WO2007080197A2

WO2007080197A2 - Immobilised enzymes

Info

Publication number: WO2007080197A2
Application number: PCT/EP2007/050402
Authority: WO
Inventors: Allan Svendsen; Michael Skjot; Jesper Brask; Jesper Vind; Shamkant Anant Patkar; Leonardo De Maria
Original assignee: Novozymes A/S
Priority date: 2006-01-16
Filing date: 2007-01-16
Publication date: 2007-07-19
Also published as: WO2007080197A3

Abstract

The invention relate to variant enzymes having one or more mutations with respect to the wild type of said variant enzyme, wherein said mutations selectively spatially orientating the active region of the variant enzyme towards the substrate when covalently immobilised.

Description

Immobilised enzymes

Sequence listing

The present invention comprises a sequence listing.

Field of invention

The invention relates to variant enzymes suitable for immobilisation on solid support having one or more mutations with respect to the wild type of said variant enzyme. The invention further relates to a method of producing a variant enzyme suitable for spatially orientationally selective immobilisation on a solid support.

Background of the invention

Enzymes are widely used in an immobilised form in the pharmaceutical and chemical industry. Though the specific activity of the relevant enzyme may be impaired by the enzyme being in an immobilised state, use of immobilized enzymes has several advantages compared to the use of enzymes free in solution. Specifically the process control may be greatly enhanced by use of immobilised enzymes.

Conventionally immobilisation of enzymes is accomplished by attaching the enzymes to a solid support by use of ionic bonding or hydrophobic interactions between the enzymes and the solid support. However, due to this relatively weak chemical attachment the enzymes may leak, i.e. the enzyme may leave the matrix or enzyme layer and go into solution.

To avoid unwanted leaking of enzymes it is known to attach the enzymes to a solid support by covalently linking the enzyme to the surface. This is preferably accomplished by linkage of specific amino acid residues present on the surface of the enzyme to the solid support. The chemical processes suitable to attach these specific amino acid residues differ according to the nature of the solid support and the preferred amino acid residue to link to the solid support. These chemical processes are generally well known in the art.

By covalent attachment, the amount of enzyme leaving the matrix or enzyme layer will be very restricted, and thus the control of the enzymatic reactions may be greatly enhanced.

However, due to the impaired specific activity inherent in the immobilised state of the relevant enzymes, there exists a need in the art for increasing the specific activity of immobilised enzymes. Definitions

"Selectively spatially orientated" refers to a selective spatial positioning with respect to the solid support, of one or more amino acid residues or of the amino acid residues constituting the active region on the surface of the enzyme.

"Mutation" refers to an artificially introduced change in the amino acid sequence of the enzyme as compared to the original amino acid sequence of said enzyme. Such change may for example be an insertion, deletion, substitution or any combination of these, of one or more amino acids. Such mutation may in principle be accomplished by any suitable tech- nique. However, mutations are preferably introduced by changing the nucleic acid sequence of the DNA encoding the enzyme using conventional techniques.

"Wild type enzyme" refers to an enzyme in its original state prior to the introduction of the mutations according to the present invention. As such, a "wild type enzyme" according to the present invention is thus not restricted to naturally occurring enzymes, and may be an enzyme that has been otherwise artificially modified prior to the introduction of the mutations according to the present invention.

"Variant enzyme" refers to a variant of a wild type enzyme, said variant having at least one mutation according to the invention.

"Active region" or "active site" refers to the region comprising the catalytically active residues of the enzyme. The active region consist of the catalytically relevant amino acid residues and may further comprise co-factors if present, i.e. heme groups, metal atoms, etc. In a very general way the "active region" or "active site" can be any set of atoms in the structure of a protein considered as relevant for the catalytic function of it.

The "non-active" area on the surface of the variant enzyme comprises in the broadest definition any position on the surface of the enzyme that is not part of the active site. In one em- bodiment of the invention the "non-active" area is defined as the "lower" area according to the definitions below.

A "selected remote region" may comprise any position on the surface of the enzyme that is part of the non-active area. In one embodiment a "selected remote region" comprises only one or a few amino acid residues within the non-active area. In one particular embodiment the selected remote region may be defined as the positions that are directly opposite to the active region. The "selected non-remote region" may in the oroadest definition comprise any position on the surface of the enzyme that is not part of a selected remote region. The "selected non-remote region" thus always comprise the active site but may further comprise any part of the lower or upper part that is not part of a selected remote region.

A "lower" position on the surface of the variant enzyme with respect to the active site refers to any position on the surface of the enzyme that is not part of the half-region of the enzyme containing the active site. Said half-region not containing the active site may be determined by dividing the enzyme in two halves by a diagonal-plane passing through the geometrical centre, said plane being arranged such that the radius vector from the geometrical centre of the enzyme to the geometrical centre of the catalytically active region is perpendicular to the plane.

An "upper" position on the surface of the variant enzyme with respect to the active site refers to any position on the surface of the enzyme that is part of the half-region of the enzyme containing the active site. Said half-region containing the active site may be determined by dividing the enzyme in two halves by a diagonal-plane passing through the geometrical centre, said plane being arranged such that the radius vector from the geometrical centre of the enzyme to the geometrical centre of the catalytically active region is perpendicular to the plane.

The region "opposite" to the active site is defined as the region on the lower part of the enzyme, said region comprising the positions on the surface of the enzyme bounded by projecting a circle on the surface of the enzyme, said circle being arranged such that a vector from the centre of the circle, through the geometrical centre of the enzyme, to the geometrical centre of the catalytically active region is perpendicular to the circle. The preferred size of the radius of said circle may vary with respect to the size of the given enzyme. Thus the radius may be of a size so that the opposite position comprises 3/4 of the lower surface area. More preferably the radius may be of a size so that the opposite position comprises 1/2 of the lower surface area. Even more preferably the radius may be of a size so that the opposite position comprises 1/4 of the lower surface area. Typically the radius may be of a size of less than 30 Angstrom (A). More typically the size of said radius is less than 25 A, even more preferred less than 20 A.

