Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1991-04-24
1994-08-30
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435135, 435176, C12P 764, C12P 762, C12N 1114
Patent
active
053427688
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Extensive research activities have been directed in order to provide immobilized lipase preparation, in consideration of the growing use for immobilized lipases to interesterification. Some of the first industrially applicable immobilized lipase preparations for interesterification are described in U.S. Pat. No. 4,275,081, col. 7, lines 28-35, from which it appears that the immobilized lipase preparations are prepared by acetone precipitation of an aqueous lipase solution on Celite.RTM. diatomaceous earth.
However, the half life of these immobilized lipase preparations are relatively low. Also, the obtainable specific lipase activity is relatively low. Furthermore, a dust problem during storage and loading into the columns is present, and also, solvents are necessary during use of these prior art immobilized lipase preparations.
Thus, the research activities have more or less been focused on immobilization of lipase on carriers with hydrophobic binding sites. Reference can be made to Biotechnology and Bioengineering, Vol. XXIV, pp. 1007-1013 (1982), from which it appears that it is generally assumed that in the preparation of immobilized lipase preparations with high half life and high activity for industrial applications a carrier with hydrophobic binding sites more or less was a conditio sine qua non. Also, reference is made to published Danish patent No. 152763, which describes an immobilized lipase preparation with a carrier consisting of a particulate macroporous weak anion exchange resin, e.g. a resin belonging to the Duolite.RTM. series. Carrier resins of this kind have hydrophobic binding sites, and the preparations exhibit a high half life and are very well suited for industrial interesterification.
Even so, however, these anion exchange resins do have drawbacks. In the first place they are very expensive, and this has a great bearing on the price of the immobilized lipase preparations. In the second place it has been found that during use in organic media extractables from the anion exchange resins are transferred to the organic medium, and even if the amount of the extractables is low, this often represents a serious disadvantage, especially if the end product of the enzymatic process is intended for human consumption. It is possible to wash the resins with organic solvents prior to immbilization, but this is a costly step.
Thus, a need exists for a cheap immobilized lipase preparation with high half life and possibility of high specific activity and without any possibility for transfer of extractables from the carrier to an organic phase.
SUMMARY OF THE INVENTION
Now, according to the invention it has been found that the above need can be fulfilled if--against the prejudice in regard to the above indicated diatomaceous earth carrier--a carefully defined class of inorganic carriers is used.
Thus, the particulate immobilized lipase according to the invention with macroporous silica or silicates as a carrier material is characterized by the fact that more than 90% of the particles have particle sizes between 100 and 1000 .mu.m, wherein more than 80% of the pores in the particles exhibit a diameter between 10 and 45 times the diameter of the lipase globules, and wherein the water content of the particulate immobilized lipase is between 1 and 20%, preferably between 2 and 20%, more preferably between 5 and 20%.
From an article in Advances in Colloid and Interface Science, 25 (1986) 235-248, "Macroporous Silica in Chromatography and Immobilization of Biopolymers" it appears that the maximum capacity for the immobilization of enzymes is found with silicas having average pore sizes of 5 to 10 times the size of the protein globules. Suprisingly, we have found that the maximum capacity for the immobilization of lipases appears with silicas or silicates with pore sizes between 10 and 45 times the size of the lipase globules. This article does not focus on lipase at all, but is concerned with enzymes in general, even with proteins in general. The invention is exclusively dir
REFERENCES:
patent: 4472503 (1984-09-01), Matsuo et al.
patent: 4798793 (1989-01-01), Eigtved
Lavayre et al., Biotechnology and Bioengineering, vol. 24, pp. 1007-1013 (1982).
Artyomova et al., Advances in Colloid and Interface Science, vol. 25, pp. 235-248 (1986).
Hansen Tomas T.
Pedersen Sven
Agris Cheryl H.
Naff David M.
Novo Nordisk A S
Zelson Steve T.
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