,
) or the pGAPPamHNL5a plasmid (
), using the PaHNL4L1Qendf3/Ocyctermrv1 primer pair.
[0107] The PCR was carried out in a 100 µl reaction volume (60 ng of plasmid DNA, 1x Phusion HF buffer, 0.2 mM dNTPs, 0.4 µM of each primer, 1.2 U of Phusion High-Fidelity DNA polymerase from Finnzymes (Cat.No. F530L)) in an Applied Biosystems thermocycler.
[0108] A denaturing step of 30 s at 98°C was followed by 30 cycles of amplification (10 s at 98°C, 20 s at 60°C, 1 min at 72°C) and a final step of 7 min at 72°C.
[0109] After the PCR, the DNA template was removed by means of DpnI digestion. The PCR product was then purified either by means of the QIAquick PCR Purification Kit from Qiagen (Cat. No. 28106) or according to the purification protocol of the QIAquick Gel Extraction Kit (Cat. No. 27106).
Assembly of the linear expression cassette:
[0110] Assembly by means of overlap extension PCR was carried out in 2 steps.
[0111] In step 1, 45-60 ng of the product of PCR I, 25-50 ng of the product of PCR II and 25-30 ng of the product of PCR III were used as template and at the same time as primers for completing a contiguous product. Extension was carried out in a 50 µl reaction mixture containing 1x Phusion HF buffer, 0.2 mM dNTPs and 0.6 U of Phusion High-Fidelity DNA Polymerase from Finnzymes (Cat.No. F530L)) in an Applied Biosystems thermocycler. A denaturing step of 30 s at 98°C was followed by 10 cycles of amplification (10 s at 98°C, 20 s at 60°C, 1 min at 72°C) and a final step at 72°C for 7 min.
[0112] In step 2, the following components were added to the PCR mixture of step 1: 34.7 µl of water (purest quality), 10 µl of 5x Phusion HF buffer, 1 µl of 10 mM dNTPs, 2 µl of pGAP158for primer (20pmol/µl), 2 µl of Ocyctermrv1 primer (20pmol/µl) and 0.6 U of Phusion High-Fidelity DNA Polymerase from Finnzymes (Cat.No. F530L). The mixture was denatured at 98°C for 30 s. This was followed by 20 cycles of amplification (10 s at 98°C, 20 s at 60°C, 1 min at 72°C) and a final step at 72°C for 7 min.
Transformation of the linear expression cassettes into Pichia pastoris:
[0113] The overlap extension PCR products were purified according to the purification protocol of the QIAquick PCR Purification Kit from Qiagen (Cat. No. 28106) or the QIAquick Gel Extraction Kit (Cat. No. 27106). This was followed by digestion by means of BlnI (AvrII) (Roche, Cat. #11558161001) or XmaJI (AvrII) (Fermentas, Cat. #ER1561). The cleavage reaction mixture was purified by means of ethanol precipitation, and approx. 1 µg of the prepared DNA was then transformed into Pichia pastoris X33 according to the Invitrogen standard protocol. 1 µg of DNA yielded 1000-6000 transformants.
Primer sequences:
[0114]
- A111Mutfor: 5'-ATCCTCGGTGGCACGACCATAATCNNKGGANNKGTCTACGCCAGAGCTAACA-3'
- A111rev: 5'-GATTATGGTCGTGCCACCGAGGATC-3'
- Ocyctermrv1: 5'-TGCTCACATGTTGGTCTCCAGCTTGC-3'
- PaHNL4L1Qendf3: 5'-GTCAGATAGCGAGGTCACTCAGTCCGAACAAAAACTCATCTCAGAAG-3'
- PaHNL5endBr: 5'-GACTGAGTGACCTCGCTATCTGACTCACATGGACTCTTGAAT-3'
- pGAP158for: 5'-CCTTCTCTCTCCTTCCACC-3'
- V113Xshortfwd: 5'-TACGCCAGAGCTAACATTTCATTC-3'
- V113Xlongrev: 5'-GAATGAAATGTTAGCTCTGGCGTAMNNGCCTGCATTGATTATGGTCGTGCC-3'
- V317Xlongfwd:
- V317Xshortrev: 5'-AACAGAGGCTTCAATTGGATTTGG-3'
- V329Xlongfwd:
- V329Xshortrev: 5'-TTGATAATAATCACTTCTAATGCCTAAAACAG-3'
- V360Xlongfwd: 5'-CCAAATTCGACTTTTGCTCATATTNNKAGCCAAGTTCCAGGACCATTGT-3'
- V360Xshortrev: 5'-AATATGAGCAAAAGTCGAATTTGG-3'
Example 3: Cultivation of Pichia pastoris transformants in deep well plates and screening of PaHNL5 mutants
[0115] A) Search for improved activity of converting (R)-2-chloromandelo-nitrile:400 µl of BM5D medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/I yeast nitrogen base; 50 g/l D-glucose; 0.4 mg/l biotin) in 2 ml deep well plates were inoculated with single colonies of transformants and incubated with shaking at 320 rpm and 28°C, 80% humidity. After an incubation period of 96 hours, the cells were removed by centrifugation and the culture supernatant was used directly for measuring enzyme activity.The activity assay was carried out in 96 well PS microtiter plates (Greiner Bio-One GmbH, D; Cat. No. 655101). Into each well 130 µl of 0.1 M phosphate-citrate buffer (pH 5.0) were introduced and then admixed with 20 µl of a suitable dilution of culture supernatant. The enzyme reaction was started by adding 50 µl of substrate solution (10 mg/ml (R)-2-chloromandelonitrile in 0.1M phosphate-citrate buffer (pH 3.5)). The slope of the absorbance profile at 300 nm was monitored on a Plate Reader Spectramax Plus 384 (Molecular Devices, D) at room temperature for 5 min (
).
The influence of different MnCl2 concentrations on the quality of the R-HNL random mutagenesis libraries was characterized by assaying in each case 96 transformants per MnCl2 concentration used for activity of converting (R)-2-chloromandelonitrile. The results are summarized in table 1. The largest proportion of active clones was achieved without addition of MnCl2. This condition was chosen in order to produce 10 000 transformants.
Approx. 10 000 transformants with random mutations, introduced by means of error prone PCR by means of HotStarTaq DNA polymerase and based on pGAPZAPaHNL5αL1Q, A111G plasmid DNA, or 1000 saturation mutagenesis transformants (based on the pGAPZAPaHNL5αL1Q, A111G plasmid and containing mutations in positions N110 and G112) were tested for improved conversion of (R)-2-chloromandelonitrile. R-HNL transformants with improved activity with respect to conversion of (R)-2-chloromandelonitrile were selected firstly for rescreening and then for further characterization. For rescreening, the selected transformants were streaked out individually. Of each streaked-out transformant, 4-8 individual colonies were again used for inoculating in each case 400 µl of BM5D medium in 2 ml deep well plates. Culturing and subsequent analysis were carried out in a manner similar to the first round of screening.B) Analysis of PaHNL5 muteins by means of converting hydroxypivalaldehyde to (R)-hydroxypivalaldehyde cyanohydrin.
[0116] Single colonies of transformants were used for inoculating 700 µl of BM7,5D medium (0.4 M potassium phosphate, pH 6.0; 13.4 g/I yeast nitrogen base; 75 g/I D-glucose; 0.4 mg/l biotin; 25 µg/ml zeocin) in 2 ml deep well plates and incubated with shaking at 320 rpm and 28°C, 80% humidity. After an incubation period
of 120 hours, the cells were removed by centrifugation and the culture supernatant was used directly for measuring enzyme activity.
