Title of Invention	An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer
Abstract	An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer by treating aqueous extract of barley rootlet with ethylenediaminetetraacetic acid (EDTA) in the concentration range of 1 to 500 mm at a temperature in the range of 4°C to 60°C for a period in the range of 0.5 to 12 hours and recovering phospomonoesterase free 5'-phosphodiesterase aqueous.

Title of Invention

An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer

Abstract

An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer by treating aqueous extract of barley rootlet with ethylenediaminetetraacetic acid (EDTA) in the concentration range of 1 to 500 mm at a temperature in the range of 4°C to 60°C for a period in the range of 0.5 to 12 hours and recovering phospomonoesterase free 5'-phosphodiesterase aqueous.

Full Text	The present invention relates to an improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer. 5'-Phosphodiesterase enzyme is an exonuclease that acts on single stranded nucleic acid from the 3' end releasing 5'-nucleotides. 5'-Phosphodiesterase degrade ribonucleic acid to 5'-ribonucleotides. Phosphomonoesterase is a non-specific enzyme that removes terminal phosphate groups from nucleotides and oligonucleotides. 5'-Ribonucleotides are high value added products used in food and pharmaceutical industries. 5'=lnosine monophosphate and 5'-guanosine monophosphate find application as flavour enhancer. Redently, 5'-guanosine have been implicated in cellular and humoral immunity. Reference may be made to US Patent US5700590 and US5492899 wherein infant/enteral nutritional formulae with 5'-ribunucleotidies is described. Reference may also be made to US Patent US5712256 wherein a ribonucleotide preparation has been used for the promotion of wound healing in animals. 5'-Ribonucleotides find a number of application in food and pharmaceutical industries. 5'-Ribonucleosides, produced as a result of phosphomonoesterase action on 5'ribonucleotides have negative effect on the function of 5'-ribonucleotides. 5'-Ribonucleotides are produced by the hydrolysis of ribonucleic acid using 5'-phosphodiesterase . However, 5'-phosphodiesterase is usually found in association with phosphomonoesterase. Inhibition of phosphomonoesterase is, therefore, important for the various applications of 5'-phosphodiesterase, for prevention of formation of nucleosides. In addition, 5'-phosphodiesterase is used as an analytical reagent in the structure/ sequence determination of nucleic acid. For these applications also purified 5'-phosphodiesterase free of phosphomonoesterase is required. Reference may be made to Japanese patent JP1980000098810 wherein a method for the preparation of 5'-phosphodiesterase free of phosphomonoesterase is given. The process involves treating the enzyme preparation with sulfated or sulfonic acid type anionic detergent. The drawback of this procedure is that detergents used in the process should be removed before it can be used for its various applications. Reference may also be made to Benaiges, Lopez-Satin and Sola in Enzyme Microbial Technology, volume 11 pages 444-451 (1989) wherein a method for the preparation of 5'-phosphodiesterase from barley rootlet is described. The authors have carried out acetone precipitation and ion-exchange chromatography on DEAE-Sephadex to get a purified 5'-phosphodiesterase preparation. Protein purification procedures removed phosphomonoesterase only to a significant extent (7:1). The drawback of this process is that still the final preparation of 5'-phosphodiesterase contained phosphomonoesterase activity to a considerable extent. Reference may also be made to US patent US0409836 wherein a method for the preparation of 5'-phosphodiesterase from barley rootlet is given. The method involves heating the acidic solution of crude enzyme extract at high temperature to inhibit phosphomonoesterase. The main drawback of this process is that the final preparation contains 5'-phosphodiesterase and phosphomonoesterase in the ratio of 7:1. It was observed while repeating the process that this process also inhibited 5'-phosphodiesterase activity to a certain extent (35% loss in activity). The main object of the present invention is to provide an improved process for preparation of phosphomonoesterase free 5'-phosphodiesterase which obviates the drawbacks as detailed above. Another object of the present invention is to prepare phosphomonoesterase free 5'-phosphodiesterase by using simple chelating agents like ethylenediaminetetraacetic acid. Still another object of the present invention is to provide an irreversible process wherein inhibition of 5'-phosphodiesterase is avoided. Yet another object of the present is to provide a process which is simple to operate and in which ethylenediaminetetraacetic acid can be removed by mere gilfilteration or dialysis. Accordingly, the present invention provides an improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer which comprises treating aqueous extract of barley rootlet with ethylenediaminetetraacetic acid (EDTA) in the