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WO2004039845A1 - Anticorps de lipase antimicrobiens, sequences nucleotidiques et d'acides amines de ces derniers, utilisations correspondantes - Google Patents

Anticorps de lipase antimicrobiens, sequences nucleotidiques et d'acides amines de ces derniers, utilisations correspondantes Download PDF

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Publication number
WO2004039845A1
WO2004039845A1 PCT/EP2002/012149 EP0212149W WO2004039845A1 WO 2004039845 A1 WO2004039845 A1 WO 2004039845A1 EP 0212149 W EP0212149 W EP 0212149W WO 2004039845 A1 WO2004039845 A1 WO 2004039845A1
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lipase
polypeptides
antibody
homology
antibodies
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WO2004039845A8 (fr
Inventor
Luigi Frati
Mauro Biffoni
Aurelia Rughetti
Hassan Rahimi Koshkaki
Serena Barachini
Marianna Nuti
Cleofe Paolacci
Simonetta Soro
Enrico Cernia
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Universita degli Studi di Roma La Sapienza
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Universita degli Studi di Roma La Sapienza
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Priority to AU2002368305A priority patent/AU2002368305A1/en
Publication of WO2004039845A1 publication Critical patent/WO2004039845A1/fr
Publication of WO2004039845A8 publication Critical patent/WO2004039845A8/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • Lipases are enzymes widely studied in the field of digestive mechanisms and they have been proposed as drugs for digestive diseases. These enzymes are also used in enantio- and regio-selective biocatalysis for the selective transformations of natural and synthetic substrates. Such Lipolytic enzymes hydrolyse in nature triglycerides being able also to synthesise primary and secondary esters and to accept as substrates esters with and without insaturations and with different acylic chain length.
  • Lipases have attracted a considerable amount of research mainly because of some their characteristics features, such as their amphipatic nature, their ability to act at hydrophobic-hydrophilic interface and their ability to catalyse chemical reactions in organic solvents. As a matter of fact these enzymes have been exploited also in many industrial processes for example in the field of agro- chemical, detergency or fine-chemical industry.
  • lipases of microbial and mammalian origin are known.
  • the amino acid sequences of many of these lipases have been elucidated and analysed with respect to structural and functional elements which are important for their catalytic function, as described for iihstance in Winker et al., 1990 and Schay et al. 1991.
  • Very-low homology has been found between microbial and mammalian lipases, except for G-X-S-X-G lipase consensus sequence.
  • Candida rugosa One of the most widely used lipolytic enzymes is the lipase from Candida rugosa (Benjiamin and Pandey,1998).
  • Candida rugosa yeast produces different isoenzymes: five of them, completely sequenced, have been also expressed with high yield (Lotti et al., 1993).
  • the isoform 1 is the enzyme more investigated in the open, closed form and in the presence of substrate analogues.
  • This enzymatic protein is a single polypeptidic chain of 543 aa with a MW of 60 kDa, with a catalytic triad (residues Ser209, His449 and Glu341) and an oxyanionic cavity (defined by the NH atoms of the main chain and the residues Gly124, Ala210 and Gly123); the active site is closed by a short polypeptidic chain defined as "lid" (composed by 31 aa: hydrophobic ones in the internal region, near the active site, and hydrophilic ones in the external surface).
  • the long tunnel positioned up to the active site and the region near the catalytic site, is also hydrophobic and composed by the Phe296, Leu297, Phe344 and 345 residues.
  • Structural data demonstrated that the enzyme during activation gives rise to a conformational change related to the rearrangement of a peptidic loop of 26 aa residues which are located in the protein portion called lid.
  • This amino acidic loop does not move as a rigid body but during rotation around 2 aa (Glu66 and Pro92) as hinges, there is an inner spatial rearrangement and a change in the organisation of the lid alpha helix.
  • mAbs monoclonal antibodies
  • Fig 1 Immunoreactivity of BF11 against C. rugosa lipases.
  • the immunoreactivity of BF11 hybridoma supernatant (containing the whole immunoglobulin molecule and the light chain) was assessed in a solid phase ELISA (Enzyme Linked ImmunoSorbent Assay) performed versus C. rugosa lipases. Reactivity was measured as absorbance at 492 nm wavelength and plotted on a graph.
