AU2003304117B2 - Extracting and purifying enzymes - Google Patents

Description

WO 2004/101613 PCT/AU2003/001618 1 Extracting and purifying enzymes Field of the invention The invention relates to extracting and purifying an enzyme from a cell, particularly, but not exclusively, to extracting and purifying a 5' phosphodiesterase from 5 a barley cell. Background of the invention Phosphodiesterase 1 (orthophosphoric diester phosphohydrolase, EC 3.1.4.1; herein "PDE-1") is an enzyme that catalyses the hydrolysis of a phosphodiester bond at the 3' hydroxyl end of ribopolynucleotide to yield a 5' ribonucleotide. 10 PDE-1 is particularly important to food and pharmaceutical industries because the 5' ribonucleotides produced by PDE-1 -mediated cleavage of yeast RNA are useful as flavour enhancers. Barley cells or rootlets are used in the food industry as a source of PDE-1. The processes for purification of PDE-1 from barley rootlets tend to be difficult to 15 operate on a commercial scale, in terms of requiring sophisticated extraction and separation techniques, multiple steps and expensive reagents and equipment. Some processes are characterised by an unacceptable loss or wastage of PDE-1. Other processes tend to produce a final product that has a sub-optimal specific activity. In view of the above, there is a need for improved processes for purification of 20 PDE-1. Summary of the invention The invention seeks to at least minimise one or more of the above identified problems or limitations and/or to provide an improved process for purification of PDE- 1. In one aspect, the invention provides a process for purifying PDE-1 from a cell. 25 The process includes the step of heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of PDE-1 in the extract.

WO 2004/101613 PCT/AU2003/001618 2 In another aspect, the invention provides a process for purifying PDE-l from a barley cell. The process includes the following steps; (a) releasing PDE-1 from the cell into a solution including calcium and magnesium to form an extract; and 5 (b) heating the extract to increase the specific activity of PDE-1 in the extract. In another aspect, the invention provides a process for purifying PDE-1 from a barley cell. The process includes the following steps: (a) releasing PDE-1 from the cell into a solution including calcium and magnesium to form an extract; 10 (b) heating the extract to increase the specific activity of PDE-1 in the extract; and (c) utilising anion exchange chromatography to purify PDE-1 from the heated extract. Typically the cell is a barley cell, such as a cell derived from a barley rootlet. 15 In another aspect, the invention provides PDE-1 produced by the process of the invention. In another aspect, the invention provides a cell including PDE-l produced by the process of the invention. In another aspect, the invention provides a process for producing a ribonucleotide. 20 The process includes the step of contacting a polyribonucleotide with PDE-1 produced by the process of the invention, to produce the 5' ribonucleotide. In another aspect, the invention provides a ribonucleotide produced by the above described process. Detailed description of the embodiments 25 As described herein, the inventor has found that heat treatment of an extract of a barley cell or rootlet in a solution comprising at least one divalent cation permits the specific activity of the extract to be increased. For example, the specific activity of a heat treated extract of a barley rootlet formed from a solution comprising calcium and WO 2004/101613 PCT/AU2003/001618 3 magnesium was observed to increase 2.6 fold over a non heat treated sample (312.6 x10 3 compared with 122 x 10-3 pmoles/min/mL). Further, a heat treated extract containing calcium and magnesium was observed to have an improved specific activity (78 x 10 tmoles/min/mL) compared with a heat treated extract containing no calcium and 5 magnesium (46 x 10- 3 pimoles/min/mL). This is a significant finding because it permits heat treatment, a purification step that is relatively simple to operate on a commercial scale, to be implemented with minimal loss of activity of PDE-1. It is believed that the specific activity of the extract is increased because the 10 divalent cation protects PDE-1 from denaturation at temperatures at which other proteins in the extract, including phosphomonoesterase, are degraded. Typically, the at least one divalent cation in the solution may be magnesium and/or calcium. For example, the solution may contain MgCl 2 and/or CaC1 2 . The magnesium and calcium ions may be included in the extract in an amount to 15 permit control of the denaturation of PDE-1 when the extract is heated. Typically, magnesium and calcium are included in the extract in an amount to at least limit the denaturation of PDE-1 when the extract is heated. For example, the concentration of calcium may be less than 100 mM and the concentration of magnesium may be less than 100 mM. 20 A concentration of calcium and magnesium in a range between about 10 to 50 mM is particularly useful as further down stream processing of the extract for further purification, such as anion exchange chromatography, may require removal of calcium and magnesium. Accordingly a concentration of calcium ions of about 50 mM and a concentration of magnesium ions of about 50 mM is particularly useful. 25 It is believed that phosphomonoesterase is a major constituent of an extract of a barley rootlet. Thus, the extract is typically heated to a temperature that permits denaturation of phosphomonoesterase, or otherwise, destruction of phosphomonoesterase activity in the extract. As described herein, temperatures less than 80C are suitable for this purpose.