concentration range of 1 to 500 mm at a temperature in the range of 4°C to 60°C for a period in the range of 0.5 to 12 hours and recovering phospomonoesterase free 5'-phosphodiesterase aqueous. In an embodiment of the present invention, the barley rootlet extract used may be a crude extract or purified extract through conventional methods such as ion-exchange chromatography and gel-filteration. In another embodiment of the present invention, the concentration of EDTA used may be in the range of 1-500 mm. In still another embodiment of the present invention, based on the above observation, is an improved process for preparation of 5'-phosphodiesterase free of phosphomonoesterase involving extracting barley rootlet enzyme in an aqueous medium and subjecting to DEAE-chromatography, eluting the enzyme with an elution buffer containing EDTA, concentration the extract by ultrafilteration and subjecting to gel filteration to obtain 5'-phosphodiesterase free of phosphomonoesterase and EDTA. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. EXAMPLE 1 One gram of barley rootlet was suspended in 25.0ml water for 30 mins and stirred in a rotorary shaker at 100 rpm at room temperature. The suspended matter was filtered through a double layer cheese cloth, filterate centrifuged and used as a crude source of enzyme. Extraction of 5'-phosphodiesterase with or without grinding of barley rootlet did not increase 5'-phosphodiesterase activity in the supernatant. On the contrary, the specific activity of 5'-phosphodiesterase greatly reduced (Table I). A portion of crude extract was dialysed against water for 12 hrs with three changes of water. Dialysed and undialysed crude extract was treated with l0mM EDTA for 30 mins at room temperature. As shown in Table II, 5'-phosphodiesterase activity was not effected whereas phosphomonoesterase activity was significantly inhibited by EDTA. Inhibition in dialysed crude preparation was more compared to nondialysed preparation. TABLE I: EXTRACTION OF PHOSPHODIESTERASE FROM BARLEY ROOTLET (Table Removed) TABLE H: EFFECT OF EDTA TREAMENT ON BARLEY ROOTLET ENZYMES (Table Removed) EXAMPLE 2 Crude dialysed extract of barley rootlet was treated with different concentrations of EDTA for 30 mins, at room temperature. As can be seen from Table III, the activity of phosphomonoesterase decreased with increasing concentrations of EDTA while 5'-phosphodiesterase activity was not affected. TABLE III: EFFECT OF CONCENTRATION OF EDTA ON BARLEY ROOTLET ENZYMES (Table Removed) EXAMPLE 3 Crude dialysed extract of barley rootlet was treated with l0mM EDTA for various time periods at room temperature. There was complete inhibition of phosphomonoesterase after 6 hours. No change in 5'-phosphodiesterase activity was observed at the end of 6 hours (Table IV). Enzyme preparation treated with EDTA for 6 hrs was dialysed against water for 12 hrs with three changes to remove EDTA. This preparation also showed no phosphomonoesterase activities indicating that inactivation ofphosphomonoesterase by EDTA is non-reversible (Table V). TABLE IV: EFFECT OF TIME OF EDTA TREATMENT ON BARLEY ROOTLET ENZYMES (Table Removed) TABLE V: EFFECT OF DIALYSIS ON EDTA TREATED ENZYMES (Table Removed) EXAMPLE 4 Crude extract of barley rootlet ( 430 ml) was loaded on to a DEAE-cellulose column previously equilibrated with 50mM Tris HC1 buffer pH 8.0. The loaded column was washed with the same buffer till no protein eluted out. The bound enzymes were eluted with 300mM NaCl in 50mM Tris HC1 buffer pH 8.0 containing l0mM EDTA. The eluted enzyme was pooled together and concentrated by ultrafilteration and loaded on to Sephadex G-150 column, previously equlibrated with 50mM Tris HC1 pH 8.0. The final 5'-phosphodiesterase preparation did not have any phosphomonoesterase activity (Table VI). At the gel Alteration step, EDTA was also removed from the 5'-phosphodiesterase preparation. At the end of the process, 5'-phosphodiesterase was purified by 14 fold with 51% recovery. The main advantages of the present invention are: 1. The improved method described involves simple, ecofriendly, cost effective, extraction of barley rootlet enzyme with water without grinding/ homogenisation. 2. 5'-Phosphodiesterase without any phosphomonoesterase activity is obtained which is suitable for analytical and preparative applications. 3. Ethylenediaminetetraacetic acid which is used in the present invention to inhibit phosphomonoesterase activity was removed from the 5'-phosphodiesterase activity. 4. The process is irreversible. We claim : 1. An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer which comprises treating aqueous extract of barley rootlet with ethylenediaminetetraacetic acid (EDTA) in the concentration range of 1 to 500 mm at a temperature in the range of 4°C to 60°C for a period in the range of 0.5 to 12 hours and recovering phospomonoesterase free 5'-phosphodiesterase aqueous. 2. A process as claimed in claim 1 wherein the concentration of EDTA used is in at least 1 mm. 3. An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer substantially as herein described with reference to the examples.