  • MoAb BF11 recognises all the different lipase isoforms tested (partially purified lipase 1 (lane 1), lipase 2 (lane 2) and lipase 3 (lane 3)) and the immunogen (lane 4) from Sigma containing a mixture of all of them.
  • BF11 Immunocharacterization of the recognized lipase isoforms was carried out using the western blotting methodology.
  • Antigen preparations containing different C. rugosa lipase isoforms (lipase 1, lipase 2 and lipase 3) were run on a SDS-PAGE in reducing condition, blotted onto nitrocellulose and tested with BF11 hybridoma supernatant (containing the whole immunoglobulin molecule and the light chain).
  • the MoAb BF11 recognises all of the different isoforms tested that have a molecular weight ranging from 58 to 64 kDa. The presence or absence of each isoform in the sample tested is indicated as + or -. Lane 1 : lip 2 and 3, lane 2: lip
  • lane 3 lip 3
  • lane 4 lip 2
  • lane 5 lip 1.
  • the MOPC21 antibody is an isotype matched control that was run in parallel. Lane 1 : hybridoma supernatant, lane 2: affinity purified antibody, lane 3: the 50 kDa cut-off supernatant ultrafiltrate) were run on SDS-PAGE in reducing condition and blotted with anti-mouse kappa light chain as secondary antibody.
  • the MOPC21 antibody is an isotype matched control was run in parallel (lane 4) and was developed with anti-mouse IgG antibody.
  • Fig. 4 Lipolytic activity in the presence of BF11 hybridoma supernatant and in presence of BF11 light chain.
  • the enzymatic activity was measured as described in the experimental part (see example 2 and 3).
  • E enzyme (mg/ml), -•- lipase; -0- lipase + BF11 supernatant; - ⁇ - lipase + BF11 light chain.
  • Fig. 5 Relative increase of lipolytic activity in the presence of BF11 supernatant and in presence of BF11 light chain.
  • the enzymatic activity has. been measured as described in the experimental part (see example 2 and 3).
  • E enzyme (mg/ml), - ⁇ - lipase; -0- lipase + BF11 supernatant; -•- lipase + BF11 light chain.
  • Lane 1 BF-11 supernatant + lipase 1:1 (1 panel A or 2 panel B), lane 2: BF-11 supernatant + lipase 1 :2 (1 or 2), lane 3 lipase alone.
  • Fig 7. RT-PCR of BF11 light chain mRNA.
  • the hybridoma mRNA was subjected to RT-PCR amplification using specific primers.
  • the amplification produced a band of the expected size ( 667 bps) when run on an agarose gel.
  • Lane 1 Molecular weight marker (100 bps ladder)
  • lane 2 amplification without template
  • Recombinant and monoclonal antibodies recognizing microbial lipases showing an homology of at least 75% to at least one of the Candida rugosa lipase isoforms 1 or 2, or 3, or 4 or 5 are disclosed by the inventors. These reagents preferably recognize Candida rugosa lipase isoforms 1-5 either in their native and SDS- denatured form.
  • nucleotide and aminoacid sequences of the light and heavy chains of the mAb have been disclosed as seq IDN1-2 and 3-4 respectively (see Sequence listing).
  • One of the preferred embodiment of the invention is the light chain, which is able to increase significantly the hydrolytic activity of the enzyme.
  • inventions are represented by recombinant and chimeric polypeptides and antibodies comprising at least one of the CDRs of the light chain, preferably the 3 rd CDR, immunoconjugates and immunocomplexes.
  • a further embodiment of the invention is the hybridoma BF11 n°PD1001 deposited on the 20/2/2001 at the Centra Biotecnologie Avanzate, Genova
  • the invention also comprises a process to prepare recombinant antibodies which makes use of the sequences IDN 1 and 3, or their fragments and a process to enhance the activity of C. rugosa lipases.
  • the invention refers to the use of the antibodies and polypeptides disclosed for biocatalysis in bioconversion reactions, such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis, for the preparation of fragrances, flavours and for the chemical modification of alcohols, acids, esters, fats in particular in the cosmetic, pharmaceutical, food or detergents industry.
  • the invention refers to polypeptides and antibodies which are able to recognize microbial lipases characterized by the fact that they enhance the activity of these enzymes.
  • an antibody For the ability of an antibody to recognize a given antigen it is usually intended the ability of an antibody to bind or to interact with the antigen in a way which can be revealed and measured for example by immunoassays, i.e. ELISA, immunoprecipitation, or by surface plasmon resonance and the like.
