Biocatalyst composition

Abstract

The present invention relates to a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure. The present invention also relates to a method for producing the said composition.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure. The present invention also relates to a method for producing the composition. [Background technology]

[0002] Proteins such as enzymes are frequently required for industrial, diagnostic, or therapeutic applications. In the prior art, to stabilize proteins and / or confer resistance to various types of stress, it has been proposed to immobilize proteins on the surface of a support and protect them with a layer of protective material. Such methods are described, for example, in International Publication 2015 / 014888, which discloses a biocatalytic composition comprising a solid support, a functional component such as an enzyme, and a protective layer for protecting the functional component by at least partially embedding it, as well as a process for producing such a biocatalytic composition. Nevertheless, there is still a need to provide enzymes such as lipases that are protected while simultaneously possessing high enzymatic activity. [Overview of the project]

[0003] The present invention provides a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure.

[0004] The present invention also provides a method for producing a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure, and this method comprises the following steps: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, (c) A step of providing a substance that interacts with the lid domain of lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof onto a solid carrier. (f) A step of forming a protective layer on the surface of a solid carrier in order to protect the lipase or fragment thereof immobilized on the solid carrier.

[0005] Surprisingly, as described in International Publication No. 2015 / 014888, the activity of immobilized and protected lipases was found to be significantly increased by substances that interact with the lid domain of the lipase. [Brief explanation of the drawing]

[0006] [Figure 1] The process for producing the composition of the present invention is schematically shown: a) A lipase or fragment thereof having a closed lid and a substance (shown as a full circle) that interacts with the lid domain of the lipase or fragment thereof are supplied to a solid carrier to immobilize the lipase or fragment thereof having an open lid onto the solid carrier; b) and c) A protective layer is grown around the immobilized lipase or fragment thereof having an open lid to embed the immobilized lipase or fragment thereof. [Figure 2]Figure 2b shows the 3D structure of pancreatic lipase in two states: a) the inactive conformation (lid closed) and b) the active conformation (lid open). The active site of pancreatic lipase is covered by a lid that prevents substrates from reaching the enzyme's active site (Figure 2a). The opening of the lipase lid is induced by interaction with substances that interact with the lipase lid domain, such as bile salts and / or protein cofactors called collipases, which allows for the stabilization of the active conformation of pancreatic lipase (Figure 2b). [Figure 3] This shows the rate of hydrolysis of lipase substrates by recombinant human pancreatic lipase (HRL) with or without colipase (CLPS) immobilized on the surface of silica nanoparticles (SNPs) and protected by an organosilica layer consisting of APTES, TEOS, and benzyltriethoxysilane (ATB), a) in its free form; and b) with or without colipase (CLPS). [Figure 4] This shows the rate of hydrolysis of lipase substrates by porcine pancreatic lipase (PL) with and without colipase (CLPS) immobilized on the surface of silica nanoparticles (SNPs) and protected by an organic silica layer consisting of APTES, TEOS, and benzyltriethoxysilane (ATB). [Figure 5] The rate of hydrolysis of lipase substrates by free human recombinant lipase (HRL) is shown using gradually increasing concentrations of sodium taurocholate (NaTc). [Figure 6] This shows the rate of hydrolysis of lipase substrates by porcine pancreatic lipase (PL), immobilized on the surface of silica nanoparticles (SNPs) and protected by an organosilica layer consisting of APTES, TEOS, and benzyltriethoxysilane (ATB), with or without sodium taurocholate (NaTc). [Figure 7] This shows the 3D structure of pancreatic lipase activated by a peptide that mimics colipase. [Figure 8]This shows the added value of the covalent bond between the enzyme surface and the protective layer. (A) Quantification of proteins performed on the reaction supernatants of NP-1, NP-1(1), and NP-1(2). (B) HRL loading per dry weight of SNPs. (C) SNP specific activity expressed in UμM / min / g. (D) HRL specific activity expressed in UμM / min / g. [Figure 9] The absorbance of nanoparticles NP-1, NP-1(1), and NP-1(2) at a wavelength of 460 nm is shown. [Modes for carrying out the invention]

[0007] The present invention relates to a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure.

[0008] For the purposes of interpreting this specification, the following definitions apply, and wherever used in the singular, the plural form is also included, and vice versa. It should be understood that the terms used herein are intended solely to describe and not to limit specific embodiments.

[0009] Features, integers, properties, and compounds described in relation to specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, insofar as they do not contradict each other. All features and / or steps of methods or processes disclosed herein (including the claims, abstract, and drawings) can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of the embodiments described above.

[0010] The terms "comprise", "comprises", and "comprising" and their variants are generally used in the sense of "include", that is, "including but not limited to", that is, allowing the presence of one or more features or components.

[0011] The singular forms "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise.

[0012] The term "about" refers to a range of values that is ±10% of the specified value. For example, the expression "about 200" includes ±10% of 200, that is, from 180 to 220.

[0013] As used herein, the term "solid support" generally refers to particles. Preferably, the solid support is monodisperse particles or polydisperse particles, more preferably monodisperse particles. The solid support usually includes organic particles, inorganic particles, organic-inorganic particles, self-organizing organic particles, silica particles, gold particles, titanium particles, preferably silica particles, more preferably silica nanoparticles (SNP). The particle size of the solid support is usually from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, particularly about 50 nm.

[0014] The terms "linker" or "crosslinker" used synonymously herein refer to any linking reagent that includes a group capable of binding to a specific functional group (e.g., primary amine, sulfhydryl, etc.). The linker related to the present invention usually connects the surface of the solid support and lipase. For example, a linker can be immobilized on the surface of the solid support, such as a silica surface as a carrier substance, and then lipase can be bound to the unoccupied binding site of the linker. Alternatively, the linker can first be bound to lipase, and then the linker bound to lipase can bind its unoccupied binding site to the solid support. Various types of linkers are known in the art, including but not limited to straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as tags.

[0015] As used herein, the term "protective layer" refers to a layer for protecting the functional properties of lipase or a fragment thereof, such as lipase or a fragment thereof immobilized on the surface of a solid support. The protective layer of the present invention is usually constructed of building blocks, and at least a part of the building blocks are monomers that can interact with the immobilized lipase, usually by covalent bonds with each other and usually by non-covalent bonds. The protective layer is formed on the surface of the solid support in order to protect the lipase or a fragment thereof immobilized on the solid support. The protective layer is usually a homogeneous layer in which at least 50%, preferably at least 70%, more preferably at least 90% of the lipase or a fragment thereof, for example, is embedded in the protective layer.

