AU700398B2 - Phenylalanine dehydrogenase production - Google Patents

Description

Purification of Phenylalanine Dehydrogenase This invention relates to a method of purification of phenylalanine dehydrogenase.

Phenylalanine dehydrogenase catalyses the reversible conversion of L-phenylalanine to phenylpyruvate, with the concomitant reduction of NAD+ to NADH, as shown by the equation; L-phenylalanine H20 NAD+ Phenylpyruvate

NH

3 H+ NADH The enzyme has been demonstrated in a number of microorganisms, including; Brevibacterium sp. (Hummel W. et al. (1984). Arch. Microbiol., 137, 47-52), Rhodococcus sp. M4 (Hummel W. et al. (1986). Appl. Microbiol. Biotechnol.; 25, 175- 185), Sporosarcina ureae (Asano Y. and Nakazawa A. (1985). Agric. Biol. Chem., 49, 3631-3632), Bacillus sphaericus (Asana Y. et al. (1987). J. Biol. Chem., 262, 10346- 10354), Bacillus badius (Asano Y. et al. (1987). Eur. J. Biochem., 168, 153-159), Rhodococcus maris K-18 (Misano H. et al. (1989). J. Bacteriol., 171, 30-36), Corynebacterium equi. EVA 5 (Evans C. T. et al. (1987). Biotechnology, 5, 818-823), Micrococcus luteus (Matsunaga T. et al. (1987). Appl. Microbiol. Biotechnol., 27, 11- 14), Nocardia sp. 239 (de Boer L. and Dijkhuizen L. (1988). Arch. Microbiol., 149, 459-465) and Thermoactinomyces intermedius (Oshima T. et al. (1991). J. Bacteriol., S 20 173, 3943-3948).

Phenylalanine dehydrogenase can be used for the synthesis of L-phenylalanine by the reductive amination of phenylpyruvic acid in the presence of ammonium ions and the reductive amination of several other a-ketocarboxylic acids to their corresponding aminocarboxylic acids as disclosed by US 4590161. It can also be used to monitor levels of phenylalanine in biological fluids such as blood or sera (eg. Hummel W. et al. (1988) Anal. Biochem., 170, 397-401., Oshima T. et al. (1988). Anal. Letters, 21, 2205-2215., Wendel U. et al. (1990). Clin. Chim. Acta., 192, 165-170, Cooper A. et al. (1989).

Analyt. Biochem., 183, 210-214 and Campbell R. S. et al. (1992). Clin. Chim. Acta. 210 pp 197-210. The quantification of serum phenylalalanine is important in the diagnosis 30 and treatment monitoring of phenylketonuria (PKU) and hyperphenylalaninaemia

(HPA).

Classical PKU arises from an autosomal recessive disorder which results in a deficiency or absence of the hepatic enzyme, phenylalanine hydroxylase 1.14.16.1).

Screening programmes for the detection of elevated serum phenylalanine levels in neonates permit such cases of HPA to be treated through dietary control. The condition and its causes is reviewed by Chang et al. (1991., J. Int. Fed. Clin. Chem., 3, 58-65).

The strain, originally called Streptomyces sp. 239 (and containing the phenylalanine dehydrogenase described herein) was first described by Kato et al. in 1974. The isolate was later renamed Nocardia sp. 239. The phenylalanine dehydrogenase from this strain was first described by de Boer et al. (1988) Arch. Microbiol., 149, 459-465.

[N:\LIBH]00048:RRB The enzyme has previously been extracted and purified (de Boer et al. (1989).

Arch. Microbiol., 153, 12-18).

The growth medium used by de Boer for the cultivation of Nocardia sp. 239 was as follows:- Component g/L

K

2

HPO

4

(NH

4 2

SO

4 MgSO 4 .7H 2 0 0.2 L-phenylalanine 1.65 plus trace salts solution.

The above medium was sterilised prior to inoculation by autoclaving at 121 0 C, psi for 20 minutes and was adjusted to pH 7.0. However, using this medium de Boer was only able to obtain rather low yields of phenylalanine dehydrogenase. It was also found that yield would not increase as the L-phenylalanine content was increased above an initial concentration of about 1 mM, and that above 2% yield actually decreased.

The object of the invention is to provide an improved method of purification of phenylalanine dehydrogenase.