  • immunoassays i.e. ELISA, immunoprecipitation, or by surface plasmon resonance and the like.
  • molecular recognition at least a micromolar affinity is usually implied.
  • polypeptides (a generic term which comprises proteins, antibodies and their functional fragments) of the invention have ' both the specificity to recognize as the antigen microbial lipases (also called lipolytic enzymes) together with the capacity to positively modulate the activity of such enzymes.
  • lipolytic enzymes hydrolyse natural triglycerides and synthesise primary and secondary esters to accept as substrates esters with and without insaturations and with different acylic chain length.
  • Candida rugosa lipases show peculiar activity and stability in water-restricted systems which represent particular advantages among lipases.
  • the antibodies and polypeptides of the invention preferably recognize microbial lipases which show at least a 75% homology to at least one of the C. rugosa lipase isoforms chosen in group consisting of: 1, 2, 3 4 5, corresponding respectively to SwissProt data base accession n°: P20261 , P32946, P32947, P32948, P32949. More preferably the homology to at least one of the C. rugosa lipase isoform is of at least 80%, even more preferably of at least 85%.
  • polypeptides of the invention recognize C.rugosa lipase 1-5 isoforms, which show an overall homology of about 78%, even more preferably such polypeptides recognize C.rugosa lipases 1-3, corresponding to the SwissProt data base accession n°: P20261 , P32946, P32947.
  • a further advantage of the antibodies and polipeptides of the invention is that they recognize both the denatured and the native form of the enzyme.
  • the antibodies and polypeptides according to the invention are characterized in that they modulate the enzymatic activity in lipase functions, namely, the hydrolytic activity and the hydrophobic interaction with natural and synthetic substrates.
  • the invention also refers to a process to enhance the activity of microbial lipase wherein the antibody according to the invention is preferably added to the enzyme before addition of the substrate in order to obtain an immunocomplex.
  • Immunocomplexes are formed for example upon incubation of the enzyme with antibodies of the invention, preferably the light chain together with the complete immunoglobuiin, for at least 5', more preferably for at least 2 hours.
  • Formation of the immunocomplexes is preferably obtained at a temperature below 20°C, comprised between 2 and 4 °C in a physiologic buffer such as phosphate buffer, wherein the enzyme concentration is preferably comprised from 0.1- 5mg/ml, even more preferably is comprised between 0.125 - 3mg/ml.
  • the antibodies of the invention are preferably comprised in the hybridoma (PD1001 deposited on the 20/2/2001 at the Centra Biotecnologie Avanzate (CBA) in Genova) supernatant at a concentration of at least 10 ⁇ g/ml.
  • the enhanced hydrolysis of the triglyceride is achieved in a time of at least 10 min.
  • an increase in the lipolytic activity of at least 2 fold as compared to the lipase used alone is obtained.
  • Suitable substrates are also represented by triglycerides with C4 - C24 acylic chain length.
  • the present invention further comprises immunocomplexes with an enhanced activity with respect to the lipase alone. This enhancement ranges from 100 to 500 %, expressed as relative increase in enzyme activity, and depending on the experimental conditions.
  • Preferred immunocomplexes comprise a microbial lipase chosen in the group consisting of: C. rugosa lipase 1-5, and at least one of the antibody, polypeptides or chimeric proteins of the invention, preferred immunocomplex is represented by the polypeptide with aminoacid sequence IDN2 and a C. rugosa lipase chosen among lipase 1 , 2 or 3.
  • the antibody according to the invention comprises a light chain with aminoacid sequence corresponding to seqlDN2 or showing a homology of at least 90%, more preferably 95% or even more preferably 98% to seqlDN2.
  • the antibody consists only of a light chain with aminoacid sequence corresponding to seqlDN2 or light chains with a homology of at least 90%, 95% or more, preferably 98% to seq IDN2.
  • the light chain is secreted by the hybridoma BF11 n°PD1001 deposited on the 20/2/2001 at the Centra Biotecnologie Avanzate di Genova, and is purified from the culture medium according to methods known in the art.
  • the light chains is preferably a kappa chain , isotype.
  • the heavy chain when present, is preferably a gamma chain and even more preferably its aminoacid sequence corresponds to seq IDN4.
  • any other heavy chain may be associated to the light chain of the invention.
