[go: up one dir, main page]

WO2016020528A1 - Nouvelles hydroxynitrile lyases - Google Patents

Nouvelles hydroxynitrile lyases Download PDF

Info

Publication number
WO2016020528A1
WO2016020528A1 PCT/EP2015/068262 EP2015068262W WO2016020528A1 WO 2016020528 A1 WO2016020528 A1 WO 2016020528A1 EP 2015068262 W EP2015068262 W EP 2015068262W WO 2016020528 A1 WO2016020528 A1 WO 2016020528A1
Authority
WO
WIPO (PCT)
Prior art keywords
hnl
protein
recombinant
seq
cyanohydrin
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.)
Ceased
Application number
PCT/EP2015/068262
Other languages
English (en)
Inventor
Elisa LANFRANCHI
Kerstin Steiner
Margit WINKLER
Anton Glieder
Tea PAVKOV-KELLER
Matthias DIEPOLD
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.)
ACIB GmbH
Original Assignee
ACIB GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ACIB GmbH filed Critical ACIB GmbH
Publication of WO2016020528A1 publication Critical patent/WO2016020528A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
    • C12P13/004—Cyanohydrins
    • 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
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to novel hydroxynitrile Iyases (HNL), which are capable to catalyze the asymmetric cyanohydrin reaction and wherein the HNLs have a tertiary protein structure comprising a dimer of 7 anti-parallel beta-strands, 2 alpha- helices and a large cavity in each monomer (Allergen BV1 -like type fold).
  • HNL novel hydroxynitrile Iyases
  • the invention further relates to the use of hydroxynitrile Iyases for producing enantiopure cyanohydrin compounds.
  • HNLs Hydroxynitrile Iyases
  • cyanogenic pathway a defense mechanism widespread in the plant kingdom. More importantly, they are valuable tools in biocatalysis, due to their ability to synthesize chiral a-cyanohydrins by a C-C bond forming condensation reaction. A chiral center is formed, the carbon chain is prolonged by one carbon atom and an additional versatile functional group - the nitrile - is introduced to the molecule.
  • Enantiopure cyanohydrins are versatile building blocks and intermediates that serve as starting material for many enzymatic and chemical follow-up reactions, which find application in pharmaceutical, agrochemical and cosmetic industries (e.g.
  • the enzymes need to fulfill several criteria: (i) availability of sufficient quantities of proteins with constant quality and batch-to-batch reproducibility at low cost, (ii) broad substrate range, (iii) high stability under acidic pH and high solvent stability and (iv) activity at low temperatures because the unselective chemical background reaction is significantly suppressed at low pH ( ⁇ 4.5), low temperature and in the absence of water.
  • PhaWUL Phlebodium aureum
  • novel HNL enzymes which are capable to catalyze the asymmetric cyanohydrin reaction. It is a further objective of the present invention to provide novel HNL enzymes comprising the following properties:
  • molecular weight 20 ⁇ 5 kDa when measured by SDS-polyacrylamide electrophoresis; and tertiary protein structure: dimer of 7 anti-parallel beta-strands, 2 alpha-helices and a large cavity in each monomer (Allergen BV1 -like type fold).
  • the method comprises the steps of providing an aldehyde or ketone compound and converting the compound to the corresponding asymmetric cyanohydrin compound in the presence of a cyanide donor and an HNL enzyme.
  • the invention relates in particular to a selective Z7 HNL, which can stereoselectively catalyze the asymmetric cyanohydrin addition.
  • Fig 1 Silver-stained BN-PA gel and related filter of an in-gel HNL activity assay. Extracted and QFF-purified D. teyermanii p rote i n s : Raw protein extract (Extr), Flow through (FT), Active fractions (A9-B2). Substrate: racemic mandelonitrile - 100 mM citrate buffer, pH 4.5.
  • Fig. 2 SDS-PA gel of £>/HNL1 expression in E. co//(pMS and pET system) and P. pastoris).
  • S soluble fraction
  • P insoluble fraction
  • T4.F1 , T4.B7 and B1.G4 three different P. pastoris clones expressing /HNL1.
  • Marker Page Ruler, pre-stained protein ladder (Thermo Scientific). The arrow indicates the protein of interest.
  • Fig. 4 Protein sequence of DMHL2
  • Fig. 7 Primers for cloning £>/HNL1 -4 in the pEHISTEV vector.
  • Fig. 8 Size exclusion chromatogram.
  • Solid line Gel Filtration Standard.
  • X-axis elution volume, y-axis UV absorption at 280 nm. Peak eluting at 7.7 ml: thyroglobulin (bovine) 670 kDa; at 12.77 ml: ⁇ -globulin (bovine) 158 kDa; at 14.38 ml: ovalbumin (chicken) 44 kDa; myoglobin (horse) 17 kDa; at 20.23 ml: vitamin B12 1.35 kDa.
  • Fig. 9a Ribbon diagram of birch pollen allergen BET V 1 L (PDB Code 1 FM4);
  • Fig. 9b Ribbon diagram of £ ⁇ 4HNL1 soaked with benzaldehyde.
  • Fig. 10 Representation of the active site of 3 ⁇ 4HNL1 and its bound substrate benzaldehyde. Residue numbering refers to the sequence including HIS-tag, TEV cleavage site and spacer residues.
  • the invention relates to a protein with hydroxynitrile lyase (HNL) activity comprising the following properties:
  • tertiary protein structure dimer of 7 anti-parallel beta-strands, 2 alpha-helices and a large cavity in each monomer (Allergen BV1 -like type fold);
  • the protein fold of the protein is related to the major Birch pollen allergen Bet v 1.
  • the protein fold is characterized by 7 anti-parallel beta-strands, 2 alpha-helices and a large cavity.
  • the tertiary structure of the allergen BV1 can be retrieved from the protein data bank server under the pdb code 1 FM4 and is depicted in Fig. 9 (top ribbon diagram)
  • amino acid sequence shows similarity to the SRPBCC superfamily of proteins.
  • SRPBCC means
  • SRPBCC START/RHO alpha C/PITP/Bet v1/CoxG/CalC
  • steroidogenic acute regulatory protein StAR
  • STARD1 - STARD15 C-terminal catalytic domains of the alpha oxygenase subunit of Rieske-type non-heme iron aromatic ring-hydroxylating oxygenases (RHOs alpha C)
  • RHOs alpha C Rieske-type non-heme iron aromatic ring-hydroxylating oxygenases
  • PITPs phosphatidylinositol transfer proteins
  • Bet v 1 the major pollen allergen of white birch, Betula verrucosa
  • CoxG CalC
  • Other members of this superfamily include PYR/PYL/RCAR plant proteins, the aromatase/cyclase (ARO/CYC) domains of proteins such as
  • Streptococcus mutans Smu.440 and related proteins are related proteins.
  • a further aspect of the invention is an isolated polypeptide, wherein said polypeptide is derived from a plant, in particular from Polypodiades plants, in particular from the Davalliaceae family, and has at least activity of a hydroxynitrile lyase.
  • Hydroxynitnle lyase activity could be determined as described in Example 1 1.
  • the protein has a hydroxynitnle lyase activity of at least 10 U/mg, preferably 100 U/mg, preferably 300 U/mg, preferably of at least 500 U/mg enzyme.
  • a further aspect of the invention is the isolated polypeptide as described herein, wherein the plants are of genus Davallia, in particular of species Da va Ilia teyermanii.
  • a further aspect of the invention is the HNL as described herein, wherein said HNL is a recombinant HNL.
  • a further aspect of the invention is the recombinant hydroxynitrile lyase (HNL) as described above, comprising SEQ ID NO: 1 or a variant amino acid sequence having at least 35% sequence identity with SEQ ID NO:1.
  • a further aspect of the invention is the recombinant hydroxynitrile lyase (HNL) as described above, comprising SEQ ID NO: 1 or a variant amino acid sequence having at least 50% sequence identity with SEQ ID NO: 1.