De Boer describes purification of the enzyme using anion exchange chromatography and reports, in a single step process, an apparent 110-fold purification of crude enzyme 15 product with 95% recovery. While the present invention also relates to enzyme activity and purification, direct comparison of improvements in the present invention over the prior published data by de Boer are extremely difficult because: De Boer's enzyme activity is based on measurement of the reductive amination of phenylpyruvate to phenylalanine, measured at pH 7.8 and 37°C. In the present 20 invention, enzyme activity is based on the oxidative deamination of phenylalanine to phenylpyruvate the reverse reaction), measured at pH 10.8 and 25 0 C, and De Boer's enzyme levels are expressed as specific activities, in nmol.

min-l.mg- 1 of protein. In the present invention enzyme levels are expressed as units of activity per litre of culture, or units per gram of cell paste (wet weight), where one unit 25 catalyses the deamination of 1plmol.min- 1 substrate under the conditions outlined in Nevertheless, during the course of investigations in the present invention, the fermentation and purification processes described in the de Boer publication were repeated, and it is possible to compare our results using de Boer's methods with the results obtained using our own methods.

Despite repeated efforts, following de Boer's method, the inventors of this application have only been able to obtain, at best, a yield of enzyme of 40 units per litre.

Following de Boer's methods we achieved a 25-fold purification of the enzyme, with maximum recovery of 82%. It is thus seen that the known growth media, methods and microorganisms only enable production of phenylalanine dehydrogenase at rather low yield and do not provide an efficient production procedure for crude enzyme from culture.

[N:\LIBH]00048:RRB The present invention seeks to overcome or at least mitigate the problems in the prior art, resulting in an improved purification of phenylalanine dehydrogenase.

The invention provides a method of purification of phenylalanine dehydrogenase from a homogenised cell supernatant containing phenylalanine dehydrogenase comprising the steps of:loading the supernatant onto an affinity chromatography column preequilibrated with buffer at basic pH, washing the column with the buffer to elute any unbound substances, and eluting phenylalanine dehydrogenase using a solution of NADH in the buffer or by combination of increased pH and increased ionic strength. In an embodiment, the column is pre-equilibrated at pH 8-9 preferably pH 8.3-8.7.

Affinity columns that are of use in the present invention are those having sufficient affinity for phenylalanine dehydrogenase such that the enzyme binds to the column. The enzyme has greater affinity for NADH than for the column matrix, so use of NADH enables recovery of enzyme from the column matrix. Examples of suitable columns are those employing triazine dye-Sepharose matrices. In an embodiment hereinafter described the affinity column is a Procion Red He-3B/Sepharose column, preferably Sepharose 4B, 6B, CL-4B or CL-6B.

i Preferably the method of purification further comprises the step of gel filtration of the purified product to remove NADH therefrom.

In an embodiment of the invention the elution is by an increased NaC1 solution at pH preferably at about pH 10. In a preferred embodiment described below the elution is by a solution of NADH in the buffer, preferably at strength of 0-5mM, more preferably about ImM.

A further embodiment of the invention provides a method of purification of phenylalanine dehydrogenase comprising the steps of:loading a homogenised Nocardia cell supernatant onto an affinity column, the S affinity column comprising a trazine dye-Sepharose matrix, the affinity column being preequilibrated with buffer at basic pH; 30 washing the column with the buffer to elute any unbound substances; and eluting phenylalanine dehydrogenase from the column using an elutant selected from a solution of NADH in the buffer, the NADH solution being of strength from 0and (ii) a solution having pH higher than pH of the buffer and an ionic strength higher than that of the buffer.

The purification method of the invention enables a single step purification of phenylalanine dehydrogenase from the crude supernatant with an effective 30 fold purification of the final product compared to the crude solution. In embodiments of the invention described below, 35 fold purification is achieved.

There now follows some Examples of embodiments of the invention in which Nocardia sp. 239 was grown on a minimal salts medium containing the following:- [N:\LIBH]00048:RRB Component g/L

K

2

HPO

4

KH

2 P0 4

NH

4 C1 CaCl 2 .2H 2 0 0.01

K

2 S0 4 2.6 NaCl L-phenylalanine 16.5 (ie. 100 mM) Mazu DF8005 antifoam 0.5 ml/L The above medium was sterilised prior to inoculation by autoclaving at 121 0 C, psi for 20 minutes and was adjusted to pH Post-sterilisation:- 1M MgCl 2 .6H 2 0 1 ml/L Trace element solution 10 ml/L Vitamin solution 1 ml/L The trace salt and vitamin solutions were as follows:- Trace salts:- Component g/L FeCl3.6H20 3.90 ZnSO 4 .H20 0.58 CoC1 2 .6H 2 0 1.00 Na 2 MoO 4 .2H 2 0 1.00 CuS04.5H20 1.16