[0016] The term “lipase or its fragment” includes naturally occurring lipases or their fragments, as well as artificially engineered lipases or their fragments. Artificially engineered lipases or their fragments are, for example, lipase variants or functionally active fragments. Therefore, in this specification, the terms “lipase fragment,” “its fragment” in relation to lipase, and “functionally active fragment of lipase” are used synonymously. In relation to the lipases of this invention, “variant or functionally active fragment” means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as lipase. Such variants include naturally occurring allelic variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the lipase, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The lipase fragments as defined herein typically have the same functional properties as the lipase, i.e., the full-length enzyme from which it is derived, and include at least a lid domain and a substrate-binding region. The lipase fragments typically contain 100 to 450 amino acids, preferably 150 to 400 amino acids, and more preferably 200 to 350 amino acids.

[0017] As used herein, the term “partially embedded lipase” means that the lipase is not completely covered by the protective layer, and therefore not completely embedded in the protective layer. In one embodiment, less than 50% of the lipase of interest is covered by the protective layer, but typically more, at least 70%, is covered, thereby improving the protection of the lipase. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the lipase of interest is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, and most preferably about 90% to about 95, 96, 97, 98, or 99% of the lipase is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more specifically about 90%, and most specifically about 95% of the lipase of interest is covered by the protective layer. In a particularly more preferred embodiment, about 70%, especially about 80%, more specifically about 90%, and most specifically about 95% of the lipase is covered by a protective layer, while the active site is not covered.

[0018] As used herein, the term “fully embedded lipase” means that the lipase according to the present invention is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered. Preferably, the lipase or a fragment thereof is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered.

[0019] As used herein, the term “at least partially embedded lipase” means that the lipase is at least partially embedded and may be completely embedded by a protective layer. Thus, “at least partially embedded lipase” means that the protective layer covers about 30% to 100%, preferably about 50% to 100%, more preferably about 80% to 100%, even more preferably about 90% to 100%, and most preferably about 95% to 100% of the lipase or fragment thereof, and the active site is preferably covered.

[0020] As used herein, “substances that interact with the lid domain of lipase or its fragments” typically refers to substances that bind to and / or to the region surrounding the lid domain of lipase or its fragments, thereby causing the lid domain to transition the lipase or its fragments to an open structure and / or to maintain the open structure of the lipase or its fragments. The lid domain of lipase is usually amphiphilic; in the closed structure, their hydrophilic side faces the solvent and their hydrophobic side faces directly toward the catalyst pocket (Brocca S., Secundo F., Ossola M., Alberghina L., Carrea G., Lotti M. (2003). Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes. Protein Sci. 12, 2312-2319.10.1110 / ps.0304003). When lipase transitions to an open structure, its hydrophobic surface is exposed and contributes to the substrate-binding region. Preferably, a substance that interacts with the lid domain of the lipase or its fragment locks the lipase or fragment into its active conformation. Once locked into its active conformation, the lipase is usually fully activated. Substances that interact with the lid domain of the lipase or its fragment to cause it to transition to an open structure include collipase or its fragment, collipase-mimicking peptides, and amphiphilic molecules.

[0021] As used herein, the term “amphiphilic molecule” refers to molecules such as chemical compounds that have both a polar (water-soluble) and a nonpolar (water-insoluble) moiety in their structure. The term may also refer to molecules such as chemical compounds that have both hydrophobic and hydrophilic regions. Examples of amphiphilic molecules include bile salts, phospholipids, and nonionic detergents.

[0022] The terms “open structure” or “open structure of lipase or its fragment” as used interchangeably herein refer to a structure of lipase or its fragment in which a substrate can enter and be converted into the active site of the lipase. In a closed structure, the entry of a substrate into the active site of the lipase or its fragment and its conversion are restricted or impossible. The structure of lipase or its fragment, i.e., whether the lipase is in an open or closed structure, can be determined by X-ray crystallography, enzyme activity testing, or site-directed spin labeling (SDSL) method. It can also be determined from electron paramagnetic resonance (EPR).

[0023] As used herein, the term “colipase or fragment thereof” includes naturally occurring colipases or fragments thereof, as well as artificially engineered colipases or fragments thereof. Artificially engineered colipases or fragments thereof are, for example, lipase variants or functionally active fragments. In relation to the colipases of this invention, “variant or functionally active fragment thereof” means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the colipase. Such variants include naturally occurring allelic variants and variants that do not exist naturally. Addition, deletion, substitution, and derivatization of one or more amino acids are intended, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the lipase, more preferably at least about 90% sequence identity, even more preferably at least about 95% sequence identity, and most preferably at least about 98% sequence identity. The collipase fragments defined herein have the same functional properties as the collipase from which they are derived. The preferred collipase is the collipase having uniplot number: P02703.

[0024] As used herein, the term “colipase-mimicking peptide” refers to a peptide consisting of 10 to 40 amino acids, which allows specific amino acid residues to be geometrically positioned in appropriate locations to interact with the amino acids of the pancreatic lipase structure, thereby inducing conformational elongation and lid opening of the lipase, and thereby possessing the same functional properties as a lipase. The lipase-mimicking peptide that can be used in the present invention is preferably the peptide shown in SEQ ID NO: 1.

[0025] As used herein, the term “bile salt” refers to bile acids conjugated with taurine or glycine, and includes sodium taurocholate, sodium glycocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, sodium glycochenodeoxycholate, and sodium taurochenodeoxycholate.

[0026] As used herein, the term “nonionic detergent” refers to a surfactant, and includes tetraethylene glycol monooctyl ether, octyl-bD-glucopyranoside, N,N-dimethyldodecylamine-N-oxide, and b-octylglucomaltoside.

[0027] As used herein, the term “phospholipid” refers to a class of lipids whose molecules have a hydrophilic “head” containing a phosphate group, linked by an alcohol residue (usually a glycerol molecule), and two hydrophobic “tails” derived from fatty acids. Phospholipids include lecithins and lysolecithins.

[0028] In a first embodiment, the present invention provides a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure.

[0029] Lipase or its fragments can be immobilized on the surface of a solid support by non-covalent or covalent bonds. Non-covalent bonds include electrostatic interactions such as pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and ionic interactions. Preferably, lipase or its fragments are immobilized on the surface of a solid support by covalent bonds or by covalent bonds via a linker.

[0030] A solution of lipase or its fragments typically contains the protein or its fragments in a buffer. Commonly usable buffers include phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. In addition, the solution may contain sugar alcohols or nonionic surfactants, as described herein. A solution of lipase or its fragments can be prepared, for example, by dissolving the lipase or its fragments in water to reconstitute a stock buffer for the lipase or its fragments.

[0031] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, and titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size is usually measured by measuring the diameter of the particles and is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is monodisperse particles, its size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is polydisperse particles, its size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, and particularly 50 nm to 50 μm. In one embodiment, the composition comprises a solid support, with U per gram of solid support μM / min The relative enzyme activity of lipase on a solid carrier, as measured, is at least 20 U per gram of solid carrier. μM / min Preferably, at least 30U per gram of solid carrier. μM / min In particular, 20 to 50 U per gram of solid carrier μM / min More specifically, 30 to 40 U per gram of solid carrier μM / min That is the case.

[0032] Typically, monodisperse particles or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in the present invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In a more preferred embodiment, the polydisperse particles are non-spherical polydisperse particles.