  • polypeptides comprising at least one of the polypeptides chosen in the group consisting of:
  • polypeptides with at least 90% homology with sequence IDN2, preferably at least 95 % homology, even more preferably at least 98%
  • IDN2 preferably at least 95 % homology, even more preferably at least 98%), characterized in a suitable context, by the ability to recognize microbial lipases.
  • the invention further comprises:
  • fragments of at least 20 consecutive amminoacids of seq IDN2 and 4 fragments of at least 20 consecutive amminoacids of the polypeptides with at least 90% homology with sequences IDN2 and 4, preferably at least 95 % homology, even more preferably at least 98% homology.
  • a protein may accept aminoacid changes, particularly when they are conservative, ie. when the replacement aminoacid is similar in structure or in the hydropathic properties, or which is non-conservative, but the change involves regions of the molecules that are non-essential or that may be modified without altering the functionality of the molecule.
  • Homologues of the light chain BF11 i.e. homologs of the sequences disclosed as seqlDN2 and seqlDN4 are also comprised in the present invention. It also well accepted that even though CDRs are the active antigen binding loops, frameworks (FR) may also participate in this binding determining its efficiency.
  • framework aminoacid sequences corresponding to aa: 1-26 (FR1), 32- 48 (FR2), 52-87 (FR3), 97-108 (FR4) of seq IDN2 and aminoacids immediately adjacent to them, are also comprised in the present invention.
  • the antibody may be in its native form or in a Fab form, as well as in all the other possible recombinant forms, such as dsFv, scFv, which for the purpose of the present invention may grouped be under the format definition of "single chain antibody” as most commonly obtained by expression in recombinant prokaryotic systems, according to methods well known in the art.
  • a preferred realization of a recombinant antibody is the one obtained by cloning and expressing a full-length cDNA polynucleotide molecule encoding for the light chain aminoacidic sequence of seq IDN2, such polynucleotide corresponding in its preferred embodiment to seq IDN1, in appropriate bacterial expression vectors such as pTrc (Amersham), pBAD (Invitrogen), pGEMEX (Promega).
  • the invention also relates to immunoconjugates derived by way of recombinant methods or chemical cross-linking or by way of non-covalent linking with the antibodies and polypeptides of the invention in their different realization forms, such as for example the polypeptide with aminoacidic seqlDN 2 or the light chain secreted by the hybridoma BF11 n°PD1001 deposited on the 20/2/2001 at the Centra Biotecnologie Avanzate di Genova.
  • examples of some of these immunoconjugated are represented by HRP (Horseradish Peroxidase) AP (alkaline phosphatase), biotin-avidin complexes etc. Therefore according to this embodiment the polypeptides of the invention comprise also particular chimeric proteins.
  • the chimeric protein of the invention comprises at least two functional domains: the one with antilipase functions which has an aminoacid sequence corresponding to the polypeptides and antibodies of the invention, preferably corresponding to seq IDN2, its homologs, fragments or preferred embodiments and the second which carries a different activity, such as for example a ligand binding domain for purification and for immobilisation purposes, such as a polyhistidine stretch or a Maltose Binding Protein domain, a Glutathion S-Transferase domain, or organic and inorganic solid matrices domains, known to the expert in the field.
  • a ligand binding domain for purification and for immobilisation purposes such as a polyhistidine stretch or a Maltose Binding Protein domain, a Glutathion S-Transferase domain, or organic and inorganic solid matrices domains, known to the expert in the field.
  • the invention also comprises chimeric antibodies carrying as CDR (Complementarity Determining region) at least one of the CDR herein disclosed and corresponding to seqs IDN 6,8,10, preferably seq ID 10 corresponding to the 3 rd CDR.
  • CDR Complementarity Determining region
  • Such CDR peptides may accept point aminoacid changes, which do not alter their binding ability and their biological activity and which are therefore comprised within the scopjeof the present application.
  • chimeric proteins and chimeric antibodies are produced by means of recombinant DNA technologies as described in Sambrook and Maniatis (CSH edition, 1988). According to these techniques the nucleotides encoding the polypeptides of the invention, are produced for example by PCR or cut out with appropriate restriction enzymes from isolated DNA molecules and cloned into cloning vectors such as plasmids etc. in frame with the polynucleotides encoding for the second domain in order to express the chimeric antibody or protein.
  • isolated polynucleotides encoding for the polypeptides of the invention i.e.