Full Text

The present invention relates to an improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer. 5'-Phosphodiesterase enzyme is an exonuclease that acts on single stranded nucleic acid from the 3' end releasing 5'-nucleotides. 5'-Phosphodiesterase degrade ribonucleic acid to 5'-ribonucleotides. Phosphomonoesterase is a non-specific enzyme that removes terminal phosphate groups from nucleotides and oligonucleotides.
5'-Ribonucleotides are high value added products used in food and pharmaceutical industries. 5'=lnosine monophosphate and 5'-guanosine monophosphate find application as flavour enhancer. Redently, 5'-guanosine have been implicated in cellular and humoral immunity.
Reference may be made to US Patent US5700590 and US5492899 wherein infant/enteral nutritional formulae with 5'-ribunucleotidies is described.
Reference may also be made to US Patent US5712256 wherein a ribonucleotide preparation has been used for the promotion of wound healing in animals.
5'-Ribonucleotides find a number of application in food and pharmaceutical industries. 5'-Ribonucleosides, produced as a result of phosphomonoesterase action on 5'ribonucleotides have negative effect on the function of 5'-ribonucleotides.
5'-Ribonucleotides are produced by the hydrolysis of ribonucleic acid using 5'-phosphodiesterase . However, 5'-phosphodiesterase is usually found in association with phosphomonoesterase. Inhibition of phosphomonoesterase is, therefore, important for the various applications of 5'-phosphodiesterase, for prevention of formation of nucleosides. In addition, 5'-phosphodiesterase is used as an analytical reagent in the structure/ sequence determination of nucleic acid. For these applications also purified 5'-phosphodiesterase free of phosphomonoesterase is required.
Reference may be made to Japanese patent JP1980000098810 wherein a method for the preparation of 5'-phosphodiesterase free of phosphomonoesterase is given. The process involves treating the enzyme preparation with sulfated or sulfonic acid type anionic detergent. The drawback of this procedure is that detergents used in the process should be removed before it can be used for its various applications.
Reference may also be made to Benaiges, Lopez-Satin and Sola in Enzyme Microbial Technology, volume 11 pages 444-451 (1989) wherein a method for the preparation of 5'-phosphodiesterase from barley rootlet is described. The authors have carried out acetone precipitation and ion-exchange chromatography on DEAE-Sephadex to get a purified 5'-phosphodiesterase preparation. Protein purification procedures removed phosphomonoesterase only
to a significant extent (7:1). The drawback of this process is that still the final preparation of 5'-phosphodiesterase contained phosphomonoesterase activity to a considerable extent.
Reference may also be made to US patent US0409836 wherein a method for the preparation of 5'-phosphodiesterase from barley rootlet is given. The method involves heating the acidic solution of crude enzyme extract at high temperature to inhibit phosphomonoesterase. The main drawback of this process is that the final preparation contains 5'-phosphodiesterase and phosphomonoesterase in the ratio of 7:1. It was observed while repeating the process that this process also inhibited 5'-phosphodiesterase activity to a certain extent (35% loss in activity).
The main object of the present invention is to provide an improved process for preparation of phosphomonoesterase free 5'-phosphodiesterase which obviates the drawbacks as detailed above.
Another object of the present invention is to prepare phosphomonoesterase free 5'-phosphodiesterase by using simple chelating agents like ethylenediaminetetraacetic acid.
Still another object of the present invention is to provide an irreversible process wherein inhibition of 5'-phosphodiesterase is avoided.
Yet another object of the present is to provide a process which is simple to operate and in which ethylenediaminetetraacetic acid can be removed by mere gilfilteration or dialysis.
Accordingly, the present invention provides an improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer which comprises treating aqueous extract of barley rootlet with ethylenediaminetetraacetic acid (EDTA) in the concentration range of 1 to 500 mm at a temperature in the range of 4°C to 60°C for a period in the range of 0.5 to 12 hours and recovering phospomonoesterase free 5'-phosphodiesterase aqueous.
In an embodiment of the present invention, the barley rootlet extract used may be a crude extract or purified extract through conventional methods such as ion-exchange chromatography and gel-filteration.
In another embodiment of the present invention, the concentration of EDTA used may be in the range of 1-500 mm.
In still another embodiment of the present invention, based on the above observation, is an improved process for preparation of 5'-phosphodiesterase free of phosphomonoesterase involving extracting barley rootlet enzyme in an aqueous medium and subjecting to DEAE-chromatography, eluting the enzyme with an elution buffer containing EDTA, concentration the extract by ultrafilteration and subjecting to gel filteration to obtain 5'-phosphodiesterase free of phosphomonoesterase and EDTA.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
EXAMPLE 1
One gram of barley rootlet was suspended in 25.0ml water for 30 mins and stirred in a rotorary shaker at 100 rpm at room temperature. The suspended matter was filtered through a double layer cheese cloth, filterate centrifuged and used as a crude source of enzyme. Extraction of 5'-phosphodiesterase with or without grinding of barley rootlet did not increase 5'-phosphodiesterase activity in the supernatant. On the contrary, the specific activity of 5'-phosphodiesterase greatly reduced (Table I). A portion of crude extract was dialysed against water for 12 hrs with three changes of water. Dialysed and undialysed crude extract was treated with l0mM EDTA for 30 mins at room temperature. As shown in Table II, 5'-phosphodiesterase activity was not effected whereas phosphomonoesterase activity was significantly inhibited by EDTA. Inhibition in dialysed crude preparation was more compared to nondialysed preparation.
TABLE I: EXTRACTION OF PHOSPHODIESTERASE FROM BARLEY
ROOTLET (Table Removed)
TABLE H: EFFECT OF EDTA TREAMENT ON BARLEY ROOTLET
ENZYMES (Table Removed)
EXAMPLE 2
Crude dialysed extract of barley rootlet was treated with different concentrations of EDTA for 30 mins, at room temperature. As can be seen from
Table III, the activity of phosphomonoesterase decreased with increasing concentrations of EDTA while 5'-phosphodiesterase activity was not affected. TABLE III: EFFECT OF CONCENTRATION OF EDTA ON BARLEY ROOTLET ENZYMES
(Table Removed)
EXAMPLE 3 Crude dialysed extract of barley rootlet was treated with l0mM EDTA for various time periods at room temperature. There was complete inhibition of phosphomonoesterase after 6 hours. No change in 5'-phosphodiesterase activity was observed at the end of 6 hours (Table IV). Enzyme preparation treated with EDTA for 6 hrs was dialysed against water for 12 hrs with three changes to remove EDTA. This preparation also showed no phosphomonoesterase activities
indicating that inactivation ofphosphomonoesterase by EDTA is non-reversible (Table V).
TABLE IV: EFFECT OF TIME OF EDTA TREATMENT ON BARLEY ROOTLET ENZYMES
(Table Removed)
TABLE V: EFFECT OF DIALYSIS ON EDTA TREATED ENZYMES