[0033] Solid carriers are usually provided in suspension. The suspension of solid carriers can be done, for example, in water, a buffer, or a nonionic surfactant, or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. Nonionic surfactants are typically ethoxylated sorbitan esters such as EG-40 diisostearate P-sorbitan, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated esters such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The material is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80).The buffers that can be used in the method of the present invention include phosphates, piperazine-N,N′-bis(2-ethanesulfonic acid), 2-hydroxy-3-morpholinopropanesulfonic acid, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), (3-(N-morpholino)propanesulfonic acid), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3-(N,N-bis[2-hydroxy These are ethyl]amino)-2-hydroxypropanesulfonic acid, N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, diglycine, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0034] In one embodiment, a substance that interacts with the lid domain of the lipase or fragment thereof is added to a suspension of the solid carrier, preferably, the substance that interacts with the lid domain of the lipase or fragment thereof is added to the suspension of the solid carrier together with the lipase or fragment thereof before the lipase or fragment thereof is immobilized on the solid carrier.

[0035] The immobilization of lipase onto a solid carrier is typically carried out by adding a solution of lipase to a suspension of the solid carrier. In a preferred embodiment, the immobilization of lipase onto a solid carrier is carried out by providing a suspension of the solid carrier and a solution of lipase, and the suspension of the solid carrier is incubated with the solution of lipase so that the lipase can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of lipase onto a solid carrier is carried out by providing a suspension of the solid carrier, a solution of lipase, and a solution of a substance that interacts with the lid domain of the lipase or a fragment thereof, and the suspension of the solid carrier is incubated with the solution of lipase and the solution of a substance that interacts with the lid domain of the lipase or a fragment thereof so that the lipase can bind to the surface of the solid carrier.

[0036] In a more preferred embodiment, the lipase is immobilized on a solid carrier by a linker, preferably a bifunctional crosslinker that binds to the surface of the lipase and the solid carrier.

[0037] In one embodiment, the surface of a solid support is modified to introduce molecules or functional chemical groups as anchor points, i.e., anchor points for lipase or for linkers connecting lipase to the solid support. Preferably, the anchor points are amine functional chemical groups or parts thereof. In a non-limiting example, an amino-modified surface of a solid support, such as an amino-modified silica surface, can be used as a modified solid support. Such an amino-modified surface of a solid support can be obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support having a silica surface containing an amino-modified surface, and more preferably a solid support obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Such a modified support can form amide bonds between lipase and amine groups on the surface of the support material, or amide bonds between linkers and amine groups on the surface of the support material. In one embodiment, the molecules or functional chemical groups introduced as anchor points are uniformly distributed on the surface of the solid support.

[0038] In a more preferred embodiment, the lipase is immobilized on a solid carrier by introducing the molecule described above as an anchor point for the lipase, or by modifying the surface of the solid carrier at least partially by using a linker, preferably a bifunctional crosslinker that binds to the anchor point of the solid carrier and the lipase or a fragment thereof.

[0039] In one embodiment, the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, a colipase-mimicking peptide, and an amphiphilic molecule. Preferably, the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, a colipase-mimicking peptide, and a bile salt; more preferably, it is selected from the group consisting of colipase or a fragment thereof, a colipase-mimicking peptide, and sodium taurocholate; and even more preferably, it is selected from the group consisting of colipase or a fragment thereof, the colipase-mimicking peptide shown in SEQ ID NO: 1, and sodium taurocholate.

[0040] In one embodiment, a substance interacting with the lid domain of the lipase or fragment thereof specifically interacts with the lid domain of the lipase or fragment thereof such that the lipase or fragment thereof transitions to an open structure and / or maintains an open structure.

[0041] In one embodiment, about 50% to 100%, preferably about 80% to 100%, more preferably about 90% to 100%, and even more preferably about 100% of the lipase or fragment thereof immobilized on the surface of the solid carrier is in an open structure.

[0042] In some embodiments, the protective layer has a specified thickness of about 1 to about 200 nm, typically about 1 to about 100 nm, preferably about 1 to about 50 nm, more preferably about 1 to about 25 nm, even more preferably about 1 to about 20 nm, and particularly about 1 to about 15 nm. The most preferred specified thickness is about 1 to about 10 nm. In some embodiments, the layer has a specified thickness of about 5 to about 100 nm, preferably about 5 to about 50 nm, more preferably about 5 to about 25 nm, even more preferably about 5 to about 20 nm, and particularly about 5 to about 15 nm. The most preferred specified thickness is about 5 to about 10 nm. The protective layer is usually porous, with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0043] The thickness of the protective layer can be measured using a microscope such as a scanning electron microscope (SEM), transmission electron microscope (TEM), or scanning probe microscope (SPM), or by light scattering or ellipsometry.

[0044] In one embodiment, the lipase or fragment thereof is partially embedded by the protective layer. In a preferred embodiment, the lipase or fragment thereof is at least partially embedded by the protective layer. In a more preferred embodiment, the lipase or fragment thereof is completely embedded by the protective layer. In one embodiment, the protective layer is embedded with a solid carrier and the lipase or fragment thereof immobilized on the surface of the solid carrier. Preferably, the protective layer is completely embedded with a solid carrier and the lipase or fragment thereof immobilized on the surface of the solid carrier. When the solid carrier is completely embedded in the protective layer and the lipase or fragment thereof immobilized on the surface of the solid carrier is completely embedded, the lipase or fragment thereof is completely, i.e., 100%, covered by the protective layer, i.e., the active site is also covered, and the solid carrier is completely, i.e., 100%, covered by the protective layer.

[0045] In preferred embodiments, the lipase or fragment thereof is recombinant human pancreatic lipase (HRL) or fragment thereof, or porcine pancreatic lipase or fragment thereof, preferably recombinant human pancreatic lipase (HRL) or fragment thereof, more preferably full-length recombinant human pancreatic lipase (HRL).