WO 2004/101613 PCT/AU2003/001618 4 It is particularly advantageous to heat the extract to between about 45 and 75'C because at temperatures approaching 70'C and above, PDE-1 activity may be lost. Accordingly, a temperature of about 60 0 C is particularly useful. The inventor has also found that the purification of PDE-1 from a barley cell 5 extract can be improved by extracting a barley cell homogenate at 4'C in a solution including magnesium and calcium. Specifically, as described herein, the specific activity of an extract comprising calcium and magnesium after maintenance at 4'C was found to be 92 x 10 3 pmoles/min/imL as compared with the activity of an extract maintained at 4 0 C in the absence of calcium and magnesium (75 x 103 pmoles/min/mL) . 10 It is believed that maintenance of such an extract at 4'C is important because it permits PDE-1 to disassociate from solids in the extract, and accordingly, to solubilise into the liquid phase of the extract, prior to further processing of the extract, such as a heat treatment step or a chromatographic separation step. The calcium and magnesium are believed to be important for limiting hydrolysis of the enzyme during the maintenance 15 of the extract at 4'C. Thus in accordance with the invention, a process for purifying PDE-l from a barley cell includes the following steps: (a) releasing PDE-1 from the cell into a solution including calcium and magnesium to form an extract; and 20 (b) heating the extract to increase the specific activity of PDE-l in the extract. Typically, the extract is maintained in conditions for promoting solubilisation of the phosphodiesterase in the extract prior to heating the extract. The extract may be maintained at less than 10'C for less than 5 days. For example, the extract may be maintained between 0 to about 4'C for between about 1 to 25 48 hours. It is particularly advantageous to maintain the extract for 24 hours at 4'C as this improves the speed of purification protocols that comprise further purification steps. The inventor has further found PDE-l can be purified to virtual homogeneity from a barley cell extract by a process including the following steps: WO 2004/101613 PCT/AU2003/001618 5 (a) releasing PDE-1 from the cell into a solution including calcium and magnesium to form an extract; (b) heating the extract to increase the specific activity of PDE-1 in the extract; and 5 (c) utilising chromatography to purify PDE-1 from the heated extract. As described herein, PDE-l can be further purified from a heat treated barley cell extract by anion exchange chromatography. Accordingly, typically, in step (c), anion exchange chromatography is utilised to purify PDE-1 from the heated extract. The inventor has found that calcium and magnesium ions tend to limit binding of 10 PDE-1 during anion exchange chromatography. Accordingly, typically the extract is desalted before anion exchange chromatography. One way of desalting to remove magnesium and calcium ions is by ultrafiltration. Alternatively, a preparative de-salting column, such as a Hi trap 26/10 desalting column can be used. It is particularly advantageous to remove substantially all of the calcium and magnesium from the extract 15 prior to anion exchange chromatography for the purpose of maximising the yield of PDE 1 purified from the anion exchange column. Typically, the extract is maintained in conditions for promoting solubilisation of the phosphodiesterase in the extract prior to heating the extract. In the processes of the invention described above, the extract of the barley cell is 20 typically produced by homogenising barley rootlets in an appropriate buffer. One way of homogenizing rootlets is by use of a blender, such as a Waring blender. Alternatively, the extract may be produced by milling barley rootlets in an appropriate buffer. The solution into which the PDE-1 from the cell is released to form an extract is typically a buffer for controlling pH. Solutions prepared from Trisma base are examples 25 of such a solution. A solution having a concentration of no more than about 100mM Tris is suitable, however a solution having a concentration between about 20 to 50 mM Tris, for example, 25 mM Tris is particularly advantageous because as described herein, this concentration of Tris is compatible with, and permits separation of PDE-1 on, an anion exchange column.