  • polypeptides with anti-microbial lipase specificity and characterized by their ability to enhance the hydrolytic activity of the enzyme upon incubation, binding or interaction with it.
  • the isolated nucleotide sequences corresponds to seqs IDN 1, 3, 5, 7.
  • polynucleotides may encode for the polypeptides of the invention.
  • polypeptides with at least 90% homology with sequence IDN2, preferably at least 95 % homology, even more preferably at least 98%
  • IDN2 preferably at least 95 % homology, even more preferably at least 98%) are in addition comprised within the scope of the present invention.
  • Vectors comprising the polynucleotides disclosed are easily prepared according to protocols well known in the art and are therefore comprised in the present invention.
  • Such vectors are bacterial expression or cloning vectors such as plasmids (i.e. pUC18, pBR322, pGEX, pTrc, pBAD), phagemids, cosmids, or eukaryotic expression vectors as many commercially available plasmids.
  • Isolated cells or microorganisms transformed with the vectors comprising the polynucleotides of the invention are as well obtained according to well known technologies; therefore genetically modified cells or microorganisms carrying such vectors represent further embodiments of this invention.
  • the invention comprises a process for the production of recombinant antibodies able to recognize microbial lipases showing a homology of at least 75%, preferably 80% or 85% homology to at least one of Candida rugosa lipases and characterized in that they enhance the hydrolytic activity of the enzyme, comprising essentially the following steps:
  • step 1) genetic modification of the polynucleotides in step 1) is meant any modification introduced, preferably at the 5' or at the 3' of said polynucleotides, including cutting out few nucleotides, modifying the extremities for cloning, or all the modifications which are usually performed to achieve easier cloning, or to optimize translation or transcription in the host cell.
  • modification also comprise the alteration of codon usage to achieve a better expression in a particular organism.
  • a further aspect of the invention is related to a process for the enhancement of the catalytic activity of C.
  • rugosa lipases essentially in which the antibodies and/or the polypeptides of the invention are placed, in basic experimental conditions for example in a physiologic solution, such as phosphate buffer at pH comprised between 3 and 9 together with the enzyme, letting the immunocomplexes form for a time of at least 5 min, preferably at least 10 min.
  • a physiologic solution such as phosphate buffer at pH comprised between 3 and 9 together with the enzyme
  • the titration curve of the lipolytic activity shows an increase of at least 10%, preferably 20% and more preferably 40%, ⁇ ; ⁇ f the straight line slope if compared with the curve in absence of BF11 supernatant (see figure 4). This corresponds to an increase in the lipolytic activity of at least 2-fold as compared to the lipase used alone.
  • Preferred conditions for the formation of the immunocomplexes are preferably obtained at temperature below 20°C, preferably below 10°C, even more preferably comprised between 2 and 4 °C in a physiologic buffer such as phosphate buffer, wherein the enzyme concentration is preferably comprised from 0.1- 5mg/ml, even more preferably is comprised between 0.125 -3mg/ml.
  • the antibodies are preferably those secreted by the hybridoma (PD1001 deposited on the 20/2/2001 at the Centra Biotecnologie Avanzate (CBA) in Genova) and comprised in the hybridoma supernatant at a concentration of at least 10 ⁇ g/ml.
  • the enhanced hydrolysis of the triglyceride by microbial lipases is achieved in a time of at least 10 min. In these conditions an increase of at least 2 fold of the lipolytic activity as compared to the lipase used alone, is obtained.
  • polypeptides and the antibodies of the invention are used to prepare immunocomplexes with microbial lipases. Such complexes are characterized by a higher enzymatic activity than that of the enzyme alone and are used to achieve higher catalytic yields in lipase reactions.
  • polypeptides and the antibodies of the invention are useful in the identification, measurements and purification, for example by affinity chromatography, of the above mentioned enzymes. Examples of such application are represented for example by immunoassays making use of such polypeptides and antibodies, or immunoaffinity column carrying the antibodies on a solid or liquid phase.
  • microbial lipases have been exploited in many industrial processes such as the agro-chemical, detergency or fine-chemical industry food and cosmetic industry. Fall therefore within the boundaries of the present invention the use of the polypeptides and immunocomplexes comprising at least one of the antibodies or the polypeptides disclosed, in all the industrial applications making use of microbial lipases, in particular in the detergent industry, in the cosmetic industry, in the flavour and feed industry or for applications in biocatalysis.