[0046] The compositions of the present invention are typically produced in a reaction vessel, such as a reactor. The reaction vessel typically contains a suspension comprising lipase or fragments thereof immobilized on a solid support and a substance that interacts with the lid domain of the lipase or fragments thereof. The formation of the protective layer is typically carried out by forming each protective layer with building blocks, which construct the protective layer in a polycondensation reaction. The polycondensation can be carried out in different solvents, preferably aqueous solutions. The polycondensation can be easily controlled and stopped as needed, and it is possible to achieve a specified thickness of the protective layer. The selection of building blocks that can be used to construct the protective layer may depend on the known structure of the lipase to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building blocks for the protective layer, structural building blocks and protective building blocks are typically used to construct the protective layer. A structural building block that can be used is, for example, tetraethyl orthosilicate (referred to herein as "TEOS" or "T"). Suitable protective building blocks may include, for example, 3-aminopropyltriethoxysilane (referred to herein as "APTES" or "A"), propyltriethoxysilane (referred to herein as "PTES" or "P"), isobutyltriethoxysilane (referred to as "IBTES"), hydroxymethyltriethoxysilane (referred to herein as "HTMEOS" or "H"), benzyltriethoxysilane (referred to herein as "BTES"), ureidopropyltriethoxysilane (referred to as "UPTES"), or carboxyethyltriethoxysilane (referred to herein as "CETES"). Structural building blocks are typically precursors of inorganic silica and can form four covalent bonds in the resulting layer. Protective building blocks are typically organosilanes and retain an organic moiety that is well-suited to interacting with lipases. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes.More preferred structural building blocks are mixtures of tetravalent and trivalent silanes, particularly mixtures of tetra-alkoxysilanes and tri-alkoxysilanes. Even more preferred structural building blocks are selected from the group consisting of tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate.A more preferred protective building block is selected from the group consisting of carboxyethylsilanetriol, benzylsilane, propylsilane, isobutylsilane, n-octylsilane, hydroxysilane, bis(2-hydroxyethyl)-3-aminopropylsilane, aminopropylsilane, ureidopropylsilane, (N-acetylglycyl)-3-aminopropylsilane, and hydroxy(polyethyleneoxy)propyl]triethoxysilane, and is particularly selected from benzyltriethoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, and (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimethoxysilane, Selected from sobutylmethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltriethoxysilane, (N-acetylglycyl)-3-aminopropyltrimethoxysilane, or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureidopropyltrihydroxyethoxysilane, (N-acetylglycyl)-3-aminopropyltrihydroxymethoxysilane.

[0047] Particularly preferred building blocks are TEOS as a structural building block, and APTES, benzyltriethoxysilane BTES, and / or HTMEOS, preferably APTES or benzyltriethoxysilane, as protective building blocks. In particular, TEOS as a structural building block and APTES or benzyltriethoxysilane as protective building blocks are used to construct the protective layer.

[0048] The reaction time between the building block and the solid support bearing the immobilized lipase or its fragments may depend on the length of the linker, if one is used, and the size of the lipase. The reaction typically takes place over a period of 0.5 to 10 hours, preferably 1 to 8 hours, more preferably 2 to 6 hours, and even more preferably 5 hours, preferably in an aqueous solution, preferably at room temperature at about 5 to about 25°C or about 20°C. The formation of the protective layer can be stopped by actively halting the polycondensation reaction, for example, by removing unreacted building blocks through a washing process, or by the self-termination of the polycondensation reaction caused by a limited amount of building blocks.

[0049] In a more preferred embodiment, the lipase is immobilized on a solid support by at least partially modifying the surface of the solid support by introducing the molecule described above as an anchor point for the lipase, and by using a linker, preferably a crosslinker that binds to the anchor point and the lipase.

[0050] In one embodiment, the molecules and / or linkers introduced as anchor points are uniformly distributed on the surface of the solid support.

[0051] In preferred embodiments, the crosslinker may be glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) Selected from the group consisting of HCl), DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl (e.g., sulfhydryl-reactive 2-pyridyldithio), and a colipase-mimicking peptide, the colipase-mimicking peptide may be functionalized with a chemical group that enables covalent bonding to the surface of a solid support.In a more preferred embodiment, the crosslinker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2HCl), DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated sulfhydrils (e.g., sulfurhydryl-reactive 2-pyridyldithio). Most preferably, glutaraldehyde is selected.

[0052] After the protective layer is formed, the resulting suspension can be washed to remove any excess substances that interact with the lid domains of the lipase or its fragments. In one embodiment, after the protective layer is formed, the solid carrier containing the lipase and the protective layer are stored. Storage is usually achieved, for example, by washing the formed composition with a buffer and storing it suspended or dissolved in the buffer for a desired period of time. In a preferred embodiment, the solid carrier containing the lipase and the protective layer are stored at a constant temperature of 2 to 25°C. In a more preferred embodiment, the solid carrier containing the lipase and the protective layer are stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing the lipase and the protective layer are stored for 10 to 30 hours at a constant temperature between 2 and 25°C, preferably at room temperature.

[0053] In a further embodiment, the present invention provides the aforementioned compositions for use as pharmaceuticals.

[0054] In further embodiments, the present invention provides compositions for use in enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in methods for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI). In preferred embodiments, the present invention provides compositions for use in methods for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI). In further preferred embodiments, the present invention provides compositions for use in enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy.

[0055] Furthermore, the use of the composition described herein for the manufacture of a medicament for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided. Furthermore, the use of the composition described herein for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided. Furthermore, a method for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject. Furthermore, the use of the composition described herein for the manufacture of a medicament for a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, is provided. Furthermore, the use of the composition described herein in a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject is provided. Furthermore, a method for enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject.

[0056] The compositions according to the present invention are preferably pharmaceutical compositions comprising a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions according to the present invention are suitable for oral administration to a subject. Unless otherwise indicated, the pharmaceutical compositions according to the present invention are prepared by known methods.

[0057] Exemplary treatment regimens involve administration once daily, twice daily, three times daily, every other day, twice a week, or once a week. The composition, for example, the pharmaceutical composition of the present invention, is usually administered in multiple doses. The interval between doses can be, for example, less than one day, daily, every other day, twice a week, or weekly. The composition, for example, the pharmaceutical composition of the present invention, may be administered as a continuous, uninterrupted treatment. The composition, for example, the pharmaceutical composition of the present invention, may be given in a regimen in which the subject undergoes treatment cycles interrupted by drug-free or non-treatment periods. Thus, the composition, for example, the pharmaceutical composition of the present invention, may be administered according to the selected intervals above over a continuous period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks, and then stopped for a period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks. The composition, for example, the pharmaceutical composition of the present invention, may conveniently be administered in unit dose form. The unit of enzyme activity ("U") can be expressed as the weight or mass of substrate hydrolyzed per unit time.

[0058] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of the composition of the present invention that is capable of producing one or more desired effects in a subject to which it is administered. Determining the therapeutic effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0059] As used herein, the terms “treatment” and “to treat” include: (1) delaying the onset of clinical symptoms of a condition, disorder, or pathology in animals, particularly mammals, particularly humans, that are suffering from or susceptible to a condition, disorder, or pathology but have not yet experienced or manifested clinical or subclinical symptoms of the condition, disorder, or pathology; (2) inhibiting a condition, disorder, or pathology (e.g., stopping, reducing, or delaying the onset of the disease with respect to at least one clinical or subclinical symptom, or stopping, reducing, or delaying its recurrence in the case of maintenance treatment); and / or (3) reducing a pathology (i.e., causing a regression of the condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms). The benefit to the patient being treated is statistically significant or at least perceptible to the patient or physician. However, it will be understood that when a patient is given medicine to treat a disease, the result is not always an effective treatment.

[0060] As used herein, “slow progression” means extending the time until the onset of symptoms. Furthermore, as used herein, “slow progression” includes a setback or inhibition of disease progression. “Inhibition” of disease progression or disease complications in a subject means preventing or mitigating disease progression and / or disease complications in a subject.