WO 2004/101613 PCT/AU2003/001618 6 It will be understood that the processes of the invention are useful for purifying PDE-1 from cells other than barley cells. Further, it will be understood that processes of the invention are useful for isolating barley PDE-1 from cells that contain a recombinant nucleic acid molecule that encodes barley PDE-l. Examples of such cells include 5 bacterial cells and yeast cells. In another aspect, the invention provides a process for producing a ribonucleotide. The process includes the step of contacting a polyribonucleotide with PDE-1 produced by the process of the invention, to produce the ribonucleotide. Preferably the polyribonucleotide is derived from yeast RNA, such as yeast ribosomal RNA. 10 Example 1: Materials and equipment. Germinating barley seeds (Schooner variety) were obtained from Barrett Burston Malting, (Thornleigh, NSW, Australia). Thymidine 5' monophosphate p-nitrophenyl ester, Trisma base, MgCl 2 , CaCl 2 , p-nitrophenyl-phosphate, Baker's yeast RNA, bovine serum albumin, guanosine 5'-monophosphate, potassium dihydrogen orthophosphate 15 were all supplied by Sigma Aldrich (Castle Hill, NSW Australia). Yeast rRNA was prepared as a 3mg/mL standard solution in RNAse free. Milli Q water (pH7.0) in a clean room environment and inunediately frozen to minus 80'C and stored until required. Guanosine 5'-monophosphate was prepared as a 1% (w/v) stock by dissolving in 20 Milli Q water and storing at -20'C. Working solutions were prepared by diluting the stock solution to 0.01% (w/v). The buffer used for FPLC was 25mM Tris HCl (pH 8.9). The fluent buffer included 1 M NaCl. For the HPLC analysis, solvent (A) was 0.02M potassium dihydrogen 25 orthophosphate (pH 6.5) solution while solvent (B) was a 40% methanol (HPLC grade) and 60% Milli Q water mixture (pH 6.5). All solvents and buffers were degassed and filtered prior to use by passing each solution through a Millipore membrane filter, pore size 0.45 pm under vacuum for 5 minutes.

