EP1012301A1 - Synthese totale et surexpression fonctionnelle d'un gene candida rugosa lip1 codant pour une lipase industrielle majeure - Google Patents

Synthese totale et surexpression fonctionnelle d'un gene candida rugosa lip1 codant pour une lipase industrielle majeure

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Publication number
EP1012301A1
EP1012301A1 EP97940483A EP97940483A EP1012301A1 EP 1012301 A1 EP1012301 A1 EP 1012301A1 EP 97940483 A EP97940483 A EP 97940483A EP 97940483 A EP97940483 A EP 97940483A EP 1012301 A1 EP1012301 A1 EP 1012301A1
Authority
EP
European Patent Office
Prior art keywords
lipase
sequence
rugosa
nucleic acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97940483A
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German (de)
English (en)
Inventor
Stefania Brocca
Claudia Schmidt-Dannert
Marina Lotti
Lilia Alberghina
Rolf Schmid
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Loders Croklaan BV
Original Assignee
Unilever NV
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Publication date
Application filed by Unilever NV filed Critical Unilever NV
Publication of EP1012301A1 publication Critical patent/EP1012301A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • Lipases catalyse the hydrolysis of triglycerides at water/oil interfaces. In vitro, however, they are versatile enzymes because of their ability to catalyse the hydrolysis and synthesis of a great variety of esters (i.e. the hydrolysis and transesterification of triacylglycerols) , or the resolution of racemic mixtures. They find applications also as industrial detergent (i.e. due to their ability to remove fat and ink stains and in food industry where they are used for fat retailoring of triglycerides.
  • Candida rugosa lipases are among the commercial lipases most often employed in hydrolysis and synthesis of a wide range of esters of commercial interest x . In most biocatalytic applications crude enzyme preparations, obtained by TCA precipitation of the culture supernatant, are applied 2 .
  • Candida rugosa is a dimorphic yeast in which, as in some other phylogenetically related Candida species 9 , the triplet CUG, a universal codon for leucine, is read as serine i0 . This unusual amino acid assignment of the CUG codon relies on the presence in C. rugosa of an unusual tRNA Ser with an anticodon CAG n .
  • CUG is extremely rare, with the notable exception of Candida rugosa where it accounts for about 40% of the total serine codons 3 , h . Accordingly, multiple copies of the genes coding for tRNA Ser CAG have been isolated from the genome of this yeast u . As a consequence of this unusual codon-usage, the heterologous expression of LIPl in S. cerevisiae resulted in an inactive lipase 12 . Hence the exchange of the CUG by universal serine triplets is required for the expression of a functional protein in heterologous hosts. This has been carried out previously for the heterologous expression of two other genes encoding cytochrome P450 cloned from another Candida microorganism i.e. C.
  • CTG is used with a high frequency (3$ of the codons) , including that corresponding to the catalytic Ser. 209
  • LIPl gene ORF 1647 nt
  • 20 out of 42 serine residues are encoded by CUG triplets .
  • Alberghina and Lotti U997) 26 describe an attempt to express LIPl in S. cerevisiae and faced unexpected issues concerning the translatability/stability of the recombinant protein in host cells and the non universal genetic code utilized by Candida cells . Insertion of the lipase gene in S. cerevisiae resulted in abundant mRNA production as witnessed by Northern blot but no trace of recombinant protein in either cells or medium. Replacement of the first 15 amino acids of lip 1 with another leader sequence i.e. that of killer toxin from Kluyveromyces lactis gave mRNA and intracellular accumulation. This is also disclosed in reference 12 which also states it was inactive.
  • S. cerevisiae does not process the native C. rugosa lipase presequence, we constructed a hybrid form for the mutant lipase genes, analogously to what was reported by the same authors .
  • the hybrid lipase mutant genes were cloned into pEMBLyex4, a shuttle expression vector for S. cerevisiae cells containing the inducible UAS GAL sequence 20 .
  • Recombinant yeast cells grown under inducing conditions produced high levels of protein, but the resulting recombinant lipase was found to accumulate intracellularly at about 10-20 mg/1 culture in a non active form. The same result was obtained for all 4 mutants, independently of the number of Ser residues restored.
  • the protein glycosylation provided indications in favour of the correct targeting of the chimeric mutated protein to the endoplasmatic reticulum, its secretion failed, suggesting difficulties at the posttranscriptional level, which might be connected with the mutations (13/19) still present in the molecule. These mutations could affect the folding of the protein, hampering in turn, its correct processing through the secretory pathway. It is also possible the signal sequence is relevant for activity thus explaining inactivity upon replacement of the signal sequence by a non native presequence. If this is the case then expression in a heterogenous host cell of an active C. rugosa lipase would be impossible. Production would then only be possible using a C. rugosa host cell with concomitant contamination with other lipases of C.
  • the subject invention comprises the chemical-enzymatic synthesis of a gene coding for a natural C. rugosa lipase and the expression thereof in a heterologous host cell in an active form, moreover the secretion thereof to such a degree that a supernatant comprising lipase with contamination of less than 20% with other protein is obtained.
  • a supernatant comprising lipase with contamination of less than 20% with other protein is obtained.
  • the synthetic gene synthesis represents one of the most ambitious gene syntheses to date. Any commercial preparations known in the art comprise active lipase from C. rugosa in numerous isoforms, other proteins and stabilisers.
  • C. rugosa lipase In general purity levels are lower than 8 ⁇ #, thus the instant invention offers a major step forward in providing substantially purer C. rugosa lipase.
  • the C. rugosa lipase according to the invention will be free of other C. rugosa lipase isoforms. It is now possible to produce C. rugosa lipase I e.g. free of lipase II-V on an industrial scale.
  • the subsequent recombinant gene was introduced into and expressed in a heterogenous yeast.
  • P. pastoris a methylotrophic yeast that has become increasingly attractive as host in the production of heterologous proteins 13 .
  • the system has been well developed for industrial-scale fermentations and Pichia pastoris can be grown to high cell density in relatively inexpensive media. No stable multicopy vectors exist for P. pastoris and foreign genes are integrated into the genome for expression.
  • the secretion by Pichia cells requires the presence of a signal sequence fused to the expressed protein.
  • have been found to be effective previously in P. pastoris .
  • the secretion signal sequence from the S. cerevisiae ⁇ -factor prepro peptide forms a preferred embodiment in a gene construct according to the invention. This presequence has been used with success previously in other situations 15 .
  • the expression of the synthetic lipase gene was carried out in S. cerevisiae. Hansenula is also a host cell which is attractive for commercial production purposes .
  • the deduced aa sequence corresponds to a protein of 549 aa with an N-terminal stretch of 15 hydrophobic aa, encoding a signal peptide.
  • the LIPl mature protein contains 42 Ser residues, 19 of them encoded by CUG triplets 3 .
  • the nucleic acid sequence according to the invention can be further adapted by removing restriction enzyme sites from the coding sequence that are common to expression vectors in which the gene is to be expressed.
  • the Hindlll site is e.g. a site common to numerous expression vectors thus it simplifies handling the sequence if the Hindlll recognition sequence present in the coding sequence is removed. It is preferable to replace such a sequence by nucleotides that maintain the amino acid identity of the non manipulated restriction enzyme recognition site.
  • Another suitable adaptation of the sequence according to the invention lies in the introduction of restriction enzyme recognition sites at points of interest thereby maintaining the original amino acid identity.
  • any such introduced restriction site will be unique to the sequence, thus if multiple sites are introduced each will be different.
  • a favorable alternative comprises additional estriction enzyme recognition sites at the 5' and 3' termini of the nucleic acid sequence according to the invention thereby enabling introduction of the sequence encoding lipase according to the invention into an expression vector.
  • nucleic acid sequences comprising slight variations on the native sequence for each specific lipase can be designed. Such variants fall within the scope of the subject invention.
  • Each native lipase can be characterised by its specific activity.
  • a variant sequence encoding a protein having such specificity is covered by the invention.
  • Such a variant will encode a protein exhibiting at least the degree of activity of the corresponding native protein as expressed from a nucleic acid sequence encoding the native protein.
  • the specificity can be determined analogously to the method illustrated in the examples, e.g. Natural variants in gene sequence may also occur and synthetic nucleic acid sequences encoding such natural variants are also envisaged as falling within the scope of the invention.
  • a recombinant substantially pure lipase as expressed by a heterologous host cell from nucleic acid in which at least 60% of the serine encoding CTG codons have been replaced by universal serine codons aswell as the corresponding nucleic acid sequence and applications thereof are covered by the invention.
  • the expression product of said sequence should exhibit lipase activity equal to or better than that of the corresponding native lipase.
  • Preferably more than 70% and suitably more than 80% of the CTG codons at serine encoding positions are replaced.
  • An embodiment where all serine codons have been replaced has been found to function exceedingly well.
  • the amino acid sequences and nucleic acid sequences of C. rugosa lipases 1-5 are known in the art. It is thus clear what native nucleic acid sequences are for all 5 lipases.
  • the subject invention covers nucleic acid sequences encoding one isoform of C. rugosa lipase either in its native form or as a variant with the amended serine codons according to the invention as already disclosed.
  • the variant nucleic acid sequence can have a different amino acid sequence than the native lipase but in addition a suitable embodiment should be capable of hybridising to the native sequence under stringent conditions as defined in the art 29 .
  • a further particular embodiment of a variant lipase 1 sequence will exhibit amino acid identity with the known lipase 1 sequence of more than 88%, preferably more than 90%.
  • a suitable embodiment will exhibit 100% identity at amino acid level .
  • the sequence according to the invention does not only merely have to consist of the mature encoding sequence but it may also comprise a precursor sequence of choice or a part thereof. Preferably such a precursor sequence will be removed during protein production, however it is envisaged that precursor fragments of the mature protein can remain present without affecting activity. Such a remaining preceding sequence should not be longer than 10 amino acids preferably not longer than amino acids in length and should not affect activity or specificity in a negative manner.
  • the host cell processes the protein during culture to the mature protein per se for a most cost effective efficient production process. Obviously it is also preferable the host cell secretes the protein in order to facilitate isolation of the protein.
  • a nucleic acid sequence encoding a ripening form i.e. a mature, pre, pro, prepro protein or even a mature protein preceded by a short sequence of less than 10 amino acids is envisaged as falling within the scope of the invention.
  • the 5' terminus of the encoding sequence for the mature protein can be preceded by a leader sequence heterologous to C. rugosa .
  • a leader sequence is derived from a yeast cell in particular if the sequence is to be expressed in a yeast host cell.
  • suitable sequences are available e.g. from S. cerevisiae .
  • nucleic acid sequences per se according to the invention are considered to fall within the scope ofthe invention but also an expression vector or a heterologous host cell comprising such a sequence i considered to be covered, in particular when the sequence is operatively linked to a promoter and is thus capable of being expressed.
  • a sequence can be single or multiple copy depending on the desired degree of expression.
  • the sequence according to the invention can differ in regard of the host microorganism. It is preferable to optimise codon usage per expression host. Culturing a microorganism according to the invention can lead to production of lipase to a degree such that said lipase is contaminated at the most by 20% of other protein in the supernatant of a culture of said microorganism.
  • composition obtainable can be preferred for particular processes. It has now become possible in an economically feasible manner to achieve contamination with less than 10% and even less than 5% other protein. More specifically one can achieve C rugosa free of other C. rugosa lipases. Thus lipase 1 free of 2-5 and lipase 2 free of lipase 1 and 3"5 etc can now be obtained without requiring extensive and expensive working up procedures . In particular a lipase 1 free of 2-5 wherein lipase 1 is defined according to sequence id nr 1 has been illustrated in the Examples.
  • Pure lipase 1 according to the invention has been illustrated as exhibiting higher activity towards caprinate (CIO) than towards palmitate (Cl6) as can be determined by pH stat assay and is illustrated in the examples.
  • CIO caprinate
  • Cl6 palmitate
  • a number of characteristics of a lipase 1 according to the invention can be seen in addition in Table II .
  • the object of the invention was to provide pure lipase on an industrial scale at a reasonable price.
  • a process for industrial scale production of a lipase according to the invention in any of the embodiments or combinations of embodiments disclosed comprising cultivation of a microorganism according to any of the embodiments disclosed followed optionally by isolation of the resulting expression product in a manner known per se for other protein production processes in industry is also considered to fall within the scope of the invention.
  • the lipase is present in the culture supernatant in an amount comprising over 80% of the total C. rugosa protein present or even of the heterologous host cell.
  • the synthetic gene containing its natural leader sequence was modified via PCR and a hybrid version of the recombinant LIPl gene (rLIPl ) was obtained by fusion of the nucleotide sequence coding for the mature LIP protein, with the S. cerevisiae prepro ⁇ -factor peptide (prepro-r IPl) .
  • the subsequent recombinant lipase gene was inserted into the vector pPICZ ⁇ B generating the plasmid pPICppLIP for the expression in P. pastoris .
  • Pichia pastoris is a yeast capable of metabolising methanol as its sole carbon source.
  • the enzyme alcohol oxidase (AOX) is involved in the metabolism of methanol and its transcription is tightly regulated and strongly induced by methanol (more than 30% of the total soluble protein in cells grown with methanol as carbon source) .
  • the rLIPl was cloned in pPICZ ⁇ B vector, a shuttle expression vector that contains the A0X1 promoter as control element of the gene expression 23 .
  • Naturally other vectors can suitably be used.
  • An alternative vector is for example pGAPZ for P. pastoris.
  • Selected mutants were further grown in small volume cultures (20ml) in a shaking flask until they reached an 0D 600 of 2-6 and were then transferred to BMMY medium for induction of lipase expression.
  • lipolytic activity was detected in the supernatant by pNPP assay, showing a significant variability among clones (1 to 20 U/ml lipase).
  • This variability in secretion level could be related to a gene dosage effect, since it has been found that P. pastoris strains containing many integrated copies of a foreign gene can yield much higher levels of foreign protein .
  • This phenomenon could rely on the multiple insertion occurring by a mechanism of in vivo circularisation of transforming DNA and accounts for a strong difference of expression in shake flasks, also reported by other authors 25 .
  • the recombinant host cell can thus comprise multiple copies of the nucleic acid according to the invention
  • High producing clones were selected and grown in 2 1 Erlenmeyer flasks containing 200 ml BMGY medium. After days induction, the lipase activity of the supernatants was 85 U/ml.
  • the clone with the highest level of lipase secretion, selected from the transformants harbouring the plasmid pPICpreproLIP was employed for the production on a larger scale of the recombinant CRL (rCRL) .
  • 1 1 culture in rich standard medium was maintained at pH 7-8 and followed for 100 h.
  • the final lipolytic activity in the supernatant was 125 U/ml (Fig. 3a) .
  • the deduced amino acid sequence of rCRL contains three potential N- glycosylation sites 3 ° at position 291, 314 and 351. Hence, after deglycosylation a decrease in the molecular weight of ca. 3 kDa was observed, showing that the recombinant protein has a carbohydrate content of 5%. Lipl was deglycosylated before and after denaturation; identical molecular weights were observed, but the deglycosylation reaction on the protein in native state required an almost double incubation time.
  • the recombinant and commercial CRL showed rather different specificity profiles when their activities were measured on acylglycerides with a wider range of chain-length.
  • the assays were carried out on acylglycerides methyl esters (m.p. 20-54 °C) and revealed a maximum activity towards palmitate (C16) for the commercial CRL and towards caprinate (CIO) for the recombinant one. Eventhough the commercial CRL shows a rather high relative activity on tricaprinate
  • Restriction enzymes, DNA-modifying enzymes, T4-DNA ligase and Taq polymerase were from MBI Fermentas (St. Leon-Rot, Germany), Taq Dye Cycle Sequencing Kit was from Applied Biosystems (Weiterstadt, Germany), DNA Gel-Extraction Kit, Midi Plasmid Kit and Prepspin Plasmid Kit were from Qiagen (Hilden, Germany), lipase type VII from C.
  • ⁇ (argF laczya) U169) was the host for plasmid amplification.
  • Pichia pastoris GS115 (his4) Invitrogen
  • S cerevisiae INVSC2 Invitrogen
  • x4 ⁇ 4 ⁇ 3A MAT ⁇ , lys5. met2, ura3, trpl
  • Plasmid pUC19 was purchased from Pharmacia; PCYTEP1 (pTl) was obtained from the expression vector pTl-OmpAROL (25,26).
  • pYES2 and pPICZ ⁇ B were supplied from Invitrogen.
  • E. coli was grown at 37°C in Luria-Bertani medium (LB) containing 100 ⁇ g/ml ampicillin for selection of clones transformed with the vector pYES2 or 25 g/ml zeocin for selection of clones transformed with the vector pPICZ ⁇ C.
  • LB Luria-Bertani medium
  • yeast pastoris was grown in shaking flasks at 30 °C, in a medium containing 1% yeast extract, 2% peptone, 100 mM potassium buffer pH 6.0, 1.34% yeast nitrogen base, 4*10 ⁇ 5 % biotin, 1% glycerol (BMGY) before the induction, or 0.5% methanol (BMMY) for the induction.
  • YEPD medium 1% yeast extract, 2% peptone, 2% dextrose
  • P. pastoris transformants YEPDS (YEPD + 2% sorbitol) plates containing zeocin (100 ⁇ g/ml) were used.
  • the procedure for culture of the transformed S. cerevisiae cells, protein extraction and detection have been described elsewhere 12 .
  • Standard recombinant DNA methods were carried out according to the methods described in Sambrock et al . 29 and Ausubel et al . 21 . Sequencing was performed by the fluorescence-based dideoxy DNA cycle sequencing method.
  • the Taq Dye DeoxyTM Cycle Sequencing Kit and the 373A DNA Sequencing System were purchased by Applied Biosystems and used according to the manufacturer's instructions.
  • the gene was divided into 4 fragments of ca. 400 bp each, which were separately cloned.
  • fig. 1 the lengths and the positions of oligonucleotides are indicated covering the 1688 b of the LIPl gene.
  • Each pair of oligonucleotides included a 20 bp duplex segment to promote the annealing stability during the mutually primed synthesis
  • cassette I and cassette IV the synthesis of starting (5" -terminus in cassette I) and terminal (3' -terminus in cassette IV) extremities were extended beyond their normal end point to generate ends containing convenient cloning sites also present in pUC19 vector polylinker.
  • the four cassettes were synthesised in one step, by using the mutually priming long oligonucleotides in PCR. This procedure allowed the synthesis of ca. 400 bp fragments corresponding to cassette I to IV, that were cloned as blunt-end into pUC19 vector linearised at the Smal site.
  • the cassette sequences were checked by automated sequencing. Although the mutation rate observed was rather low (2/400 nt) , it was necessary to carry out a repair strategy in order to remove sequence variants that appeared in the final cloned products.
  • a number of restriction sites present in the synthetic gene and in the cloning vector pUC19 were used in order to combine in one copy error-free, different sequences coming from distinct clones of the same cassette.
  • the four error-free subassemblies, cloned into pUC19 vector were designated as pUC-I to pUC-IV.
  • the complete codon-optimised synthetic lipase gene was assembled into pUC19 vector.
  • the Xm ⁇ l/blunt-S ⁇ ll fragment from pUC-II was purified and ligated into pUC-III, which had been linearized by double digestion with Pstl/blunt and Sail.
  • the thus-created plasmid, containing the cassette II and the cassette III were joined at the Sail site, and was designed as pUC- (II-III) .
  • the Xm ⁇ l-EcoRI fragment from pUC- (II-III) was purified and ligated into pUC-I, which had been linearized by double digestion with Xm ⁇ l and EcoRI.
  • the synthetic CRL gene was transferred into the pCyTexPl vector 3 ° as intermediate step for the in frame fusion of the gene with the
  • S. cerevisiae ⁇ -factor leader sequence The B ⁇ mHI fragment from pUC(I-IV) was ligated in B ⁇ mHI linearised and dephosphorylated pCytexPl vector. The resulting plasmid was designated pCyLIP.
  • PCR was performed in order to put the sequence encoding the mature lipase directly in frame with the ⁇ -factor signal sequence present in the Pichia pastoris expression vector pPICZ ⁇ B.
  • the oligos used in the PCR were LI (5' -CTG ACA GTT TAA ACG CTG TCT TGG-3'), complementary to the pPICZ ⁇ B sequence including the Pmel site and L2 (5'-AGC TTC AGC CTC TCT TTT CTC TCC GAC TTC GAC GGG GTT GGC
  • GGT GAA ACC GAT TGC-3' complementary to the 3 'end of the ⁇ -factor signal sequence and including the 5 'sequence of the mature lipase along with a Ball site.
  • the PCR product was directly cloned in pCytexPl Sphl/blunt linearised giving the plasmids pCyprepro ⁇ factor.
  • the Bgll fragment from pCyLIP, containing all the mature form of the lipase synthetic gene was ligated with the complementary Bgll fragments from the plasmid pCyprepro ⁇ factor. The efficiency of this cloning was improved by the fact that the ligations restored the Ampicillin resistance gene contained into the vector.
  • the obtained plasmid was called pCyppLIP.
  • the plasmid pCyppLIP containing the gene for the mature synthetic lipase, in frame with prepro ⁇ -factor leader sequence was B ⁇ mHI/blunt- Hindlll digested and the resulting fragment was ligated into pPICZ ⁇ B linearised with Xb ⁇ l/blunt-Hindlll, giving pPICppLIP.
  • pYES2 As the expression vector in S. cerevisiae cells, we employed pYES2, a 2 ⁇ - based vector containing the GAL1 -GAL10 promoter, able to induce high level expression of genes cloned in its proximity upon growth in galactose medium 31 .
  • pPICnlLIP was B ⁇ mHI digested and ligated into pYES2 linearised with the same enzyme and dephosphorylated, giving pYnlLIP.
  • pYnlLIP was used in turn for the cloning of the synthetic lipase gene preceded by prepro- ⁇ - factor leader sequences into vector pYES2.
  • the Hindlll-Bstell fragment containing the recombinant gene was isolated from pPICppLIP and inserted in pYES2 digested with the same enzymes.
  • the plasmid thus obtained was designated as pYppLIP.
  • Pichia pastoris GS115 cells were transformed with pPICnlLIP, pPICppLIP and pPICpreLIP, by electroporation and transformations were plated onto solid selective medium (YEPD containing zeocin) . Positive transformants selected on the basis of their ability to grow in the presence of zeocin, were checked for lipase activity. Colonies from initial transformation plates were picked out in replica onto minimal tributyrin-methanol plates . The tributyrin-methanol plates were incubated at 30 °C for 48 hrs with 0,1 ml of methanol being added to the lid of each plate every 24hrs.
  • S. cerevisiae Invsc2 cells were transformed with pYppLIP by electroporation. Transformations were plated onto solid minimal medium containing tributyrin and galactose. Transformants, selected on the basis of their ability to grow in the absence of leucine, were directly screened for lipase production and secretion, by being the positive colonies surrounded by a transparent halos as compared to the opaque background of the medium containing tributyrin emulsion.
  • Fermentat i ons The fermentation was carried out in a 1 1 bioreactor (Braun) at 30 °C, in a rich standard medium at pH 6.0 containing 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 4*10 ⁇ 5 % biotin and 0.5 % methanol.
  • the culture broth was maintained at constant pH by adding 2 M HC1 and 2 M NaOH.
  • the stirring rate was 350 rpm and the aeration rate was 1 1/min.
  • the lipolytic activity of the supernatants and the cell wet weight were monitored throughout the fermentation. 5 nil of methanol were added daily to the bioreactor. In the case of high density fermentation, methanol was added after the cell culture reached 75 mg/ml of wet weight.
  • Enzyme assay The fermentor broth was clarified by centrifugation at 30.000 g for 20 min at 4°C. Sodium azide was added to the fermentor broth at a final concentration of 0,03% w/v as bacteriostatic agent, before adjusting to pH 7-5 with sodium hydroxide. All the measurements for the physical- chemical, as well as for the catalytic characterisation, were carried out directly on the clarified fermentor broth, without any kind of further purification.
  • Lipase activity was routinely measured by a pH-stat. 66 mM tributyrin were emulsified in distilled water containing gum arabic (20 mg/ml) as stabiliser using a homogeniser (Ultraturrax T25, Janke & Kunkel) for 7 min at maximum speed. 20 ml of the substrate solution were heated to 30°C and adjusted to pH 7.2. After the addition of 10-200 ⁇ l of the enzyme solution, the activity was measured with a pH-stat (Metrohm) . One unit was defined as the amount of enzyme which released 1 ⁇ mol fatty acid per minute.
  • substrate specificity 20 mM of triacylglycerols , and 5% w/v of cocoa butter and 100 mM of fatty acid methyl esters were each emulsified in distilled water, containing gum arabic (20 mg/ml), and used as substrate solution in the pH-stat assay.
  • the tristearin emulsion was obtained adding 5% v/v aceton, being verified that, under the same conditions the lipases retained 47% of their full activity on tributyrin.
  • N-te ⁇ rminal sequencing Amino terminal sequence analysis was performed on the recombinant lipases produced during the cultivation in flasks. The cells were separated by centrifugation and the resulting supernatants (180 ml) were ultrafiltered through a 50 kDa membrane. The resulting lipase solutions had a final volume of 20 ml and a concentration of 800 U/ml for ppLIP and preLIP. For the protein sequencing a gas-phase sequencer 470A (Applied Biosystems)was used following the manufacturer's instructions. After blotting, the PVDF membranes were stained with Coomassie Brilliant Blue R-250, and the lipase bands cut out and used for N-terminal sequence determination .
  • Protein concentration was determined with the bicinchoninic acid (BCA) protein assay kit (Pierce) using the enhanced method according to the manufacturer's instructions (Pierce, Instructions 23220/23225) and bovine serum albumin as standard.
  • BCA bicinchoninic acid
  • Protein samples were incubated for 12 h with endo- ⁇ -N- acetylglucosaminidase H (25 mU/mg protein) at 37 °C in a 50 mM potassium acetate buffer, pH 5-5. containing 0.5 mM phenylmethylsulphonyl fluoride to prevent proteolysis .
  • endo- ⁇ -N- acetylglucosaminidase H 25 mU/mg protein
  • a 50 mM potassium acetate buffer pH 5-5. containing 0.5 mM phenylmethylsulphonyl fluoride to prevent proteolysis .
  • the protein was first incubated with 0.01% (m/v) SDS at 95°C for 3 min.
  • Isoelectric focusing and SDS-polyacrylamide electrophoresis Analytic SDS-PAGE (8-25%) and IEF (pH 3-9) were performed with a Pharmacia Phast System according to the manufacturer's recommendations.
  • the gels were stained for protein detection by a silver staining procedure 32 .
  • Preparative gel electrophoresis was carried out in a 12.5% polyacrylamide gel according to Laemmli 33 and proteins were stained with Coomassie Brilliant Blue R-250.
  • the effect of pH on the enzyme activity was determined by pH-stat assay at 30°C using tributyrin as substrate.
  • the effect of pH on lipase stability was determined by incubating aliquots of lipase solution for 20 h at 4°C in 0.1 M phosphate buffers at different pHs. Residual activity was measured by pH-stat assay.
  • the optimum temperature for enzyme activity was determined at pH 7-2 and various temperatures with tributyrin as substrate.
  • the effect of temperature on lipase stability was determined by incubating aliquots of lipase solution for 30 min in 25 mM Tris-buffer, pH 7-5 at various temperatures. Residual activity was measured by pH-stat assay.
  • the heat-inactivation curves were determined measuring the residual activity after a different incubation time at 50°C in 25 mM Tris-buffer, pH 7.5
  • CRL Candida rugosa lipase
  • N.D. not determined
  • MW molecular weight
  • r opt temperature optimum of activity
  • r stab temperature stability after 30 min of incubation
  • pH opt pH optimum of activity
  • pH stab pH stability
  • pNPP p-nitrophenyl palmitate.
  • Fig. 1 Design of the synthetic gene. Positions and lengths of oligonucleotides covering the Lip 1 gene (1647 bp) are indicated by arrow. Each cassette was synthesised using 2 or 3 couples of long overlapping oligonucleotides, represented as solid bars. Separately synthesised gene cassettes were cloned into pUC19 vector and ligated in order to assemble the entire gene.
  • Fig. 2 SDS-PAGE analysis of recombinant Candida rugosa lipase.
  • Lane 1 molecular weight standard in kDa
  • lane 2 lipase from 0,5 ⁇ l culture supernatant was withdrawn after 5 days induction
  • lane 3 0,5 ⁇ l of supernatant as withdrawn from 5 days non-induced culture.
  • Fig. 3 Recombinant lipase production during cultivation with different media. Fermentative production of lipl in a 11 bioreactor using (A) standard conditions: BMMY medium, 30°C and pH 6.0 and (B) high cell density conditions are reported by Payne et al. at 30°C and pH 6.0. The lipase activity was measured with a pH-stat, using tributyrin as substrate, at 30°C and pH 7.2. The lipase activity was measured with a pH-stat, using tributyrin as substrate, at 30 °C and pH 7-2.
  • Fig Substrate specificity of recombinant and commercial C. ugosa lipase. Activity towards various triacylglycerides (A) and fatty acid methyl esters (B) of different chain length of the acyl group (C2 to C22) . Cocoa butter (C16-18) contains predominantely palmitic acid (Cl6) in the sn-1, oleic acid (Cl8:l) in the sn-2 and stearic acid (Cl8) or palmitic acid (C16) in the sn ⁇ 3 position of the tracylglycerool . Relative activities were determined by pH-stat assay at pH 7.2 and 30°C for triacylglycerols and 50°C for fatty acid methyl esters.
  • the original sequence has no 98522 • Below this sequence the synthetic gene of the examples is illustrated.
  • the original sequence corresponds to the sequence id no 1 giving a nucleotide sequence for native lipase 1 and the corresponding amino acid sequence upon translation of said sequence.
  • the synthetic gene is sequence id. no. 2.

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Abstract

La levure dimorphe Candida rugosa présente un usage inhabituel du codon qui fait obstacle à l'expression fonctionnelle de gènes provenant de ladite levure dans un hôte hétérologue classique. Les lipases produites par cette levure sont largement utilisées dans des bioconversions industrielles, mais des échantillons de lipase commerciale contiennent plusieurs isoformes différentes codées par la famille des gènes de type LIP. Dans une premier essai difficile, le gène LIP1 codant l'isoforme majeure des lipases C. rugosa (CRL) a été modifié systématiquement par des mutagènes dirigées pour obtenir une expression fonctionnelle dans S. cerevisiae. Dans une approche de substitution, le gène (1688 bp) a été complètement synthétisé à l'aide d'une séquence nucléotidique optimisée en termes d'expression hétérologue dans la levure et de manipulation génétique simplifiée. Le gène synthétique a été fonctionnellement surexprimé dans Pischia pastoris. La CRL recombinante a été produite à un niveau et un degré de pureté élevés de l'ordre de 90 à 95% des protéines secrétées. Les propriétés physico-chimiques et catalytiques de la lipase recombinante ont été comparées avec celles d'une préparation commerciale, non recombinante de la lipase C. rugosa.
EP97940483A 1997-09-16 1997-09-16 Synthese totale et surexpression fonctionnelle d'un gene candida rugosa lip1 codant pour une lipase industrielle majeure Withdrawn EP1012301A1 (fr)

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GB9907082D0 (en) * 1999-03-26 1999-05-19 Chirotech Technology Ltd The preparation of carboxylic acid derivatives
EP1130100A1 (fr) * 2000-02-14 2001-09-05 Unilever N.V. Enzymes lipolytiques modifiées et leur utilisation
US6706474B1 (en) 2000-06-27 2004-03-16 Board Of Trustees Of The University Of Illinois Nucleic acid enzyme biosensors for ions
DE10100913A1 (de) * 2001-01-11 2002-07-25 Haarmann & Reimer Gmbh Verfahren zur Herstellung von L-Menthol
CN100336906C (zh) * 2001-01-15 2007-09-12 广州市绿巨人生物环保技术有限公司 脂肪酶基因序列及其在酵母中的应用
US7052879B2 (en) 2001-08-31 2006-05-30 Academia Sinica Recombinant Candida rugosa lipases
EP1601332A4 (fr) 2003-03-07 2012-05-02 Verenium Corp Hydrolases, acides nucleiques les codant, et procedes de fabrication et d'utilisation correspondants
US20080070291A1 (en) 2004-06-16 2008-03-20 David Lam Compositions and Methods for Enzymatic Decolorization of Chlorophyll
CN104498450B (zh) * 2014-12-18 2017-02-22 上海交通大学 褶皱假丝酵母脂肪酶1突变体及其基因
WO2017211930A1 (fr) 2016-06-10 2017-12-14 Dsm Ip Assets B.V. Lipase mutante et utilisation de cette dernière
CN106047917B (zh) * 2016-07-25 2019-10-29 北京化工大学 产脂肪酶的毕赤酵母基因工程菌株的构建方法
WO2019219903A2 (fr) 2018-05-18 2019-11-21 Dsm Ip Assets B.V. Lipase mutante et utilisation associée
CA3100615A1 (fr) 2018-05-18 2019-11-21 Dsm Ip Assets B.V. Lipase mutante et utilisation de cette derniere

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