[0061] Preventive measures include prophylactic treatment. For preventive use, the drug combination of the present invention is administered to subjects suspected of having or at risk of developing the above-mentioned disease or disorder. For therapeutic use, the drug combination is administered to subjects, for example, patients already suffering from the above-mentioned disease or disorder, in an amount sufficient to cure or at least partially cessate the symptoms of the disease. The effective dose for such use will depend on the severity and course of the disease, previous treatments, the subject's health status and response to the drug, and the judgment of the attending physician.

[0062] If the target condition does not improve, the pharmaceutical combination of the present invention may be administered chronically, i.e., over a long period including the subject's entire life, to improve, or otherwise control or limit the symptoms of the target disease or condition.

[0063] If the patient's condition improves, the drug combination can be administered continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily stopped over a specific period (i.e., a “drug-free period”). Once the patient’s condition improves, a maintenance dose of the drug combination of the present invention may be administered as needed. Thereafter, the dose, or the frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved disease is maintained.

[0064] In a further embodiment, the present invention provides a method for producing a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure, the method comprising the following steps: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, (c) A step of providing a substance that interacts with the lid domain of lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof onto a solid carrier. (f) A step of forming a protective layer on the surface of a solid carrier in order to protect the lipase or fragment thereof immobilized on the solid carrier.

[0065] Step (a) is usually carried out by providing the solid carrier suspended in water or a buffer, preferably in water, a nonionic surfactant or buffer or a mixture thereof, preferably in a buffer, more preferably suspended in water and / or a nonionic surfactant, even more preferably suspended in water and / or a nonionic surfactant without a buffer in the suspension, particularly suspended in a mixture of water and a nonionic surfactant, and more specifically suspended in a mixture of water and a nonionic surfactant without a buffer in the suspension. The suspension can be stirred, for example, at 400 rpm, 20°C for 30 minutes. Steps b) and c) can usually be carried out separately or at the same time, for example, both lipase and substance can be provided in a single solution. Immobilization of lipase onto the solid carrier in step e) of this method is usually carried out by adding a solution of lipase, or a solution containing lipase and substance, to the suspension of the solid carrier. Preferably, a linker connecting the solid carrier and the lipase or its fragments is added to the suspension of the solid carrier before adding the solution of the lipase or its fragments to the suspension of the solid carrier. In a preferred embodiment, the immobilization of the lipase to the solid carrier is carried out by providing a suspension of the solid carrier and adding a solution of the lipase or a solution containing the lipase or a substance, and the suspension to which the solution of the lipase has been added is incubated so that the lipase can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of lipase or its fragments onto a solid carrier in step (e) is carried out by i) adding a linker to the solid carrier provided in step a), preferably adding the linker to a suspension of the solid carrier provided in step a), and ii) adding the lipase or its fragments provided in step (b), preferably adding a solution of the lipase or its fragments provided in step (b) to the solid carrier and linker, or to a suspension containing the solid carrier and linker, wherein in step (e), the linker connects the solid carrier to the lipase or its fragments.In one embodiment, a building block of the protective layer, preferably a monomer of the building block of the protective layer, more preferably an organosilane, even more preferably a triethoxysilane, particularly APTES, is added to the solid carrier and linker, or to a suspension containing the solid carrier and linker, before the addition of a solution of lipase or its fragments.

[0066] In preferred embodiments, the surface of the solid carrier is at least partially modified to improve the immobilization of lipase to the solid carrier. In particular, the surface of the solid carrier is at least partially modified before the lipase is immobilized. The surface of the solid carrier can be at least partially modified by adding molecules to the surface of the solid carrier as anchor points for the lipase, as described above.

[0067] The suspension containing the solid support is typically incubated after each of the above addition steps so that the lipase or fragment thereof is linked to the solid support, preferably the surface of the solid support, and to the lipase or fragment thereof via a linker, preferably by covalent bonds, thereby immobilizing the lipase or fragment thereof to the solid support, respectively, enabling reactions between, for example, the solid support and the molecule as an anchor point, the solid support and the linker, and the solid support containing the linker and the lipase or fragment thereof.

[0068] In one embodiment, in step (e), the lipase or fragment thereof is immobilized on the solid carrier by linking the solid carrier and the lipase or fragment thereof via a linker, preferably by linking the solid carrier and the lipase or fragment thereof via a linker, and the solid carrier is linked to the lipase or fragment thereof by covalent bonds between the linker and the solid carrier and between the linker and the lipase or fragment thereof. Preferably, i) a linker is added to the solid carrier provided in step (a), ii) the lipase or fragment thereof provided in step (b) is added to the solid carrier and the linker, and in step (e), the linker links the solid carrier and the lipase or fragment thereof. The linker used is as described above and preferably links the surface of the solid carrier and the lipase or fragment thereof by covalent bonds. More preferably, the linker is added to the solid carrier provided in step (a) in a molar excess relative to the lipase or fragment thereof provided in step (b), preferably, the linker is added to the solid carrier in step (b) in a molar excess of 1 to 1000 times relative to the lipase or fragment thereof provided in step (b), more preferably, the linker is added to the solid carrier in step (b) in a molar excess of 2 to 300 times relative to the lipase or fragment thereof provided in step (b), and even more preferably, the linker is added to the solid carrier in step (b) in a molar excess of 4 to 250 times relative to the lipase or fragment thereof provided in step (b), and in particular, the linker is added to the solid carrier in step (b) in a molar excess of 205 times relative to the lipase or fragment thereof provided in step (b).

[0069] In a preferred embodiment, linkers that did not connect the solid carrier to the lipase or its fragments in step (e) are present while a protective layer is formed on the surface of the solid carrier in step (f). In a more preferred embodiment, linkers, or a portion thereof, that did not connect the solid carrier to the lipase or its fragments in step (e) covalently bond the protective layer to the lipase or its fragments in step (f). In an even more preferred embodiment, linkers that did not connect the solid carrier to the lipase or its fragments in step (e) are not removed in step (e) or step (f), or between steps (e) and (f). In a particular embodiment, linkers that did not connect the solid carrier to the lipase or its fragments in step (e) are not removed in step (e) or step (f), or between steps (e) and (f), and linkers, or a portion thereof, that did not connect the solid carrier to the lipase or its fragments in step (e) covalently bond the protective layer to the lipase or its fragments in step (f). ii) After adding the protein, the amount of linker that did not link the solid carrier and the lipase or its fragment in step (e) is usually 30% to 70%, preferably 40% to 60%, and more preferably 50%, of the amount of linker added to the solid carrier in step (e). In one embodiment, there is no washing step between adding the linker to the solid carrier provided in step (a) in (i) and adding the lipase or its fragment to the solid carrier and linker in (ii). In one embodiment, there is no washing step between any of steps (a) to (f). In one embodiment, there is no washing step between adding the linker to the solid carrier provided in step (e) in (i) and adding the lipase or its fragment to the solid carrier and linker in ii), and there is no washing step between any of steps (a) to (f).