(Table Removed)
EXAMPLE 4
Crude extract of barley rootlet ( 430 ml) was loaded on to a DEAE-cellulose column previously equilibrated with 50mM Tris HC1 buffer pH 8.0. The loaded column was washed with the same buffer till no protein eluted out. The bound enzymes were eluted with 300mM NaCl in 50mM Tris HC1 buffer pH 8.0 containing l0mM EDTA. The eluted enzyme was pooled together and concentrated by ultrafilteration and loaded on to Sephadex G-150 column, previously equlibrated with 50mM Tris HC1 pH 8.0. The final 5'-phosphodiesterase preparation did not have any phosphomonoesterase activity (Table VI). At the gel Alteration step, EDTA was also removed from the 5'-phosphodiesterase preparation. At the end of the process, 5'-phosphodiesterase was purified by 14 fold with 51% recovery.
The main advantages of the present invention are:
1. The improved method described involves simple, ecofriendly, cost effective, extraction of barley rootlet enzyme with water without grinding/ homogenisation.
2. 5'-Phosphodiesterase without any phosphomonoesterase activity is obtained which is suitable for analytical and preparative applications.
3. Ethylenediaminetetraacetic acid which is used in the present invention to inhibit phosphomonoesterase activity was removed from the 5'-phosphodiesterase activity.
4. The process is irreversible.

We claim :
1. An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer which comprises treating aqueous extract of barley rootlet with ethylenediaminetetraacetic acid (EDTA) in the concentration range of 1 to 500 mm at a temperature in the range of 4°C to 60°C for a period in the range of 0.5 to 12 hours and recovering phospomonoesterase free 5'-phosphodiesterase aqueous.
2. A process as claimed in claim 1 wherein the concentration of EDTA used is in at least 1 mm.
3. An improved process for the preparation of phospomonoesterase free 5'-phosphodiesterase useful as flavour enhancer substantially as herein described with reference to the examples.

Documents:

1224-del-2003-abstract.pdf

1224-del-2003-claims.pdf

1224-del-2003-complete specification (granted).pdf

1224-del-2003-correspondence-others.pdf

1224-del-2003-correspondence-po.pdf

1224-del-2003-description (complete).pdf

1224-del-2003-form-1.pdf

1224-del-2003-form-19.pdf

1224-del-2003-form-2.pdf

1224-del-2003-form-3.pdf

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Patent Number

199848

Indian Patent Application Number

1224/DEL/2003

PG Journal Number

37/2008

Publication Date

12-Sep-2008

Grant Date

24-Nov-2006

Date of Filing

30-Sep-2003

Name of Patentee

Council of Scientific & Industrial Research

Applicant Address

RAFI MARG,NEW DELHI-110001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	Santhoor Gururaja Bhat	Central Food Technological Research Institute, Mysore-570013,INDIA.
2	Jessy Abraham	Central Food Technological Research Institute, Mysore

PCT International Classification Number

C12N 3/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA