertion is produced by standard recombinant DNA techaniques. For example, DNA fragmeents coding for the different polypeptide sequences- are ligated together in-frame in amccordance with conventional techniques, for examples by employing blunt-ended or stageger-ended termini for ligation, restriction enzyme dige stion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkalire phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using aanchor primers which give rise to complementary ovesrhangs between two consecutive gene fragments which can subsequently be annezaled and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds - Ausubel et al. John Wiley &

Sons: 1992). Moreov=er, many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide). A lipolytic enzyme-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the It polytic enzyme protein.

Functional equivalents

The terms “functional equivalents” and “fumnctional variants” are used interchangeably hereimn. Functional equivalents of lipolytic enzyme encoding DNA are isolated DNA fragmenrats that encode a polypeptide that exhibits a particular function of the Aspergillus niger lipolytic enzyme as defined hereirm. A functional equivalent of a lipolytic enzyme polypeptide according to the invention iss a polypeptide that exhibits at least one function of =n Aspergillus niger lipolytic enzyme as defined herein. Functional equivalents therefore also encompass biologically active f ragments.

Functional protein or polypeptide equivalents rmay contain only conservative substitutions of one om more amino acids in the amino acid sequences selected from the .. group consisting of SSEQ ID NO: 3, 6, 9, 12, 15, 18, 21 , 24, 27, 30, 33, 36 and 39 or . substitutions, insertioms or deletions of non-essential amino acids. Accordingly, a non-

-26 = essential amino acid is a residue that can be =iftered in the amino acid sequences selected from the group consisting of SEQ 1D NO: 3, 6,9, 12, 15, 18, 21, 24. 27,30, 33, 36 and 39 without substantially altering the biolog ical function. For example, amino acid residues that are conserved among the fipoly=tic enzyme proteins of #he present invention, are Predicted to be particularly unamermable to alteration. Furtherrmore, amino acids conserve -d among the lipolytic enzyme protesins according to the present invention and other lipolystic enzymes are not likely to be amenable to alteration.

The team “conservative substitution” is imtended to mean that a stubstitution in which the amiro acid residue is replaced with a.n amino acid residue having a similar side chain. These families are known in the art a_nd include amino acids wilh basic side chains (e.g.lysiine, arginine and hystidine), acidic sside chains (e.g. aspartic amcid, glutamic acid), uncharged polar side chains (e.g., gly=cine, asparagines, glutarmnine, serine, threonine, tyrosine, cysteine), non-polar side «chains (e.g., alanine, val ine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonime, valine, isoleucine) and aromatic side chains (e.-g., fyrosine, phenylalanine tryptophan, histidine).

Functional nucleic acid equivalents may typically contain silent mutations or mutations thal do not alter the biological functiosn of encoded polypeptide Accordingly, the invention provides nucleic acid molecules encoding lipolytic enzyme proteins that contain changes in amino acid residues that are not essential for a particrular biological activity. Such lipolytic enzyme proteins differ in _amino acid sequence sele=cted from the group consisting of SEQ ID NO: 3,6, 9 12, 15, 18, 21, 24, 27, 30, 33, 336 and 39 yet retain at leasst one biological activity. In one embodiment the isolated nucleic acid molecule comprises a nucleotide sequence emncoding a protein, wherein the protein comprises a ssubstantially homologous amino acd sequence of at least about 60%, 65%, 70%, 75%, 8 0%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid se2quence selected from the group consisting of SEQ ID NO: 3,6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39.

For example, guidance concerning how to make phenotypicalimy silent amino acid substitutions Is provided in Bowie, J.U. et al, Science 247:13063-1310_ (1990) wherein the authors indicate that there are two main approaches forr studying the tolerance of =n amino acid sequence to changes. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second a_pproach uses genetic engineering to introduce amino acid changes at =specific positions of a cloned gene and selects or screens to identify sequences that renaintain functionaslity. As the authors state, these studies have revealed that prote ins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be pemissive at a certain position of the protein. For example, most buried amino acid residues require non-p olar side chains, whereas few feamtures of surface side chains are generally conserved. Other such phenotypicallzy silent substitut ions are described in Bowie et al, supra, and the references cited thereirm.

An isolated nucleic acid molecule encoding a protein homologous to thea protein selected. from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27", 30, 33, 36 and 339 can be created by introducing one or more nucleotide substitutions, &xdditions or deleti ons into the corresponding coding nucleotide sequences (Table 1) such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein. Such mutations may be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis.

The term “functional equivalents® also encompasses orthologues of the

Aspergillus niger lipolytic enzymes provided h erein. Orthologues of the Aspergillus niger lipolytic enzymes are proteins that can be isolated from other strains or spe=cies and possesss a similar or identical biological activity. Such orthologues can readily be identified as comprising an amino acid sequemce that is substantially homologous to the amino cid sequences selected from the grou p consisting of SEQ ID NO: 3, 6, =8, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39.

As defined herein, the term “substa ntially homologous” refers to a fi rst amino acid or nucleotide sequence which contains & sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequerces have a common domain. For example, amino acid or nucleotide ssequences which contain a common domain having about 80%, preferably 65%, more preferably 70%, e=ven more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 993% identity or more are defined herein as sufficiently identical. ’

Also, nucleic acids encoding other lEpolytic enzyme family members, which thus have & nucleotide sequence that differs frorm a nucleotide sequence selected from the group consisting of SEQ IDNO: 1,2, 4, 5,7, 8,10, 11,13, 14,16, 17,19, 20, =22, 23, 25,

26, 28, 29, 31, 322, 34, 35, 37 and 38, are within thee scope of the invention. Moreover, nucleic acids enc=oding lipolytic enzyme proteins fron different species which thu=s have a nucleotide sequence which differs from a nucleotides sequence selected from thea group consisting of SECA ID NO: 1, 2, 4, 5,7, 8,10, 11, 13 , 14,16, 17, 19, 20, 22, 23, 225, 26, 28,29, 31, 32, 341, 35, 37 and 38 are within the scope of the invention.

Nucleic acid molecules corresponding to v=riants (e.g. natural allelic variants) and homologues. of the polynucleotides of the invermtion can be isolated based on their homology to the nucleic acids disclosed herein usirig the cDNAs disclosed herein or a suitable fragmemmt thereof, as a hybridisation probe according to standard hybricdisation techniques preferably under highly stringent hybridis=ation conditions.

In addition to naturally occurring allelic variants of the Aspergillu-s niger sequences prov-ided herein, the skilled person will recognise that changes can be introduced by mautation into the nucleotide sequencess selected from the group consisting of SEQ ID NO: 1,2, 4, 5,7, 8, 10, 11, 13, 14, 16, 1 7, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38 thereby leading to changes in the amino acid sequencee of the lipolytic enzyme protein without substantially altering the function of the protein.

In anot:her aspect of the invention, impro—ved lipolytic enzymes are provided. improved lipolytic enzymes are proteins whereirs at least one biological activity is improved. Such proteins may be obtained by randormly introducing mutations alosng all or part of the lipolwytic enzyme coding sequence, such as by saturation mutagene=sis, and the resulting nmutants can be expressed recombminantly and screened for bwiological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of lipolytic enzymes and thus improved proteins may easily be selected.

In a preferred embodiment the lipolytic emnzyme has an amino acid s-equence selected from the group consisting of SEQ ID NO: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39. In another embodiment, the lipolytic erizyme is substantially homot ogous to - the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 9,12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 and retain s at least one biological activity of a polypeptide seleected from the group consisting of SEQ iD NO: 3, 6, 9, 12, 15, 188, 21, 24, 27, 30, 33, 36 and 39, yet differs in amino acid sequence due to natural va_riation or mutagenesis ass described above. . in a Further preferred embodiment, the lipolytic enzyme has an armino-acid "sequence enceoded by an isolated nucleic acid —fragment capable of hybridi=sing to a nucleic acid selected from the group consisting of SEQ ID NO: 1, 2,4,5,7,8,10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38, preferably under highly stringent hybridisation conditions.