[0070] In one embodiment, the linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) A selection is made from the group consisting of HCl, DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), preferably glutaldehyde.

[0071] In preferred embodiments, the linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2 It is selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), and is preferably glutaldehyde.

[0072] The formation of the protective layer in step (f) of this method is usually carried out by forming each protective layer using building blocks, and the building blocks construct the protective layer through the polycondensation reaction described above.

[0073] In one embodiment, the method includes the following steps: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, (c) A step of immobilizing lipase or a fragment thereof onto a solid carrier. (d) A step of providing a substance that interacts with the lid domain of a lipase or a fragment thereof, (e) A step of interacting the lipase or fragment thereof from (c) with the substance from (d), (f) A step of forming a protective layer on the surface of a solid carrier in order to protect the lipase or fragment thereof immobilized on the solid carrier.

[0074] Steps (a) through (f) can be carried out similarly to steps (a) through (f) of the method provided in a further aspect of the present invention, considering that step (c) of the above method corresponds to step (e) of the method provided in a further aspect of the present invention.

[0075] In one embodiment, the protective layer is formed by building blocks, where structural building blocks and protective building blocks are used to form the protective layer, the structural building blocks being inorganic silica precursors capable of forming four covalent bonds in the layer being formed, and the protective building blocks being the aforementioned organic silanes.

[0076] In one embodiment, approximately 30% to 100% of the lipase is embedded in the protective layer.

[0077] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles, and titanium particles, preferably silica particles, and more preferably silica nanoparticles (SNPs).

[0078] A preferred method of the present invention is a method for producing a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure, and this method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspended state, preferably in a suspended state in a buffer, water and / or a nonionic surfactant, and more preferably in a suspended state in a mixture of water and a nonionic surfactant. (b) A step of providing lipase or a fragment thereof, (c) A step of providing a substance that interacts with the lid domain of lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof onto a solid carrier, preferably wherein the surface of the solid carrier is at least partially modified before the lipase or fragment thereof is immobilized onto the solid carrier, i) a linker is added to the suspension of the solid carrier provided in step (a), or i) a linker is added to the suspension of the solid carrier provided in step (a) after at least partially modifying the surface of the solid carrier, and ii) a solution of the lipase or fragment thereof provided in step (b), preferably a solution of the lipase or fragment thereof provided in step (b), is added to the suspension of the solid carrier and the linker, the linker linking the solid carrier and the lipase or fragment thereof, (f) A step of forming a protective layer on the surface of a solid carrier in order to protect lipase or a fragment thereof immobilized on the solid carrier, wherein a linker, or a part thereof, that did not connect the solid carrier and the lipase or fragment thereof in step (e), covalently bonds the protective layer to the lipase or fragment thereof. Alternatively, steps (a) through (f) can be carried out as similar to steps (a) through (f) of the aforementioned method, considering that step (e) of the above method corresponds to step (c) of the aforementioned method.

[0079] Furthermore, a composition is provided comprising a solid carrier, an open-structured lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure, and this composition can be obtained by the present method, particularly by the preferred method of the present invention described above. [Examples]

[0080] Materials and methods reagent: - 99% tetraethyl orthosilicate (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), polysorbate 80, recombinant human pancreatic lipase (HRL, certified reference substance), porcine pancreatic lipase (4x USP standard), 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate), Tris base, collipase, and sodium taurocholate hydrate were purchased from Sigma-Aldrich. HRL, porcine pancreatic lipase, and collipase were dissolved in water to reconstitute the stock buffer. - Benzyltriethoxysilane (B, 96%) was purchased from abcr GmbH. - The trifluoroacetate salt of peptide Glu-Leu-Gly-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Glu-Gly-Gly-Gly-Glu-Arg-Gly-Gly-Gly-Gly-Asn-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 1), which has an E-azide-Nle-OH at its carboxyl terminus, was purchased from Bachem.

[0081] Synthesis of silica nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20000 g for 20 minutes and subsequently washed with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with the image analysis software Olympus Stream Motion.

[0082] Free recombinant human pancreatic lipase activity assay: A solution of recombinant human pancreatic lipase (3.43 μL, 1 mg / mL) in Tris buffer (0.1 M, pH 8.4, 56.6 μL) was added to 1,2-di-O-lauroyl-rac-glycero-3-(6-methylresorufin glutarate) (60 μL, 100 μM). The kinetics of lipase activity were monitored by steady-state fluorescence measurements (λ ex / λ em = 529 / 600 nm) at 37 °C for 30 min in a black 96-well plate.

[0083] Free recombinant human pancreatic lipase activity assay in the presence of colipase: A solution of recombinant human pancreatic lipase (3.43 μL, 1 mg / mL) and colipase (0.72 μL, 3 μg / mL) in Tris buffer (0.1 M, pH 8.4, 55.8 μL) was added to 1,2-di-O-lauroyl-rac-glycero-3-(6-methylresorufin glutarate) (60 μL, 100 μM). The kinetics of lipase activity were monitored by steady-state fluorescence measurements (λ ex / λ em = 529 / 600 nm) at 37 °C for 30 min in a black 96-well plate.

[0084] Immobilized and protected recombinant human pancreatic lipase activity assay: A solution of immobilized and protected recombinant human pancreatic lipase (20 μL, 10 mg / mL) in Tris buffer (0.1 M, pH 8.4, 40 μL) was added to 1,2-di-O-lauroyl-rac-glycero-3-(6-methylresorufin glutarate) (60 μL, 100 μM). The kinetics of lipase activity were monitored by steady-state fluorescence measurements (λ ex / λ em = 529 / 600 nm) at 37 °C for 30 min in a black 96-well plate.

[0085] Immobilized and protected porcine pancreatic lipase activity assay: To a solution of immobilized and protected porcine pancreatic lipase (15 μg / mL enzyme) in Tris-buffer (0.1 M, pH 8.4), 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (50 μM) was added. The kinetics of lipase activity were measured by steady-state fluorescence (λ) in a dark 96-well plate at 37°C for 30 minutes. ex / λ em Monitoring was performed using (=529 / 600nm).

[0086] Example 1 A) Immobilization and shielding of recombinant human pancreatic lipase (HRL) SNP (10 mg / mL) in H2O / PS80 (8 mg / L) was mixed with APTES (3.8 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Recombinant human pancreatic lipase solution (525 μg / mL, 11 μM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized lipase using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-HRL-ATB was cured overnight in a 20°C water bath.

[0087] B) Co-immobilization and shielding of recombinant human pancreatic lipase (HRL) and colipase (CLPS): SNPs (10 mg / mL) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. A solution containing recombinant human pancreatic lipase (525 μg / mL, 11 μM) and colipase (110 μg / mL) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized protein using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-HRL-CLPS-ATB was cured overnight in a 20°C water bath.