[0117] The activity assay was carried out in 2 steps. Firstly, hydroxypivalaldehyde was enzymatically converted to the corresponding (R)-hydroxypivalaldehyde cyanohydrin. This was followed either by determining conversion by way of a color reaction of the remaining aldehyde with 4-hydrazino-7-nitrobenzofurazane (NBDH) or by determining the enantiomeric excess after derivatization of the reaction mixture by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0118] R-HNL transformants with improved activity with respect to converting hydroxypivalaldehyde to (R)-hydroxypivalaldehyde cyanohydrin were selected first for rescreening and then for further characterization. For rescreening, the selected transofmants were streaked out individually. Of each streaked-out transformant, 4-8 individual colonies were again used for inoculating in each case 700 µl of BM7,5D medium in 2 ml deep well plates. Culturing and subsequent analysis were carried out in a manner similar to the first round of screening.
I) Conversion of hydroxypivalaldehyde:
[0119] The cyanohydrin reaction was carried out in 2 ml Scienceware® 96 deep-well plates (Scienceware/Bel-Art) at room temperature. 400 µl of culture supernatant were introduced into each well and admixed with 150 µl of 3 M citrate-phosphate buffer (pH 2.4). Subsequently, 200 µl of a hydroxypivalaldehyde substrate solution and a magnetic stir bar (Cat. No. VP 734-2, V&P Scientific, CA, USA) were added to each well. The hydroxypivalaldehyde substrate solution was prepared by adding 1.2 ml of briefly heated, liquid hydroxypivalaldehyde to 46.8 ml of 3 M citrate-phosphate buffer (pH 2.4). Finally, 22 µl of a 12 M NaCN solution were added in order to start the reaction. To prevent HCN from escaping, the deep well plates were covered with SILVERseal™ Sealer aluminum foils (Greiner Bio-One GmbH, D, Cat. No. 676090). The microreaction mixtures were mixed using an Alligator Tumble Stirrer and magnetic stir bars (Cat. No. VP 734-2) from V&P Scientific (CA, USA). A mixing frequency of 25 Hz was chosen during the reaction.
[0120] After one hour, the reaction was stopped by removing the SILVERseal™ Sealer aluminum foils and by adding 50 µl of a 50% (v/v) sulfuric acid solution. As a result, the pH of the reaction solutions was shifted to pH < 1.0, conversion of hydroxypivalaldehyde was stopped, and the HCN cosubstrate was removed in this way. The reaction mixture was subsequently analyzed.
II) Determination of conversion by way of a color reaction with 4-hydrazino-7-nitrobenzofurazan (NBDH):
[0121] This screening method allows the remaining aldehyde concentration in the hydroxypivalaldehyde reaction mixtures to be determined. The FLUOstar OPTIMA plate reader from BMG LABTECH GmbH (Offenburg, D) was used for fluorimetric analysis. The 485-P filter was used as excitation filter and the 520-P filter was used as emission filter. Fluorimetric analysis was carried out in 96 well PP microtiter plates from Greiner Bio-One (Cat. No. 651201). Each well was charged with 150 µl of 3 M citrate-phosphate buffer (pH 2.4) to which 10 µl of the 1:10 diluted hydroxypivalaldehyde reaction mixtures were then added, with finally 60 µl of a saturated NBDH/ethanol solution being added in order to start conversion of NBDH to the corresponding hydrazone. The increase in the highly fluorescent hydrazone was monitored over 3 minutes and the slope of this reaction was directly correlated to the amount of aldehyde remaining in the reaction mixtures.
III) Determination of the enantiomeric excess by means of chiral GC:
[0122] Hydroxypivalaldehyde and the corresponding cyanohydrins formed during enzymatic conversion were extracted with methyl tert-butyl ether (MTBE): the hydroxypivalaldehyde reaction mixtures in the 96 well plates were admixed with 500 µl of MTBE and stirred at 20 Hz for 5 min by means of the Alligator Tumble Stirrer from V&P Scientific (CA, USA). After another 10 min, 35 µl of the organic phase were transferred to 96 well PP microtiter plates from Greiner Bio-One (Cat. No. 651201). Subsequently, 140 µl of CH2Cl2, 23 µl of acetoanhydride and 23 µl of pyridine were added, and the microtiter plates were sealed with SILVERseal™ Sealer aluminum foils (Greiner Bio-One GmbH, D, Cat. No. 676090). Derivatization was carried out on a Titramax 1000 shaker from Heidolph Instruments (D) at 450 rpm and room temperature for 30 min. The derivatized samples were then analyzed by means of a Hewlett Packard 6890 GC on a cyclodextrin column (CP-Chirasil-Dex CB (25m x 0.32 mm, 0.25 µm film)). A GC PAL, CTC-10022 Autosampler from CTC Analytics was used as autosampler. It was possible to cool the samples at 4°C during measurements. Further settings for the detector and inlet and the temperature program used and the retention times obtained for R- and S-hydroxypivalaldehyde cyanohydrin are listed below:
- Inlet:
- Setting: split
- Gas: H2
- Temperature: 250°C
- Pressure: 1.0 bar
- Split ratio: 20:1
- Temperature program:
- 110°C-0 min
- 10°C/min to 130°C
- 20°C/min to 170°C
- 170°C-0.5 min
- Time: 4.5 min
- FID detector:
- Temperature: 250°C
- H2: 20 ml/min
- Air: 200 ml/min
- N2: 30 ml/min
- Retention times:
- R-Hydroxypivalaldehyde cyanohydrin: 2.44 min
- S- Hydroxypivalaldehyde cyanohydrin: 2.55 min
Example 4: Sequence check by means of "colony PCR"
[0123] The best Pichia transformants were characterized by amplifying and sequencing the integrated, mutated hnl5 gene by means of PCR.
[0124] For this purpose, a Pichia pastoris single colony was resuspended in 50 µl of Y-Per® and heated to 65°C for 10 min. The mixture was cooled briefly on ice, then admixed with 60 µl of phenol/chloroform/isoamyl alcohol (25/24/1) and mixed well. This reaction mixture was centrifuged at 13 200 rpm in a bench centrifuge for 30 min. After centrifugation the aqueous phase was transferred to a fresh Eppendorf tube and purified by means of ethanol precipitation. The precipitated DNA was then dissolved in 50 µl of water (purest quality), and 2-5 µl of this DNA preparation were used as template for PCR amplification of the mutated hnl5 gene. The reaction mixture with a total volume of 100 µl moreover included the two primers, pGAPZA484f and HNL5alp21, with a concentration of in each case 0.4 µM, 0.2 mM dNTPs, 1x Phusion HF buffer and 1.2 U of Phusion High-Fidelity DNA Polymerase from Finnzymes
(Cat.No. F530L). The 3-stage PCR was carried out in an Applied Biosystems thermocycler and comprised the following steps:
- 30 s at 98°C, followed by 35 cycles of in each case 10 s at 98°C, 20 s at 60°C and 1 min at 72°C, and finally a single incubation of 7 min at 72°C.
[0125] After two purifications by means of the QIAquick PCR Purification Kit from Qiagen (Cat. No. 28106), the PCR product was used for sequencing.
Primer sequences:
[0126]
- HNL5alp21: 5'-ATGGTACCGAATTCTCACATGGACTCTTGAATATTATGAATAG-3`
- pGAPZA484f: 5'-TTCGAAACGAGGAATTCACGATGAG-3'
[0127] The integrated hnl5 genes of the random mutagenesis R-HNL transformants with improved activity of converting (R)-2-chloromandelonitrile and, respectively, hydroxypivalaldehyde, were checked by amplification and sequencing according to the colony PCR method.
Example 5: Recloning of isolated variants of the Pahnl5 gene into the pHILD2 vector and cultivation of Pichia pastoris pHILD2 transformants
[0128] To confirm that the randomly generated mutations in the Pahnl5 gene have contributed to improving the corresponding Pahnl5 genes, to purify "multicopy transformants" and to improve expression, the integrated mutated hnl5 gene was amplified by means of PCR, cloned by means of EcoRI cleavage sites into the pHILD2 vector (Invitrogen, San Diego, CA) and checked for correct orientation. Amplification of the integrated mutated Pahnl5 genes from genomic DNA of the Pichia pastoris transformants was described in example 4. The two primers used, HNL5alp21 and pGAPZA484f, comprised EcoRI cleavage sites.