Accordingly, the lipolytic enzyme Is a protein which cormprises an amino acid sequence at least about 60%, 85%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence selected from the group consisting of SEQ 1D NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 and retains at least one functional activity of the polypeptide selected from the g roup consisting of SEQ

ID NO: 3, 6, 9, 12, 15, 18,21, 24,27, 30, 33, 36 and 39. in particular, the lipolytic enzyme is a protein which comprises an amino acid sequence at least abo ut 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 3 or the lipolytic enzyrme is a protein which comprises an amino acid sequence at least about 55%, 60%, 65%s, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 7%, 98%, 99% or more 16 homologous to the anuino acid sequence shown in SEQ ID NO: &, or the lipolytic enzyme is a protein which cormprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO : 9, or the lipolytic enzyme is a protein whiich comprises an amino acid sequence at leasst about 40%, 45%, 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 972k, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 12 or the lipolytic enzyme is a protein whaich comprises an amino acid sequence at least about 40%, 45%, 50%, 56%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to thee amino acid sequence shown in SEQ ID NO»: 15, or the lipolytic enzyme is a protein winich comprises an amino acid sequence at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more homologous to trae amino acid sequence shown in SEQ ID NO: 18 or the lipolytic enzyme is a protein which comprises an amino acid sequence at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more’ homologous to the amino acid sequence shown in SEQ ID NO: 21, or the lipolytic enzyme is a protein which comprises an amino acid sequence at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 24 or the lipolytic enzyme is a protein which comprises an amino acid sequence at least absout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%4, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 27, or the lipolytic enzyme is a protesin which comprises an amimno acid sequence at least abwout 40%, 45%, 50%, 55%, 60%. 85%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, Om8%, 99% or more homologous to the amino acid sequen ce shown in SEQ ID NO: 30m or the lipolytic enzyme is a protein which comprises an ami no acid sequence at least aloout 40%, 45%, 50%, 55%, 60% , 65%. 70%, 75%, 80%, 85-%., 90%, 95%, 96%, 97%, 8%, 99% or more homologous to the amino acid sequernce shown in SEQ ID NO: 33_, or the lipolytic enzyme is a prot=ein which comprises an am3no acid sequence at least atoout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 8%, 99% or more homologouss to the amino acid sequernce shown in SEQ ID NO: 36 or the lipolytic enzyme is a protein which comprises an amino acid sequence at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, *98%, 99% or more homologouss to the amino acid sequemnce shown in SEQ ID NO: 39 .

Functional equivalents of a protein according to the invention can also be identified e.g. by screenimng combinatorial libraries of mutaants, e.g. truncation mutantss, of the protein of the inventicon for lipolytic enzyme activity. In. one embodiment, a variegated library of variants is gene=rated by combinatorial mutagenesis at the nucleic acid level. A variegated library of variants can be produced by, for exxample, enzymatically ligatirg a mixture of synthetic oligonucleotides into gene sequence=s such that a degenerate set of potential protein sequen ces is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods that can be used to pr=oduce libraries of potential variznts of the polypeptides of™ the invention from a dege=nerate oligonucleotide sequerwce. Methods for synthesizing degenerate oligonucleotides are known in the art (see, .9., Narang (1983) Tetrahe=dron 39:3; ltakura et al. (19834) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Scieence 198:1056; Ike et al. (19833) Nucleic Acid Res. 11:477). + In addition, libr=aries of fragments of the coding sequence ofa polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants. For example, a libramy of coding sequence fragrenents can be generated by treating a double stranded PCR fragment of the coding sequaence of interest with a nuclease under conditions wherein nicking occurs only about onc=e per molecule, dematuring the double stranded DN A, renaturing the DNA to forms double stranded DNA which can include sense/antiserse pairs from different nicked products, removing sinsgle stranded portions from refcmed duplexes by treatment with S1 nuclease, andl ligating the resulting fragment INbrary into an expression vector. By this method, an expression library can be derived which encodes N-terminal and intemal fragments of -various sizes of the protein of intewrest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutatiors of truncation, and for screening cDNA libraries for cmene products having a selected pwroperty. The most widely used te=chniques, which are a menable to high through-put an=alysis, for screening large gene libraries typically inclaude cloning the gene library inte replicable expression vectors, traansforming appropriate cells with the resulting library of —vectors, and expressing the cormbinatorial genes undemr conditions in which detection of a desired activity facilitates isolamtion of the vector encending the gene whose produsct was detected. Recursive ensemble 16 mutagenesi=s (REM), a technique which enhamnces the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. US=A 89:7811- 7815; Delgr-ave et al. (1993) Protein Engineewring 6(3):327-331). it will be apparent for the person skilled in the art that DNA=a sequence polymorphi sms that may lead to changes i n the amino acid sequence of the lipolytic enzyme ma=ay exist within a given populatiorm. Such genetic polymorphisms rmay exist in cells from different populations or within a. population due to natural allelic variation.

Allelic varisants may also include functional e quivalents.

Faragments of a polynucleotide according to the invention may al=so comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as=s probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether they encode functional. or non-functional polypeptides... can be used as hybridizatiosn probes or -. polymerasse chain reaction (PCR) primers . Uses of the nucleic acid molecules of the: present imvention that do not encode a peolypeptide having a lipolytic ermzyme activity include, i nter alia, (1) isolating the gene ercoding the lipolytic enzyme prostetn, or allelic. variants &hereof from a cDNA library e.g. freom other organisms than Aspercgillus niger; (2) - in situ hybridization (e.g. FISH) to metaptmase chromosomal spreads to p rovide precise chromosomal location of the lipolytic enzy/me gene as described in Verma et al., Human

Chromossomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3)

Northern blot analysis for detecting expression of lipolytic enzyme mRNA in specific tissues andlor cells and 4) probes and primers that can be used as a diagrmostic tool to analyse= the presence of a nucleic acid thybridisable to the lipolytic enzymes probe in a given biological (e.g. tissue) sample.

Also encompassed by the invention is a method of obtaining a functional equivaleent of a lipolytic enzyme-encociing gene or cDNA. Such a me thod entails * obtainirg a labelled probe that includes an isolated nucleic acid which enceodes all ora portion of the sequence selacted from the group consisting of SEQ iD NO= 3,6, 9, 12, 15, 18. 21, 24, 27, 30, 33, 36 and 39 or a variant thereof; screening a nucleic acid fragme nt library with the labelled probe under conditions that allow hybridi sation of the probe to nucleic acid fragments in the liberary, thereby forming nucleic acid d uplexes, and preparing a full-length gene sequence From the nucleic acid fragments in any labelled duplex to obtain a gene related to the lipolytic enzyme gene.

In one embodiment, a nucleic acid of the invention is at least 40%, 45%, 50%, 55%, E30%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 39%, or more homologous to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 4, 5,7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 222, 23, 25, 26, 28,29 ,31,32 34,35 37and 38 or the complement thereof. in another preferred embodiment a polypeptide of the invention is at least 40%, 45%, %50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%%, 94%, 95%, 96%, "97%, 98%, 99%, or more homologous to an amino acid sequence sselected from the greoup consisting of SEQ ID NO: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39.

Host cells

In another embodiment, the invention features cells, e.g., transforrmed host cells or recombinant host cells that contain a nucleic acid encompassed by th_e invention. A "transsformed cel" or “recombinant cell” is a coll into which (or into an ancestor of which) has koeen introduced, by means of recombinant DNA techniques, aa nucleic acid according to the invention. Both prokaryotic and eukaryotic cells are #ncluded, e.g., bacteeria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi,

in particular Aspergillus niger.

A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gen_e product in a specific, desired fashion.

Such modificzations (e.g., glycosylation) and processing (e.g. cleavage) ©f protein products may facilitate optimal functioning of the= protein.

Vari-ous host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skilll in the art of molecular biology and/or microbiology= can be chosen to ensure the desired and correct modificzation and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess thes cellular machinery for proper processing of the primary transcript, glycosylatior, and phosphorylation of the gene product can be used. Such hosst cells are well known i nthe art.

Hosst cells also include, but are not Bimited to, mammalian cell lines such as

CHO, VERO, BHK, Hela, COS, MDCK, 293, 37T3, WI38, and choroid plexus cell lines.

If desired, the polypeptides according to the invention can be produced by a stably-trans®ected cell line. A number of veactors suitable for stable trarmsfection of mammalian cells are available to the public, mu ethods for constructing such ceili lines are also publicly known, e.g., in Ausubel et al. (supmra).

Antibodies

Th-e invention further features antibeodies, such as monoclonal or polyclonal antibodies, that specifically bind lipolytic enzyrmne proteins according to the inwention.