[0088] Activation of recombinant human pancreatic lipase (HRL) by colipase The activation of recombinant human pancreatic lipase by a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester), was investigated (Figure 3a). The faster rate of hydrolysis of the lipase substrate by lipase in the presence of a lipase compared to lipase alone indicates appropriate activation of the lipase enzyme by the lipase.

[0089] To generate activated lipase nanoparticles, recombinant human pancreatic lipase was co-immobilized with a colipase and protected with a hydrophobic organic silica shield on the surface of the silica nanoparticles, as described in B) above. The activation of lipase by the colipase was evaluated using a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate) (Figure 3b). The fact that the hydrolysis rate of the lipase substrate by the lipase co-immobilized with the colipase, as described in B), was faster than that of lipase immobilized without the colipase, as described in A), indicates that the activation of the lipase enzyme by the colipase in the shielded nanoparticles was surprisingly more than three times faster than that of lipase without the colipase. These results confirm the strategy of co-immobilizing lipase and colipase on the surface of silica nanoparticles to generate nanoparticles that retain immobilized and protected lipase maintained within its active conformation.

[0090] Example 2 A) Immobilization and shielding of porcine pancreatic lipase (PL): SNP (10 mg / mL) in H2O / PS80 (8 mg / L) was mixed with APTES (3.8 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Porcine pancreatic lipase solution (1.9 mg / mL, 39 μM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lipase using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-PL-ATB was cured overnight in a 20°C water bath.

[0091] B) Co-immobilization and shielding of porcine pancreatic lipase (PL) and colipase (CLPS): SNPs (10 mg / mL) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. A solution containing porcine pancreatic lipase (1.9 mg / mL, 39 μM) and colipase (150 μg / mL) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized protein using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-PL-CLPS-ATB was cured overnight in a 20°C water bath.

[0092] Activation of porcine pancreatic lipase (PL) by colipase To generate activated lipase nanoparticles, porcine pancreatic lipase was co-immobilized with a colipase and protected with a hydrophobic organic silica shield on the surface of the silica nanoparticles, as described in B) above. The activation of lipase by the colipase was evaluated using a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate) (Figure 4). As described in B) above, the rate of hydrolysis of the lipase substrate by the lipase co-immobilized with the colipase was faster than that of lipase immobilized without the colipase, as described in A) above, indicating that the activation of the lipase enzyme by the colipase in the shielded nanoparticles was, surprisingly, three times greater than that of the lipase enzyme without the colipase. These results confirm the strategy of co-immobilizing lipase and colipase on the surface of silica nanoparticles to generate nanoparticles that hold immobilized and protected lipase maintained within its active conformation. This also demonstrates the versatility of this method in terms of the origin of the enzyme used.

[0093] Example 3 Immobilization and shielding of porcine pancreatic lipase (PL) in the presence of sodium taurocholate (NaTc): SNP (10 mg / mL) was added to a mixture of NaTc (2 mM), H2O, and H2O / PS80 (8 mg / L), to which APTES (3.8 mM) was added. The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Porcine pancreatic lipase (1.9 mg / mL, 39 μM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized lipase using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-PL-NaTc-ATB was cured overnight in a 20°C water bath.

[0094] Activation of recombinant human pancreatic lipase (HRL) by sodium taurocholate (NaTc) The activation of recombinant human pancreatic lipase by sodium taurocholate was investigated using the fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (Figure 5). The higher rate of hydrolysis of the lipase substrate by lipase in the presence of gradually increasing concentrations of sodium taurocholate compared to lipase alone indicates appropriate activation of the lipase enzyme by bile salts.

[0095] Activation of porcine pancreatic lipase (PL) by sodium taurocholate (NaTc) To generate activated lipase nanoparticles, porcine pancreatic lipase was immobilized in the presence of sodium taurocholate and protected with a hydrophobic organic silica shield on the surface of silica nanoparticles. The activation of lipase by sodium taurocholate was evaluated using a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate) (Figure 6). The rate of hydrolysis of the lipase substrate by lipase immobilized in the presence of sodium taurocholate was faster than that of lipase immobilized without bile salts, indicating that the activation of the lipase enzyme by sodium taurocholate in the shielded nanoparticles was, surprisingly, almost three times faster than that of lipase without sodium taurocholate. These results confirm the strategy of immobilizing lipase on the surface of silica nanoparticles in the presence of sodium taurocholate to generate nanoparticles that retain immobilized and protected lipase maintained within its active conformation.

[0096] Example 4: Activation of pancreatic lipase (PL) by a colipase-mimicking peptide Analysis of the lipase-colipase complex made it possible to identify the amino acid residues responsible for the lipase-colipase interaction. Based on these findings, the inventors designed a peptide represented by Sequence ID No. 1 (Glu-Leu-Gly-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Glu-Gly-Gly-Gly-Glu-Arg-Gly-Gly-Gly-Gly-Asn-Gly-Gly-Gly-Gly-Gly (Figure 7). This peptide was chemically modified at its carboxyl terminus by adding an E-azide-Nle-OH group, enabling crosslinking of the lipase enzyme on the surface of silica nanoparticles by click chemistry.

[0097] Example 5: Generation of NP-1 variants: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and enzyme activity.

[0098] In the initial experiment, nanoparticles (NP-1(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L) was mixed with APTES (3.1 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Sodium taurocholate (2 mM) and human recombinant lipase (0.732 g / L, 15.2 μM) were added sequentially, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized HRL using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then removed from H2O / The sample was washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a 20°C water bath.

[0099] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and nanoparticles (NP-1(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. APTES (3.1 mM) was added to SNP (10 mg / mL, 69 nm) and PS80 (8 mg / L) in phosphate buffer (25 mM, pH 7.5). The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Sodium taurocholate (2 mM) and human recombinant lipase (0.732 g / L, 15.2 μM) were added successively, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of immobilized HRL using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-1(2) was cured overnight in a water bath at 20°C.

[0100] In the third initial experiment, nanoparticles (NP-1) were generated in H2O / PS80 (8 mg / L). The nanoparticles were not washed between each chemical step to retain excess glutaraldehyde in the reaction mixture, which did not bind the solid support to the manipulated HRL. Therefore, glutaraldehyde remained present during layer growth, inducing covalent bonding between the protective layer and the human recombinant lipase. APTES (3.1 mM) was added to SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.1 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Sodium taurocholate (2 mM) and human recombinant lipase (0.732 g / L, 15.2 μM) were added sequentially, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized HRL using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then subjected to H2O / The NP-1 was washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). NP-1 was cured overnight in a 20°C water bath.

[0101] The covalent bond between the protective layer and HRL can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of imine bonds through the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine amino acids in the HRL and organosilica layers. The absorbance of nanoparticles NP-1(1), NP-1(2), and NP-1 at a wavelength of 460 nm was measured after the formation of the organosilica layer and the final particle washing (see Figure 9A). The results showed that NP-1 absorbed much more light than NP-1(2) at this wavelength. Interestingly, NP-1 and NP-1(1) appeared darker to the naked eye, but their absorbances were similar. This suggests that the instrument could not distinguish the formation of imine bonds due to strong interference from the particles themselves (due to the small amount of enzyme used).