[0129] The resulting plasmids were transformed into Pichia pastoris GS115 according to the Invitrogen standard protocol. In each case approx. 100 transformants were transferred to deep well culture plates for inoculation, using sterile toothpicks, and cultivated for screening for active transformants.
[0130] To this end, 250 µl of BM0,5G medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/l yeast nitrogen base; 5 g/I glycerol; 0.4 mg/l biotin) in 2 ml deep well plates were inoculated with single colonies of transformants and incubated with shaking at 320 rpm and 28°C, 80% humidity. Expression via the AOX1 promoter was induced by adding 250 µl of BMM2 medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/I yeast nitrogen base; 10 ml/l methanol; 0.4 mg/l biotin) after 60-70 hours. After
a further 10, 24 and 48 hours, more methanol was added by way of adding in each case 50 µl of BMM10 medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/l yeast nitrogen base; 50 ml/l methanol; 0.4 mg/l biotin). Approx. 72 hours after the first MeOH induction, the cells were removed by centrifugation and the culture supernatant was directly used for measuring enzyme activity.
[0131] The best Pichia transformants were characterized by amplifying again and sequencing the integrated, mutated hnl5 gene by means of PCR. This was carried out in a manner similar to example 4.
[0132] Table 2 depicts an overview of the particular expression strains of the best mutants:
[0133] Example 6: Cultivation of Pichia pastoris R-HNL mutants of the first round of random mutagenesis in 2 I shaker flasks and characterization of PaHNL5 muteins using (R)-2-chloromandelonitrile and 2-chlorobenzaldehyde
A) Cultivation of recloned R-HNL transformants in 2 I shaker flasks
[0134] Precultures comprising 50 ml of YPD medium (10 g/I Bacto™ yeast extract, 20 g/l Bacto™ peptone, 20 g/l D-glucose) were inoculated with large single colonies and incubated with shaking at 120 rpm and 28°C, 80% humidity, overnight. Subsequently, 225 ml of BM0,5G medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/I yeast nitrogen base; 5 g/l glycerol; 0.4 mg/l biotin) in 2 I flasks with baffles were inoculated with the precultures to give a starting OD of 0.05 and incubated with shaking at 120 rpm and 28°C, 80% humidity. Expression via the AOX1 promoter was induced by adding 25 ml of BMM10 medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/l yeast nitrogen base; 50 ml/l methanol; 0.4 mg/l biotin) after 60-70 hours. Further methanol additions of 2.5 ml per shaker flask (250 ml) were carried out after 10, 24 and 48 hours.
[0135] Approx. 72 hours after the first methanol induction, the cells were removed by centrifugation and the culture supernatant was used directly, or in diluted or concentrated form for measuring enzyme activity.
B) Characterization of PaHNL5 variants
[0136] To determine the specific activity of the particular mutants, the expression clones were cultured in each case in a plurality of shaker flasks. The culture supernatant was concentrated by ultrafiltration (10 kDa cutoff) using 20 ml Vivaspin PES centrifugation columns from Sartorius (Göttingen, D). Protein concentration was subsequently carried out using a Biorad (Hercules, Ca) protein assay (Bradford). The standard used for producing a calibration line was native PaHNL from Sigma (M-6782 Lot 41 H4016).
[0137] Samples containing approx. 3 µg of total protein were taken from the enzyme samples and applied directly to a gel (protein gel NuPAGE 4-12% Bis Gel 1.0 mm X 15 well; Invitrogen). Furthermore, samples containing approx. 1.5 µg of total protein were deglycosylated by endoglycosidase H (Cat. #P0702L, NEB) according to the protocol provided by the supplier and then applied. The standard used was SeeBlue Plus2 Pre-Stained Standard from Invitrogen (Carlsbad, USA).
C) Cleavage of (R)-2-chloromandelonitrile:
[0138] Firstly, the enzyme activities of the mutants of the invention with regard to cleavage of the substrate (R)-2-chloromandelonitrile (DSM Fine Chemicals Linz, A) were determined photometrically. To this end, 0.7 µl of 0.1 M phosphate-citrate buffer (pH 5.0) were introduced to quartz cuvettes and admixed with 100 µl of a suitably diluted enzyme solution. The reaction was started by finally adding 200 µl of an (R)-2-chloromandelonitrile substrate solution (10 mg/ml (R)-2-chloromandelonitrile in 0.1 M phosphate-citrate buffer (pH 3.5)). The slope of the absorbance profile at 300 nm was monitored for 5 min. The specific activities of the R-HNL muteins of the first random mutagenesis library are summarized in table 3.
[0139] The muteins A111G4_E10, N31A111G, I108MA111G and N110SA111G showed improved specific activity of (R)-2-chloromandelonitrile cleavage.
[0140] I304VA111G showed high activity directly in the culture supernatant but no improved specific activity, suggesting increased expression.
D) Synthesis of (R)-2-chloromandelonitrile
[0141] To compare the selectivities of the mutants of the invention for (R)-2-chloromandelonitrile synthesis, 15 mmol of 2-chlorobenzaldehyde were dissolved in 2.1 ml of tert-butyl methyl ether (MTBE). The corresponding PaHNL preparation (0.5 mg) was diluted with 50 mM phosphate-citrate buffer (pH 3.4) to give a final volume of 3.7 ml. The buffered solution was again adjusted to pH 3.4 and then mixed with the substrate dissolved in MTBE in 30 ml glass vials. The solution was cooled to 10°C, and 1.2 ml of liquid HCN were metered in using a syringe. A Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D) was used for stirring at 700 rpm to produce an emulsion.
[0142] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0143] Table 4 depicts the results for conversion (%conv) and enantiomeric excess (%ee) of the R-HNL variants of the invention after different points in time.
[0144] The variants A111G4_E10, N31A111G and I108MA111G produced increased conversion compared to PaHNL5αL1Q, A111G and showed improved activity of (R)-2-chloromandelonitrile synthesis. N110SA111G had reduced activity with regard to (R)-2-chloromandelonitrile synthesis but improved activity with regard
to (R)-2-chloromandelonitrile cleavage. This comparison indicates that the N110S mutation results only in an increased catalytic rate of the cleavage reaction.
Example 7: Site-specific mutagenesis for combining advantageous mutations
[0145] In each case 10 ng of the expression plasmids pHILD2-PaHNL5αL1Q, I108M, A111G, 2_A11*; pHILD2-PaHNL5αL1Q, A111G, 4_E10; pHILD2-PaHNL5αL1Q, N110S, A111G, 16_A3 or pHILD2-PaHNL5αV317A (see example 5) were used as template for the mutagenesis reaction by means of the QuikChange XL Site Directed Mutagenesis Kit from Stratagene (Cat. #200516). In each case 200 ng of the particular two mutagenesis primers were used for the reaction.
[0146] The following temperature program was used:
- A) Denaturation at 95°C for 1 min
- B) 8 cycles, each of 50 s at 95°C, 50 s at 60°C and 10 min at 68°C
- C) Extension at 68°C for 7 min.
[0147] The template DNA was removed by DpnI digestion as described in the kit's protocol, and 2 µl of the reaction mixture were used as described for transforming ultracompetent E. coli XL10 Gold cells. Plasmid DNA was prepared from the transformants and sequenced. Plasmids of mutants having the correct sequence in the region of the coding DNA insert were amplified and transformed into Pichia pastoris GS115 with the aid of the Invitrogen standard protocol.
[0148] Approx. 100 histidine-autotrophic Pichia transformants were cultured in deep well plates, and the activity of the culture supernatants was tested by means of the (R)-2-chloromandelonitrile cleavage reaction (see example 3). In each case clones having the highest enzyme activity among the individual mutants were selected for shaker flask experiments.