As= used herein, the term "antibody™' (Ab) or "monoclonal antibody" (Mab) is meant to irmclude intact molecules as well as =antibody fragments (such as, for example,

Fab and F(ab"), fragments) which are capable of specifically binding to lipolytic enzyme protein. Faub and F(ab’), fragments lack the Fc fragment of intact antibody, clear more rapidly frorm the circulation, and may have lesss non-specific tissye binding of an intact _ 30 antibody (Wvahl et al, J. Nucl. Med. 24:3165-325 (1983). Thus, these fragments are preferred. . i The antibodies of the present invent_ion may be prepared by any ofa variety of methods. For example, cells expressing the lipolytic enzyme protein or an antigenic ~~ fragment thereof can be administered to an animal ir order to induce the productiosn of sera containing polyclonal antibodies. In a preferred method, a preparation of lipolytic enzyme protein. is prepared and purified to rend-er it substantially free of natural contaminants. Ssuch a preparation is then introduced. into an animal in order to prosduce polyclonal antisera of greater specific activity. in the most preferred method, the antiboedies of the present inventiory are monoclonal antibodies (or lipolytic enzyme proteirm binding fragments thereof). Such monoclonal an®ibodies can be prepared using hybridoma technology (Kohler ext al,

Nature 256:495 (1975); Kohler et al, Eur. J. Immurwol. 6:511 (1976); Hammerling et al.,

In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).

In general, suczh procedures involve immunizing an animal (preferably a mouse) with a lipolytic enzymee protein antigen or, with a lipolytic enzyme protein expressing cel 1. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myelo ma cell line may be employed in accordance with the present inveentoin; however, it is goreferably to employ the parent mye loma cell line (SP20), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cellls are selectively maintained in HAT medium, and then cloned by 1 imiting dilution as described by Wands et al. (Gastro-eanterology 80:225-232 (1981)). The hybridoma cells obtained through such a selectior are then assayed to identify clones which secrete antibodies capable of binding the lipolytic enzyme protein antigen. In general, the p olypeptides can be coupled to a cari er protein, such as KLH, as desscribed in Ausubel et -al., supra, mixed with an adjuvant, arad Injected into a host mammal.

In particular, various host animals cam be immunized by injection of a polypeptide of interest. Examples of suitable host animals include rabbits, mice, guinea pigs, and ratss. Various adjuvants can be used to increase the immunological response, depending omh the host species, including but n ot limited to Freund's (complete and incomplete), adjuvant mineral gels such as aluminum hydroxide, surface actve substances ssuch as lysolecithin, pluronic polyolss, polyanions, peptides, oil emulsions, keyhole lim pet hemocyanin, dinitrophenol, B3CG (bacille Calmette-Guer@n) and .-

Corynebacte=rium parvum. Polyclonal antibodie=s are heterogeneous populations of antibody moRecules derived from the sera of the immunized animals.

Suczsh anfibodies can be of any immuno=globulin class including 19G, 1gM, IgE, .

IgA, 1gD, and any subclass thereof. The hyberidomas producing the mAbs of this invention can be cultivated in viatro or in vivo.

Once produced, polyclonal or monoclonal antibodieas are tested for specifi ¢ recognition of a protein according to the invention or function=al equivalent thereof in a n immunoassay, such as a Westen blot or immunoprecipitatio n analysis using standard techniques, e.g., as described in Ausubel et al., supra. Antibcodies that specifically birmd to a protein according to the imvention or functional equivalents thereof are useful in the invention. For example, such antibodies can be used in an immunoassay to detect a protein according to the invention in pathogenic or non-pathoegenic strains of Aspergilless (e.g., in Aspergillus extracts).

Preferably, antibodies of the invention are producced using fragments of a protein according to the inveration that appear likely to be armtigenic, by criteria such =as high frequency of charged residues. For example, such fragments may be generated by standard techniques of PCR, and then cloned into the pGEX expression vector (Ausutoel et al., supra). Fusion proteins may then be expressed in £. coli and purified usings a 145 glutathione agarose affinity rnatrix as described in Ausubel, et al., supra. If desired, several (e.g., two or three) fussions can be generated for each protein, and each fusion can be injected into at least tuo rabbits. Antisera can be raissed by injections in a seri- es, typically including at least three booster injections. Typically, the antisera are checked for their ability to immunoprecipsitate the protein according to the invention or functional equivalents thereof whereas wnrelated proteins may serve a sa control for the specifi city of the immune reaction.

Alternatively, technicyues decribed for the productican of single chain antibodies (U.S. Patent 4,946,778 and 4,704,692) can be adapted to produce single ciain antibodies against a protein -according to the invention or fuanctional equivalents ther—eof. 5 Kits for generating and screening phage display libraries are commercially available e.g. from Pharmacia.

Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can bez found in, for example, U.S. .T Patent No. 5,223, 409; PCT Publication No. WO 92/1861. 9; PCT Publication No. WO 91/17271; PCT Publication No. WO 20791; PCT Publication No. WO 92/20791; PCT

Publication No. WO 92/156°79; PCT Publication No. WO 933/01288; PCT Publicatiorm No. : WO 92/01047; PCT Publicastion No. WO 92/09690; PCT P=ublication No. WO 90/02809,

Fuchs et al. (1991) Bio/Teachnology 0:4370-1372; Hay et al (1992) Hum. Ant-ibod.

Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et aml. (1993)

EMBO J. 12:725-734.

Polyclonal and monoclonal antibodies that specifically bind a protein according to the invention of functional equivalents thereof can be used, for example, to detect expression of a gene encoding a protein according to the invention or a Functional equivalent thereof e.g. in another strain of Aspergillus. For example, a protein &xccording to the invention can be readily detected in conventional immunoassays of Aspergillus colls or extracts. Examples of suitable assays include, without limitation, Western blotting, ELISAs, radicimmune assays, and the like.

By "specifically binds" is meant that an antibody recognizes andi binds a particular antigen, e.g., a protein according to the invention, but does not suBostantially recognize and bind other unrelated molecules in a sample. :

Antibodies can be purified, for example, by affinity chromatography methods in which the polypeptide antigen is immobilized on a resin.

An antibody directed against a polypeptide of the invention (e.g., maonoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supematant) in order to ev-aluate the ) abundance and pattern of expression of the polypeptide. The antibodies caan also be used diagnostically to monitor protein levels In cells or tissue as part of a clinical testing procedure, ©.g., to, for example, determine the efficacy of a given treatment r-egimen or in the diagnosis of Aspergillosis..

Detection can be facilitated by coupling the antibody to a detectable substance.

Examples of detectable substances include various enzymes, prosthetlic groups, fluorescent materials, luminescent materials, bioluminescent materials, and wradioactive materials. Examples of suitable enzymes include horseradish peroxidases, alkaline phosphatase, B-galactosidase, or acetylcholinesterase; examples of suitable Eluorescent materials include umbelliiferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a. luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive mater als include 125) 131) 35g por °H. .

Preferred epitopes encompassed by the antigenic peptide are regio=ns that are located on the surface of the protein, e.g., hydrophili=c regions. Hydrophobicity lots of the proteins of the irvention can be used to identify hysdrophilic regions.

The antigenic peptide of a protein of the= invention comprises at Meast 7 (preferably 10, 15., 20, or 30) contiguous amino aacid residues of the amiro acid sequense selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 and encompasses an epitope of the protein such that an =antibody raised against the peptide forms a specific immune cosmplex with the protein.

Preferred epitopes encompassed by the artigenic peptide are regions=s of the protein according ®o the invention that are located on the surface of the prote=in, e.g., hydrophilic regionss, hydrophobic regions, alpha regions, beta regions, coil regions, turn regions and flexible regions. immunoassays

Qualitative or quantitative determination of a polypeptide according to the present invention in a biological sample can ocecur using any art-known method.

Antibody-based te chniques provide special advantagges for assaying specific polypeptide levels in a biologic=al sample.

In these, —the specific recognition is providedll by the primary antibody (p=olyclonal or monoclonal) but the secondary detection systema can utilize fluorescent, en zyme, or other conjugated ssecondary antibodies. As a result, &an immunocomplex is obtaimned.

Accordingly, the invention provides a meth od for diagnosing whether a certain organism is infected with Aspergillus comprising the steps of: » Isolating am biological sample from said organism suspected to be infe=cted with

Aspergillus, « reacting s=aid biological sample with an antibody according to the inventieon, e determinirg whether inmunecomplexes are —formed.

Tissues can also be extracted, e.g. with urea and neutral detergert, for the liberation of protzein for Western-blot or dot/slot =assay. This technique car aiso be applied to body flmids. :

Other artibody-based methods useful for detecting a protein accord-ing to the invention includes immunoassays, such-as the ernzyme linked immunosorbeent assay (ELISA) and the radioimmunoassay (RIA). For exaample, monoclonal antibodiees against a protein according to the invention can be used both as &n immunoabsorbent and as an enzyme-labeled probme to detect and quantify the protein =according to the invention. The amount of protein present in the sample can be calculated by reference to the amount present in a standeard preparation using a linear regression computer algorithm. In another ELISA assawy, two distinct specific monoclonal antibodies can be used to detect a protein according to the invention in a biological fluid. In this assay, one of the antibodies is used ass the immuno-absorbent and the other as the enzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or "two- step” assay. The one-step” assay involves contactimg a protein according to the invention with immobilized antibody and, without washiing, contacting the mixture with the labeled antibocly. The “two-step” assay involves washing before contacting the mixture with the labeled antibody. Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one comp onent of the assay system on a support, thereby allowing other components of the syste m to be brought into contact with the component and readily removed from the sample.

Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the goroduction of hydrogen peroxide by reacting with substrate. Activity of an oxidase label maay be assayed by measuring the comcentration of hydrogen peroxide formed by the enzy me-labelled antibody/substrate reaction.

Besides emzymes, other suitable labels inclucie radioisotopes, such as iodine (1251, 121), carbon (* 4C), sulphur (8), tritium (3H), indiumm (***In), and technetium >" Tc), and fluorescent lab-e€ls, such as fluorescein and rhodam ine, and biotin.

Specific b&nding of a test compound to a protein according to the invention can be detected, for example, in vitro by reversibly or irreversibly immobilizing the protein according to the [invention on a substrate, ©.g., thes surface of a well of a 96-well 8 polystyrene microtitre plate. Methods for immobilizirg polypeptides and other small molecules are weEl known in the art. For example, th e microtitre plates can be coated with a protein according to the invention by adding the protein in a solution (typically, ata. concentration of 0v.05 to 1 mg/ml in a volume of 1-100 ul) to each well, and incubatings the plates at rooms temperature to 37 OG for 0.1 to 36 Tours. Proteins that are not bound to the plate can toe removed by shaking the excess solution from the plate, and them washing the plates (once or repeatedly) with water or a buffer. Typically, the protein is contained in water or a buffer. The plate is then waashed with a buffer that lacks thes bound protein. To block the free profein-binding sites on the plate s, the plates are blockead with a protein that is unrelate=d to the bound protein. For example, 300 ul of bovine serum albumin (BSA) at a comncentration of 2 mg/m! in Tri=s-HCl is suitable.

Suitable substrates include those smubstrates that contain a defined cross-linking chemiisiry (e.g., plastic substrates, such as polystyrene, styrene, or polypropylene substrates from Corning Costar Corps. (Cambridge, MA), for examyple). If desired, a beaded particle, eg. beaded agarosse or beaded sepharose, carm be used as the substmrate.

Binding of the test compound to the polypeptides according te the invention can be detected by any of a variety of arti<nown methods. For example, =a specific antibody can Tbe used in an immunoassay. If desired, the antibody can be labeled (e.g. fluorescently or with a radioisotope=) and detected directly (see, eg., West and

McMsahon, J. Cell Biol. 74:264, 1977) — Alternatively, a second antibo=dy can be used for detection (e.g., a labeled antibody thak binds the Fc portion of an anti—AN97 antibody). In an alternative detection method, the protein according to the invention is labeled, and the 1.abel is detected (e.g., by labeling a protein according to thee invention with a radioisotope, fluorophore, chromopho re, or the like). In still another mmethod, the protein accoerding to the invention is produced as a fusion protein with a =protein that can be ‘dete=cted optically, e.g., green fluore=scent “protein (which can be ~detected under UV light). In an alternative method, the pwotein according to the invention can be covalently attached to or fused with an enzymes having a detectable enzyma. tic activity, such as horse radish peroxidase, alkaline pho=sphatase, alpha-galactosidase, or glucose oxidase.

Genses encoding all of these enzymes have been cloned and are readily available for use by those of skill in the art. If desired, the fusion protein can incl ude an antigen, and suck an antigen can be detected and measured with a polycleonal or monoclonal "antibody using conventional methods. Suitable antigens include e nzymes (e.g., horse radi sh peroxidase, alkaline phospha tase, and alpha-galactosidase) and non-enzymatic polypeptides (e.g., serum proteins, such as BSA and globulins, anc milk proteins, such as caseins). ie . ; : - Epitopes, antigens and immunogens.

In another aspect, the inve=ntion provides a peptide or polypeptide comprising an epitogpe-bearing portion of a polypeptide of the invention. The epitope of this polypepti-de portion is an immunogenic or santigenic epitope of a poBypeptide of the invention . An "immunogenic epitope” is deflined as a part of a protei n that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On t-he other hand, a region off a protein molecule to which an antibody can bind is defined as an “antigenic epitope."™ The number of immunogenic epitcepes of a protein generally™ is less than the number of antigenic epitopes. See, for instasce, Geysen, H. M. et al., WProc. Natl. Acad.

Sci. USA 81:3998-4002 (1984).

As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., tha_t contain a region of a protein molecsule to which an antibody caan bind), it is well known i n that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. Ses, for instance, Sutcliffe, J. G. et al., Science 21:660-666 (1984).

Peptidess capable of eliciting protein-reactlive sera are frequently re=presented in the primary sequence of a protein, can be characterized by a set of simp le chemical rules, and are confined neither to immunodominant regions of intaect proteins (i.e., immunogenic epitopes) nor to the amine or carboxyl terminals. Peptides that are extremezly hydrophobic and those of six or~ fewer residues generally are ineffective at inducineg antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residuess, usually are effective. Sutcliffe et al., supra, at 661 . For instance, 18 of 20 peptides designed according to these guidelines, contaiming 8-39 residues covering 75% of the sequence of th e influenza virus hemag-glutinin HA! polypeptide chain, indwiced antibodies that reacsted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase =and 18/18 from the rables glycoprotein induced antibodies that precipitated the respective proteins.

Antigenic epitope-bearing peptidees and polypeptides of the invention are therefore useful to raise antibodies, ircluding monoclonal anti bodies, that bind specifi-cally to a polypeptide of the .inven®ion. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with a n antigen epitope- pearin-g peptide generally secrete antibody reactive with the native protein. Sutcliffe et al., supra, at 663. The antibodies raise«d by antigenic epitope b-earing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes posttraansiation processing. The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assaays for the mimicked protein, for instance in competition assays since it has been shown that even short pepticies (e.g, about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson, LA. etal., Cell 37:767-778 at 77 (1984). The anti-peptides antibodies of the invention also aree useful for purification of the mimicked protein, for instance, by adsorption chromatograp hy using methods well known in the art.

Antigenic epitcepe-bearing peptides and polypepticies of the invention desigmned according to the above guidelines preferably contain a seqmuence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino a=cids contained within the armino acid sequence of a polypeptide of the invention. Howe=ver, peptides or polypeptidess comprising a larger portion of &n amino acid sequence -of a polypeptide of the invemntion, containing about 30 to about 50 amino acids, or any lemngth up to and including thee entire amino acid sequence of a= polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epilope-bearing peptide is sselected to provide substamntial solubility in aqueous seolvents (i.e., the sequence includes- relatively hydrophilic resiciues and highly hydrophobi.c sequences are preferably avoide=d); and sequences conta ining proline residues are particularly preferred.

The epitope—bearing peptides and polypeptides of the invention maw be produced by any cormventional means for making peptides or polypeptides including recombinant means ussing nucleic acid molecules of the i nvention. For instance, a short épitope-bearing amino acid sequence may be fused to = larger polypeptide whicha acts as a carrier during recombinant production and pumrification, as well as during immunization to produsce anti-peptide antibodies. oo Epitope-bearing peptides also may be synthessized using known metho.ds of chemical synthesis. For instance, Houghten has described a simple metho=d for synthesis of large nu mbers of peptides, such as 10-20 mg of 248 different 13 re=sidue : peptides representing single amino acid variants of a segment of the HA! polypesptide which were prepared and characterized (by ELISA-type binding studies) in less tha.n four weeks. Hoaughten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1285). This "Simultanesous Multiple Peptide Synthesis (SMIPS)” process is further describ ed in U.S.

Patent No. 4,631,211 to Houghten et al. (1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods.

A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously. Houghten et al., supra, at 5134.

Ewpitope-bearing peptides and polypeptides of the invention are use to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wil=son et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:9t 0-914; and

Bittle, F.J. et al., J. Gen. Virol. 66:2347-2354 (1985).

GSenerally, animals may be immunized with free peptide; however, ==nti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecmuilar carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instanc-e, peptides containingg cysteine may be coupled to carrie 1 using a linker such as maleim-idobenzoy}-

N-hydroxyysuccinimide ester (MBS), while other peptides may be coupled to Carrier using a more general linking agent such as glutaraldehyde.

Mnimals such as rabbits, rats and mice are immunized with either free or camiercotupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsionss containing about 100 ug peptide» or carrier protein and Freundl's adjuvant.

