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WO 2016/020528 Al 11 February 2016 (11.02.2016) P O P C T

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WO 2016/020528 Al 11 February 2016 (11.02.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/020528 Al 11 February 2016 (11.02.2016) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 9/88 (2006.0 1) C12P 13/00 (2006.0 1) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/EP20 15/068262 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 7 August 2015 (07.08.2015) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (25) Filing Language: English PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 14180247.0 7 August 2014 (07.08.2014) EP (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: ACIB GMBH [AT/AT]; Petersgasse 14, A- GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 8010 Graz (AT). TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (72) Inventors: LANFRANCHI, Elisa; Hans Brandstettergasse DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, 9/2/9, A-8010 Graz (AT). STEINER, Kerstin; Hochstein- LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, gasse 15/14, A-8010 Graz (AT). WINKLER, Margit; SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Rudolf-Hans-Bartsch Strasse 32, A-8042 Graz (AT). GW, KM, ML, MR, NE, SN, TD, TG). GLIEDER, Anton; Wetzawinkel 20, A-8200 Hofstatten an der Raab (AT). PAVKOV-KELLER, Tea; Eck- Published: ertstrasse 72, A-8020 Graz (AT). DIEPOLD, Matthias; — with international search report (Art. 21(3)) Pluddemanngasse 75d, A-8010 Graz (AT). (74) Agents: GASSNER, Birgitta et al; Donau-City-Strasse 11, A-1220 Vienna (AT). 0 0 o © v © (54) Title: NEW HYDROXYNITRILE LYASES (57) Abstract: The present invention relates to novel hydroxynitrile lyases (HNL) which have a molecular weight of about 20 kDa and a protein fold of the allergen BVl -like type and to methods of producing them. The invention also relates to the use hydroxyni- trile lyases for producing enantiopure cyanohydrin compounds. New hydroxynitrile iyases Field of the Invention [0001] 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). The invention further relates to the use of hydroxynitrile Iyases for producing enantiopure cyanohydrin compounds. Background Art [0002] Hydroxynitrile Iyases (HNLs) take part in the 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. Gruber-Khadjawi, M., Fechter, M.H., Griengl, 2012; Lanfranchi et al., 201 3 ; Winkler, Glieder, & Steiner, 2012). To meet the requirements for industrial application, 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. [0003] A number of (R)- and (S)- selective HNLs have been identified so far (see Tables in Dadashipour & Asano, 201 1; Winkler et al., 2012). Currently, the (/^-selective hydroxynitrile Iyases from Prunus amygdalus ( HNL), Prunus mume (Pm NL), Eriobotrya japonica HNL, Arabidobsis thaliana (-4 /HNL), Linum usitatissimum (LuHNL) and the bacterial G N from Granulicella tundricola can be heterologously expressed. Whereas /HNL, Lu and /H NL can be more or less successfully expressed in E. coli, the glycosylated and disulfide bridge containing enzymes from Rosaceae, e.g. Pa N , Pm , and HNL, can only be expressed with reasonable yield in Pichia paston's as soluble and active protein. However, the main disadvantage of - HNL is its low stability at pH below 5.4. L L displays a very narrow substrate spectrum for only aliphatic aldehydes and methylketones as {R)- selective enzyme. Compared to other HNLs, HNL is significantly less active (Hussain et al., 2012). Thus, there is still a need for highly active and stable HNLs, which are easily expressed as recombinant proteins. [0004] One member of the (/^-selective HNL group is the hydroxynitrile lyase from Phlebodium aureum (P a ), isolated by Wajant et al., 1995. The protein was purified, and a first biochemical characterization was performed. Pha was described as a multimer of 20 kDa subunits. It was described as a new enzyme, not related to other known HNLs. Its specific activity was determined to be exceptionally high (19,000 U/mg), which revealed a great potential of this enzyme as biocatalyst (Wajant et al., 1995). However, the protein sequence was never determined and no further studies were reported. [0005] Inspired by the results of Wajant et al., ferns were the first target of choice for the discovery of new HNL sequences. Since the HNLs known to date are a perfect example of convergent evolution belonging to four different structural folds to date and sharing no sequence identity between the different subgroups, basic sequence comparison tools (such as BLAST) are not a suitable tool to identify distinct and unique HNL enzymes as the algorithms are based on the homology and similarity between sequences. [0006] Therefore, a unique strategy was developed to visualize HNL enzymes (Lanfranchi et al., 201 3 , Fig. 1). By combining this strategy with transcriptome and mass spectrometry (MS) data, a new protein family with HNL activity was identified. Summary of invention [0007] It is the objective of the present invention to provide 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: action: catalyzing stereoselective asymmetric cyanohydrin formation; 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). [0008] It is a further objective of the present invention to provide a method for the recombinant production of the novel HNL enzyme in prokaryotic and eukaryotic expression systems. [0009] It is a further objective of the present invention to provide a method for producing asymmetric cyanohydrin compounds utilizing the novel HNL enzyme. 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 H N L, which can stereoselectively catalyze the asymmetric cyanohydrin addition. Brief description of drawings [0010] Fig 1: Silver-stained BN-PA gel and related filter of an in-gel HNL activity assay. Extracted and QFF-purified D. teyermanii prote ins: Raw protein extract (Extr), Flow through (FT), Active fractions (A9-B2). Substrate: racemic mandelonitrile - 100 mM citrate buffer, pH 4.5. [001 1] Fig. 2 : SDS-PA gel of £ H N L 1 expression in E. // (pMS and pET system) and P. pastoris). S : soluble fraction, P : insoluble fraction; T4.F1 , T4.B7 and B 1.G4: three different P. pastoris clones expressing H NL1. Marker: Page Ruler, pre-stained protein ladder (Thermo Scientific). The arrow indicates the protein of interest. [0012] Fig. 3 : Protein sequence of H N L 1 [0013] Fig. 4 : Protein sequence of D 2 [0014] Fig. 5 : Protein sequence of Z H NL3 [0015] Fig. 6 : Protein sequence of Z¾HNL4 [0016] Fig. 7 : Primers for cloning H NL1-4 in the pEHISTEV vector. [0017] 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: γ-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. Dotted line: Peak eluting at 15.36 ml: D [0018] Fig. 9a: Ribbon diagram of birch pollen allergen BET V 1L (PDB Code 1FM4); Fig. 9b: Ribbon diagram of H N L 1 soaked with benzaldehyde. [0019] Fig. 10: Representation of the active site of H N L1 and its bound substrate benzaldehyde.
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