Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1998-09-08
2000-10-31
Achutamurthy, Ponnathapu
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4352523, 4353201, 435134, 435913, 435917, 530350, 536 232, 536 2374, C12N 920, C12N 120, C12N 1500, C07H 2104, C07K 100
Patent
active
061400947
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a protein having phospholipase activity which has the mature sequence of Aspergillus lysophospholipase or a sequence derived therefrom and which may be cleaved at at least one site, wherein the restriction fragments are optionally either linked by means of at least one bond cleavable under reducing conditions or at least one of the unlinked restriction fragments has phospholipase activity. The invention furthermore relates to a process for the production of this protein and to the use of this protein for degumming vegetable oils and as a baking auxiliary.
When degumming edible oil, non-hydratable phospholipids are rendered water soluble by phospholipase and thus removed from the edible oil gently, at low cost and in an environmentally friendly manner. European patent application 0 513 709 (Rohm/Lurgi) for the first time presents an effective enzymatic degumming process. In this process, an edible oil, previously degummed with water, is emulsified with an aqueous solution of a phospholipase to yield droplets smaller than 10 .mu.m. After hydrolysis (pH 3 to 6, temperature 50 to 70.degree. C.), the aqueous phase is separated. Lurgi has introduced this enzymatic degumming process into the edible oils industry as the "EnzyMax process". DE 43 39 556 describes a further variant of this process involving reuse of the enzyme by dissolving the enzyme out of a spent aqueous phase containing gum by adding surfactants or solubilising agents and reusing it as a substantially gum-free solution containing enzyme.
Producing sufficient quantities of enzyme for operating the process on a large industrial scale is possible only by using microorganisms. There is thus a requirement for a microbial source which allows production of unlimited quantities of the enzyme phospholipase. DE-OS 195 27 274.9 (Rbhm/Lurgi), dated 26.07.1995, states that a suitable phospholipase has been found in Aspergillus niger. This phospholipase cleaves lecithin to lysolecithin, but is also capable of cleaving lysolecithin further to yield phosphatidylcholine. Pure lysophospholipases from Aspergillus which are only capable of cleaving lysolecithin are ineffective in the degumming process. This also applies to the non-acyl-cleaving phospholipases C and D.
Phospholipases may furthermore be used as baking auxiliaries to improve dough processing.
The object underlying the present invention is to provide a low-cost phospholipase at elevated purity. It should be possible to produce the phospholipase in large quantities by means of a transformed host organism. Using the enzyme, it should be possible to produce preparations which are particularly suitable for hydrolysing phospholipids and thus for clarifying starch hydrolysates and for producing baking auxiliaries.
This object is achieved according to the invention by a protein having phospholipase activity, which is characterised in that it has the mature sequence of the Aspergillus lysophospholipase or a sequence derived therefrom and it may be cleaved at at least one site, wherein, in the event of cleavage, the restriction fragments are either linked by means of at least one bond cleavable under reducing conditions or at least one of the unlinked restriction fragments has phospholipase activity. This object is furthermore achieved by a protein having phospholipase activity, which is characterised in that it is recognised by an antibody against purified phospholipase from Aspergillus foetidus RH 3046.
It has surprisingly been found that a microorganism transformed with the deoxyribonucleic acid (DNA) isolatable from Aspergillus according to DE-OS 196 20 649.9 does not merely code for a lysophospholipase, but, under certain culture conditions, also continues processing to yield a phospholipase. The phospholipase thus has the same primary structure as the lysophospholipase, but a different secondary and tertiary structure and thus different physiological properties. The corresponding sequence is represented in SEQ ID no. 1 of DE-OS 196 20 649.9. Another phospholipase coding sequence h
REFERENCES:
Process Biochemistry. 30(5) : 393-401, 1995.
Jungschaffer Gerald
Khanh Quoc Nguyen
Loffler Fridolin
Schuster Erwin
Sprossler Bruno
Achutamurthy Ponnathapu
Rohm GmbH
Saidha Tekchand
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