Several bwooster injections may be needed, for instance, at intervals of about two weeks, to providez a useful titer of anti-peptide antibcady which can be detected, for example, by

ELISA asssay using free peptide adsorbed #0 a solid surface. The titer of anti-peptide antibodie s in serum from an immunized ani mal may be increased by selection of anti- peptide antibodies, for instance, by adsorption to the peptide on a solid support’ and elution off the selected antibodies according to methods well known in the art. : “Jmmunogenic epitope-bearing peptides of the invention, i.e., thosse parts of a protein tlnat elicit an antibody response whean the whole protein is the immunogen, are identified according to methods known in the art. For instance, Geysen et al, 1984, supra, cdiscloses a procedure for rapid concurrent synthesis on solid supports of hundredss of peptides of sufficient purity to react in an enzyme-linked im munosorbent assay. Imiteraction of synthesized peptides with antibodies is then easily detected without

A removing them from the support. in this manner a peaptide bearing an immunogenic epitope of a desired protein may be identified routinely ky one of ordinary skill in the art.

For instance, the immunologically important epitope in thee coat protein of foot-and-umouth disease virus was locate=d by Geysen et al. with a resolution of seven amino ac ids by synthesis of an overlapping set of all 208 possible hexa. peptides covering the entime 213 amino acid sequence of the protein. Then, a completes replacement set of pepti des in which all 20 amino acid s were substituted in tum at every position within the e=pitope were synthesized, and t he particular amino acids conferring specificity for the rezaction : with antibody were determined. Thus, peptide analogs Of the epitope-bearing peptides of the invention can be nade routinely by this method. . U.S. Patent No. 4,708, 781 to

Geysen (1987) further describes this method of idientifying a peptide bearing an immunogenic epitope of adesired protein.

Further still, U.=S. Patent No. 5,194,392 to Gemysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids cer other compounds) which Is a ftopological equivalent of the ep-itope (i.e., a "mimotope”) vvhich is complementary to a particular paratope (antigen bindirg site) of an antibody of imterest.

More generally, U.S. P=atent No. 4,433,092 to Geyse=n (1989) describes a me=thod of detecting or determining a sequence of monomers whieh is a topographical equiv-alent of a ligand which is complementary to the ligand bindirg site of a particular receptor of interest. Similarly, U.S . Patent No. 5,480,971 to Heoughten, R. A. et al. (1996) on

Peralkylated Oligopepotide Mixtures discloses Ilnear C1-C7-alkyl pera lkylated oligopeptides and sets sand libraries of such peptides, as well as methods for usi ng such oligopeptide sets andl libraries for determining the sequence of a peramlkylated oligopeptide that prefe rentially binds to an acceptor molecule of interest. Thuis, non- peptide analogs of the apitope-bearing peptides of the invention also can be made routinely by these methmods. ‘

Usse of lipolytic enzymes in industrial processes

The invention also relates to the use of the= lipolytic enzyme accordimg to the invention in a selected number of industrial processes. Despite the long-term ex_perience obtained with these processes, the lipolytic enzyme a-ccording to the invention features a number of significant advantages over the enzymes currently used. Dependin_g on the:

A specific application, these advantages can include aspects like lower production costs, higher specificity towards the substrate, less antigenic, les s undesirable side activities, higher yields wher produced in a suitable microorgani sm, more suitable pH and temperature ranges, better tastes of the final product as weell as food grade and kosher aspects.

The preset invention also relates to methods for [oreparing a dough or a baked product comprising incorporating into the dough an effective amount of a lipolytic enzyme of the present invention which improves one or more properties of the dough or the baked product obtained from the dough relative to a dough or a baked product in which the polypepti de is not incorporated.

The phrase “incorporating into the dough” is d efined herein as adding the lipolytic enzyme according to the invention to the dough, any ingredient from which the dough is to be maddie, and/or any mixture of dough ingredie mts form which the dough is to be made. In other words, the lipolytic enzyme according teo the invention may be added in any step of the clough preparation and may be added ir one, two or more steps. The lipolytic enzyme according to the invention is added to them ingredients of a dough that is kneaded and baked to make the baked product using maethods well known in the art.

See, for example, U.S. Patent No. 4,567,046, EP-A-426,2211, JP-A-60-78529, JP-A-62- © 111629, and JP-A—63-258528.

The term ‘effective amount” is defined herein as an amount of the lipolytic enzyme according to the invention that is sufficient for providing a measurable effect on at least one property of interest of the dough and/or baked product.

The term. “improved property” is defined hereim as any property of a doughs : andlor a product obtained from the dough, particularl y a baked product, which iss improved by the action of the lipolytic enzyme according) to the invention relative to aa -dough or product in which the lipolytic enzyme accoerding ‘to the invention is not incorporated. The improved property may include, but is not limited to, increased strength of the do ugh, increased elasticity of the dough, imncreased stability of the dough , reduced stickiness of the dough, improved extensibility of" the dough, improved flavour o f the baked product, improved anti-staling of the baked pro=duct. N

The improved property may be determined by c=omparison of a dough and/or = : baked product prepared with and without addition of a polypeptide of the preserat invention in acco rdance with the methods of present in~vention are described below im the Examples. Organoleptic qualities mays be evaluated using procedures well establishe d in the baking industry, and may include, for example, the uses of a panel of trained tasste-testers.

The term “increased strength of the «jough” is defined herein as the property of a dough that has generally more elastic progperties and/or requires mores work input to mould and shape.

The term “increased elasticity of the dough” is defined herein as the property of a dough vvhich has a higher tendency to rega in its original shape after bei ng subjected to a certain physical strain.

The term “increased stability of the cough” is defined herein as tlhe property of a dough thaatis less susceptible to mechanical =abuse thus better maintainirmg its shape and volume. ~The term “reduced stickiness of the dough” is defined herein ass the property of a dough that has less tendency to adhere to surfaces, e.g., in the dough production machinery, and is either evaluated empirically by the skilled test baker «or measured by the use ofa texture analyser (e.g., TAXT2) a-s known in the art. ~The term “improved extensibility of the dough” is defined hereime as the property of a dough that can be subjected to increase d strain or stretching without= rupture. ~The term “improved machineabilityy of the dough” is defined herein as the property of a dough that is generally less sticsky and/or more firm and/or rmore elastic.

The term “increased volume of the baked product” is measuread as the specific volume of a given loaf of bread (volume/we=ight) determined typically Ioy the traditional rapeseed displacement method.

The term “improved crumb structure of the baked product” is clefined herein as the property of a baked product with finer =and/or thinner cell walls in tThe crumb and/or more uriform/homogenous distribution of cells in the crumb and is umsually evaluated . empirically by the skilled test baker.

The term “improved softness of the baked product’ is the oppamsite of “firmness” and is d efined herein as the property of a b=aked product that is more e==asily compressed and is esvaluated either empirically by the silkilled test baker or measure d by the use of a texture zanalyzer (e.g., TAXT2) as known in —the art. ’ The term “improved flavor of the toaked product’ is evaluated by a trained test panel.

The term “improved anti-staling of the baked product” is defined hereirm as the properti es of a baked product that have a reduced rate of deterioration of quality parame=ters, e.g., softness and/or elasticity, during storage.

The term “dough” is defined herein as a mixture of flour and other ingmredients firm eneugh to knead or roll. The dough may be fresh, frozen, pre-bared, or pre=-baked.

The pr-eparation of frozen dough is described by Kulp and Lorenz in Frozen and

Refrige-rated Doughs and Batters.

The term “baked product’ is defined herein as any product preparecl from a dough, either of a soft or a crisp character. Examples of baked products, whether of a white, light or dark type, which may be advantageously produced by the present invention are bread (in particular white, whole~meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pasta, pita bread, tortillas, tacos, cakes, pancal=es, biscuits, cookies, pe crusts, steamed bread, and crisp bread, and the= like.

Lipolytic enzyme of the present invention and/or additional enzymes to be used in the methods of the present invention may be in any form suitable for th e use in question, e.g., in the form of a dry powder, agglomerated powder, or grarulate, in particualar a non-dusting granulate, liquid, in particular a stabilized liquid, or orotected enzymme such described in WO01/11974 and WO002/26044. Granula—tes and agglormerated powders may be prepared by conventional methods, e.g., by spraying the lipolyti c enzyme according to the invention onto a carrier in a fluid-bed granul ator. The carrier may consist of particulate cores having a suitable particle size. The camiTier may be soBuble or insoluble, e.g., a salt (such as NaCl or sodium sulphate), sugar (such as sucrosse or lactose), sugar alcohol (such as sorbitol), starch, rice, com grits, or~ soy. The lipolytdc enzyme according to the invention and/or additional enzymes may be contained in slown-release formulations. Methods for preparing slow-release formulations are well knowr in the art. Adding nutritionally acceptable stabilizers such as sugar, sugar alcohol, or another polyol, and/or lactic acid or another organic acid according to esstablished methods may for instance, stabilize liquid enzyme preparations.

The lipolytic enzyme according to the invention may also be incorporated in. yeast comprising ‘compositions such as disclosed in EP-A-0619947, EP-A-0659344 and

WO00:2/49441. .

For inclusion in pre-mixes of flour it is advantageous that the p olypeptide according to the invention is in the form of a dry product, e.g., a non-dusting granulate,

oo WO 2004/018660 PCT/EP2003/009145 whereas for inclusion together with a liquid it is advantageously in a liquid form.

One or more additional eenzymes may also be Incorporated into the dough. The additional enzyme may be of any” origin, including mammalian aned plant, and preferably of microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art.

In a preferred embodiment, the additional enzyme may bee an amylase, such as an alpha-amylase (useful for providing sugars fermentable bw yeast and retarding staling) or beta-amylase, cyclodextrin glucanotransferase, pepti dase, in particular, an exopeptidase (useful in flavour enhancement), transgiutaminases, lipase (useful for the modification of lipids present in the dough or dough constituermts so as to soften the dough), phospholipase, cellulase, hemicellulase, in particular a pentosanase such as xylanase (useful for the partial hydrolysis of pentosans which increases the extensibility of the dough), protease (useful for gluten weakening in particular when using hard wheat flour), protein disulfide isomerase, .9., a protein disulfide isomersase as disclosed in WO 95/00636, glycosyltransferase, peroxidase (useful for improving tthe dough consistency), laccase, or oxidase, e.9., an glucose oxidase, hexose oxisdase, aldose oxidase, pyranose oxidase, lipoxygenase or L-amino acid oxidase (use=ful in improving dough consistency).

When one or more additional enzyme activities are to Ibe added in accordance with the methods of the present invention, these activities may be added separately or together with the polypeptide according to the invention, optiormally as constituent(s) of the bread-improving and/or dough-improving composition. The other enzyme activities may be any of the enzymes described above and may be do=sed in accordance with established baking practices.

The present invention also relates to methods for pre=paring a baked product comprising baking a dough obtained by a method of the preserat invention to produce a baked product. The baking of the dough to produce a baked preaduct may be performed using methods well known in the art. ut The present invention also relates to doughs and bake-d products, respectively, produced by the methods of thes present invention. Co : The present invention further relates to a pre-mix, e.c3, in the form of a flour . composition, for dough and/or baked products made from dou gh, in which the pre-mix comprises a polypeptide of the present invention. The term “pre--mix’ is defined herein to be understood in its conventional meaning, i.e., as a mix of baking age=nts, generally including flour, which may be used not only in industrial bread-baking plan-ts/facilities, but also in retail bakeries. The pre-muix may be prepared by mixing the polypeptide or a bread-improving and/or dough-imgproving composition of the invention scomprising the : polypeptide with a suitable carrier such as flour, starch, a sugar, or a salt. The pre-mix may contain other dough-improving andlor bread-improving additives, e=.g., any of the additives, including enzymes, mentioned above.

The present invention further relates to baking additives in the form of a granulate or agglomerated powder, which comprise a polypeptide of the present invention. The baking additive preferably has a narrow particle size distrib=ution with more than 95% (by weight) of the particles in the range from 25 to 500 pm.

In dough and bread making the present invention may be used in combination with the processing aids defined hereinbefore such as the chemical proc=essing aids like oxidants (e.g. ascorbic acid), reducing agents (e.g. L-cysteine), oxidor=eductases (e.g. glucose oxidase) and/or other enzymes such as polysaccharide modifying enzymes (e.g. a—amylase, hemicellulase, branching enzymes, etc.) and/or protein modifying enzymes (endoprotease, exoprotease, branching enzymes, etc.).

EXAMPLE 1 . Fermentation of Aspergillus niger

Lipolytic enzymes encoded by the nucleotide sequence as provi ded herein were obtained by constructing expression plasmids containing the DENA sequences, transforming an A. niger strain with this plasmid and growing the AspergFilus niger strains in thw following way.

Fresh spores (10°-107) of A. niger strains were inoculated in 20 mt CSL-medium (100 mi flask, baffle) and grown for 20-24 hours at 34°C and 170 rpm. After inoculation . of 5-10 mi CSL pre-culture in 100 ml CSM medium (500 ml flask, baffle) the strains were fermented at 34°C and 170 rpm for 3-5 days. :

Cell-free supematants vwere obtained by centrifugation in 50 rl Greiner tubes ’ (30 minutes, 5000 mpm). The supematants were pre-filtered over a GF/AL Whatman Glass microfiber filter (150 mm /E) to remove the larger particles, adjusted to pH 5 with 4 N

KOH (if neczessary) and sterile fitrated over a O.2 pm (bottle-top) filter with suction to remove the —fungal material. The supernatants wesfe stored at 4°C (or -20°C).

The CSL medium consisted of (in amosunt per litre): 100 g Com Steep Solids (Roquette), 1g NaH,PO4*H.0, 0.5 g MgS0.=7H.,0, 10 g glucose*H0 and 0.25 g

Basildon (awntifoam). The ingredients were dissolved in demi-water and &he pH was adjusted to pH 5.8 with NaOH or H,SO4; 100 rw flasks with baffle and foasm ball were filled with 2€ ml fermentation broth and sterilized for 20 minutes at 120°C after which 200 ul of a soluttion containing 5000 IU/ml penicillin 2nd 5 mg/ml Streptomycin was added to each flask afer cooling to room temperature.

Th e CSM medium consisted of (in amount per litre): 150 g maltos=e*H20, 60 g

Soytone (psepton), 1 g NaH,PO4*H.0, 15 g M1gS0,*7TH.0, 0.08 g Tweer 80, 0.02 g _ Baslidon (amntifoam), 20 g MES, 1g L-arginine. The ingredients were dissolved in demi- water and &he pH was adjusted to pH 6.2 with NaOH or H,SO4; 500 ml flask<s with baffle and foam Ioall were filled with 100 ml fermentation broth and sterilized for 220 minutes at 120°C after which 1 mi! of a solution contaiming 5000 IU/ml penicillin 2nd 5 mg/mi

Streptomycin was added to each flask after cooling to room temperature.

EXAMPLE 2

Purification of the lipolytic enzymes of the invention

Step 1 - Preparation of ultrafittrates

Thine supernatants of the cultures, as obtained in Example1, were uilrafiltrated to remove thee low molecular contaminations that could interfere with the enzymatic activity determinafitions and the baking tests. Ultrafiltrati on of 30 ml supernatant wass performed in a Millipore: Labscale TFF system equipped witha a filter with a 10 kDa cut-off=.

Deepending on their colour, the samples were washed 3-5 times with 40 ml volumes of cold 100 mM phosphate buffer pH 6.0 including 0.5 mM Ca.Cl, The final volume of the enzyme solution was 30 ml and is further referred to as “ultramfitrate’.

Step 2 - [Determination of the lipolytic enzymes= concentration by A280 and HPSEC.

The concentration of the lipolytic enzy-mes in the ultrafiltrate was c=alculated from the extincstion at 280 nm (A280) attributable to the lipolytic enzymes and the calculated : molecular extinction coefficient of the lipolytic enzymes. Measurement of the A280 was

J

Performed in an Uvikon XL Secomam spectrophotometer (Beun de Ronde, Abcoude, “Yhe Netherlands).

The molecular extinction coefficient of an enzyme can be calculated from the mumber of tyrosine, tryptophan and cysteine residues per enzyme mo Tecule (S.C. Gill =and P.H. von Hippel, Anal. Biochem. 182, 319-326 (1989)). The molecular extinction .<oefficient of these amino acids are 1280, 5690 and 120 M*.cm™ respectively. The number of tyrosine, tryptophan and cysteine residues in the lipolytic enzymes of the invention can be deduced frorm the protein sequences selected from the group consisting of SEQ ID NO: 3, 6. 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39. The calculated extinction coefficients of the lipolytic enzymes of the invention are summarized in Table 2.

Table 2 ] ] alculated e inction eee snayme (Da) om rem | 7g" al CT al A Bl IA

EEE Ee

NE . ; —— 15 . The extinction of the ultrafiitrate at 280 nm (A280) that is attributable to the lipolytic enzymes depends on the purity of the enzyme sample=. This purity was determined using HPSEC (High Performance Size Exclussion Chromatography) with a

TSK SW-XL column (300*7,8 mm; MW range 10-300 kDa>). The elution buffer consisted of 25 mM sodium phosphate buffer pH 6.0 and was used at a flow of 1 mi/min. Samples of 5—100 pl were imjected. The absorbance at 280 nm wass measured.

The A280 in the ultrafiltrate attributable to the lipolytic enzyme of the invention was obtained from he ratio of the peak surface of the respective lipolytic enzyme peak in the chromatogram and the total surface of the peaks absorbing at 280 nm. The lipolytic enzyme concentration in the ultrafiltrate was then calculated by multiplying the A280 of the ultrafiltrate by the ratio described above and divided by the calculated extinction coefficient (1 mg/m1 solution — Table 2 most right column) £or each lipolytic enzyme.

EXAMPLE 3

Activity measurements

The cell-free supernatants obtained in Example “1 were subjected to the lipase, phospholipase and galactolipase assays as summarized ir Table 3.

Table 3. Lipolytic e nzyme activities in the cell free supernamnts as prepared in Example 1. lyso

I ICI I EA CE

I IA I A EER

—weeo® | o | + | 0 | 0 3 = not different from blanc; +/+++++ = higher than blanc, }

Lipase activity was determined spectrophotomet rically by using 2,3-mercapto-1- propanol-tributyrate (TBDMP) as a substrate. Lipase hydrolyses the sulphide bond of

:

TBDMP thereby liberating thio-butanoic acid which in a subsequent reaction wit 4,4,- dithiodipyriidine (DTDP) forms 4-thiopyridone. “The latter is in a tautomeric equil Tbrium with 4-mer—capthopyridine which absorbs at 334- nm. The reaction is carried out in 0.1 M acetate busffer pH 5.0 containing 0.2 % Triton-X-100, 0.65 mM TBDMP and 0.2 mM DTDP § at 37°C. One lipase unit is defined as the amount of enzyme that liberates 1 micromole of 4 thio-brutanoic acid per minute at the reactiom conditions stated.

P*hospholipase A was determined spectrophotometrically by using 1,2- dithiodiockanoyl-phosphatidyicholine as a subestrate. Phospholipase A hydrolysses the sulphide bond at the 1 position (PLA1) or the 2 position (PLA2) thereby liberating 4 thio- octanoic =acid which, in a subsequent reactiors reacts with 4,4'-dithiopyridine to orm 4- thiopyridosne. The latter is In tautomeric equilibwium with 4-mercaptopyridine that absorbs at 334 nr. The reaction is carried out in 0.1 IM acetate buffer pH 4.0 containing 0.2 %

Triton-X1%00, 0.65 mM substrate and 0.2 mM IDTDP at 37°C. One phospholipase A unit (PLA) is defined as the amount of enzyme that liberates 1 micromole of 4 thio-oOctanoic acid per rminute at the reaction conditions statexd. m_ysophospholipase activity was de€ermined with 3'P-NMR spectroscopy by using lys ophosphatidyl-choline as a substrate. Lysophospholipase hydrolyses tke ester bond theereby liberating the fatty acid from the glycerol moiety. The sca-formed glycerolpshosphacholine is quantified using NMR.

The reac=tion is carried out in 50 mM acetic acid buffer pH 4.5 further containing 1 mg/ml lysophossphatidyicholine and 5 mM CaCl, for 30 minutes at 55°C.

One lyscophospholipase unit (LPC) is defined as the amount of enzyme that forms 1 micromaele of 4 glycerolphosphocholine per m inute at the reaction conditions stated.

Galactolipase activity was determi ned with H-NMR spectroscopy boy using digalacteosyldiglyceride as a substrate, according to the method described by Hirayama and Ma-tsuda (1972) Agric. Biol. Cheri. 36a 1831. Galactolipase hydrolyses -the ester bond between the fatty acids and the glycerol backbone thereby liberating onee or both fatty acsids. The reaction is carried out in 50 mM acetic acid buffer pH 4.5 further containi ng 4 mM CaCl, 0.2% Triton X-100 and 1 mg/ml digalactosylidiglycersde (Lipid

Products) for 30 minutes at 30°C. One gal&ctolipase unit is defined as the aamount of enzyme that forms 1 micromole of fatty ackd per minute at the reaction conditiorms stated.

The uitrafiltrates obtained in Example 2, were subjected to the FALJ enzyme activity measurement. The activity of the fungal alpha-amylase was measu red using

Phadebas Amylase test tablets (Pharmacia). Phadebas tablets contain a water™ insoluble stearch substrate and a blue dye, bound bys cross-linking to the substrate. The sebstrate is hydolysed by fungal amylase, releasin g dyed soluble maltodextrines that go into solution. A calibration curve was prepare d with a solution containing a referemnce fungal al pha amylase activity. From the referen«ce and unknown samples appropriate dilutions weere prepared in 50 mM malic acid buffear pH 5.5. Samples of 5 ml were incu bated with 30°C for 5 minutes, a Phadebas tablet was added and after 15 minutes the reaction was sEopped by the addition of 1.0 mi 0.5 N sodium hydroxide. The mixtures were allowed to cool down to room temperature for 5 rminutes after which 4.0 m! water wras added, shaken by hand and after 15 minutes the samples were centrifuged at 4700 rpm for 10 minutes. The extinction of the top layers was measured at 620 nm. The OD 620 nm is a measure for fungal alpha amylase actiwity. One fungal amylase unit (FAU) is defined h erein as the amount of enzyme that converts 1 gram of starch (100% dry wnatter) per twour into a product having a transmissio n at 620 nm after reaction with a iodine solution of known strength at the reaction conditions stated. “able 4. FAU and protein in the ultrafiltraates as prepared in Example 2.

Protein (mg/ml) fungal lipolyti frormn the 280 nm ngai amy~iase polytic enzyme analysis (FAU/mY)

A NE J EN

Ec EE CN EN

BSc IC I I wees on [es]

FT | es eww | os wesw [0 m wesw wees wm [ ew mes wees Te |e ews | ww

I RL EE

In addition to the activities mentioned in Table 4, minor activities of glucoamylase was al=so present, however in such low amounts that these enzymes did not interfere in the ba king experiments described in example 4.

EXAMPLE 4

Baking experiments 1 — pup loaves

Pup loaves were baked from 150 gram dough pieces obtained by mixing 200 g 40 flour (Kolibri™/Ibis™ in a ratio of 80/20), 1,4 g dried baker's yeast (Fermipan®), 4 g salt, 3 g sugar, 10 mg asscorbic acid, 116 g water and 2 g fat. After mixing for 6 minutes and seconds in a pin mmixer, the dough was divided into pieces of 150 grams and proofed for 45 minutes at 30=C, punched, proofed for another 25 minutes, moulded and panned.

Proofing took place ext a relative humidity of 90-100%. After a final proof of 70 minutes at 15 30°C, the dough wass baked for 20 minutes at 225°C.

The variouss effects (Tables 5 and 6) of the different lipolytic enzymes in the baking experiments =were compared with a control containing the same amount of fungal amylase that was =xdded otherwise by the dosage of the ultrafiltrate (for the fungal amylase activity in te ultrafiltrates see Table 4). This was necessary since the amounts - of fungal amylase acided with the lipolytic enzymes in particular affected the: loaf volume, not the other pararmeters. The volume of the breads with the control amount of fungal amylase added was taken as 100%. : 2

Table 5. effect mee

IEREREN ENE dough stick A i ticky control much excellent ough stickine oo stic stic g SS ky bread better dry ££ 2 horter o] control

OQ | dough extensibility | Toos short | than the bread good too long control non- contro crumb structure good excellent uniform bread - Nearly . control © crust colour too light excellert too dark £ wrhite bread 3 Far too too contro absolutely x crumb colour excellent . 2 yeliow yellow bread white

Far too contro softer excellent firm bread

Loaf volume was detersmined by the Bread Volume Measurer B™VM-3 (R! Cards

Instruments AB, Viken, Swedem). The principle of this measurement i=s based on the reflection of ultrasound measured by a sensor around a rotating bread. .A measurement time was taken of 45 seconds.

Dough stickiness and esxtensibility were evaluated by a qualified baker using the scale depicted in Table 5. The average of 2 loaves per object was measwred.