PCR-primers for site-specific mutagenesis:
[0149] For the N3I mutation, based on the pHILD2-PaHNL5αL1Q, I108M, A111G, 2_A11* plasmid or the pHILD2-PaHNL5αL1Q, I108M, A111 G, 4_E10 plasmid:
- N3If: 5'-GCTGAAGCTCAAGCCATTACTTCTGCTCATGAT-3'
- N3Ir: 5'-ATCATGAGCAGAAGTAATGGCTTGAGCTTCAGC-3'
- I108Mf: 5'-CTCGGTGGCACGACCATGATCAGTGGAGGCGTC-3'
- I108Mr: 5'-GACGCCTCCACTGATCATGGTCGTGCCACCGAG-3'
- I108M2f: 5'-CTCGGTGGCACGACCATGATCAATGGAGGCGTC-3'
- I108M2r: 5'-GACGCCTCCATTGATCATGGTCGTGCCACCGAG-3'
- N225Sf: 5'-GAAGATCCTCTTCTCTTCCTCTACATCAAATTTGTCAGCTATTG-3'
- N225Sr: 5'-CAATAGCTGACAAATTTGATGTAGAGGAAGAGAAGAGGATCTTC-3'
- I108M2f: 5'-CTCGGTGGCACGACCATGATCAATGGAGGCGTC-3'
- I108M2r: 5'-GACGCCTCCATTGATCATGGTCGTGCCACCGAG-3'
[0150] The PaHNL combination variants prepared in this way were thus: I108MA111G4_E10, N3II108MA111G, N3II108MA111G4_E10, I108MN110SA111G, I108MA111 GN225S and I108MA111GV317A.
Example 8: Production and characterization of PaHNL5 enzyme variants for conversion of 2-chlorobenzaldehyde
[0151] To determine the specific activity of the particular mutants, each of the expression clones described in example 7 was cultured in a plurality of 2 I shaker flasks. Cultivation was carried out in a manner similar to example 6A.
[0152] The culture supernatant was concentrated by ultrafiltration (10 kDa cutoff) using 20 ml Vivaspin PES centrifugation columns from Sartorius (Göttingen, D) and then used for determining enzyme activity.
[0153] To determine substrate specificities, the protein concentration of the enzyme preparations was measured using the Biorad protein assay (Hercules, Ca). Conversion and enantiomeric excess with regard to (R)-2-chloromandelonitrile synthesis were determined by means of GC:
- For this purpose, 15 mmol of 2-chlorobenzaldehyde were dissolved in 2.1 ml of methyl tert-butyl ether (MTBE). 0.5 mg of the corresponding PaHNL preparation was diluted with 50 mM phosphate-citrate buffer (pH 3.4) to give a final volume of 3.7 ml. The buffered solution was again adjusted to pH 3.4 and then mixed with the substrate dissolved in MTBE in 30 ml glass vials. The solution was cooled to 10°C, and 1.2 ml of liquid HCN were metered in using a syringe. A Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D) was used for stirring at 700 rpm to produce an emulsion.
[0154] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0155] Table 5 depicts the results for conversion (%conv) and enantiomeric excess (%ee) of the R-HNL variants of the invention after different points in time.
[0156] The combination of I108MA111 G with N225S showed no further improvement in conversion or enantiomeric excess. However, by combining the best mutations of the first random mutagenesis library, it was possible to further increase conversion compared to the I108MA111G variant. Virtually complete conversion was achieved already after 6 hours, using only 33.3 mg of unpurified enzyme preparation per mole of substrate. The N3II108MA111G4_E10 variant, for example, achieved a conversion of 99.7% after 6 h.
Example 9: Production of A111G (WO 2004/083424), A111G4 E10, N31A111G, I108MA111G, N311108MA111G and N3II108MA111G4 E10 muteins in a 5 I bioreactor
[0157] A sufficient amount of enzyme of the clones Pichia pastoris GS115 pHILDPaHNL5alphaL1Q, A111G of
, and the clones described in examples 6-8, Pichia pastoris GS115 pHILD2-PaHNL5αL1Q, A111G, 4_E10; Pichia pastoris GS115 pHILD2-PaHNL5αL1Q, N3I, A111G, 25_H2; Pichia pastoris GS115 pHILD2-PaHNL5αL1Q, I108M, A111G, 2_A11*; Pichia pastoris GS115 pHILD2-PaHNL5αL1Q, N3I, I108M, A111G and Pichia pastoris GS115 pHILD2-PaHNL5αL1Q, N3I, I108M, A111G4_E10, was produced in a 5 I fermenter for further characterizations. Muts strains were chosen for culturing.
[0158] First, a preculture of each clone comprising 50 ml of YPD medium (10 g/I Bacto™ yeast extract, 20 g/l Bacto™ peptone, 20 g/l D-glucose) was inoculated with a large single colony and incubated at 28°C and 120 rpm for 24 h. The first
preculture was used to inoculate 2 x 200 ml YPD medium in 2 I flasks with baffles. Incubation was carried out for approx. 20 h at 28°C and 120 rpm.
[0159] Chemicals 1-7, amounts calculated for 3.5 liters, were dissolved in deionized water and introduced into a 5 I bioreactor (Biostat®CT from Sartorius, Göttingen, D). After in situ sterilization, the pH of the medium was adjusted to pH 5.0 with 25% ammonia by way of sterile addition through a feed pump. This was followed by introducing into the bioreactor 13.5 ml of sterile-filtered trace element solution comprising vitamin H by means of a sterile syringe.
[0160] Approx. 400 ml of preculture from in each case two 2 I shaker flasks were used for inoculation. The starting OD600 in the fermenter was 1-2. With an operating temperature of 28°C, an aeration rate of 2.5-10 liters of air/min and a stirring speed between 500 and 1500 rpm, the partial pressure of oxygen (pO2) was maintained at a value >30% of the saturation concentration. The pH of the culture medium was kept constant at pH 5.0 by sterile addition of 25% ammonia through a feed pump.
[0161] After the initially introduced glycerol had been consumed, after a fermentation time of 15-20 hours, metering-in of the glycerol medium was started, initially at 45 ml/h. The metering-in rate was increased in steps to 90-100 ml/h and continued for approx. 12 hours, until approx. 150 OD600 were reached. Metering-in of glycerol was then stopped.
[0162] The third phase of fermentation was initiated by inducing expression by metering in methanol. The metering-in rate was adjusted initially to 10-15 ml/h. This rate was increased in steps to 45-60 ml/h over 12-15 hours and maintained for another 60 hours.
[0163] After approx. 72 hours of methanol induction, the cells were harvested by two centrifugations at 4000 rpm in a Beckman Coulter™ (Fullerton, Ca, USA) Avanti™ J-20XP centrifuge with the Beckman JLA 8.1000 rotor at 4°C for 20 min. The culture supernatant was collected and the enzyme activity of (R)-2-chloromandelonitrile cleavage was determined. The fermentation supernatant was diluted appropriately with 0.1 M phosphate-citrate buffer (pH 5.0) and, after addition of the (R)-2-chloromandelonitrile substrate solution (10mg/ml (R)-2-chloromandelonitrile in 0.1 M phosphate-citrate buffer (pH 3.5)), the absorbance profile at 300 nm was monitored in microtiter plates for 5 min. Enzyme activity in the culture supernatant after centrifugation was approx. 10-15 times greater than after fermentation in 2 I shaker flasks.
[0164] The culture supernatant was concentrated by means of crossflow ultrafiltration using 30 kDa cutoff modules from Sartorius (VIVASCIENCE Vivaflow 50 from Sartorius, Göttingen, D). Thus enzyme preparations with a protein concentration of 1-3 mg/ml were produced which were stored at 4°C for a short time and at -20°C for a longer period. Since Pichia pastoris secretes hardly any of its own proteins into the culture supernatant, the enzyme produced and concentrated in this way was already very pure.
