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Extraction and purification of phosphodiesterase

a technology of phosphodiesterase and extraction, which is applied in the field of extracting and purifying an enzyme from a cell, can solve the problems of inability to operate on a commercial scale, the loss or wastage of pde-1 in the process,

Inactive Publication Date: 2006-01-19
PROTECH RES PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The invention provides a process for purifying PDE-1 from a cell, which involves heating an extract of the cell containing divalent cations to increase the specific activity of PDE-1. The concentration of divalent cation in the extract is typically at least about 20 mM. The purified PDE-1 can be used to produce ribonucleotides. The technical effects of the invention include improved purification of PDE-1 and increased production of ribonucleotides."

Problems solved by technology

The processes for purification of PDE-1 from barley rootlets tend to be difficult to 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.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Equipment

[0053] Germinating barley seeds (Schooner variety) were obtained from Barrett Burston Malting, (Thornleigh, NSW, Australia). Thymidine 5′ monophosphate p-nitrophenyl ester, Trisma base, MgCl2, CaCl2, p-nitrophenyl-phosphate, Baker's yeast RNA, bovine serum albumin, guanosine 5′-monophosphate, potassium dihydrogen orthophosphate were all supplied by Sigma Aldrich (Castle Hill, NSW Australia).

[0054] Yeast rRNA was prepared as a 3mg / mL standard solution in RNAse free Milli Q water (pH7.0) in a clean room environment and immediately frozen to minus 80° C. and stored until required.

[0055] Guanosine 5′-monophosphate was prepared as a 1% (w / v) stock by dissolving in Milli Q water and storing at −20° C. Working solutions were prepared by diluting the stock solution to 0.01% (w / v).

[0056] The buffer used for FPLC was 25 mM Tris HCl (pH 8.9). The eluent buffer included 1 M NaCl.

[0057] For the HPLC analysis, solvent (A) was 0.02M potassium dihydrogen orthophosphate (...

example 2

Preparation of a standard curve for p-nitrophenol to determine PDE-1 activity

[0062] A solution of 10 mg / 100 mL of p-nitrophenol was prepared in 50 mM Tris HCl (pH 8.9) to yield a concentration of 0.719 μmoles / ml of p-nitrophenol. Dilutions were prepared in 50 mM Tris HCl (pH8.9) and read as a 4.1 mL sample size at 410 nm to 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

[0063] 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.

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Abstract

A process for purifying a phosphodiesterase 1 (PDE-1) from a cell including heating an extract of a cell formed from a solution including at least on divalent cation, to increase the specific activity of PDE-1 in the extract.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part of U.S. Pat. No. 10 / 733,857, filed Dec. 11, 2003, incorporated herein expressly by reference.FIELD OF THE INVENTION [0002] The invention relates to extracting and purifying an enzyme from a cell, particularly, but not exclusively, to extracting and purifying a 5′ phosphodiesterase from a barley cell. BACKGROUND OF THE INVENTION [0003] 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. [0004] 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. [0005] Barley cells or rootlets are used in the food industry as a source of PDE-1. [0006] The processes for purification of PDE-1 from barley root...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/16C12N15/55
CPCY10S435/814C12N9/16
Inventor PATANE, MICHAEL
Owner PROTECH RES PTY LTD