  • a further aspect of the invention is the recombinant HNL as described above, wherein said variant amino acid sequence having at least 75%, preferably at least 90 % identity with SEQ ID NO: 1.
  • the invention relates to the recombinant HNL as described above, wherein the amino acid sequence of the HNL is at least 35%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to SEQ ID NO: 1.
  • a further aspect of the invention is the recombinant HNL as described above having the SEQ ID NO: 1 (0/HNL1 ), SEQ ID NO:2 (£3 ⁇ 4HNL2), SEQ ID NO:3 (£>/HNL3), or SEQ ID NO:4 (£3 ⁇ 4HNL4).
  • the invention encompasses proteins, in particular fusion proteins or truncations of the protein or parts of the protein with HNL activity, which comprise at least one polypeptide of the invention.
  • a further aspect of the invention is an isolated polynucleic acid molecule encoding a recombinant HNL as described above.
  • the present invention also relates to nucleic acids coding for the HNL as described above, which are complementary to such nucleic acid sequences which hybridize with coding nucleic acids under stringent conditions.
  • a further aspect of the invention is a method to obtain further members of the BetV1 family with HNL activity.
  • the method comprises the use of the information from SEQ ID NO: 1 , 2, 3 or 4 or the tertiary structure of these proteins as query to search for related sequences in sequence databases, structure databases or sequences obtained by DNA sequencing projects.
  • SEQ ID NO: 1 , 2, 3 and 4 can be used to produce primers for the identification and cloning of directly homologous forms using PGR.
  • probes to investigate further naturally occurring functional variants of the / /HNL genes and thus the corresponding coding enzyme variants can be used.
  • SEQ ID NO: 1 or from allelic or naturally occurring functional variants thereof, e.g. via PGR using a deficient DNA polymerase a bank of artificially produced functional enzyme variants can be produced.
  • standard methods can be used to introduce individual point mutations into the DNA sequence which lead to amino acid exchanges; this means that the protein's properties such as substrate specificity can be changed, or the protein sequence can be changed without changing the fold and activity of the protein.
  • sequences coding for the protein can also be used in form of synthetic DNA constructs which are optimized with respect to their codons, DNA/RNA stability, facilitate translation or in order not to hybridize with sequences of SEQ ID NO: 1 , 2, 3 or 4.
  • the coding DNA sequences can be cloned into routine vectors and, after transforming host cells with said vectors, can be expressed in cell culture.
  • suitable expression vectors are pET-26b(+) and pEHISTEV for E. coli, and also expression vectors of other prokaryotic single cell organisms can be used.
  • suitable vectors for P. pastor is ar the pPpT4 S and pPpB1 shuttle vectors.
  • the gene may be integrated into the genome of the host cell. Also vectors of other eukaryotic single cell organisms can be used.
  • the expression vectors of the invention may contain further functional sequence regions, such as a replication start point, operators, termination signals, coding sequences for antibiotic resistance, tags which facilitate purification (for example a His-tag or a Strep-tag) or other peptide sequences which are produced by the fusion proteins.
  • a shuttle vector may contain functional sequence regions as described above and further homologous sequences for correct integration into the genomes.
  • a further aspect of the invention is a vector comprising an isolated DNA molecule as described above.
  • the vector comprises all regulatory elements necessary for efficient transfection as well as for efficient expression of proteins.
  • Such vectors are well known in the art and any suitable vector can be selected for this purpose.
  • a further aspect of the present invention relates to a recombinant host cell which is transfected with a vector as described above.
  • the host cell is a non-human cell, in particular an E. col i O P. pastoris ce ⁇ .
  • Transfection of cells and cultivation of recombinant cells can be performed according to well-known methods in the art.
  • Such a recombinant cell as well as any therefrom descendant cell comprises the vector.
  • a cell line is provided which expresses the inventive HNL protein either continuously or upon activation depending on the vector.
  • host cells can be transformed using the usual methods, such as for example the heat shock method or electroporation.
  • a further aspect of the present invention is constituted by expression systems which comprise host cells or host cell cultures which are transformed with the vector systems described above.
  • Preferred hosts are single-cell prokaryotic organisms, in particular E coli.
  • eukaryotic genes it may be advantageous to use eukaryotic expression systems in order, for example, to introduce post- translational modifications or to facilitate folding of enzymes from eukaryotes.
  • Particularly suitable eukaryotic host cells are yeast cells, in particular P. pastoris cells.
  • a further aspect of the invention is a recombinant non-human cell obtained by introducing a vector as described herein.
  • the expression systems can be cultivated using standard protocols which are known to the skilled person. After expression of a protein with HNL activity of the invention, it is purified, for example by centrifuging and/or using chromatography. In order to carry out catalytic reactions, the purified enzymes and also the raw extracts or centrifugation residues or fractions can be used directly.
  • a further aspect of the invention is a culture obtained by culturing the recombinant cell as described herein.
  • a further aspect of the invention is a recombinant HNL recovered from the culture as described herein.
  • the invention relates to an HNL recovered from the culture as described herein.
  • the protein can be isolated by disrupting the cells and recovering the protein from the supernatant.
  • the whole culture broth including cells and supernatant culture media
  • the culture supernatant can be used as active catalyst preparation without any further isolation or purification or the culture supernatant can be used as active catalyst.
  • the invention relates to a method for producing an HNL, comprising recovering the HNL from a culture as described herein. Said isolation or purification from the cell culture can be performed by methods known in the art.
  • lyophilization, precipitation, affinity tagging or co-precipitation, affinity chromatography, anion-exchange chromatography and size exclusion chromatography can be used to isolate said protein.
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein an aldehyde or ketone compound is converted to the corresponding cyanohydrin compound in the presence of a cyanide donor and an HNL as described herein.
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein an aldehyde or ketone compound is converted to the corresponding cyanohydrin compound in the presence of a cyanide donor and a recombinant HNL as described herein.
  • a further aspect of the invention is the method for producing enantiopure cyanohydrin compounds, wherein a compound of general formula I is reacted with a compound of general formula II in the presence of an HNL to yield an asymmetric cyanohydrin compound III
  • R 1 and R 2 are independently from one another H, Ci-2oalkyl, C2-2oalkenyl, C2-2oalkynyl, Ca-iocycloalkyl, C 4- 2ocycloalkylalkyl, Ce-uaryl, C7-2oarylalkyl, 3-14 membered heterocycioaikyi, 4-20 membered heterocycloalkylalkyl, 5-20 membered heteroaryl or 6-20 membered heteroarylalkyl, optionally substituted by one or more R a ;
  • R 3 is H, an alkali metal, C(CH 3 )2OH , or Ci-2oalkyl; and
  • each R a is independently H, halogen, -CF 3 , -OR b , -NR b R b , -(CH 2 ) n COOR b ,
  • each R b is independently H or optionally substituted Ci-2oalkyl, C2-2oalkenyl, or
  • n 0, 1 , 2 or 3.
  • a further aspect of the invention is the method for producing enantiopure cyanohydrin compounds, wherein a recombinant HNL is used.
  • alkyl group if not stated otherwise, denotes a linear or branched
  • Ci-2oalkyl preferably a linear or branched chain of one to twenty carbon atoms.
  • alkenyl group if not stated otherwise, denotes a partially unsaturated linear or branched C2-2oalkenyl, preferably a linear or branched chain of two to twenty carbon atoms that contains at least one double bond.
  • alkynyl group if not stated otherwise, denotes a partially unsaturated linear or branched C2-2oalkynyl, preferably a linear or branched chain of two to twenty carbon atoms that contains at least one triple bond.
  • a cycloalkyi group denotes a monocyclic non-aromatic hydrocarbon ring containing three to ten carbon atoms, preferably four to six carbon atoms, or a bicyclic non-aromatic hydrocarbon ring system containing seven to ten carbon atoms, preferably eight to ten carbon atoms, wherein the cycloalkyi group optionally comprises one or more double bonds.
  • a heterocycioaikyi group denotes a monocyclic non-aromatic hydrocarbon ring containing three to fourteen carbon atoms, preferably four to eight carbon atoms, or a bicyclic non-aromatic hydrocarbon ring system containing seven to fourteen carbon atoms, preferably eight to ten carbon atoms, wherein in the heterocycioaikyi group one or more of the carbon atoms of the hydrocarbon ring or ring system is replaced by a group selected from the group comprising -N-, -O-,
  • An aryl group preferably denotes a mono- or bicyclic, preferably monocyclic aromatic hydrocarbon group having six to fourteen carbon atoms; the aryl group is preferably phenyl.
  • a heteroaryl group denotes an aromatic 5- or a 6- membered monocyclic hydrocarbon group wherein at least one of the carbon atoms is replaced by a heteroatom like O, N, and/or S, and wherein the aromatic monocyclic 5- or 6- membered cyclic hydrocarbon group is optionally fused to a further monocyclic 5- to 7-membered, preferably 5- or 6-membered, aromatic or nonaromatic hydrocarbon ring, wherein in the further monocyclic aromatic or nonaromatic hydrocarbon ring one or more, preferably one or two carbon atoms may be replaced by a heteroatom like O, N, and/or S.
  • a halogen group is chlorine, bromine, fluorine or iodine.
  • the method according to the invention can be carried out in a mono- or biphasic system or in an emulsion.
  • the monophasic reaction solution comprises an aqueous or an organic solvent.
  • aqueous solutions are for example water, a hydroxynitrile lyase containing solution, or a buffer solution.
  • buffer solutions are phosphate buffer, citrate buffer, acetate buffer, borate buffer, MES, HEPES, Tris buffer, or mixtures thereof.
  • the pH of these solutions can be between pH 1 and 8, preferably from 2 to 5.
  • Appropriate organic solutions can be slightly water-miscible or water immiscible aliphatic or aromatic hydrocarbons, which are optionally halogenated, alcohols, ethers or esters or mixture thereof or the substrate itself.
  • Suitable organic solutions are for example, but not limited to ethyl acetate, butyl acetate, methyl tert- butyl ether, diisopropyl ether, dibutyl ether, carbon tetrachloride, benzene, toluene, cyclohexane, hexadecane, hexane, heptane, chloroform, xylene, pentanol, hexanol, octanol and dodecanol, benzaldehyde, or mixtures thereof.
  • neoteric solvents which refers to ionic liquids and supercritical fluids that have remarkable new properties. These neoteric solvents are characterized by physical and chemical properties that can be finely tuned for a range of applications by varying the chemical constituents in the case of ionic liquids and by varying physical parameters in the case of supercritical fluids.
  • the advantages of conducting bioconversions in aqueous - organic solvent two-liquid phase systems are well known in the art.
  • the biphasic system consists of two phases mutually not miscible, e.g. an aqueous and an organic phase.
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein the reaction is carried out in a mono- or biphasic system or in an emulsion.
  • the invention relates to a method for producing enantiopure cyanohydrin compounds, wherein the biphasic system comprises aqueous and organic solution as described herein.
  • the conversion reaction moreover takes place at temperatures of from -10°C to +50°C, preferably at 0°C to 25°C, more preferred at 0°C to 10°C.
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein the recombinant HNL is a purified enzyme or is contained in cleared lysate.
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein the cyanohydrin compound is obtained with at least 50%, preferably with at least 60%, more preferred with at least 75% enantiomeric excess (e.e.).
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein the asymmetric cyanohydrin compound is obtained with at least 50%, preferably with at least 75%, more preferred with at least 90% enantiomeric excess (e.e.).
  • a further aspect of the invention is a method for producing enantiopure cyanohydrin compounds, wherein the cyanohydrin compound is obtained with a conversion rate of at least 15%, preferably with at least 20%, more preferred with at least 50%.
  • the invention relates to a method for producing enantiopure cyanohydrin compounds, wherein the used Z7 HNL is a purified enzyme, or contained in whole cells or in the fermentation broth or in a cleared lysate or in immobilized form, for example on a support such as Celite®, Avicel, etc. or as cross-linked enzyme crystals or as cross-linked enzyme aggregates among other immobilization methods.
  • a support such as Celite®, Avicel, etc. or as cross-linked enzyme crystals or as cross-linked enzyme aggregates among other immobilization methods.
  • the active site of the HNL comprises an arginine, an aspartate, a serine and three tyrosine residues.
  • amino acid residues corresponding to R69, D85, S87, Y101 , Y1 17 and Y161 of SEQ ID NO: 1 - 4 contribute to the HNL activity of the novel HNL enzymes.
  • Alignment refers to the process or result of matching up the nucleotide or amino acid residues of two or more biological sequences to achieve maximal levels of identity and, in the case of amino acid sequences, conservation, for the purpose of assessing the degree of similarity and the possibility of homology.
  • conserved substitution refers to a change at a specific position of an amino acid or, less commonly, DNA sequence that preserves the physico-chemical properties of the original residue or achieves a positive score in the governing scoring matrix.
  • Identity or sequence identity refers to the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, often expressed as a percentage identity of the amino acid residues, based on the total number of amino acid residues of the amino acid sequence. If the two sequences, which are being compared, have a different number of amino acid residues, the percentage is based on the number of the shorter amino acid sequence.
  • Homology refers to similarity attributed to descent from a common ancestor. Homologous biological components (genes, proteins, structures) are called homologs. Often the term homolog is used interchangeably with the term variant, meaning rather a certain degree of identity between two sequences without implying a common ancestor. Therefore within the meaning of the invention, the term variant of a sequence means a sequence with a certain degree of identity to the sequence, if not otherwise stated.
  • Similarity refers to the extent to which nucleotide or protein sequences are related. Similarity between two sequences can be expressed as percent sequence identity and/or percent positive substitutions.
  • sequence identity, similarity and positives values provided herein refer to the value obtained using the BLAST suite of programs using default parameters, a description is found in Altschul et al., J. Mol. Biol., 1990, 215,
  • sequence can mean a nucleotide sequence, which constitutes a polynucleotide, or an amino acid sequence, which constitutes a peptide.
  • Heterologous nucleic acid sequence or "nucleic acid sequence heterologous to a host” means a nucleic acid sequence which encodes e. g. an expression product such as a polypeptide that is foreign to the host ("heterologous expression” or
  • heterologous product e.g. a nucleic acid sequence originating from a donor different from the host or a chemically synthesized nucleic acid sequence which encodes e. g. an expression product such as a polypeptide that is foreign to the host.
  • heterologous protein means a protein that is foreign to the host.
  • Form to the host can mean, that the nucleic acid or protein originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition or genomic locus by deliberate human intervention.
  • a heterologous nucleic acid sequence as referred herein encompasses also nucleic acids, which are codon optimized for the host according to the codon usage of the host.
  • a cell which contains or shall contain a vector or an isolated nucleic acid sequence, and supports the replication and/or expression of the vector or of the nucleic acid. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells.
  • a “vector”, “vector expressible in a host” or “expression vector” are used interchangeably and is a polynucleic acid construct that is used in transfection or transformation of a host cell and into which a polynucleotide can be inserted, it is generated recombinantly or synthetically, with a series of specified polynucleic acid elements that permit transcription of a particular nucleic acid sequence in a host cell.
  • this vector includes a transcriptional unit comprising a particular nucleic acid sequence to be transcribed operably linked to a promoter.
  • Vectors are often replicons.
  • a vector expressible in a host can be e. g. an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus.
  • the term "introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • Transduction is usually used for the process of injection of foreign DNA by a virus into the host bacterium.
  • Transfection is the process of deliberately introducing nucleic acids into cells.
  • the term is often used for the introduction of foreign DNA into eukaryotic cells, notably for non-viral methods.
  • Competence is the ability of a cell to take up extracellular DNA from its environment.
  • the terms “transformation”, “transformed” or “introducing a nucleic acid into a host cell” are used interchangeably and denote any process wherein an extracellular nucleic acid like a vector, with or without accompanying material, enters a host cell.
  • the term “cell transformed” or “transformed cell” means the cell or its progeny into which the extracellular nucleic acid has been introduced and thus harbors the extracellular nucleic acid.
  • the nucleic acid might be introduced into the cell so that the nucleic acid is replicable either as a chromosomal integrant or as an extra chromosomal element. Transformation of appropriate host cells with e. g.
  • an expression vector can be accomplished by well-known methods such as microinjection, electroporation, particle bombardment or by chemical methods such as Calcium phosphate-mediated transformation, described e. g. in Maniatis et al. , 1982, Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, in Ausubel et al. , 1994, Current protocols in molecular biology, John Wiley and Sons, or in "Molecular Cloning: A laboratory Manual, Third edition (3 volume set)", Cold Spring Harbor Laboratory Press, 2001.
  • nucleic acid or nucleic acid sequence or polynucleotide might be DNA, RNA, or DNA/RNA hybrid. Unless otherwise indicated, the terms include reference to the specified sequence as well as the complementary sequence thereof. In case the nucleic acid sequence is located on a vector it is usually DNA.
  • DNA which is referred to herein can be any polydeoxynuclotide sequence, including, e.g.
  • double-stranded DNA single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double- stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently- closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radio labeled DNA and fluorochrome-labeled DNA, DNA containing one or more non-naturally occurring species of nucleic acid.
  • DNA sequences can be synthesized by standard chemical techniques, for example, the phosphotriester
  • RNA which is referred to herein can be e.g. single-stranded RNA, cRNA, double stranded RNA, double stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double- stranded RNA wherein the RNA strands are fully complementary, double-stranded RNA wherein the RNA strands are only partially complementary, covalently cross- linked RNA, enzyme digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi-synthetic RNA, biosynthetic RNA, naturally isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non-naturally occurring
  • variants or “variants of a sequence” is meant a nucleic acid sequence that vary from the reference sequence by nucleic acid substitutions, preferably by conservative nucleic acid substitution, whereby one or more nucleic acids are substituted by another with same characteristics. Variants encompass as well degenerated sequences, sequences with deletions and insertions, as long as such modified sequences exhibit the same function (functionally equivalent) as the reference sequence.
  • polypeptide refers to a polymer of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • isolated and purified nucleic acid sequence refers to the state in which the nucleic acid sequence will be free or substantially free of material with which they are naturally associated such as other nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e. g. cell culture) when such preparation is by recombinant technology practiced in vitro or in vivo.
  • LB broth and LB medium are used interchangeable.
  • the "origin of replication" (also called the replication origin) is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. DNA replication may proceed from this point bidirectionally or unidirectionally.
  • Promoter refers to a nucleic acid sequence that regulates expression of a transcriptional unit.
  • a “promoter region” is a regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter region will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA
  • polymerase such as the putative -35 region and the Pribnow box.
  • Signal sequence refers to a nucleic acid sequence which encodes a short amino acid sequence (i.e., signal peptide) present at the NH2-terminus of certain proteins that are normally exported by cells to non- cytoplasmic locations (e.g., secretion) or to be membrane components. Signal peptides direct the transport of proteins from the cytoplasm to non-cytoplasmic locations.
  • 'Translation initiation region is a signal region which promotes translation initiation and which functions as the ribosome binding site such as the Shine Dalgarno sequence.
  • transcription terminator and “transcription termination region” are used interchangeably and indicate a section of genetic sequence that marks the end of gene or operon on genomic DNA for transcription. It causes RNA polymerase to terminate transcription.
  • the transcription termination region is usually part of a transcriptional unit and increases the stability of the mRNA.
  • [001 1 1] 'Transcriptional unit refers to a nucleic acid sequence that is normally transcribed into a single RNA molecule.
  • the transcriptional unit might contain one gene (monocistronic) or two (dicistronic) or more genes (polycistronic) that code for functionally related polypeptide molecules.
  • a nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a signal sequence is operably linked to DNA for a protein if it is expressed as a preprotein that participates in the secretion of the protein;
  • a promoter is operably linked to a coding sequence if it affects the transcription of the sequence;
  • a translation initiation region such as a ribosome binding site is operably linked to a nucleic acid sequence encoding e. g. a polypeptide if it is positioned so as to facilitate translation of the polypeptide.
  • Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the assay detects the cyanogenesis reaction. It was performed on microtiter plate format. A final volume of 150 L contained 10 - 30 pL of sample and 20 pL of racemic mandelonitrile (30 - 90 mM stock). The reaction was performed in 100 mM sodium citrate buffer at pH 4.5 and gaseous cyanide was detected through a Feigl- Anger test paper (Krammer et al., 2007) by the development of blue spots.
  • RNA was isolated from Da va Ilia teyermanii f o 11 o w i n g the protocols provided by the SpectrumTM Plant Total RNA Kit (Sigma Aldrich). Quality assessment to ensure RNA integrity was performed with an Agilent 2100 Bioanalyzer (Agilent Technologies) and agarose gel electrophoresis (1 % agarose gel, running conditions: 80V, 40 min). The transcriptome sequence was obtained via a commercial service from Microsynth, by De Novo Next generation sequencing of a normalized
  • transcriptome library (Roche sequencing system). The resulting transcriptome data were deposited on an in-house server for analysis with a WEB based tool developed for enzyme identification from transcriptome data.
  • a suitable activity screening procedure had to be developed: a sandwich assembly was used, where the polyacrylamide gel was in direct contact with a filter paper soaked with substrate (racemic mandelonitrile 4 pL/mL) and reaction buffer (sodium citrate, 100 mM, pH 4.0). On top of the gel, a Feigl-Anger test paper was placed, separated from the gel by a permeable nylon tissue (Krammer et al., 2007). BN-PAGE gels were subsequently stained by silver staining (Blum, et al., 1987). A sharp 20-kDa-band was observed in the elution fractions, which corresponded to the HNL activity signal (Fig. 1 ).
  • One of the six targets displayed HNL activity, determined as described above, and was termed £ ⁇ 4HNL1.
  • Neither the gene nor the protein sequence showed similarities higher than 31 % when subjected to a blastp search in NCBI (Basic Local Alignment Search Tool of NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi, max target sequences: 100; expect threshold: 10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gap costs: existence 1 1 , extension 1 ; computational adjustments; conditional compositional score matrix adjustment; database: non-redundant protein sequences (nr)).
  • NCBI Basic Local Alignment Search Tool of NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi, max target sequences: 100; expect threshold: 10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gap costs: existence 1 1 , extension 1
  • the genes coding for proteins with HNL activity from Da va Ilia teyermanii ⁇ not contain any known signal sequences like those of other HNLs from plant species. In other species, the HNLs are separated from the substrate for example by transport to the extra- or intercellular space. DtHHL isoenzymes appear to be intracellular in the natural host and also if the protein is expressed employing its natural unmodified protein sequence in P. pastoris, it remains intracellular. However, it is known in the art from many other examples that natural intracellular proteins can be expressed as secreted proteins by adding a secretion signal sequence to the N-terminal end of the protein sequence or by fusion to other carrier proteins.
  • the amino acid sequence of 3 ⁇ 4HNL1 was employed as a template for the identification of similar sequences within the D. teyermanii tra n scri ptom e (Protein- nucleotide BLAST with standard parameters). Three nucleotide sequences were identified as putative Z3 ⁇ 4HNL1 isoenzymes. D. teyermanii g D N A wa s isolated with the PowerPlant ® Pro DNA Isolation Kit (MO BIO Laboratories, Inc.) according to the provided manual.
  • Putative isoenzyme sequences were amplified by PGR with 100 ng of gDNA as template (98°C 30 sec initial denaturation, 98°C 7 sec denaturation, 58°C 15 sec annealing, 72°C 30 sec extension, 72°C final elongation). PGR products were isolated from a 1 % agarose gel after electrophoresis and sequenced (LGC Genomics). Identified sequences were confirmed or corrected according to the sequencing results.
  • PGR products were cloned into the pJET1.2 vector with the CloneJETTM PGR Cloning Kit (Life Technologies) according to the provided manual.
  • E. col HOP 10 F' competent cells were transformed with a standard method. Clones were selected on LB agar with ampicillin (100 mg/L), screened by colony PGR and confirmed by sequencing (LGC Genomics).
  • E. co// ' GeneArt® Gene synthesis, Life Technologies.
  • the gene was cloned into the pET-26(b)+ vector using standard techniques (restriction sites: Nddi HindW) and confirmed by sequencing (LGC Genomics).
  • E. co// BL21 -Gold (DE3) competent cells were transformed with a standard method. Clones were selected on LB agar plates with kanamycin (50 mg/L). HNL expression was performed according to the following protocol: A single colony of E.
  • a synthetic gene was ordered after codon optimization for expression in P. pastoris (GeneArt® Gene synthesis, Life Technologies). The gene was cloned into the pPpT4 S and pPpB1 shuttle vectors, under the control of the AOX1 promoter
  • E. co//TOP10 F' competent cells were transformed with a standard method. Clones were selected on LB agar plates with Zeocin (25 mg/L). Plasmid DNA was isolated with the GeneJET Plasmid Miniprep Kit (Life Technologies) according to the provided protocol and the sequence of interest was confirmed by sequencing (LGC Genomics). The DNA preparation was linearized with SmA
  • CBS7435MutS was performed as described by Lin-Cereghino et al. (2005). Clones were selected on YPD agar plates with Zeocin (100 or 600 mg/L for low and high copy number integration, respectively). Selected P. pas ton ' s CBS 7435 MutS clones were screened for the best Z3 ⁇ 4HNL1 expression in micro-scale cultivation (96-deep well plates).
  • the expression was performed according to the following protocol: cells from single colony were transferred to 250 ⁇ _ in a buffered minimal dextrose medium BMD1 % (1 % glucose; (1.34 g/L yeast nitrogen base with ammonium sulfate and without amino acids, 200 mM potassium phosphate buffer at pH 6, 0.4 mg/L biotin, 2 g/L glucose)) at 28°C, 320 rpm and 80% humidity.
  • BMD1 % (1 % glucose; (1.34 g/L yeast nitrogen base with ammonium sulfate and without amino acids, 200 mM potassium phosphate buffer at pH 6, 0.4 mg/L biotin, 2 g/L glucose)
  • a first induction was performed with 250 pL BMM2 (0.5% final concentration of methanol; (1.34 g/L yeast nitrogen base with ammonium sulfate and without amino acids, 200 mM potassium phosphate buffer at pH 6, 0.4 mg/L biotin, 1 % v/v methanol)) after 60 h of cultivation. Further inductions were performed with 50 pL BMM10 (1.34 g/L yeast nitrogen base with ammonium sulfate and without amino acids, 200 mM potassium phosphate buffer at pH 6, 0.4 mg/L biotin, 5% v/v methanol (0.5% final concentration of methanol)) 10, 24 and 48 h after the first induction. The cultivation was stopped 72 h after the first induction.
  • the cells were harvested by centrifugation, disrupted with Y-PERTM Plus Dialyzable yeast protein extraction reagent (Life Technologies) and screened for HNL activity.
  • Small scale cultivation of the best P. pastor is CBS 7435 MutS clones expressing £ ⁇ 4HNL1 was performed in shake flasks. A single colony was inoculated in 180 mL (or 50 mL) BMD1 % for 60 h at 28°C, 120 rpm. Induction of protein expression was done by addition of 20 mL (or 5.5 mL) BMM10 and further addition of 2 mL (or 0.5 mL) methanol 10 h, 24 h and 48 h after the first induction. 72 h after the first induction, the cells were pelleted by centrifugation and disrupted as described in Example 7. Cell free lysate was assayed for HNL activity (as described in Example 10).
  • DM L isoenzymes 1 -4 were cloned into the pEHISTEV vector (Restriction sites: NcdIHindW) and the constructs confirmed by sequencing (LGC Genomics).
  • E. coli BL21 Star (DE3) competent cells were transformed with a standard method. Clones were selected on LB agar plates with kanamycin (50 mg/L). His-HNL expression was performed according to the protocol described above.
  • E. coir Cell pellets were re-suspended in 25 ml_ of 50 mM potassium phosphate buffer, pH 6.0, and disrupted by sonication (80% duty cycle, 7 output, 6 min). Samples were kept on ice during cells disruption.
  • P. pastoris Cell pellets were disrupted with Y-PER Plus Dialyzable Yeast Protein Extraction Reagent (Life Technologies).
  • the protein amounts were quantified by use of the Bradford reagent (Roti ® - Quant - Carl Roth), or the PierceTM BCA Protein Assay Kit (Life Technologies).
  • Protein purification was performed by affinity chromatography with a HisTrap FF 5 mL column (GE Healthchare) using an AKTA purifier system.
  • E. coli lysate was prepared in 20 mM sodium phosphate, 0.5 M NaCI, 10 mM imidazole, pH 7.4 as described above. Clear lysate (filtered through a 0.45 ⁇ syringe filter) was loaded on the column, previously equilibrated with 20mM sodium phosphate, 0.5 M NaCI, 10 mM imidazole at pH 7.4 (start buffer). Unbound material was washed with 10 column volumes of start buffer.
  • Superdex 200 10/300 GL column (catalog # 17-5175-01 , GE Healthcare; Bed volume: 24 ml_; Matrix: Spherical composite of cross-linked agarose and d extra n) at 4°C on an ⁇ Avant 25 (catalog # 28-9308-42, GE Healthcare) with a flow rate of 0.1 mL/min.
  • the column was pre-equilibrated with 150 mM NaCI, 10 mM Tris-HCI at pH 8. The absorbance of the eluent was monitored at 280 nm (A280).
  • a Gel-Filtration-Standard (catalog #151 -1901 , BioRad) was used in 1 : 10 dilution.
  • the apparent molecular mass of native 7 HNL1 was estimated according to the elution profile of the protein as depicted in Fig. 9 to be approximately 40 kDa, indicating that the active enzyme forms a dimer. This is in contrast to the active form of the Phlebodium aureum HNL, whose native size was estimated to be 168 ⁇ 40kDa (Wajant et al., 1995).
  • a spectrophotometric assay to detect the HNL reaction in cyanogenesis direction was performed on microtiter plate format.
  • a final volume of 200 ⁇ _ contained 20 ⁇ _ of protein sample and 130 ⁇ _ of 50 mM sodium citrate phosphate buffer, pH 5.0.
  • Blank reactions were carried out in which 50 mM sodium phosphate buffer pH 6.5 was used instead of protein sample.
  • the reaction was initiated by the addition of 50 ⁇ _ of substrate solution (60 mM racemic mandelonitrile in 3 mM sodium citrate-phosphate buffer, pH 3.5).
  • the reaction was measured spectrophotometrically following the formation of benzaldehyde at 280 nm for 10 minutes at 25°C in a Synergy Mx plate reader (BioTek). For calculations, the blank values were subtracted.
  • the values given in the tables below refer to the average of three (technical triplicates) to nine (three biological triplicates in technical triplicate) single measurements and their respective standard deviations.
  • citrate- phosphate buffer was used (pH 2.5 - 5.0).
  • Example 11 Hydroxynitrile lyase assay for determination of cyanogenic activity with any cyanohydnn
  • the reaction mix consisted of: 140 ⁇ _ of citrate phosphate buffer pH 5.0, 10 ⁇ _ of DMNL (0.001 -0.01 mg) and 10 ⁇ _ of cyanohydrin stock solution (300 mM cyanohydrin in 0.1 M citric acid). The reaction was performed at room temperature for 5-10 min.
  • reaction was stopped by the addition of 10 ⁇ _ of mix I [100 mM AAchlorosuccinimide, succinimide 10-fold excess (w/w) in water] followed by incubation at room temperature and 1 ,000 rpm for 5 min.
  • Racemic mandelonitrile was used as the positive control.
  • Z7 HNL1 prefers the natural substrate mandelonitrile, but also aliphatic substrates such as acetone cyanohydrin are converted.
  • HNL activity was measured as described above.
  • R- Mandelonitrile was used as the substrate.
  • 1 Unit (U) was defined as the amount of enzyme that produces 1 ⁇ of benzaldehyde per min under the assay conditions.
  • Each reaction was carried out in biological duplicate and technical triplicate and the blank was subtracted.
  • the specific activities were 562 U/mg for Z2/HNL1 , 746 U/mg for 0 HNL2, 607 U/mg for DMML3 and 861 U/mg for / /HNL4, respectively (with a confidence interval of 20%). This is significantly higher (4 to 6 times) than the specific activities of any other recombinant HNL (cleavage of the natural substrate
  • enzyme solution (20 ⁇ _, 0.01 mg/mL) was added to 50 mM sodium citrate- phosphate buffer pH 2.5, 3.0, 3.5, 4.0, 4.5 5.0, 5.5, 6.0, 6.5 and 7.0 and 50 mM potassium chloride buffer pH 2.0 and 2.5.
  • the reaction was started by addition of racemic mandelonitrile (15 mM final concentration).
  • HNL activity was measured as described in Example 10.
  • blank reactions were performed at each pH in which sodium phosphate buffer pH 6.5 was used instead of 9/HNL1 solution. Each reaction was carried out in biological triplicate and technical triplicate and the blank was subtracted.
  • DtHNL isoenzymes were incubated in 50 mM phosphate citrate buffer (final concentration of 1 mg/mL) at the pH values as specified in Table 2 at 5-8°C.
  • HNL activity at different temperature was measured spectrophotometrically in quartz cuvettes (1 mL). Therefore 100 ⁇ _ of enzyme solution (0.001 mg/mL final concentration) was added to 700 pL of 50 mM citrate phosphate buffer, pH 5.0. The reaction was started by the addition of 200 pL 60 mM racemic mandelonitrile (12 mM final concentration). In parallel, blank reactions were performed at each temperature in which sodium phosphate buffer pH 6.5 was used instead of £ ⁇ 4HNL solution. Enzymatic activity was measured from 10-50°C at 280 nm in a spectrophotometer (Cary Series Agilent Technologies). Each reaction was carried out in biological triplicate and technical triplicate and the blank was subtracted.
  • Table 6 shows that the activity of 3 ⁇ 4HNL isoenzymes increases with temperature.
  • the highest activity of Z3 ⁇ 4HNL1 and 7 HNL3 was observed at 40°C, of DMHL2 at 35°C, and of Z3 ⁇ 4HNL4 at 45°C.
  • the chemical background reaction decomposition of racemic mandelonitrile to benzaldehyde and HCN
  • the reactions in this system should preferably be carried out at low temperatures, e.g. below 10°C.
  • Z2 HNL1 (1 mg/mL) was incubated in sodium phosphate buffer at pH 6.5 at different temperatures as specified in Table 7. At the time-points specified in Table 7, an enzyme aliquot was diluted to 0.01 mg/mL and HNL activity was measured at 25°C as described herein. Each reaction was carried out in biological duplicate (Z7 HNL1 ) or triplicate ( 3 ⁇ 4HNL2, 3, 4) and technical triplicate and the blank was subtracted. The same experiments were carried out for 3 ⁇ 4HNL2 (Table 8), 7 HNL3 (Table 9) and Z7 HNL4 (Table 10). [00149] Z2/HNL1 (Table 7) and DMHL2 (Table 8) are not stable at 40°C and loose activity already after 2 hours. Surprisingly, DMNL3 and Z2 HNL4 are more stable at 40°C. After 48 hours, DMNL3 and Z7 HNL4 show 65% and 50% of their original activity (Table 9 and Table 10).
  • the target protein was tested for its ability to catalyze the cyanohydrin synthesis from benzaldehyde (or 2-CI-benzaldehyde, 3-Ph-propionaldehyde, cinnamaldehyde, 2- furylaldehyde, acetophenone) and HCN.
  • the reactions were performed in a two-phase system with methyl tertiary-butyl ether in a total volume of 1.5 ml_.
  • the HCN-solution ( ⁇ 2 M) was prepared as described before by Okrob et al. , 201 1. All reactions involving HCN were performed in a ventilated hood with gas trap and equipped with a calibrated HCN detector. A stock solution was prepared
  • Table 12 summarizes the results of Z7 HNL1 catalyzed synthesis of cyanohydrins.
  • Z2 HNL1 is more active than /3 ⁇ 4HNL in terms of mandelonitrile synthesis.
  • 0.096 mg/mL of commercial /3 ⁇ 4HNL (Sigma) preparation is used in the same reaction setup, 96.4% of conversion with 98.1 % e.e. is obtained (0.096 mg/mL /3 ⁇ 4HNL and 0.056 mg 7 HNL1 correspond to 5 U/mL of enzyme determined in cyanogenesis direction).
  • Enzyme concentration is based on the buffer phase of which 500 ⁇ _ were emulsified with 1 ,000 ⁇ _ of MTBE/HCN. Conversion is defined as the disappearance of substrate (corrected by the internal standard). Non-enzymatic transformation is defined as the consumption of the substrate detected in the blank reaction that did not contain enzyme (racemic cyanohydrin is produced). It needs to be noted that the non- enzymatic background reaction cannot be considered as a blank because it does not occur to the same extent if enzyme is present that consumes the substrates. However, the value gives an explanation to the low e.e. of the enzyme catalyzed product. Hence, the enzyme may still be perfectly stereoselective for the particular substrate but too slow at the respective conditions.
  • the £ ⁇ 4HNL1 is superior to 3 ⁇ 4HNL (Sigma Aldrich) in synthesis direction (benzaldehyde to mandelonitrile).
  • the known HNLs typically prefer either aromatic substrates (e.g. /3 ⁇ 4HNL) or aliphatic substrates (e.g. LLMUL).
  • DM L accepts both types of substrates: it is highly active for the cleavage of mandelonitrile and it also cleaves the aliphatic cyanohydrins
  • a single colony of P. pastoris CBS 7435 MutS £>/HNL1 opt B1.G4 was incubated overnight in BMGY medium (10 g/L yeast extract, 20 g/L peptone, 700 ml_ dH2O; 100 mL/L 10x yeast nitrogen base, 100 mL/L 200 mM potassium phosphate buffer pH 6.0, 100 mL/L 10% w/v glycerol, 2 mL/L 500x biotin) at 28°C in shake flask. Cultivation was performed in 5 L BIOSTAT® CT fermenter. First, a glycerol batch phase (glycerol 4%) was performed over-night.
  • BMGY medium 10 g/L yeast extract, 20 g/L peptone, 700 ml_ dH2O; 100 mL/L 10x yeast nitrogen base, 100 mL/L 200 mM potassium phosphate buffer pH 6.0, 100 mL/L 10% w/v gly
  • the supernatant was discarded and the cell pellet was dried in a 60°C incubator for 3 days. After drying, the tubes were weighed again and the cell dry weight was calculated. At the termination of the fermentation, the entire cell culture was harvested (ca. 1.5 kg of total wet cell weight).
  • pEHisTEV £>/HNL1 was incubated overnight in LB medium with 50 mg/L kanamycin at 37°C. After 16 h, the cell pellet was collected by centrifugation and washed two times with minimal media (M9 salts, 2% glucose, 2 mM MgSO 4 , 0.01 mg/mL thiamine, 0.01 mg/mL FeCb). The main-culture (400 mL minimal media with kanamycin (50 mg/L) and selenomethionine (SeMet (50mg/L)) was inoculated with an aliquot of the pre-culture to a final ODeoo of 0.25.
  • the culture was incubated at 37°C and 150 rpm until the ODeoo reached 0.5. Then, 0.5 mM IPTG was added and the culture was incubated at 25°C and 150 rpm for 38 h. Cell free extract was prepared as described above.
  • SeMet/3 ⁇ 4HNL1 was purified by affinity chromatography (NiSepharose 6 Fast Flow resin). Elution was performed with 20 mM sodium phosphate, 0.5 M NaCI, 300 mM imidazole, pH 7.4. Fractions containing SeMetZ? HNL1 were combined and desalted (PD10 Desalting columns, GE Healthcare LifeScience). Protein was stored at -20°C in 50 mM potassium phosphate buffer pH 6.0 prior to crystallization.
  • SeMetZ?/HNL1 (3 mg/mL in 10mM Tris-HCI pH8) was used to grow crystals in 0.2 M sodium thiocyanate, 20% (w/v) polyethylene glycol 3350. A 1 :1 ratio of protein and screening solutions was used. Typically, crystals appeared after 2-3 days. After supplementation of 30% glycerol, the crystals were flash-cooled in liquid-nitrogen.
  • the SeMet Z2 HNL1 datasets were collected at 100k from single crystals on the synchrotron beamline ID29 (EMBL, Grenoble, France). The data sets were processed and scaled using the XDS program package (Kabsch, 2010).
  • the AutoSol Program (McCoy et al., 2007, Terwilliger et al., 2009) and the AutoBuild Program (Terwilliger et al., 2008) from the PHENIX software suit (Adams et al., 2010) were used to define the selenium heavy metal-atom sites using a SeMet Z7/HNL1 SAD data set, as well as to build an initial model. The resulting model was again completed manually in Coot and refined with PHENIX.
  • native crystals were also grown by mixing 1 pL protein sample (4 mg/mL in 10mM Tris-HCL pH 8) with 0.5 pL reservoir solution (0.1 M 2-(4-(2- hydroxyethyl)-1 -piperazinyl) ethanesulfonic acid pH 7.5 and 10% (w/v) polyethylene glycol; JSCG condition B4) using the same method as previously described. Typically, crystals appeared after 2-3 days.
  • the crystal structure revealed a dimer of 7 anti-parallel beta-strands, 2 alpha- helices and a large cavity in each monomer.
  • the residues important for substrate binding and catalysis were identified from the structure containing the substrate benzaldehyde and the structure containing the product mandelonitrile.
  • the substrate showed interactions with the residues arginine 94, aspartic acid 1 10 and serine 1 12, tyrosine 126 and tyrosine 142 (Fig. 10).
  • This numbering refers to the sequence that starts with the HIS-tag and the TEV cleavage site, including some spacer residues.
  • the active site residues are arginine R69, aspartate D85, serine S87 and three tyrosines Y101 , Y1 17 and Y161 in case the numbering refers to the native £ ⁇ 4HNL1 sequence.
  • a new hydroxynitrile lyase from fern from the plant to the sequence, ACIB

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne de nouvelles hydroxynitrile lyases (HNL) présentant un poids moléculaire d'environ 20 kDa et un repliement protéique du type de celui de l'allergène BV1, ainsi que leurs procédés de production. L'invention concerne également l'utilisation d'hydroxynitrile lyases à des fins de production de composés de cyanohydrine énantiomériquement purs.
PCT/EP2015/068262 2014-08-07 2015-08-07 Nouvelles hydroxynitrile lyases Ceased WO2016020528A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14180247 2014-08-07
EP14180247.0 2014-08-07

Publications (1)

Publication Number Publication Date
WO2016020528A1 true WO2016020528A1 (fr) 2016-02-11

Family

ID=51298597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/068262 Ceased WO2016020528A1 (fr) 2014-08-07 2015-08-07 Nouvelles hydroxynitrile lyases

Country Status (1)

Country Link
WO (1) WO2016020528A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1160329A2 (fr) * 2000-06-02 2001-12-05 Nippon Shokubai Co., Ltd. Procédé enzymatique et procédé pour la préparation enzymatique de la cyanhydrine optiquement active
WO2012143374A1 (fr) * 2011-04-18 2012-10-26 Teknologian Tutkimuskeskus Vtt Substances à action hypoallergénique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1160329A2 (fr) * 2000-06-02 2001-12-05 Nippon Shokubai Co., Ltd. Procédé enzymatique et procédé pour la préparation enzymatique de la cyanhydrine optiquement active
WO2012143374A1 (fr) * 2011-04-18 2012-10-26 Teknologian Tutkimuskeskus Vtt Substances à action hypoallergénique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt [online] 3 April 2013 (2013-04-03), "SubName: Full=Uncharacterized protein {ECO:0000313|EnsemblPlants:GSMUA_Achr6P14810_001};", XP002735333, retrieved from EBI accession no. UNIPROT:M0T729 Database accession no. M0T729 *
MARKOVIC-HOUSLEY Z ET AL: "Crystal Structure of a Hypoallergenic Isoform of the Major Birch Pollen Allergen Bet v 1 and its Likely Biological Function as a Plant Steroid Carrier", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 325, no. 1, 3 January 2003 (2003-01-03), pages 123 - 133, XP027091999, ISSN: 0022-2836, [retrieved on 20030103], DOI: 10.1016/S0022-2836(02)01197-X *
RADAUER CHRISTIAN ET AL: "The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands", BMC EVOLUTIONARY BIOLOGY, BIOMED CENTRAL LTD., LONDON, GB, vol. 8, no. 1, 15 October 2008 (2008-10-15), pages 286, XP021042040, ISSN: 1471-2148, DOI: 10.1186/1471-2148-8-286 *

Similar Documents

Publication Publication Date Title
Zirpel et al. Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
JP6494522B2 (ja) 代謝工学によるエルゴチオネイン生産法
CN104087560B (zh) 一种细菌漆酶突变体蛋白、其重组表达质粒、转化的工程菌株及其发酵制备方法
US11306331B2 (en) Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity
Lanfranchi et al. Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily
CN106102454B (zh) 使用o-甲基转移酶生物合成生成紫檀芪的方法
Ruwe et al. Identification and functional characterization of small alarmone synthetases in Corynebacterium glutamicum
Xu et al. Genome hunting of carbonyl reductases from Candida glabrata for efficient preparation of chiral secondary alcohols
Roiban et al. Development of an Enzymatic Process for the Production of (R)-2-Butyl-2-ethyloxirane
US20170211104A1 (en) Biosynthetic production of acetaminophen, p-aminophenol, and p-aminobenzoic acid
Hu et al. SERS-based microdroplet platform for high-throughput screening of Escherichia coli strains for the efficient biosynthesis of D-phenyllactic acid
CN104673814B (zh) 一种来自于阴沟肠杆菌的l‑苏氨酸醛缩酶及其应用
Du et al. Promotion of the asymmetric reduction of prochiral ketone with recombinant E. coli through strengthening intracellular NADPH Supply By Modifying EMP and introducing NAD kinase
CN118207171B (zh) 生物素连接酶突变体及其在生物素生产中的应用
CN106032531B (zh) 一种氰醇裂解酶、其制备方法及应用
US10036047B2 (en) Methods for hydroxylating phenylpropanoids
CN114760980A (zh) 针对10-乙酰基-3,7-二羟基吩噁嗪的过氧化物酶活性
CN112175919A (zh) 内酯水解酶突变体及其应用
Zhao et al. Development of cyclic AMP receptor protein-based artificial transcription factor for intensifying gene expression
CN108277216B (zh) S-氰醇裂解酶及其应用
CN108018265B (zh) 一种肌醇氧化酶突变体及其编码基因和应用
WO2016020528A1 (fr) Nouvelles hydroxynitrile lyases
CN103923889A (zh) 一种苯乙醛还原酶突变体
Wardenga et al. Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones
CN113322289B (zh) 制备(2r,3s)-2-氨基-3-羟基-3-(4-(甲基磺酰基)苯基)丙酸的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15750367

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15750367

Country of ref document: EP

Kind code of ref document: A1