WO2007134365A1

WO2007134365A1 - EXTRACTING AND PURIFYING β-AMYLASES

Info

Publication number: WO2007134365A1
Application number: PCT/AU2007/000627
Authority: WO
Inventors: Michael Patane; Mitchell Parker; Akshat Talwalkar
Original assignee: Protech Research Pty Ltd
Priority date: 2006-05-22
Filing date: 2007-05-10
Publication date: 2007-11-29

Abstract

The invention relates to a process for the extraction and purification of β-amylase from a cell, the process comprising releasing the enzyme from the cell into a solution comprising a reducing agent and heating the cell extract in order to increase the specific activity of the β-amylase. The invention also relates to uses for an enzyme so prepared in the production or modification of foodstuffs, beverages and detergents.

Description

Extracting and purifying B amylases

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 β-amylase. Background of the invention β-amylase (EC 3.2.1.2), otherwise known as 1, 4-α-D-glucan maltohydrolase; saccharogen amylase; beta-amylase; or glycogenase, is an enzyme that catalyses the hydrolysis of 1 , 4-α-D-glucosidic linkages in polysaccharides so as to remove successive maltose units from the non-reducing ends of the chains. β-amylase acts on starch, glycogen and related polysaccharides and oligosaccharides producing β-maltose by an inversion. Hence β-amylase is particularly important in food industries for producing maltose.

The processes for obtaining commercial quantities of β amylase tend to be difficult to operate on a commercial scale, in terms of requiring sophisticated fermentation technology, including multiple steps for extraction, separation and purification as well as high capital investment. Some processes are characterised by an unacceptable loss in activity or wastage of β-amylase. Other processes tend to produce a crude or non purified final product that may have sub-optimal specific activity with some processes utilising Genetically Modified Organisms which require additional source identification.

In view of the above, there is a need for improved processes for the extraction and purification of β-amylase.

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 β- amylase. (a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract;

(b) heating the extract to increase the specific activity of the β-amylase in the extract. In another aspect, the invention provides β-amylase produced by the process of the invention.

In another aspect, the invention provides a cell including β-amylase produced by the process of the invention.

Detailed description of the embodiments As described herein, the inventor has found that the specific activity of a β- amylase in a cell extract can be increased by releasing β-amylase from a cell such as a barley cell, rootlet or grain into a solution including a reducing agent such as cysteine and heating the solution. For example, the specific activity of a heat treated extract of barley formed from a solution containing 2OmM cysteine derived from milled grains was observed to increase 1.5 fold over a non heat treated sample (168.7μmoles/min/mL compared with 112.1μmoles/min/mL). Further, a heat treated extract containing 2OmM cysteine was observed to have an improved specific activity of 3.5 fold (47.4μmoles/min/mL) compared with a heat treated extract without Cysteine.

In certain embodiments there is provided a process for purifying β-amylase from a cell. The process includes the steps of:

(a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract;

(b) heating the extract to increase the specific activity of the β-amylase in the extract. It will be understood that the processes of the invention are useful for purifying β-amylase from cells other than barley cells. Further, it will be understood that processes of the invention are useful for isolating barley β-amylase from cells that contain a recombinant nucleic acid molecule that encodes barley β-amylase. Examples of such cells include bacterial cells and yeast cells.

Where the extract is formed from a barley grain, the grain is typically one which has not germinated. Such grains are useful as β-amylase tends to be accumulated during grain development and is not synthesised during germination.

Prior to forming an extract, the barley grain may be homogenised, for example by grinding in a unit such as a hammer mill. The grain may be milled using an apparatus employing counter rotating rollers. Otherwise, grains may be coarsely ground in a blender. In certain embodiments, a cell is immersed or otherwise soaked in a solution including a reducing agent. Typically the reducing agent is cysteine. The solution typically has a concentration of cysteine ranging between about ImM and 100 mM cysteine. Preferably the concentration of cysteine is about 2OmM. However, other concentrations may be used including 5mM, 10 mM, 15mM, 3OmM, 4OmM 5OmM, 6OmM, 7OmM, 8OmM, 9OmM, 95mM.

The solution is typically buffered to control pH. Trisma base is a suitable buffering agent. A solution having a concentration of no more than about 20OmM 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 β-amylase on an anion exchange column.

The extract may be formed by incubation at a temperature no greater than about

1O⁰C. These temperatures may be maintained for no more than about 3 days. For example, the extract may be maintained between 0 to about 10⁰C for between about 1 to 48 hours. It is particularly advantageous to maintain the extract for 4 hours at 1O⁰C as this improves the speed of purification protocols that comprise further purification steps.

The inventor has found that an extract so formed contains various components such as enzyme inhibitors, contaminating enzymes such as phosphomonoesterase and low molecular weight protein derivatives. Further, it has been found that the specific activity of β amylase in such an extract may be increased by heating the extract to a temperature that permits destruction of the activity of these components in the extract. As described herein, temperatures generally less than 8O⁰C are suitable for this purpose.

It is particularly advantageous to heat the extract to between about 45 and 75⁰C because at temperatures approaching 7O⁰C and above, β amylase activity may be lost. Accordingly, a temperature of about 6O⁰C is particularly useful.

Following heating, the extract may be cooled and subjected to further purification.

The inventor has found that β-amylase can be purified to virtual homogeneity from a barley cell extract by a process including the following steps: (a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract;

(b) heating the extract to increase the specific activity of the β-amylase in the extract; and

(c) utilising ultrafiltration and ion exchange chromatography to concentrate and purify β-amylase from the heated extract.

As described herein, β-amylase can be concentrated and purified from a heat treated barley cell extract by tangential flow ultrafiltration. Accordingly, typically, in step (c), ultrafiltration is utilised to purify β-amylase from the heated extract.

In another embodiment, β-amylase 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 β-amylase from the heated extract.

The process is particularly useful for modifying the oligo and polysaccharide content of an ingredient for use in the manufacture of foods and beverages. Accordingly, in one embodiment the process includes: (a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract; (b) heating the extract to increase the specific activity of the β-amylase in the extract; and

(c) contacting the extract with a carbohydrate -containing ingredient for use in the manufacture of a food or beverage to permit β-amylase in the extract to modify carbohydrate in the carbohydrate -containing ingredient.

The carbohydrate -containing ingredient may be a dough for use in the production of breads and bread-like products, a wort for use in the production of beer and other alcohol containing beverages; a sugar mixture for use in the production of confectionary.

The process is also useful for the production of a detergent. Accordingly, in one embodiment there is provided a process for producing a detergent including:

(b) heating the extract to increase the specific activity of the β-amylase in the extract; and (c) providing the extract to a detergent -containing composition, to produce a detergent.

Example 1 : Materials and equipment.

Germinating barley seeds (Schooner variety) were obtained from Barrett Burston Malting, (Thornleigh, NSW, Australia). Cysteine, Trisma base and hydrochloric acid were supplied by Sigma Aldrich (Castle Hill, NSW Australia). Betamyl ^-amylase test reagent kit was obtained from Megazyme International Ltd (Bray, Ireland).

In one application, non germinated barley grains were coarsely ground in a Waring blender or Perten Laboratory Hammer Mill at high speed incorporating a 2mm sieve. In another application, non germinated barley grains were immersed in Tris buffer for 2 hours and milled at 400 rpm on a Kustnel Freres & Cie roller mill at a feed rate of 2 kg per minute with a gap setting of lmm using counter rotating smooth rollers to crack the grains allowing extraction of the enzyme. The processed grains (100 kg) were immersed in buffer (300 Ltrs) containing 25mM Tris-HCL (pH 8.0) and 2OmM cysteine and agitated in a D-Tank using counter directional paddles for 4 hours at a temperature of 1O⁰C to extract the enzyme,

The crude /?-amylase extract was then screened and centrifuged in a Sharpies centrifuge at 16,000 rpm at 1O⁰C at a feed rate of 120 Ltrs per hour to remove any precipitate.

The crude /?-amylase extract was then heat processed to 65⁰C and recirculated for one hour in a SWEP plate heat exchanger to denature unwanted proteins before being rapidly cooled to <10°C by recirculation through a fabricated tubular heat exchanger immersed in a refrigerated glycol bath to avoid inactivation of the extracted ^-amylase .

The cooled extract was then re-centrifuged at 16,000rpm at 1O⁰C to remove flocculated proteins.

The heat treated and cooled extract was concentrated a using a Millipore prep scale CDUF006TG 6ft² polyethersulphone spiral wound cross flow ultrafiltration cartridge with 1OkDa nominal molecular weight cut off run on a Masterflex economy drive peristaltic pump and Masterflex Easy load II head, at a flow rate of 400 mL per minute and 18 psi inlet pressure to separate and concentrate the ^-amylase containing fractions in the retentate.

A sample of the extract was subjected to secondary purification by anion exchange chromatography using a Fast Protein Liquid Chromatography Unit (FPLC).

The buffer used for FPLC was 25mM Tris HCl (pH 8.0). The elucnt buffer included 0.5M NaCl.

All solvents and buffers were degassed and filtered prior to use by passing each solution through a Millipore membrane filter, pore size 0.45 μm under vacuum for 5 minutes.

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 Unicom analytical software was used for all protein purification. The column used for the purification was a Hi Prep 16 /10 DEAE FF column connected to a Super loop 50 (to facilitate larger injection volumes).

Isolation of /^-amylase was identified by protein bands on native electrophoresis gels and individual absorption peaks on anion exchange chromatography. An LW Scientific UV-Vis spectrophotometer was used to measure enzyme activity operating at 410nm. The system was controlled by a Celeron processor computer supporting LW Graphite version 3.1 analytical software.

Example 2: Preparation of a Betamyl reagent substrate to calculate β amylase activity. The analysis of /?-amylase used a modified Megazyme Betamyl Method utilising a substrate containing, /Miitrophenyl-α-D-maltopentose (PNPG5). Preparation involves dissolving the contents of one vial in 1OmL of distilled water and using 5OuJL per assay with the remaining volume divided in ImL aliquots and stored frozen between uses. The action of the Betamyl assay provides for the hydrolysis of p-nitrophenyl maltopentaoside to maltose and /?-nitrophenyl maltotrioside by /?-amylase, the nitrophenyl trioside is immediately cleaved to glucose and free p-nitrophenol by the α-glucosidase present in the substrate mixture. The rate of release of /7-nitrophenol relates directly to the rate of release of maltose by the ^-amylase. The reaction is stopped by the addition of ImL of a 1 % (w/v) Trisma solution (pH 11.0) and the phenolate colour is developed by addition of Trisma base solution with absorbance read at 41 Onm.

One unit of enzyme activity is defined as the amount of enzyme required, in the presence of excess α-glucosidase, required to release one micromole of p-nitrophenol from PNPG5 in one minute under the defined assay conditions.

Calculation of activity is defined as one ^-amylase unit/g of grain = (Absorbance / Time of Assay) x (Volume in Curvette / Sample Volume) x (1 / 17.8 [Extinction coefficient]) x (Extraction Volume / Sample weight [g] x Dilution)

Example 3: Preparation of a standard curve for protein to determine β -amylase 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 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: Modified Betamyl Method for the assay of /?-amylase activity.

The assay requires 5OuJL of the extracted enzyme solution to be mixed with 5OuJL of the prepared Betamyl substrate reagent containing the p-nitrophenyl maltopentaoside and σ-glucosidase which is incubated at 4O⁰C for 10 minutes. The reaction is stopped with the addition of ImL of 1 % (w/v) Trisma solution (pH 1 1.0) and the absorbance read at 410nm. Activity is determined by calculation. A reference blank is prepared by adding ImL of 1% (w/v) Trisma to 5OuJL, Betamyl substrate and 5OuJL of distilled water.

Example 5: Preparation of crude β amylase extract. 100 kg of 3 to 12 month stored barley grains were immersed in 300 Ltrs of 25mM

Tris-HCl (pH 8.0) containing 2OmM Cysteine and held for 2 hours to achieve a moisture content not exceeding 40%.