After these tests the dough pieces were rounded and a first proof was performed for 45 minutes at 30°C and hereafter the dough was purached, moulded, panned, proofed for 75 minutes: at 30°C. The relative humidity during the proofs was set at 85%. - Subsequently the stab lity of the proofed dough was judged by the presence of 1% bladders, tom side crust and ixregular curved surfaces of the crust. The dough pieces were baked for 20 minutes at 225°C. Loaf volumes were determineci by the BVM-3 method: in the table the averagye is presented of 2 breads that are bake=d from the same object.

~ WO 2004/018660 PCT /EP2003/009145 © -56-

The crumb structure was judged by a qualified baker using the scsale depicted in

Table 5. After storing the loaves for three days in polyethylene bags at roowm temperature crumb firmness was measured using a Stevens Texture Analyser. Two slices of 2 cm thickness from the centre of each loaf were analysed by the texture anealyser using a probe of 1.5 inch diameter, a compression depth of 5 mm (25%) =and a rate of compression of 0.5 mm/sec. in the table the average is shown of two meassurements.

Crust colour was Judged by a qualified baker according to the scale depicted in

Table 5. As a reference the standard recipe for Dutch tin bread was used.

Crumb colour was judged by a qualified baker according to the scsale depicted in

Table 5. The colour of the crumb of the control breads was judged as n=omal (3). As a positive control the breads of 2 objects are used with the same comyposition as the control plus 0.5% soya flour. The proofing and baking procedure are the =same as that of the control without soya flour. The latter is judged as “excellent”.

The overhanging top of the bread was judged by the hangin g of the top in relation to the baking tin, the lower the edges of the top the lower the BMudgement. The less hanging, the better the judgement,

Staling of the bread was judged by feeling the firmness of the crumb of slices of the bread. Before slicing took place, the bread was stored in a plasttic bag at room temperature for 4 days. The softer the crumb of the slices is, the better thme judgement.

Table 6. Baking performance of th e lipolytic enzymes of the invention

Lipolytic 9 2 z 0 5 5 2 5 |88|&8s|85|65| 8 | 5 [27 > » o Oo o o ego | 00 | 3 | 8 | «| 2 | ss | C1 *] eos | oe | |S | 4 [e+ [+ | 1° ees |e [3 [+ | +a & [SF] eo [fez | 8 | 2 | + | 5 | 4a [TT] 7 eeoaz | 8 | 8 | 5 | + | 2 [8 [® [P]7

Neem [os | 5 | 2 | 4 [241 T° [7

Ea I ME EN NC NC NC RC A I egoes [0 | 9 | 5 | 4 [8 | ¢ | 4 | * 1° esos [oo | 3 | 6 | 4 | 5 | 4 | 4 | °°]

I I I EE I HC NN NC NG eee [os | 3 | 8 | 4 | | 4 | 4 [os]

Neos | 10 | 3 | 8 | 4 | 4 | 8 | 4 [4 | 4 meeoiz [Mo | 8 | 4 | 4 | + [8 | «33

EXAMPLE §

Baking experiments 2 — batard

The baking performance of lipolytic enzymes according to the invention was tested in the French type of bread called “batard”. Preparation «of batards in a standard baking process was done by mixing 3000 g of wheat flou r at circa 20°C, 70 g compressed yeast, 60 g salt, 68 ppm ascorbic acid, 30 ppm Bamkezyme® HS» (fungal hemicellulase), 7 ppm Bakezyme® P500 (fungal a~amyiase) =and 1680 mi water (8— 10°C) in a spiral mixer (Diosna: 2 minutes in speed 1; 100 Wh input in speed 2). The dough temperature was 27°C. The machineability of the dough vevas analysed by hand by a baker. The dough was given a bulk proof of 15 minutes in a proofing cabinet at 32°C and 90% RH. Afterwards the dough was divided into 6 piecess of 350 g, rounded and proofed for 15 minutes at 32°C and 90% RH. At the end of this period the dough pieces were moulded and shaped and given a final proof of 90 minutess at 32°C and 90% RH.

The fully proofed doughs were cut in the length of the dough piezce and baked in an oven at 240°C for 30 minutes with initial steam addition. After cooling down to room p temperature the volumes of the loaves were determined byy the BVM-method (see example 4). 20 . . .. =

Break, shred and shape of the breads were analysed cCirectly after cooling down to room temperature by a qualified baker using the score in Table 7. After 16 hours (overnight) starage in a clo sed box at room temperature the crumb quality was assessed a qualified baker. The value for the breads (Table 8) was derived from 1 object. /

Table 7

Effect see — [2 | = y{ + [| 5 dr thin and crispy extrerm

Break and shred K i" weak and control crust crust too= thin, re n a ak and shred } weal an soft bread firm break of too haard soffit the cut contro

Crumb structure pocor not uniform good excelleent bread } control Much learger height flat medium larger than (3) bread than =(3) ) t @ control completely completely cut cut cl-osed | cut closed opene=ed; bread opened tearexd

Table 8. Baking performance of the lipolytic enzymes of t he invention lipolytic enzyme

Dosage™* “ooo Break & Crum b 9 py Sh red structuare (%) ew | 0 | w | = | @ | 3 wees @ [@ [1 0 nero | @ | © | + | 1°

ER A I A A NE

* in ppm based on flour veveight and enzyme weight determin.ed by the A280 method

Claims (24)

1. An isolated polynucleotide hybridisable to a polynucleotide selected from the group consisting of SEQ ID NO: 1, 2, 4,5,7, 8,10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38.

2. An isolated polynucleotide according to claim 1 hy bridisable under high stringency conditions to a polynucleotide selected from ~the group consisting of SEQ ID NO: 1, 2, 4, 5,7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35,37 and 38.

3. An isolated polynucleotide according to claims 1 or 2 obtainable from a filamentous fungus.

4. An isolated polynucleotide according to claim 3 obta@nable from Aspergillus niger.

5. An Isolated polynucleotide encoding a polypeptide cormprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalents timereof.

6. An isolated polynucleotide encoding at least one Functional domain of a polypeptide selected from the group consisting of SECQm ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 39 or functional equivalemts thereof. 200

7. An isolated polynucleotide comprising a nucleotide seq uence selected from the group consisting of SEQ ID NO: 1, 2, 4,5,7, 8, 10, 11,13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37 and 38 oor functional equivalents thereof.

8. An isolated polynucleotide selected from the group commsisting of SEQ ID NO: 1, 2,4,5,7,8,10, 11,13, 14, 18, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, - 37 and 38. ’

9. A vector comprising a polynucleotide sequence according to claims 1t08. .

10. A vexctor according to claim 9 wherein ssaid polynucleotide sequences according to claims 1 to 8 is operatively linked with regulatory sequences ssuitable for exp wession of said polynucleotide sequeance in a suitable host cell.

11. A vector according to claim 10 whereir said suitable host cell is a filamentous fungus.

12. A ranethod for manufacturing a polynuecieotide according to claims 1-8 ora vector according to claims 9 to 11 comprising the steps of culturingg a host cell trarsformed with said polynucleotid e or said vector and isolating said polynucleotide or said vector from said host cell.

13. "An isolated lipolytic enzyme selected from the group consisting of SSEQ ID NO: 3, 6 9, 12, 15, 18, 21, 24, 27, 30, =33, 36 and 39 or functional equivalents thereof.

14. Anx isolated lipolytic enzyme according to claim 13 obtainable fron Aspergilius nigger.

15. Ara isolated lipolytic enzyme obtai nable by expressing a polynucleotide according to claims 1 to 8 or a ve=ctor according to claims 9 fo 11 in an appropriate host cell, e.g. Aspergillus miger.

16. Re=combinant lipolytic enzyme comprising a functional domain of any of the lipwolytic enzymes according to claims 13-15.

17. A method for manufacturing a lipolytic enzyme according to cla@ims 13 to 16 comprising the steps of transformirg a suitable host cell withm an isolated polynucleotide according to claims 1 to 8 or a vector according to claims 9 to 1-1, culturing said cell under conditions allowing express-ion of said polynucleotide and optionally purifyingy the encoded polypeptide from said cell or culture medium.

18. A. recombinant host cell comprising & polynucleotide according to claims 1 to 8 o.r a vector according fo claims 9 to 1 1.

19. A recombinant host cell expressing a lipolytic enzyme according to claims 13 to

20. Purified antibodies reactive with a lipolytic enzyme according to claims 13to 1 6.

21. Fussion protein comprising a lipolytic enzyrme sequence according to claims 13 to

16.

22. A process for the production of dough comprising adding a lipolytic emnzyme according to anyone of claims 13-16.

23. A process for the production of a baked product from a dough as prepared by the promcess of claim 22.

24. Usee of a lipolytic enzyme according to anyone of claims 13-16 for the preparation of a dough and/or the baked product thereof.