H

3

BO

3 0.30 S: MnSO4.7H20 0.72 HC1 (conc.) 7.2 ml/L S Demin H20 to 1000ml .Vitamin solution:- Vitamin mg/L Pyridoxin 0.125 Biotin 0.125 Thiamine 0.025 Nicotinic acid 1.875 Riboflavin 0.125 Enzyme activities are given as Units, defining one unit as 1 micro-mole of substrate (phenylalanine) deaminated per minute at 25'C and pH 10.8 IN:\LIBH100048:RRB Preparation 1 Nocardia sp. 239 was grown in 100ml of the minimal salts medium (as described above) in a 250ml flask shaken at 200rpm for 16-24 hours at 37°C. The resultant cell material was collected by centrifugation and assayed for phenylalanine dehydrogenase activity. The yield of enzyme was equivalent to 370 units/litre of culture.

Preparation 2 The organism was grown as an 8 litre batch culture in a stainless steel reactor at 37 0 C. The culture was stirred at 400-700 rpm and the dissolved oxygen level was maintained above 40% by altering the air flow rate and impeller speed. The pH of the medium was controlled at pH 7.0 0.2 and the vessel was inoculated with a 200 ml seed culture prepared in the same medium and incubated for 16-24 hours, as described in Example 1. The 8 litre culture was incubated for 18 hours (optical density 600nm 21) and was harvested by centrifugation. The enzyme yield was 360 units/litre.

Preparation 3 Frozen ampules of Nocardia sp. 239 containing ml (7.2 x 108 CFU/ml) were used to inoculate a 20 litre seed culture which was grown in a stainless steel reactor under conditions similar to those described in Example 2.

A 400 litre vessel containing 400 litres of the minimal medium was seeded with the litre seed culture, which in mid log phase (OD at 600nm of 12-14). Growth was 20 continued at 37 C and a controlled pH of 7.0 0 0.2 with aeration at 200 litres of air per minute and stirred at 200 rpm until the percentage carbon dioxide in the exhaust gases began to fall. The duration of the culture was 18.5 hours and the final optical density at 600nm was 16.0. The culture was cooled to 8-10 0 C, transferred under top pressure to a S. holding vessel and harvested using a Westfalia centrifuge run at 50-60 litres/hour.

25 The final enzyme yield was 140,000 units from a total of 14.0 Kg of wet cell paste, Sequivalent to 350 units/litre of culture.

Example 1 All purification procedures were performed at 10 0 C or less. Phenylalanine dehydrogenase was purified in a single step from homogenised cell supernatant (crude extract) using a Procion Red HE-3B/Sepharose CL-4B column.

The Procion dye was coupled to the support matrix by the method of Atkinson et al.

((1981). Biochem. Soc. Trans., 9, 290-293) and the level of dye substitution was determined to be 3.15 mg of dye per millilitre of gel, using acid-hydrolysed samples.

Sepharose 4B, 6B and CL-6B were all found to bind similar levels of dye and were equally effective as purification matrices.

The optimum binding conditions were found to be 50 mM Tris-HCl buffer, pH The protease inhibitor, phenylmethanesulphonyl fluoride (PMSF) was routinely added to [N:\LIBH]00048:KBM all buffers at 0.1 mM. The maximum binding capacity was found to be 4.0 units of phenylalanine per millilitre of the matrix. The enzyme was eluted from the column by the inclusion of 1 mM NADH in the buffer, or by a combination of increased pH and ionic strength (eg. 500 mM NaCI and pH 10.0).

A 1 ml disposable plastic column was packed with 1 ml of the Procion matrix and equilibrated with 50 mM Tris-HCl pH 8.5 0.1 mM PMSF. A cell suspension of Nocardia sp. 239 was disrupted by sonication and the cell debris removed by centrifugation to yield a cell extract containing 0.10 units per mg of protein. A total of 2 units of enzyme were loaded to the 1 ml column and, after washing to remove unbound substances, were eluted with 1mM NADH. The purification achieved an effective 40-fold purification over crude (final specific activity 4.0 U/mg) and a recovery of 78%.