The grains were then milled using counter rotating smooth rollers at a roller speed 400 rpm and gap setting of lmm with a feed rate of 2kg of grain per minute. Extraction of /?-amylase was conducted by immersion of the milled grains in a buffered solution of 25mM Tris-HCl (pH8.0) containing 2OmM Cysteine for 4 hours at 10⁰C to facilitate solubilization of /?-amylase.

The insoluble material was removed by screening the extract through the compacted grains and a 10 micron filter. The filtrate was then centrifuged at 16,000 rpm at 1O⁰C to remove any remaining solids and then stored at 4⁰C until required. This process formed the crude ^-amylase extract.

The activity and specific activity of the crude /J-amylase extract was determined according to Examples 2, 3 and 4 above. Example 6: Purification of β amylase from the erode β amylase extract.

The first stage of the purification process involved the removal of any superfluous proteins from the erode β amylase extract with the aim of increasing the specific activity of the /?-amylase. The crude extract was heated in a SWEP plate heat exchanger to 6O⁰C and maintained at that temperature for 1 hour. The heated extract was then cooled in a tubular heat exchanger to <10°C and centrifuged to remove the denatured proteinaceous materials. The post heat treated extract was then concentrated and purified by cross flow ultrafiltration to facilitate down stream anion exchange chromatography. The activity and specific activity of the heat treated β amylase extract was determined according to Examples 2, 3 to 4 above.

Ion exchange chromatography was undertaken by a 5OmL sample of the extract injected onto a Hi Prep 16 /10 DEAE FF column connected to a Super loop 50 (to facilitate larger injection volumes) at a flow rate of 3.0 mL per minute to fractionate β amylase. The isolated fraction was removed by the elution buffer containing 0.5M NaCl and reconcentrated by ultrafiltration and stabilized in a 40% sorbitol solution. A single peak was obtained and analysed for activity and specific activity according to Examples 2, 3 and 4 above.

Example 7: Purification profile for β amylase. The results for the purification of β amylase are shown in Table 1. Table 1

Activity: μmoles/min

Specific activity: μmoles/min/mg

Claims

1. A process for purifying β-amylase from a cell including:

(a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract; (b) heating the extract to increase the specific activity of the β-amylase in the extract.

2. The process according to claim 1 wherein the reducing agent is cysteine.

3. The process according to any one of the preceding claims wherein the solution has a concentration of cysteine from about ImM to 100 mM cysteine.

4. The process according to any one of the preceding claims wherein the cell is a barley cell.

5. The process according to any one of the preceding claims wherein the cell contains recombinant β-amylase.

6. The process according to any one of the preceding claims wherein the cell is comprised in a grain.

7. The process according to claim 6 wherein the grain is not a germinated grain.

8. The process according to claim 6 wherein the grain is a homogenised grain.

9. The process according to any one of the preceding claims wherein the cell is immersed or otherwise soaked in the solution including a reducing agent.

10. The process according to any one of the preceding claims wherein the solution is buffered to control pH.

11. The process according to any one of the preceding claims wherein the extract is formed by incubation at a temperature no greater than about 1O⁰C.

12. The process according to any one of the preceding claims wherein the extract is heated to a temperature of from 45 to 8O⁰C to increase the specific activity of the β-amylase in the extract..

13. A process for purifying a β-amylase from a cell including: (a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract;

(c) utilising ultra filtration and ion exchange chromatography to concentrate and purify β-amylase from the heated extract.

14. The process according to claim 13 wherein the chromatography is anion exchange chromatography.

15. A β-amylase produced by a process according to any one of the preceding claims.

16. A cell including a β-amylase produced by a process according to any one of the preceding claims.

17. A process for modifying the oligo and polysaccharide content of an ingredient for use in the manufacture of foods and beverages including:

18. The process according to claim 17 wherein the ingredient is a dough for use in the production of bread, a wort for use in the production of alcoholic beverages, or a sugar mixture for use in the production of confectionary.

19. A process for producing a detergent including: (a) releasing β-amylase from a cell into a solution including a reducing agent to form an extract;

(c) providing the extract to a detergent -containing composition, to produce a detergent.