  • flavour esters In the flavour and feed industries the majority of flavours and fragrances are esters, some of which are fatty acid esters. Many of the naturally occurring esters are now synthesized, and lipase biocatalysis could be advantageous due to specific and mild reactions. Although many flavour esters are composed of acids and alcohols very different from fatty acids and glycerol (branched, cyclic, aromatic, very short chain) lipases may still accept these as substrates. Synthesis of terpene alcohol esters by various lipases has been reported, as well as the continuous synthesis of ethyl butyrate (pineapple/banana flavour) by immobilised Candida rugosa lipase. A number of other flavour esters are made by lipase activity.
  • Biochemical production of butter flavour can be made by alcoholysis of butter fat and ethanol, e.g. with Candida rugosa lipase.
  • This type of reaction provide a mixture of fatty acid ethyl esters, which may be combined to natural components in foods, e.g. margarine and cookies (Harwood, J. et al.1989).
  • the oil industry is wide interested towards lipase application to the oil acidity neutralisation.
  • the acidity of tropical oils is due to the presence of free fatty acids.
  • the content of these acids can be depressed by partial esterifications by lipases (Graille, J. et. al 1988).
  • the Soya-bean oil changes its flavour during storage and during frying process giving rise to a typical fish smell.
  • Soya-bean oil flavour instability is due to the linoleic acid.
  • Rhyzomucor miehei lipase has shown specificity towards polyinsatured fatty acid. It is possible to decrease at 3 % the linoleic acid content by biocatalised transesterification at 10 °C ( Karmal, T.N.B. et al. 1988). Otherwise ⁇ -linoleic acid has an high dietetic and commercial value. It is in the prostaglandin path-synthesis and it is rare in edible oils. At the moment the natural source of this fatty acid is evening primerose seeds.
  • fatty acids may be released as ethyl- or other alcohol esters with flavour potential, by lipase catalysed alcoholysis or esterification.
  • Flavour development is usually the result of a very small degree of lipid hydrolysis (few percent) whereas a higher degree may lead also to change in the physical properties of the fat.
  • Such partial hydrolysis of oils and fats can improve the palatability and digestibility of the lipid component in feeds and pet foods.
  • Hard fats may be softened due to mono- and di-glycerides and more energy supplied to the animals if the lipid is "pre-digested".
  • the emulsifiers production is another important aspect of food industry.
  • monoglycerides and sugar esterified with fatty acids are employed as emulsifiers in foods.
  • fatty acids and oil soiling are difficult to remove from fabrics at low washing temperatures.
  • Spillage of lipid containing foods and accumulation of sebum from skin are common problems. Therefore, intensive research efforts have been directed towards development not only of new detergent compounds and surfactants but also toward enzymes which could facilitate removal of this type of soiling under wash conditions (Newmark, P. 1988). Hydrolysis of triglycerides and other fatty esters does not increase the water solubility of the lipids as readily as in the case of protein and starch hydrolysis.
  • a detergent lipases require an high alkaline and thermo-stability, stability toward proteases and compatibility with common detergents (common powder detergents pH is 9-10, washing temperatures are >37°C), which may be provided by the antibodies and polypeptides of the present invention.
  • lipids constitute a very small part of wood, they may have important effects.
  • spruce about 2 % resin is found.
  • the resin is a mixture of triglycerides, free fatty acids and other components.
  • the resin content is reduced to about 0.5 % or less in existing pulping processes.
  • a more complete resin removal is possible by addition of lipase in the process with improved water absorption characteristics of cellulose fibres as results.
  • the resin content may also influence other properties, e.g. paper strength or colour and result in paper making processes (the formation of suspension of wood fibres).
  • Printed paper can be treated by lipase in order to facilitate removal of colour from the printing ink, containing vegetable oil base. This application can be useful for improvement of return paper.
  • the biocatalytic strategy to the resolution of racemic mixture can be applied to compounds containing alcohols, esters and acids moieties. If chiral or prochiral substrates are being used, it is usually just one enantiomer which undergoes reaction, thus leading to the chiral resolution of racemates (Sonomoto, K. et al.1988).
  • polypeptides and antibodies disclosed in the present application are also of use in the field of biocatalysis for example the racemic switch of farmaceutical compounds and their precursor in the synthesis pathway, or in research studies aimed at the optimization of lipase enzymatic activity: as a matter of fact, kinetic and computer modelling studies of this interaction can significantly contribute in the understanding of the enzyme-structure relationship of lipases as well as provide indications for the > generation of simple peptide enzyme activators for biocatalysis.
