Immobilized proteins and use thereof

Abstract

The invention relates to an immobilized protein material comprising a protein that is immobilized on a glass material or organic polymer through affinity tag binding. The glass material may be a porous glass material such as (hybrid) controlled porosity glass. The invention also relates to the use of an immobilized enzyme material as a heterogeneous biocatalyst in chemical synthesis. The invention further relates to a method for the immobilization of affinity tagged proteins on a glass material or organic polymer, and to a method for the purification and isolation of affinity tagged proteins by the immobilization of such proteins on a glass material or organic polymer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 15 / 112,206, filed Jul. 18, 2016, which is a national stage application of International Appl. PCT / SE2015 / 050108, filed Jan. 30, 2015, and which claims priority of Swedish Patent Appl. No. 1450822-0, filed Jul. 2, 2014, and Swedish Patent Appl. No. 1450105-0, filed Jan. 31, 2014, all of which are hereby incorporated by reference.TECHNICAL FIELD[0002]The invention relates to an immobilized protein material comprising a protein that is immobilized on a glass material or organic polymer through affinity tag binding. The glass material may be a porous glass material such as (hybrid) controlled porosity glass. The invention also relates to the use of an immobilized enzyme material as a heterogeneous biocatalyst in chemical synthesis. The invention further relates to a method for the immobilization of affinity tagged proteins on a glass material or organic polymer, and to a method f...

AI Technical Summary

Benefits of technology

[0074]Another advantage of the present invention is that the immobilized enzyme material easily can be recycled. Since the immobilized enzyme material is a heterogeneous catalyst, the material can simply be collected from the reaction mixture by filtration. The material can thereafter be re-used in a further reaction, if necessary after purification of the material. Especially for enzymes that are expensive and / or difficult to cultivate, the possibility of recycling the immobilized enzyme material is an important aspect.

[0075]The enzyme that is immobilized on the carrier may be any enzyme that is useful as a biocatalyst in organic synthetic transformations, including, but not limited to, enzymes acting as oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Thus, the immobilized enzyme materials may be used as heterogeneous biocatalysts in any organic reaction wherein the immobilized enzyme is able to specifically catalyze the reaction. Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions and enzymatic isomerization reactions. Particularly useful biocatalytic reactions are enantioselective reactions. Specific examples of biocatalytic reactions include selective acylations of alcohols or amines with lipase, transaminations with w-transaminase, monooxygenations with CYP P450 or Baeyer-Villiger monooxygenase, oxidations of alcohols or reductions of ketones / aldehydes with alcohol dehydrogenase, and oxidations of amines with monoamine oxidase.

[0076]In one embodiment, two or more different enzymes may be immobilized onto the carrier, wherein each of the different enzymes is able to catalyze a different reaction. It may then be possible to use the material containing two or more different immobilized enzymes as a heterogeneous biocatalyst in a multi-step or cascade reaction. Such a cascade reaction may for instance be a reaction wherein two or more enzyme-catalyzed reactions are performed on a substrate in two or more subsequent steps (i.e., a reaction wherein a substrate for a first enzyme is transformed into a substrate for a second enzyme, and so on), such as a transamination reaction by an w-transaminase followed by an acylation reaction by a lipase. Alternatively, such a cascade reaction may be a reaction wherein a substrate is transformed by a first enzyme and wherein a co-factor for the first enzyme is regenerated by a second enzyme, such as the selective / specific reduction of a ketone / aldehyde by alcohol dehydrogenase with the concomitant regeneration of consumed NADH by formate dehydrogenase.

[0077]When two or more different enzymes are immobilized onto the carrier, it is convenient if the different enzymes contain the same affinity tag, such as a polyhistidine tag. As the binding affinity for the chelating metal ion is equal for each of the enzymes, the different enzymes will bind equally strong to the carrier. Theoretically, therefore, when using equal amounts of n different enzymes having the same affinity tag, the amount of each different enzyme on the carrier will be 1 / n (not taking into account any diffusion effects).

[0078]For cascade reactions using an immobilized protein material with two or more different immobilized enzymes, and wherein the different enzymes show a difference in catalytic activity, it may be advantageous to immobilize larger amounts of the enzyme(s) having lower catalytic activity, in comparison to the amount of enzyme having the higher catalytic activity. This will speed up the rate-determining step, and increase the overall rate of the reaction cascade.

[0079]Alternatively, cascade reactions may be performed by mixing one or more different immobilized enzyme materials in desired ratios.

Problems solved by technology

However, since enzymes are biological molecules evolved for a cell environment, they are often unsuited for other environments.

Adsorption of enzymes to solid surfaces can lead to undesired interactions between the enzyme and the solid support.

It has been shown that protein adsorption onto silica nanoparticles may lead to changes in the secondary structure of the protein, which can result in deactivation of the enzyme (Lundqvist et al., Langmuir 2004, vol.

Although this technique can be successfully applied in chromatographic procedures for purification and isolation of proteins, the gel-immobilized enzymes are less suitable as heterogeneous catalysts in organic synthesis.

The IMAC technique is furthermore primarily restricted to aqueous conditions.

This results in a nonspecific binding of the enzyme to the CPG, often with concomitant loss of the enzymatic activity.

A further drawback of this method is that the enzyme to be immobilized must be purified from other enzymes prior to the immobilization step, in order to avoid the immobilization of a mixture of different enzymes on the CPG.

However, the high costs of enzymes and the frequently observed loss of activity upon immobilization of the enzyme on solid support are obstacles in this development.

Despite progress made in recent years, there still is no general and simple method for the preparation of heterogeneous catalysts by immobilization of enzymes.

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