Example 2 A total of 30 g of wet cells were suspended in buffer and disrupted by sonication.

The cell debris was removed by centrifugation and the crude extract (185 units, 0.09 U/mg) was loaded onto a 100 ml column (46 mm diameter, 61 mm high) packed with the Procion matrix. The column was run at 300 ml/hour and produced a recovery of 75 with a 38-fold purification (3.42 U/mg) over crude.

Example 3 2 Using material from the 400 litre culture (Preparation cells were suspended in 20 buffer and disrupted using an APV Manton restricted orifice homogeniser, operated at 8000 psi. The cell debris was removed by centrifugation and the crude extract (5000 units, 0.10 U/mg) was applied to a 2.4 litre Procion column (140 mm diameter, 155 mm high) which was run at 4.8 litres/hour (max. 8.4 litres/hour). The enzyme was eluted with 1 mM NADH and achieved a purification of 39 fold (3.9 U/mg) and a recovery of 25 The invention thus provides phenylalanine dehydrogenase from e.g. Nocardia sp.

239, in a quality and quantity suitable for the determination of phenylalanine in biological fluids. The enzyme has been purified from crude cell extracts in a single column affinity chromatography step using a method that is simple, reproducible and well suited to scaleup.

[N:\LIBH]00048:KBM The claims defining the invention are as follows: 1. A method of purification of phenylalanine dehydrogenase from a homogenised cell supernatant containing phenylalanine dehydrogenase comprising the steps of:loading the supernatant onto an affinity chromatography column preequilibrated with buffer at basic pH, washing the column with the buffer to elute any unbound substances, and eluting phenylalanine dehydrogenase using a solution of NADH in the buffer or by a combination of increased pH and increased ionic strength.

2. The method of claim 1 wherein the column is pre-equilibrated at a pH of 3. The method of claim 1 or claim 2 wherein the column is pre-equilibrated at a pH of 8.3-8.7.

4. The method of any one of claims 1 to 3 wherein the column is a Procion Red HE-3B Sepharose column.

5. The method of claim 4 wherein the column is selected from the group consisting of Sepharose 4B, Sepharose 6B, Sepharose C1-4B and Sepharose CL-6B.

6. The method of any one of claims 1 to 5 wherein phenylalanine dehydrogenase is eluted from the column by a solution of NaCl of increased strength.

7. The method of claim 6 comprising eluting phenylalanine dehydrogenase from 20 the column at a pH of 9.0 or greater.

8. The method of claim 7 wherein eluting phenylalanine dehydrogenase from the column at a pH of about 9. The method of any one of claims 1 to 5 comprising eluting phenylalanine dehydrogenase from the column by a solution of NADH in the buffer.

10. The method of claim 9 comprising eluting phenylalanine dehydrogenase from the column using a 0-5mM NADH solution.

11. The method of claim 9 comprising eluting phenylalanine dehydrogenase from S the column using a NADH solution of about 1mM.

12. The method of any one of claims 9 to 11 further comprising carrying out gel filtration to remove NADH from phenylalanine dehydrogenase eluted from the column.

13. A method of purification of phenylalanine dehydrogenase according to claim 1 the method comprising the steps of:loading a homogenised Nocardia cell supernatant onto an affinity column, the affinity column comprising a trazine dye-Sepharose matrix, the affinity column being preequilibrated with buffer at basic pH; washing the column with the buffer to elute any unbound substances; and eluting phenylalanine dehydrogenase from the column using an elutant selected from a solution of NADH in the buffer, the NADH solution being of strength from 0and (ii) a solution having pH higher than pH of the buffer and an ionic strength higher than that of the buffer.

[N:\LIBH]OOO48:RRB

Claims (1)

14. A method of purification of phenylalanine dehydrogenase, substantially as hereinbefore described with reference to any one of Examples 4-6. Dated 15 December, 1997 Microbiological Research Authority Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *to. 0 St 1 Purification of Phenylalanine Dehydrogenase ABSTRACT A method for purification of phenylalanine dehydrogenase from a homogenised cell supernatant containing phenylalanine dehydrogenase including the steps of loading the supernatant onto an affinity chromatography column, washing the column to elute unbound substances and subsequently eluting phenylalanine dehydrogenase using a solution of NADH or a combination of increased pH and increased ionic strength. The column is pre-equilibrated with buffer at basic pH. o** ft 4 a [N:\LIBH]00048:RRB