Enzymes are used on an industrial scale as catalysts for processing various crude materials. Often these processes are cost-effective only when the enzymes can be re-used many times. For recirculation the enzymes need to be separated from the process liquid. This is possible when the enzymes are attached to a carrier which can be filtrated or centrifuged.
An important group of industrial enzymes have an amphiphilic nature. These enzymes are characterized by the presence of a hydrophilic part as well as a hydrophobic part in the molecule. Lipases and phospholipases are representatives of this group of enzymes. Amphiphilic enzymes are enzymes which, when dispersed in an oil and water emulsion, will migrate and accumulate in the interface of the aqueous phase and the oil phase. This is the definition of amphiphilic enzymes in the context of the present invention. The hydrophobic part of the enzyme points into the hydrophobic phase and the hydrophilic part points into the aqueous phase.
The invention will be described with lipase as most important example of an amphiphilic enzyme. Other industrially applied amphiphilic enzymes are phospholipases of which various types are known and which are e.g. used for the hydrolysis of phospholipids to lysophospholipids.
Lipases are employed for their ability to modify the structure and composition of triglyceride oils and fats. They catalyze different types of triglyceride conversions, such as hydrolysis, esterification and transesterification. These are equilibrium reactions which in one direction result into hydrolysis of triglycerides into free fatty acids and glycerol, mono- or diglycerides, and in the other direction result into re-esterification of glycerol, monoglycerides and diglycerides into triglycerides. For the re-esterification process removal of the water which is formed in the reaction medium is necessary to shift the equilibrium in the direction of triglyceride synthesis.
The use of lipase in a substantially water-free process medium needs the dispersion of lipase in oil in an active form, which is a major problem. For that purpose preferably an immobilized lipase is used which is active in an oil which contains a slight amount of dissolved water but not any dispersed water.
Presently the main process for immobilized lipase manufacture comprises first a microbiological fermentation of suitable microorganisms which produce the enzyme under proper conditions, a removal of the micro-organisms and an optional enzyme purification. Then a solution of the obtained lipase is added to a carrier and the enzyme is allowed to get attached to the carrier surface. Such immobilization method is exemplified for an interesterification process in e.g. GB 2 159 527. The attachment of the enzyme to the carrier enables easy separation of the irreversibly immobilized enzyme from the process medium for subsequent use.
Generally, the used carrier materials are porous, particulate, water insoluble materials which provide large surface areas per unit volume are. The preparation of immobilized enzymes is described in e.g. EP 0 140 542, EP 0 382 767, WO 95/22606, EP 0 444 092 and WO 89/01032.
During the enzymatic triglyceride processing the immobilized enzyme gradually looses its activity. It has to be substituted frequently by a fresh enzyme preparation. The enzyme consumption determines for a major part the total processing costs. A great economy advantage would be obtained, when it would be possible to extend the lifetime of the enzyme.
In the usual porous carrier materials mass transfer limitations further decrease the lipase activity.
Our earlier not pre-published patent application WO 97/01632 describes a process for immobilizing an enzyme in a cheap and easy way as well as a process for re-generating and re-activating such enzyme preparation when after use it eventually has become worn-out. The first process comprises the steps
a. selecting an amphiphilic enzyme for immobilization, PA1 b. preparing an emulsion comprising a continuous hydrophobic phase and a dispersed aqueous phase in which aqueous phase are dissolved the enzyme and material suitable to act as carrier for the enzyme when the next step is carried out, PA1 c. removing water from the dispersed phase until this phase turns into solid enzyme coated particles. PA1 a. selecting an amphiphilic enzyme for immobilization, PA1 b. preparing an emulsion comprising a continuous hydrophobic phase and a dispersed aqueous phase in which aqueous phase are dissolved the enzyme and material suitable to act as carrier for the enzyme when the next step is carried out, PA1 c. removing water from the dispersed phase until this phase turns into solid enzyme coated particles,
In said application the carrier material is described as material fully soluble in the aqueous phase. No reference is made of any matter in the aqueous phase which is insoluble.