[0165] The following chemicals were utilized for preparing the culture medium (amount per 3.5 I):
Glycerol medium:
[0166] 750g of glycerol were admixed with deionized water to give 1.5 I, dissolved and sterilized. Subsequently, 12 ml/l trace element solution were added.
Methanol medium:
[0167] 0.9 I of methanol were introduced to a sterile 2 I bottle and admixed with 12 ml/l trace element solution.
Example 10: Purification and characterization of the enzyme variants produced in the 5 I fermenter
[0168] The enzyme preparations produced in the fermenter and then concentrated were chromatographically purified.
[0169] Prior to purification, the concentrated enzyme preparations were equilibrated by repeatedly diluting and concentrating with the low-salt binding buffer A (20 mM citrate-phosphate buffer, pH 5.5). This is carried out by crossflow ultrafiltration by means of 30 kDa cutoff modules (VIVASCIENCE Vivaflow 50 from Sartorius, Göttingen, D). The equilibrated enzyme preparations were then purified on an ÄKTApurifier 10 FPLC instrument from Amersham Biosciences UK Limited (Buckinghamshire, UK) via an anion exchanger Q-Sepharose Fast Flow (QFF) column with a column volume of 10 ml. Elution was carried out using elution buffer B (20 mM citrate-phosphate buffer + 1 M NaCl, pH 5.5). Prior to purification, the column was equilibrated with five column volumes of buffer A. Thereafter, the gradient profile listed in table 6 was used for the various PaHNL5 variants from heterologous production by Pichia pastoris:
[0170] A flow rate of 2 ml/min and a fraction volume of 2 ml were chosen. The protein content of those fractions which, according to evaluation of the chromatogram, should contain protein (depending on peak position), was measured by means of Biorad (Hercules, Ca) protein assay (Bradford method). The enzyme activity of cleaving (R)-2-chloromandelonitrile was also determined in these fractions. The 2-3 fractions containing the highest activity were pooled and used for further analysis of
enzyme characteristics. The protein concentration was determined by means of Biorad (Hercules, Ca) protein assay (Bradford). The standard used for preparing the calibration lines was native PaHNL from Sigma (M-6782 Lot 41 H4016).
[0171] Samples containing approx. 3 µg of total protein were taken from the enzyme samples and applied directly to a gel (protein gel NuPAGE 4-12% Bis Gel 1.0 mm X 15 well; Invitrogen). Furthermore, samples containing approx. 1.5 µg of total protein were deglycosylated by endoglycosidase H (Cat. #P0702L, NEB) according to the protocol provided by the supplier and then applied to a protein gel. The standard used was SeeBlue Plus2 Pre-Stained Standard from Invitrogen (Carlsbad, USA).
[0172] The enzyme properties were determined by using the purified R-HNL variants for the synthesis of (R)-2-chloromandelonitrile, (R)-2-bromomandelonitrile and (R)-2-fluoromandelonitrile, respectively.
[0173] For this purpose, 15 mmol of 2-chlorobenzaldehyde, 2-bromobenzaldehyde, or 2-fluorobenzaldehyde were dissolved in 2.1 ml of methyl tert-butyl ether (MTBE). The appropriate PaHNL variant (0.25 mg or 0.5 mg) was diluted with 50 mM phosphate-citrate buffer (pH 3.4) to give a final volume of 3.7 ml. The buffered solution was again adjusted to pH 3.4 and then mixed with the substrate dissolved in MTBE in 30 ml glass vials. The solution was cooled to 10°C, and 1.2 ml of liquid HCN were metered in using a syringe. A Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D) was used for stirring at 700 rpm to produce an emulsion.
[0174] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0175] Table 7 a)-d) depicts the results for conversion (%conv) and enantiomeric excess (%ee) of the R-HNL variants of the invention after different points in time.
Example 11: Cultivation of Pichia pastoris transformants in shaker flasks and characterization of the enzyme variants with regard to conversion of hydroxypivalaldehyde [Comparative Example]
[0176] A sufficient amount of enzyme was prepared from the clones Pichia pastoris GS115 pHILDPaHNL5αL1Q (
), Pichia pastoris GS115 pHILDPaHNL5αL1Q, V317G (
) and Pichia pastoris GS115 pHILD2-PaHNL5αV317A (see example 2 and example 5) in 2 I shaker cultures for initial characterizations.
A) Cultivation of Pichia pastoris transformants
[0177] In each case 135 ml of BM0,5G medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/I yeast nitrogen base, 5 g/I glycerol; 0.4 mg/l biotin) in 2 I flasks with baffles were inoculated with a large single colony and incubated with shaking at 120 rpm and 28°C, 80% humidity. Expression via the AOX1 promoter was induced by adding 15 ml of BMM10 medium (0.2 M potassium phosphate, pH 6.0;
13.4 g/I yeast nitrogen base; 50 ml/l methanol; 0.4 mg/l biotin) after 60-70 hours. Further methanol additions of 1.5 ml per shaker flask (150 ml) were carried out after 10, 24 and 48 hours.
[0178] Approx. 72 hours after the first methanol induction, the cells were removed by centrifugation and the culture supernatant was used directly, or in diluted or concentrated form for measuring enzyme activity.
B) Characterization of PaHNL5 variants
[0179] The culture supernatant from the shaker flasks was concentrated approx. 20-fold by means of ultrafiltration (10 kDa cutoff) using 20 ml Vivaspin PES centrifugation columns from Sartorius (Göttingen, D). Protein concentration was subsequently carried out using a Biorad (Hercules, Ca) protein assay (Bradford). The standard used for producing a calibration line was native PaHNL from Sigma (M-6782 Lot 41 H4016).
[0180] Samples containing approx. 3 µg of total protein were taken from the concentrated enzyme preparations and applied directly to a gel (protein gel NuPAGE 4-12% Bis Gel 1.0 mm X 15 well; Invitrogen). Furthermore, samples containing approx. 1.5 µg of total protein were deglycosylated by endoglycosidase H (Cat. #P0702L, NEB) according to the protocol provided by NEB and then applied. The standard used was SeeBlue Plus2 Pre-Stained Standard from Invitrogen (Carlsbad, USA). Figure 2 depicts SDS-PAGEs of the PaHNL5αL1Q (=WT), V317G and V317A muteins.
C) Synthesis of (R)-hydroxypivalaldehyde cyanohydrin:
[0181] To compare the selectivities of the mutants of the invention for converting hydroxypivalaldehyde to the corresponding (R)-cyanohydrin, in each case 1 mg of the corresponding PaHNL was diluted with 3 M citrate-phosphate buffer (pH 2.4) to a final volume of 5 ml. The buffered solution was adjusted again to pH 2.4 by adding 50% (w/w) citric acid solution and mixed with 120 mg of briefly heated, liquid hydroxypivalaldehyde in 30 ml glass vials. The solution was cooled to 4°C, and 100 µl of liquid HCN were metered in using a syringe. The solution was stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0182] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0183] Table 9 depicts the results for conversion (%conv) and enantiomeric excess (%ee) of the R-HNL variants of the invention after different points in time.
[0184] While the V317G variant achieved approximately the same enantiomeric excess and conversion in converting hydroxypivalaldehyde as the wild type (PaHNL5αL1Q), the V317A variant was able to achieve substantially better selectivities and conversions.
[Comparative
Example][0185] A sufficient amount of enzyme was prepared from the clone Pichia pastoris GS115 pHILD2-PaHNL5αV317A (see example 2 and example 5) in a 5 l fermenter for further characterizations. A Muts strain was chosen for culturing.
[0186] Firstly, a preculture comprising 50 ml of YPD medium (10 g/I Bacto™ yeast extract, 20 g/l Bacto™ peptone, 20 g/I D-glucose) was inoculated with a large single colony and incubated at 28°C and 120 rpm for 24 h. Said first preculture was used for inoculating 2 x 200 ml of YPD medium in 2 l flasks with baffles. This was followed by approx. 10 h of incubation at 28°C and 120 rpm.
[0187] All chemicals required for fermentation have been mentioned previously in example 9.
[0188] Chemicals 1-7, amounts calculated for 3.5 liters, were dissolved in deionized water and introduced into a 5 l bioreactor (Biostat®CT from Sartorius). After in situ sterilization, the pH of the medium was adjusted to pH 5.0 with 25% ammonia by
way of sterile addition through a feed pump. This was followed by introducing into the bioreactor 13.5 ml of sterile-filtered trace element solution comprising vitamin H by means of a sterile syringe.
[0189] Approx. 400 ml of preculture from two 2 l shaker flasks were used for inoculation. The starting OD600 in the fermenter was 1.2. With an operating temperature of 28°C, an aeration rate of 2.5-10 liters of air/min and a stirring speed between 500 and 1500 rpm, the partial pressure of oxygen (pO2) was maintained at a value >30% of the saturation concentration. The pH of the culture medium was kept constant at pH 5.0 by sterile addition of 25% ammonia through a feed pump.
[0190] After the initially introduced glycerol had been consumed, after a fermentation time of 15-20 hours, metering-in of the glycerol medium was started, initially at 45 ml/h. The metering-in rate was increased in steps to 90-100 ml/h and continued for approx. 15 hours, until approx. 180 OD600 were reached. Metering-in of glycerol was then stopped.
[0191] The third phase of fermentation was initiated by inducing expression by metering in methanol. The metering-in rate was adjusted initially to 10-15 ml/h. This rate was increased in steps to 45-60 ml/h over 12-15 hours and maintained for another 60 hours.
[0192] After approx. 72 hours of methanol induction, the cells were harvested by two centrifugations at 4000 rpm in a Beckman Coulter™ (Fullerton, Ca, USA) Avanti™ J-20XP centrifuge with the Beckman JLA 8.1000 rotor at 4°C for 20 min. The culture supernatant was collected and the enzyme activity of (rac)-mandelonitrile cleavage was determined. The fermentation supernatant was diluted appropriately with 1 M phosphate-citrate buffer (pH 5.0) and, after addition of the mandelonitrile substrate solution (40 µl (rac)-mandelonitrile + 5 ml 0.1 M phosphate-citrate buffer (pH 3.0)), the absorbance profile at 280 nm was monitored for 5 min. The enzyme activity in the culture supernatant after centrifugation was 62.4 U/ml, resulting in an enzyme yield of approx. 174 700 U, with a total yield of approx. 2.8 l of culture supernatant.
[0193] The culture supernatant was concentrated by means of crossflow ultrafiltration using 30 kDa cutoff modules from Sartorius (VIVASCIENCE Vivaflow 50 from Sartorius, Göttingen, D). In this way a V317A enzyme preparation with a protein concentration of 3.715 mg/ml and an enzyme activity of 316.1 U/ml (standard HNL assay with rac-mandelonitrile) was prepared. The enzyme preparation was divided into 1 ml or 6 ml aliquots and stored at -20°C. Since Pichia pastoris secretes hardly any of
its own proteins into the culture supernatant, the enzyme produced and concentrated in this way was already very pure (see also figure 4).
[0194] Samples were taken at various points in time during fermentation. The cells were removed by centrifugation and the protein concentration of the culture supernatants was determined by means of Biorad (Hercules, Ca) protein assay (Bradford). The standard used for preparing a calibration line was native PaHNL from Sigma (M-6782 Lot 41 H4016).
[0195] Of the individual culture supernatants, 5 µl were applied directly to a gel (protein gel: NuPAGE 4-12% Bis Gel 1.0 mm X 15 well; Invitrogen). Furthermore, in each case 3 µl of the culture supernatants were deglycosylated by endoglycosidase H (Cat. #P0702L, NEB) according to the protocol provided by the supplier, and subsequently applied. The standard used was SeeBlue Plus2 Pre-Stained Standard from Invitrogen (Carlsbad, USA). Protein gels with native and deglycosylated V317A samples at various points in time of the 5 l fermentation are depicted in figures 3 and 4, respectively.
[0196] The enzyme preparations produced in the fermenter and then concentrated are purified chromatographically in a manner similar to example 10.
Example 13: Monomerization of hydroxypivalaldehyde [Comparative Example]
[0197] The compound hydroxypivalaldehyde is a dimer at room temperature:
Equilibrium between hydroxypivalaldehyde monomer (1) and dimer (2)
[0198] For the reaction with HCN, the dimer was monomerized in order to obtain an exposed aldehyde group. The monomerization was carried out in two ways:
A) Thermal monomerization:
[0199] For thermal monomerization, the hydroxypivalaldehyde dimer was heated in undiluted form at approx. 100°C. A secondary product was found to form in the process. The longer the aldehyde was heated at temperatures of approx. 100°C,
the larger was the amount of secondary product formed. This product was produced by the "anomalous Tishchenko reaction" (
): 2,2-dimethyl-1,3-propanediol and 3-hydroxy-2,2-dimethylpropionic acid - compounds produced in a Cannizzaro reaction - fused to give 3-hydroxy-2,2-dimethylpropyl-3-hydroxy-2,2-dimethylpropionic ester (figure 6). All normal Tishchenko reactions (
) require a Lewis acid and demand the absence of water (
). In the present case, non-anhydrous conditions were used, and temperatures of approx. 100°C were sufficient in order to form the fusion product by means of "anomalous Tishchenko reaction" (
). The mixture derivatized with acetic anhydride in the presence of pyridine and dichloromethane was analyzed by means of GC (figure 5) on a cyclodextrin column (CP-Chirasil-Dex CB). It was possible to fractionate the monomer, dimer and the produced ester:
"Anomalous Tishchenko reaction" (
) with hydroxypivalaldehyde (1) to give 3-hydroxy-2,2-dimethylpropyl-3-hydroxy-2,2-dimethylpropionic ester (3)
B) Acid-catalyzed monomerization:
B) Acid-catalvzed monomerization:
[0200] Acid-catalyzed hemiacetal cleavage of the dimer is also possible in order to shift the equilibrium to the monomer's side. Due to good stability of the PaHNL5 variants in acidic medium (
), it was subsequently also possible to carry out enzyme-catalyzed addition of HCN at low pH values.
[0201] In order to test the properties of hydroxypivalaldehyde monomerization at different pH values, in each case 180 mg of liquid hydroxypivalaldehyde were admixed with 5 ml of 30 mM potassium phosphate buffer (pH 2.0-7.0) in 30 ml glass vials and stirred at 250 rpm and room temperature on a Variomag Electronicruhrer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0202] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0203] The results are depicted in figure 8 and table 10. Monomerization of the hydroxypivalaldehyde dimer was shown to progress very slowly. Comparing the proportions of monomer after 28 h, the amount of hydroxypivalaldehyde monomer increased with decreasing pH of the solution (see table 10). After approx. 4 h (see figure 6), a relatively large proportion of monomer is also observed at higher pH values. However, at these pH values stereoselectivity of HCN addition in the presence of R-HNL is only low.
Example 14: Characterization of the V317A mutant produced in a 5 l fermenter, using the substrates hydroxypivalaldehyde and pivalaldehyde [Comparative Example]
[0204] The influence of numerous reaction parameters on selectivity and activity of the V317A mutant was assayed. The V317A enzyme preparation from the 5 l fermentation was used in aqueous medium, in a two-phase system with MTBE and in immobilized form on Celite in an organic system. In the experiments below, one unit corresponds to the amount of enzyme that converts 1 µmol of mandelonitrile to benzaldehyde and HCN at pH 5.0 and room temperature in one minute.
A) Conversion of hydroxypivalaldehyde in 2 M potassium phosphate buffer at different pH values:
[0205] Reaction mixtures of 5 ml each were chosen for hydroxypivalaldehyde conversion at different pH values. 170 units of the concentrated V317A enzyme preparation (85.1 U/mg) were diluted with 2 M potassium phosphate buffer in 30 ml glass vials to give a final volume of 5 ml. For the corresponding blank reactions, the enzyme preparation was replaced with the same volume of deionized water. Reaction mixtures at pH 1.0, pH 1.5, pH 2.0, pH 2.5, pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0 and pH 8.0 were carried out. After addition of the enzyme preparation or deionized water, the buffered solutions were again adjusted to the corresponding pH. This was followed by addition of 120 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 100 µl (= 2.2 equivalents) of liquid HCN were metered in using a syringe. The solution was stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0206] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0207] Table 11 and figure 7 depict the results of conversion (%conv) and enantiomeric excess (%ee) at various points in time.
B) Conversion of hydroxypivalaldehyde in 1 M potassium phosphate buffer at different pH values:
[0208] Reaction mixtures of 5 ml each were chosen for hydroxypivalaldehyde conversion at different pH values. 170 units of the concentrated V317A enzyme preparation (85.1 U/mg) were diluted with 1 M potassium phosphate buffer in 30 ml glass vials to give a final volume of 5 ml. For the corresponding blank reactions, the enzyme preparation was replaced with the same volume of deionized water. Reaction mixtures at pH 1.5, pH 2.0, pH 2.5, pH 3.0, pH 4.0, pH 5.0 and pH 6.0 were carried out. After addition of the enzyme preparation or deionized water, the buffered solutions were again adjusted to the corresponding pH. This was followed by addition of 120 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 100 µl (= 2.2 equivalents) of liquid HCN were metered in using a syringe. The solution was stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0209] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0210] Table 12 and figure 8 depict the results of conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0211] The results of the conversions at different pH values revealed that a pH range from pH 2.0-3.0 is optimally suited to converting hydroxypivalaldehyde to the corresponding (R)-cyanohydrin. While the V317A enzyme does not have sufficient activity below pH 2.0, conversions at above pH 3.0 were adversely affected by the unselective chemical background reaction. This was indicated by decreasing enantiomeric excesses in the enzymatic reactions and by increasing conversions of the purely chemical reactions (blank reactions) with increasing pH.
C) Comparison of different buffer concentrations:
[0212] The potassium phosphate buffer concentrations were varied further. 170 units of the concentrated V317A enzyme preparation (85.1 U/mg) were diluted with 50 mM, 100 mM, 1 M or 2 M potassium phosphate buffer (pH 2.5) in 30 ml glass vials to a final volume of 5 ml. In the blank reaction mixture, the enzyme preparation was replaced with the same volume of deionized water and a buffer concentration of 2 mol/I was chosen. The buffered solutions were adjusted again to pH 2.5 after addition of the enzyme preparation or the deionized water. This was followed by addition of 120 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 100 µl (= 2.2 equivalents) of liquid HCN were metered in using a syringe. The solutions were stirred at 600 rpm on a Variomag-Electronicrohrer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0213] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0214] Table 13 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0215] When comparing conversion after 2 hours, a distinct difference was identified. The lower the buffer concentration, the lower the resulting conversion values. The enantiomeric excess remained roughly constant. With increasing reaction time, said difference disappeared almost completely. With a buffer concentration of 2 mol/l, 99.1% conversion was achieved after 22 h, while 98.7% conversion was measured with a buffer concentration of 50 mmol/l. The improved conversion with higher buffer concentrations can be explained by the fact that monomerization of the hydroxypivalaldehyde substrate proceeds faster with higher buffer concentrations, thereby providing a larger proportion of monomer for addition of HCN.
D) Variation in the amount of enzyme used:
[0216] Three different amounts of enzyme were compared: 56 units, 112 units or 316 units of the concentrated V317A enzyme preparation were diluted with 3 M citrate-phosphate buffer (pH 2.5) in 30 ml glass vials to a final volume of 5 ml. In the blank reaction, the enzyme preparation was replaced with the same volume of deionized water. The buffered solutions were adjusted again to pH 2.5 after addition of the enzyme preparation or the deionized water. This was followed by addition of 120 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 100 µl (= 2.2 equivalents) of liquid HCN were metered in using a syringe. The solutions were stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0217] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0218] Table 15 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0219] Hydroxypivalaldehyde was converted directly to the corresponding (R)-cyanohydrin with >94% enantiomeric excess and >93% conversion even within 5 h. An increase in the amount of enzyme resulted in improved enantiomeric excesses and conversions.
E) Variation in the amount of hydroxypivalaldehyde used:
[0220] 170 units of the concentrated V317A enzyme preparation were diluted with 2 M potassium phosphate buffer (pH 2.5) in 30 ml glass vials to a final volume of 5 ml. The buffered solutions were adjusted to pH 2.5 after addition of the enzyme preparation. This was followed by adding 13 mg, 56 mg or 120 mg liquid hydroxypivalaldehyde, corresponding to final concentrations of 25 mM, 110 mM and 235 mM hydroxypivalaldehyde, respectively. The reaction mixtures were cooled at 4°C, and 100 µl of liquid HCN were metered in using a syringe. The solutions were stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0221] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0222] Table 15 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0223] Conversion and enantiomeric excess were improved further by reducing the initial amount of hydroxypivalaldehyde. With an initial amount of hydroxypivalaldehyde of 13 mg, 97.0%ee and 100% conversion were achieved after 6 h. 97.5% enantiomeric excess and 100% conversion were achieved after 22 h.
F) Variation in the amount of HCN used:
[0224] 170 units of the concentrated V317A enzyme preparation were diluted with 1 M potassium phosphate buffer (pH 2.5) in 30 ml glass vials to a final volume of 5 ml. In the blank reaction mixture, the enzyme preparation was replaced with the same volume of deionized water. The buffered solutions were adjusted to pH 2.5 after addition of the enzyme preparation or the deionized water. This was followed by addition of 120 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 70 µl (1.5 equivalents), 90 µl (2.0 equivalents), 110 µl (2.5 equivalents), 140 µl (3.0 equivalents), 160 µl (3.5 equivalents) or 180 µl (4.0 equivalents) of liquid HCN were metered in using a syringe. The solutions were stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0225] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0226] Table 16 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0227] The results with different HCN amounts revealed only slight differences in enantiomeric excess. The increase in conversion with progressing reaction time was more clearly visible. Except conversion with 3.0 equivalents of HCN, it was possible to correlate a slight improvement in conversion with an initial elevated amount of HCN. Furthermore, a slight decrease in enantiomeric excess with an initial elevated amount of HCN was observed.
G) Conversion of hydroxypivalaldehyde in the two-phase system and comparison with a purely aqueous solvent system:
[0228] For the reaction mixtures with a purely aqueous system, 2 mg of the concentrated V317A enzyme preparation (= 170 units) or 2 mg of PaHNL5 (
) (= 852 units) were diluted with 50 mM phosphate buffer (pH 3.4 and pH 5.1, respectively) in 30 ml glass vials to a final volume of 5 ml. After addition of the enzyme preparation, the buffered solutions were again adjusted to pH 3.4 and pH 5.1, respectively. This was followed by addition of 180 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 100 µl of liquid HCN were metered in using a syringe. The solutions were stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0229] For the reaction mixtures with two-phase system, 2 mg of the concentrated V317A enzyme preparation (= 170 units) or 2 mg of PaHNL5 (
) (= 852 units) were diluted with 50 mM phosphate buffer (pH 3.4 and pH 5:1, respectively) in 30 ml glass vials to a final volume of 2.5 ml. After addition of the enzyme preparation, the buffered solutions were again adjusted to pH 3.4 and pH 5.1, respectively. This was followed by addition of 2.5 ml of methyl tert-butyl ether (MTBE) and 180 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were cooled at 4°C, and 100 µl of liquid HCN were metered in using a syringe. The solutions were stirred at 600 rpm on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D).
[0230] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0231] Table 17 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0232] The V317A mutant was shown to convert the hydroxypivalaldehyde substrate to the corresponding (R)-cyanohydrin more rapidly and with higher enantioselectivity than the PaHNL5 wild type, both in the purely aqueous solution and in the two-phase system (H2O/MTBE). This again demonstrated that an increase in pH results in a reduction of enantiomeric excess. The results further indicate that a purely aqueous reaction system is to be preferred over a two-phase system (H2O/MTBE) for conversion of hydroxypivalaldehyde. The two-phase system achieved smaller enantiomeric excesses and lower conversions.
®
and conversion of hydroxypival-aldehyde in the microaqueous, organic solvent system[0233] 2 g of Celite® 545 were introduced to a 50 ml glass beaker and stirred with 20 ml of 50 mM phosphate buffer (pH 7.0) on a magnetic stirrer at room temperature for 45 min. The Celite® was then filtered off and washed thoroughly with deionized water. 50 ml round-bottomed flasks were charged in each case with 140 mg of washed Celite® to which 8.6 ml of 30 mM potassium phosphate buffer (pH 3.4) were added. Furthermore, 1.41 mg of the concentrated V317A enzyme preparation (= 120 units) or 1.41 mg of PaHNL5 (
) (= 600 units) were added. The mixtures were mixed well and frozen in liquid N2 on a Rotavapor (BÜCHI Labortechnik AG, Flawil, CH) and lyophilized at 0.040 mbar and -50°C for 14 h 30 min.
[0234] The immobilized substances were transferred to 10 ml round-bottomed flasks and admixed with 2970 *l of diisopropyl ether (DIPE), 24.8 µl of 30 mM potassium phosphate buffer (pH 3.4) and 120 mg of briefly heated, liquid hydroxypivalaldehyde. The reaction mixtures were stirred at room temperature on a Heidolph Instruments (D) MR 3001 magnetic stirrer and started by adding 100 µl of liquid HCN.
[0235] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0236] Table 18 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0237] When immobilized on Celite® in the microaqueous, organic solvent system, both the V317A mutant and the PaHNL5 wild type showed comparable enantioselectivities. After 20 hours, an enantiomeric excess of approx. 97% was achieved. However, a disadvantage of this reaction system is the slow monomerization of the hydroxypivalaldehyde dimer in the organic solvent, DIPE, and the moderate conversions resulting therefrom.
I) Preparative conversion of 1.2 g of hydroxypivalaldehyde with V317A mutein in a 100 ml reactor
[0238] In a 100 ml Schmizo reactor with KPG stirrer (Schmizo AG, Zofingen, CH), 3 mg of the concentrated V317A enzyme preparation (= 255 units) were diluted with 2 M potassium phosphate buffer (pH 2.5) to a final volume of 50 ml. The pH was checked and adjusted again to pH 2.5. This was followed by adding 1.2 g of briefly heated, liquid hydroxypivalaldehyde (final concentration: 235 mM). Thus only 0.255 mg of enzyme per mmol of hydroxypivalaldehyde was used. The reaction mixture was cooled at 4°C and stirred vigorously. Finally, the reaction was started by adding 1 ml of liquid HCN.
[0239] Workup commenced after a reaction time of 20 h. The reaction solution was discharged from the reactor and extracted three times with methyl tert-butyl ether (MTBE). The organic phases were combined, dried over Na2SO4 and concentrated on a rotary evaporator.
[0240] After derivatization with acetic anhydride in the presence of pyridine and dichloromethane, analysis was carried out by means of GC on a cyclodextrin column (CP-Chirasil-DEX CB).
[0241] The yield of the reaction after 20 h was 88% with 96%ee.
J) Conversion of pivalaldehyde to (R)-2-hydroxy-3,3-dimethylbutyronitrile with V317A or PaHNL5
[0242] 2 mg of the concentrated V317A enzyme preparation (= 170 units) or 2 mg of PaHNL5 (
) (= 852 units) were diluted with 30 mM phosphate buffer (pH 3.4) in 30 ml glass vials to a final volume of 5 ml. After addition of the enzyme preparation, the buffered solutions were again adjusted to pH 3.4. This was followed by addition of 200 µl of pivalaldehyde. The reaction mixtures were cooled at 4°C and stirred at 600 rpm for 10 min. Then 100 µl of liquid HCN were metered in using a syringe. The solutions were stirred on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D) at 600 rpm during the reaction.
[0243] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0244] Table 19 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0245] With the V317A mutein, quantitative conversion with 95.7%ee was found even after a reaction time of 2 h.
Example 15: Conversion of nivalaidehyde to (R)-2-hydroxy-3,3-dimethylbutyronitrile with V317A and PaHNL5 [Comparative Example]
[0246] A sufficient amount of enzyme was prepared from the clones Pichia pastoris GS115 pHILD2-PaHNL5αV317A or Pichia pastoris GS115 pHILD2-PaHNL5
(
) in 5 l bioreactors for further characterizations. The culture supernatant was concentrated in each case approx. 6-fold by means of Crossflow Ultrafiltration using 30 kDa cutoff modules from Sartorius (VIVASCIENCE Vivaflow 50 from Sartorius, Göttingen, D).
[0247] In 30 ml glass vials, 2 mg, 3 mg or 4 mg of the concentrated V317A enzyme preparation (= 170 units, 256 units and 341 units, respectively) or 2 mg, 3 mg or 4 mg of the concentrated PaHNL5 enzyme preparation (= 545 units, 817 units and 1089 units, respectively) were diluted with 1 M phosphate buffer (pH 3.0) to a final volume of 5 ml. The buffered solutions were again adjusted to pH 3.0 after addition of the enzyme preparation. This was followed by addition of 200 µl of pivalaldehyde (Sigma-Aldrich, T71501). The reaction mixtures were cooled at 9°C. 100 µl of liquid HCN were metered in using a syringe. The solutions were stirred on a Variomag Electronicrührer Poly 15 magnetic stirrer from H+P Labortechnik (Oberschleißheim, D) at 600 rpm during the reaction.
[0248] Samples were taken at various points in time, derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by means of GC on a cyclodextrin column (CP-Chirasil-Dex CB).
[0249] Table 1 depicts the results for conversion (%conv) and enantiomeric excess (%ee) at various points in time.
[0250] With the V317A mutein, quantitative conversion with 97.5%ee was found even after a reaction time of 1 h.
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@misc
{
Glieder_Liu_Pscheidt_Gaissberger_2014,
title={R-HNL RANDOM VARIANTS AND THEIR USE FOR PREPARING OPTICALLY PURE, STERICALLY HINDERED CYANOHYDRINS},
author={Glieder, Anton and Liu, Zhibin and Pscheidt, Beate and Gaissberger, Richard},
year={2014},
month={Jan},
}
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Glieder, Anton and Liu, Zhibin and Pscheidt, Beate and Gaissberger, Richard. R-HNL RANDOM VARIANTS AND THEIR USE FOR PREPARING OPTICALLY PURE, STERICALLY HINDERED CYANOHYDRINS. EP Patent 07857382.1. Jan 22, 2014.
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Glieder, Anton and Liu, Zhibin and Pscheidt, Beate and Gaissberger, Richard. (2014). EP. Patent No.07857382.1
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Glieder, Anton and Liu, Zhibin and Pscheidt, Beate and Gaissberger, Richard. 2014. R-HNL RANDOM VARIANTS AND THEIR USE FOR PREPARING OPTICALLY PURE, STERICALLY HINDERED CYANOHYDRINS. EP Patent EP2092060B1, filed Dec 11, 2007, and issued Jan 22, 2014.
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