[0102] To overcome this limitation, the inventors used an inverted microscope to image NP (Figure 9B). At the same concentration, NP-1 appeared significantly darker than NP-1(1) under the microscope. This visual confirmation indicates that NP-1 has more imine bonds, which is likely due to covalent bonding of a protective layer against human recombinant lipase.

[0103] Improvement of enzyme stability and specific activity through covalent bonding to the protective layer In the first experiment, nanoparticles NP-1(1) were generated under unbuffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, nanoparticles NP-1(2) were generated under buffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the third experiment, nanoparticles NP-1 were generated under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0104] To determine the HRL immobilization yield on the surfaces of NP-1(1), NP-1(2), and NP-1, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield increased fourfold under conditions where the presence of glutaraldehyde was maintained (NP-1) (Figure 8A), and consequently, the enzyme load per dry weight of SNP increased fourfold compared to buffer conditions where glutaraldehyde was removed by the washing step (NP-1(2)) (Figure 8B).

[0105] The lipase biocatalytic activity of NP-1(1), NP-1(2), and HRL immobilized and protected on NP-1 was evaluated. Even more surprising than the increased enzyme immobilization load when glutaraldehyde was maintained was the 37-fold increase in the specific activity of the nanoparticles compared to buffered conditions in which glutaraldehyde was removed by the washing step, and the 2-fold increase compared to unbuffered conditions in which glutaraldehyde was removed by the washing step (Figure 8C). These astonishing 37-fold and 2-fold increases in the specific activity of the nanoparticles correspond to the HR specific activity (U per 1g of HRL) of the enzyme protected in the presence of glutaraldehyde compared to buffered and unbuffered conditions in which glutaraldehyde was removed by the washing step, respectively. μM / min This is accompanied by an extremely surprising 10-fold and 2-fold increase (Figure 8D). This result is completely unexpected, as the enzyme is expected to exhibit significantly higher activity in the presence of the buffer.

[0106] In summary, covalent bonding of a protective layer to the enzyme surface unexpectedly improves the load, stability, and specific enzyme activity compared to enzymes protected by an organic silica layer via electrostatic interactions alone.

Claims

1. A composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure.

2. The composition according to claim 1, wherein the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, colipase-mimicking peptides, and amphiphilic molecules.

3. The composition according to claim 1 or 2, wherein the substance specifically interacts with the lid domain of the lipase or fragment thereof such that the lipase or fragment thereof transitions to an open structure and / or maintains an open structure.

4. The composition according to any one of claims 1 to 3, wherein about 100% of the lipase or fragment thereof immobilized on the surface of the solid carrier is in an open structure.

5. The composition according to any one of claims 1 to 4, wherein a solid carrier is embedded in the protective layer, and lipase or fragments thereof fixed on the surface of the solid carrier are embedded.

6. The composition according to any one of claims 1 to 4, wherein the protective layer has a solid carrier embedded in it, a lipase or fragment thereof fixed on the surface of the solid carrier is embedded in it, and a substance that interacts with the lid domain of the lipase or fragment thereof is embedded in it.

7. A composition according to any one of claims 1 to 5 for use as a pharmaceutical.

8. A composition according to any one of claims 1 to 5, for use in enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in a method for the prevention, delaying the progression or treatment of pancreatic exocrine insufficiency (EPI).

9. A method for producing a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, (c) A step of providing a substance that interacts with the lid domain of a lipase or a fragment thereof. (d) A step of interacting the lipase or fragment thereof from (b) with the substance from (c), (e) A step of immobilizing lipase or a fragment thereof onto a solid carrier. (f) A step of forming a protective layer on the surface of a solid carrier in order to protect the lipase or fragment thereof immobilized on the solid carrier. A method that includes this.

10. A method for producing a composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure: (a) A step of providing a solid carrier, (b) A step of providing lipase or a fragment thereof, (c) A step of immobilizing lipase or a fragment thereof onto a solid carrier. (d) A step of providing a substance that interacts with the lid domain of a lipase or a fragment thereof, (e) A step of interacting the lipase or fragment thereof from (c) with the substance from (d), (f) A step of forming a protective layer on the surface of a solid carrier in order to protect the lipase or fragment thereof immobilized on the solid carrier. A method that includes this.

11. i) a linker is added to a solid carrier provided in step (a), ii) a lipase or fragment thereof provided in step (b) is added to the solid carrier and the linker, and in step (e), the linker connects the solid carrier and the lipase or fragment thereof, the method according to claim 9.

12. The method according to claim 11, wherein a linker that did not connect the solid carrier and the lipase or a fragment thereof in step (e) is present during the formation of a protective layer on the surface of the solid carrier in step (f).

13. The method according to claim 11, wherein there is no washing step between (i) adding a linker to the solid carrier provided in step (a) and (ii) adding lipase or a fragment thereof to the solid carrier and the linker.

14. The method according to any one of claims 9 and 11 to 13, wherein there is no washing step between any of steps (a) to (f).

15. The method according to any one of claims 9 and 11 to 14, wherein a linker, or a portion thereof, that did not connect the solid carrier and the lipase or a fragment thereof in step (e) covalently bonds the protective layer and the lipase or a fragment thereof in step (f).

16. i) a linker is added to a solid carrier provided in step (a), ii) a lipase or fragment thereof provided in step (b) is added to the solid carrier and the linker, and in step (c), the linker connects the solid carrier and the lipase or fragment thereof, the method according to claim 10.

17. The method according to claim 16, wherein a linker that did not connect the solid carrier with the lipase or a fragment thereof in step (c) is present during the formation of a protective layer on the surface of the solid carrier in step (f).

18. The method according to claim 16, wherein there is no washing step between (i) adding a linker to the solid carrier provided in step (a) and (ii) adding lipase or a fragment thereof to the solid carrier and the linker.

19. The method according to any one of claims 10 and 16 to 18, wherein there is no washing step between any of steps (a) to (f).

20. The method according to any one of claims 10 and 16 to 19, wherein a linker, or a portion thereof, that did not connect the solid carrier and the lipase or a fragment thereof in step (c) covalently bonds the protective layer and the lipase or a fragment thereof in step (f).

21. The linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) The method according to any one of claims 9 to 20, selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane).

22. The method according to any one of claims 9 to 20, wherein the linker is glutaraldehyde.

23. A composition comprising a solid carrier, a lipase or fragment thereof immobilized on the surface of the solid carrier, a substance that interacts with the lid domain of the lipase or fragment thereof, and a protective layer that protects the lipase or fragment thereof by embedding it, wherein the lipase or fragment thereof is in an open structure, and the composition can be obtained by the method according to any one of claims 12 to 15 or 17 to 20.

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