WO 2004/101613 PCT/AU2003/001618 7 An Amersham Pharmacia AKAT gradient processing FPLC system complete with a 900 model monitor, lamp and detector (set at 280nm), 920 model pump and Frac 950 fraction collector interfaced to a Compaq Deskpro Pentium III computer supporting Unicorn analytical software was used for all protein purification. The columns used 5 included a Hi Trap 26/10 desalting column connected to a Super loop 50 (to facilitate larger injection volumes), a 16/10 Hi-Load Q anion exchange column with a final purification undertaken on a Mono Q HR 5/5 column. The HPLC system used for the analysis of 5' nucleotides was a Shimadzu Liquid Chromatography unit, consisting of a Shimadzu pump (model LC-1OAi) with an on-line 10 degassing unit, SCL-1OA system controller, SPD-M1OA diode array detector and a column oven (set at 30'C), the system was controlled by a Dell Pentium III computer operating a Shimadzu class VP (version 5.03) analytical software program, The column used for the analysis of 5' ribonucleotides was an Activon Goldpak HPLC column (250mm x 4.6mm, 5mm particle size and 1 Onm pore size). Peak heights were determined 15 from chromatograms recorded at both 245 nm and 280 nm. A Pharmacia LKB UV-Visible spectrophotometer was also used to measure enzyme activity and was set at 410 nm with an aligned temperature control cell all controlled by a HP Pentium II computer operating a Biochrome 4060 enzyme kinetics software program. 20 Example 2: Preparation of a standard curve for p-nitrophenol to determine PDE-1 activity. A solution of 10 mg/100 mL of p-nitrophenol was prepared in 50mM Tris HC1 (pH 8.9) to yield a concentration of 0.719 ptmoles/ml of p-nitrophenol. Dilutions were prepared in 50mM Tris HCl (pH8.9) and read as a 4.1 mL sample size at 410 nm to 25 directly correlate with the enzyme activity absorbances at each purification step. Example 3: Preparation of a standard curve for protein to determine PDE-1 specific activity. Protein was determined using the BioRad micro assay procedure derived from the original method of Bradford utilising a standard curve produced for bovine serum WO 2004/101613 PCT/AU2003/001618 8 albumin. Each analysis was conducted in duplicate requiring incubation at room temperature for 10 minutes with the absorbance measured at 595 nm. Standards were prepared in the range of 0.2 to 1.4 mg/mL of protein. Example 4: Enzyme kinetics assay. 5 The assay solution consisted of 0.1 mL of purified PDE-1, 2.0 mL of 50mM Tris HCl (pH 8.9), lmL of lmM thymidine 5' monophosphate p-nitrophenyl ester in 50mM Tris HCl (pH 8.9) and lmL of a 50mM MgC1 2 in 50mM Tris HC1 (pH 8.9). The reaction mixture was incubated at 37'C for 30 minutes with the optical density of enzymatically produced p-nitrophenol measured at 410nm each minute to determine the PDE-1 reaction 10 kinetics. The activity was determined by extrapolation against a standard curve with 1 unit of activity defined as the micromoles ofp-nitrophenol produced from p-nitrophenyl ester per minute per mL of purified PDE-1. Specific activity was defined as the activity per milligram of purified PDE-1. 15 Example 5: Confirmation of PDE-1 activity Confirmation of enzyme activity was undertaken by reaction of the purified enzyme with 3mg/mL Baker's yeast RNA and measuring the 5'ribonucleotide products by reverse phase HPLC. The HPLC column was equilibrated with 100% buffer A for 30 minutes at a flow 20 rate of 1 ml.min ensuring the pressure remained below 180 Kpa. The ratio of solvent B in the elution system was increased linearly over 18 minutes to 40% then increased to 100% over the next 2 minutes and held for a further 2 minutes to flush any remaining strongly bound compounds. The system was automatically returned to its starting conditions over the following 8 minutes and stabilised on buffer A for a further 30 minutes before the 25 next injection. The total analysis time was 60 minutes with elution performed at 30C oven temperature. Example 6: Preparation of crude PDE-1 extract. lOg of fresh barley rootlets were obtained from seeds that had been germinating for 5 days including a 2 day steep in 5mg/kg gibberellic acid to facilitate enzyme WO 2004/101613 PCT/AU2003/001618 9 extraction. The rootlets were dispersed in l0OmL 25 mM Tris-HCl (pH7.5) containing 50mM MgCl 2 and 50mM CaCl 2 . This mixture was homogenised in a Waring blender at high speed for 1 minute and maintained at 4C for 24 hours to facilitate solubilisation of PDE-1. 5 The insoluble material was removed from the extract by filtering through double cheese cloth. The filtrate was then centrifuged at 15,000 rpm for 30 minutes at 4 0 C to remove solids and the supernatant was passed through a 0.45pLM filter and stored at 4 0 C in a sterile container with 0.01% sodium azide. This process formed the crude PDE-1 extract. 10 The activity and specific activity of the crude PDE-l extract was then determined according to Examples 2 to 4 above. Example 7: Purification of PDE-1 from the crude PDE-1 extract. The first stage of the purification process involved the removal of heat labile enzymes (primarily Acid Phosphatase [Phosphomonoesterase (PME)] EC 3.1.3.2) and 15 inhibitory proteins from the crude PDE-1 extract with the aim of reducing any loss of activity or damage to the structure of PDE-1. To inactivate PME, the crude extract was heated in a water bath to 60'C and maintained at that temperature for 1 hour. The extract was then cooled to room temperature and the pH adjusted to 8.9. The extract was then filtered through a 0.45pm filter. 20 The activity and specific activity of the heat treated PDE-1 extract was then determined according to Examples 2 to 4 above. Ion exchange chromatography was then undertaken. A 100mL sample of the extract was injected into a Super loop 50 column and desalted by FPLC on a Hi trap 26/10 desalting column at a flow rate of 7.0 mL per minute, to remove magnesium and 25 calcium. The desalted fractions were then pooled and reloaded onto the Super loop column and passed through a Hi load 16/10 anion exchange column at 3.0 mL per minute to initially fractionate PDE-1. The isolated fraction was again desalted to remove the 1 M NaCl elution buffer and was then purified by passing the fraction through the Mono Q WO 2004/101613 PCT/AU2003/001618 10 HR 5/5 column at 1.5 mL per minute. A single peak was obtained and analysed for activity and specific activity according to Examples 2 to 4 above. Example 8: Purification profile for PDE-1. The results for the purification of PDE- 1 are shown in Table 1. 5 Table I Sample Activity Specific activity Purification factor Crude extract 169 122 1 Heat treatment 156.3 312.6 2.6 Anion 267.5 1407.9 11.5 exchange Activity: x 10-3 pmoles/min/mL Specific activity: x 10-3 pmoles/min/mL/mg Example 9: Effect of calcium and magnesium on PDE-1 activity of crude extract during solubilisation at 4 0 C. 10 We sought to determine whether calcium and magnesium would have an effect on stabilisation of PDE-1 in the crude extract, or otherwise, on preserving or enhancing PDE-1 activity of the crude extract, during the step of solubilising PDE-1 at 4'C that follows the homogenisation step described in Example 6. To this end we maintained the homogenised extract at 4'C in 25mM Tris HC1 15 (pH8.9): (i) in the absence of calcium and magnesium; (ii) with 5mM MgCl 2 and 5mM CaC1 2 ; and (iii) with 50mM MgCl 2 and 50mM CaC1 2 . We found that calcium and magnesium enhanced and indeed stabilised PDE-1. At 50mM MgCl 2 and 50mM CaCl 2 , the PDE-1 activity was 22% greater than in the sample with no magnesium or calcium (92 x 103 pmoles/min/mL compared to 75 x 10 3 20 pmoles/min/mL).

WO 2004/101613 PCT/AU2003/001618 11 Example 10. Effect of calcium and magnesium on PDE-1 activity of crude extract during heat treatment. We sought to determine whether calcium and magnesium would have an effect on stabilisation of PDE- 1 in the crude extract or otherwise on preserving or enhancing PDE 5 1 activity of the crdue extract, during the step of heating PDE- 1 as described in Example 7. To this end, we incubated the crude extract at 60'C for 1 hour in 25mM Tris HCl (pH8.9): (i) in the absence of calcium and magnesium; (ii) with 5mM MgCl 2 and 5mI4 CaCl 2 ; and (iii) with 50mM MgCl 2 and 50mM CaCl 2 . 10 We found that after heating, the activity dropped significantly in samples without magnesium and calcium and in samples with 5mM MgCl 2 and 5mM CaCl 2 by over 40% compared with samples with 50mM MgCl 2 and 50mM CaCl 2 (46 x10 3 pmoles/min/mL and 48 x10 3 ptmoles/min/mL compared with 78 x10 3 tmoles/min/mL).

Claims (18)

1. A process for purifying a phosphodiesterase 1 (PDE-1) from a cell including heating an extract of a cell formed from a solution including divalent cations magnesium and calcium to increase the specific activity of PDE- 1 in the extract. 5

2. A process according to claim I wherein the concentration of the divalent cations is less than 100mM.

3. A process according to claim 2 wherein the concentration of the divalent cations is about 10 to 50 mM.

4. A process according to claim 3 wherein the concentration of the divalent cations is about 50 10 mM.

5. A process according to any one of claims 1-4 wherein the extract is heated to a temperature that permits depletion of phosphomonoesterase activity from the extract.

6. A process according to claim 5 wherein the extract is heated to less than 80'C.

7. A process according to claim 6 wherein the extract is heated to between about 45 and 75*C. 15

8. A process according to claim 7 wherein the extract is heated to about 60*C.

9. A process for purifying PDE-1 from a barley cell including: releasing PDE-1 from the cell into a solution including calcium and magnesium to form an extract; and heating the extract to increase the specific activity of PDE- 1 in the extract.

10. A process according to claim 9 wherein the extract is maintained in conditions for promoting 20 solubilisation of the phosphodiesterase in the extract prior to heating the extract.

11. A process according to claim 10 wherein the extract is maintained at less than 10*C.

12. A process according to claim 11 wherein the extract is maintained at between 0 to about 4C. 13

13. A process according to any one of claims 9-12 comprising the further step of utilising chromatography to purify PDE-1 from the heated extract.

14. A process according to claim 13 wherein anion exchange chromatography is utilised to purify PDE-1 from the heated extract. 5

15. A PDE-1 purified by a process according to any one of the preceding claims.

16. A cell including PDE-1 wherein the PDE-1 is produced by a process according to any one of claims 1-14.

17. A process for producing a ribonucleotide including contacting a polyribonucleotide with a PDE- 1 according to claim 15 to produce the ribonucleotide. 10

18. A ribonucleotide produced by a process according to claim 17.