  • the hybridomas producing specific anti-lipase antibodies were screened by solid- phase immunoassays as follows: the immunogen or other antigen in optimal concentration where allowed to absorb to plastic (polystyrene 96 well plates (Costar 3590, USA) in carbonate buffer pH 9.2 at 37 °C overnight. Wells where washed once with saline phosphate buffer (PBS, pH 7.0). Unspecific binding was blocked by incubating plates for 2 hrs at 37 °C with PBS containing 5% Bovine Serum Albumen (BSA, SIGMA -Aldrich cat.# A/4503).
  • plastic polystyrene 96 well plates (Costar 3590, USA) in carbonate buffer pH 9.2 at 37 °C overnight.
  • Wells where washed once with saline phosphate buffer (PBS, pH 7.0). Unspecific binding was blocked by incubating plates for 2 hrs at 37 °C with PBS containing 5% Bovine Serum Albumen (BSA, SIG
  • Antibodies selected where further characterized using ELISA and Western Blot Electrophoresis in the presence and absence of SDS was carried out on 8% polyacrylamide gels. The immunoreactivity of each mAb with lipase was also studied using Western Blotting technique. Proteins were transferred to a nitrocellulose membrane electrophoretically from the SDS-polyacrylamide gel. Prestained Molecular Weight markers (Invitrogen.USA cat#10748-010) where run in parallel lanes. To block the remaining free sites, the blots were incubated (solution of PBS containing BSA 3% for 1 hour at room temperature).
  • culture hybridoma supernatants or purified mAbs in PBS containing 0.05% Tween 20 were incubated for 1 hour at room temperature.
  • the strips were rinsed three times with PBS/Tween-20 and incubated for 1 hour at room temperature with peroxidase conjugated goat anti-mouse immunoglobulin.
  • the blots were washed three times with PBS/Tween-20 and the specific immunoreactivity was revealed with the substrate solution of ECL (Amersham pharmacia biotech, USA cat#2106) detection reagent.(Fig. 2 ).
  • Isotyping Isotyping of selected mAb was carried out using the Zymed Immunoassay Kit (USA, cat # 97-6550). The isotyping test showed that the heavy chain belongs to the isotype lgG1 and the light chain belongs to the kappa chain isotype.
  • Example 2 Characterization of the BF11 hybridoma supernatant
  • the BF11 supernatant was subjected to ultrafiltration using a Millipore filter of 50.000 cut off to eliminate high molecular weight proteins including the whole monoclonal antibody BF11 (heavy chain in reducing condition).
  • Different forms of the monoclonal antibody BF11 (figure 3: lane 1: culture supernatant, lane 2: affinity purified antibody, l&he 3: the 50 kDa cut-off ultrafiltrate) were run on SDS- PAGE in reducing condition and blotted with an anti-mouse kappa light chain secondary antibody.
  • the MOPC21 antibody is an isotype matched control that was run in parallel (lane 4) and developed with anti-mouse IgG antibody.
  • Example 4 Lipase immunoactivation studies employing BF11 light chain To study the ability of BF11 light chain to increase lipase lipolytic activity, 50 ⁇ l of enzyme solution (from 0.125 to 3 mg/ml) was incubated with 2ml of BF11 light chain. Incubations were performed in physiological buffer for 2 hour at 4°C. The residual activity of lipase-BF11 light chain complex was measured using the standard method of tributirryn hydrolysis (alkalimetric final titration). The assay mixture, containing 2 ml of physiological buffer, 0.5 ml of tributyrin and lipase- BF11 light chain complex solution, was incubated at 37 °C under magnetic stirring (300 rpm) for 30 min. The reaction mixture was stopped with 2.5 ml of acetone/ethanol mixture 1:1 (v/v) and titrated with 0.01 M NaOH in the presence of phenolphthalein as indicator using an automatic burette.
  • Figure 4 and 5 report the curves of relative increase of lipase activity versus enzyme concentrations for BF11 light chain (in comparison to BF11 supernatant).
  • Example 5 Lipolytic activity assay of the immuno-complex on polyacrylamide gel
  • Native electrophoresis was performed using a combination of Phastsystem with ready PhastGel media with 8-25% gradient in polyacrylamide gel, according to the manufacture's instruction. The run was performed in three steps up to 229Avh. The ⁇ -naphtyl acetate in N,N-dimethylformamide and Fast Garnet GBC were used for the specific esterasic activity staining.
  • Figure 6A and 6B show 2 gels which were loaded with enzyme solutions with and without BF11 supernatant.
  • the lipase-BF11 supernatant complex was active on gel (positive staining).
  • Increasing in BF11 supernatant concentration the equilibrium shift to quantitative complex formation and alone enzyme spot disappeared.
  • Example 6 Cloning of the full-length cDNA sequence encoding BF11 light chain.

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Abstract

La présente invention concerne des polypeptides et des anticorps qui sont capables de reconnaître les lipases microbiennes et d'accroître l'activité hydrolytique de ces enzymes. Les séquences nucléotidiques et d'acides aminés d'une forme de réalisation préférée de ces anticorps sont également déterminées en tant que seq IDN 1 - 4 dans le listage de séquences. Cette invention se rapporte également aux utilisations industrielles de ces anticorps.
PCT/EP2002/012149 2002-10-31 2002-10-31 Anticorps de lipase antimicrobiens, sequences nucleotidiques et d'acides amines de ces derniers, utilisations correspondantes Ceased WO2004039845A1 (fr)

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AU2002368305A AU2002368305A1 (en) 2002-10-31 2002-10-31 Antimicrobial lipase antibodies their nucleotide and aminoacid sequences and uses thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11459400B2 (en) 2015-07-17 2022-10-04 The University Of Chicago Methods and composition for modifying enzymes
US11680091B2 (en) 2018-02-23 2023-06-20 The University Of Chicago Methods and composition involving thermophilic fibronectin type III (FN3) monobodies
WO2024040115A3 (fr) * 2022-08-16 2024-05-02 University Of Vermont And State Agricultural College ANTICORPS POUR FCγRIIA PLAQUETTAIRE ET PROCÉDÉS D'UTILISATION ASSOCIÉS

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004569A1 (fr) * 1992-08-20 1994-03-03 The Agricultural And Food Research Council Utilisation de molecules specifiques pour potentialiser l'activite du facteur de croissance insulinoide i
EP0620276A1 (fr) * 1989-12-21 1994-10-19 Celltech Therapeutics Limited Anticorps humanisés
WO2001032712A2 (fr) * 1999-11-03 2001-05-10 Maxygen, Inc. Generation de diversite d'anticorps
WO2002006457A2 (fr) * 2000-07-13 2002-01-24 Maxygen, Inc. Genes de lipase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620276A1 (fr) * 1989-12-21 1994-10-19 Celltech Therapeutics Limited Anticorps humanisés
WO1994004569A1 (fr) * 1992-08-20 1994-03-03 The Agricultural And Food Research Council Utilisation de molecules specifiques pour potentialiser l'activite du facteur de croissance insulinoide i
WO2001032712A2 (fr) * 1999-11-03 2001-05-10 Maxygen, Inc. Generation de diversite d'anticorps
WO2002006457A2 (fr) * 2000-07-13 2002-01-24 Maxygen, Inc. Genes de lipase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AMITAL H ET AL: "CATALYTIC ANTIBODIES: GENERATION, NATURE, AND POSSIBLE ROLE AS CHEMICAL WARFARE SCAVENGERS", MILITARY MEDICINE, ASSOCIATION OF MILITARY SURGEONS OF THE US, BETHESDA, MD, US, vol. 161, no. 1, January 1996 (1996-01-01), pages 7 - 10, XP009004767, ISSN: 0026-4075 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11459400B2 (en) 2015-07-17 2022-10-04 The University Of Chicago Methods and composition for modifying enzymes
US11680091B2 (en) 2018-02-23 2023-06-20 The University Of Chicago Methods and composition involving thermophilic fibronectin type III (FN3) monobodies
US12312392B2 (en) 2018-02-23 2025-05-27 The University Of Chicago Methods and composition involving thermophilic fibronectin type III (FN3) monobodies
WO2024040115A3 (fr) * 2022-08-16 2024-05-02 University Of Vermont And State Agricultural College ANTICORPS POUR FCγRIIA PLAQUETTAIRE ET PROCÉDÉS D'UTILISATION ASSOCIÉS

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