"Linker amino acid residues" is defined as natural or non-natural amino acid residues that can be linked covalently to a solid support. Preferred linking amino acid residues are selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid. More preferably linking amino acid residues are selected from the group consisting of residues of Lysine anu Cysteine. Most preferred linking amino acid residue is Lysine.

The position of a particular amino acid residue is defined by the position of the central carbon atom (C_α) of the particular amino acid residue in the 3-dimensional enzyme structure. The C_αatom of a given amino acid is defined as the central carbon atom in the 3-dimensional structure of said amino acid.

In order to determine whether a given amino acid residue R₁ having the coordinates (x^y^Z_j), is present in the lower or upper part of the enzyme with respect to the active region a geometrical centre R_cg, having the coordinates (x^y,^) may be defined. The geometrical centre of a given enzyme is defined as the central position with respect to all C_α atoms in the enzyme. The geometrical centre can be obtained for example from calculating the centre using the C_α atoms present in a PDB computer file of the protein.

Further the geometrical centre of the active region of the enzyme R_cgcat, having the coordinates (x_Cgcat,ycgcat,z_cgca,) is defined as the central position with respect to the C_α atoms present in amino acid residues that are part of the active site. The geometrical centre of the active region can be obtained for example from calculating the centre using the C_α atoms of the ac- tive region present in a PDB computer file of the protein.

For the purpose of relative positioning of the amino acid residue R_j a unit vector specifying the direction from R_cg to R_cgcat is defined as

R — R " dir -

R eg cat R eg

R_J is said to be in the "upper" region of the enzyme with respect to the "active site" if,

(R _J - R _cg ) - n _dιr > 0

R_J is said to be in "lower" region of the enzyme with respect to the "active site" if,

(R _J - R _cg ) - n _dιr < 0 Once the position of a given residue (j) has been classified as situated in the being "upper" or "lower" part, it may further be classified as being on the surface of the enzyme or not, according to the solvent accessibility of the particular residue.

Whether a given amino acid residue is on the surface of the enzyme may be determined according to W. Kabsch and C. Sander (1983). Biopolymers 22, pp. 2577-2637. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, for example if its solvent accessible surface as calculated with the program DSSP is over 30 A².

Brief description of drawings

Fig. 1 shows a perspective view of a stylistic enzyme (1 ) divided in an upper region (2) and a lower region (3) by a diagonal-plane (4) passing through the geometrical centre of the enzyme (5), said diagonal-plane (4) being arranged such that the radius vector from the geo- metrical centre of the enzyme (5) to the geometrical centre of the catalytically active region (6) is perpendicular to the plane. The catalytically active region (7) is thus situated on the upper half of the enzyme. The enzyme further has an opposite region (8), comprising the positions on the surface of the enzyme bounded by projecting a circle (9) having a radius (10) and a centre in a position diametrically opposite to the geometrical centre of the catalytically active region (6). In the embodiment shown in fig. 1 the selected remote region is defined as the opposite region (8).

Fig. 2 shows an immobilised enzyme (21 ), wherein the selected remote region is defined as the opposite region on the surface of the enzyme according to the embodiment of fig. 1. The immobilised enzyme is thereby selectively spatially orientated such that the active site is facing outwards from the solid support.

Fig. 3 shows a perspective view of a stylistic enzyme (31 ) divided in an upper region (32) and a lower region (33) by a diagonal-plane (4) passing through the geometrical centre of the en- zyme (35), said diagonal-plane (34) being arranged such that the radius vector from the geometrical centre of the enzyme (35) to the geometrical centre of the catalytically active region (36) is perpendicular to the plane. The catalytically active region (37) is thus situated on the upper half of the enzyme. In the embodiment shown in fig. 3 the selected remote region (38) is situated in both the upper and lower part of the surface of the enzyme.

Fig. 4 shows an immobilised enzyme (41 ), wherein the selected remote region is situated in both the upper and lower part of the surface of the enzyme according to the embodiment of fig. 3. The immobilised enzyme is thereby selectively spatially orientated such that the active site is facing tangentially or downwards with respect to the solid support.

Sequence listings SEQ ID NO:1 Thermomyces lanuginosus lipase SEQ ID N0:2 Candida antarctica lipase B SEQ ID N0:3 Nocardiopsis sp. protease 1 OR

Summary of the invention The present invention provide variant enzymes having one or more mutations with respect to the wild type of said variant enzyme, wherein said mutations selectively spatially orientate the active region of the variant enzyme towards the substrate, when said variant enzyme is covalently immobilised on a solid support.

The mutation(s) according to the invention increase the number of linker amino acid residues selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid, in a selected remote region on the surface of the variant enzyme with respect to the active site and/or decrease the number of said amino acid residues on the remaining surface of the variant enzyme.

The present invention further provide a method of producing a variant enzyme suitable for spatially orientationally selective immobilisation on a solid support, by a process comprising the steps of; a) identifying the location of the active site using a 3-dimentional structure of said enzyme, b) selecting the preferred orientation of the active site relative to the solid sup- port, c) identifying the location of linker amino acid residues selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid on the surface of the enzyme, d) increasing the number of linker amino acid residues selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid, in a selected remote region of the surface of the enzyme and/or decreas- ing the number of said amino acid residues on the remaining part of the surface of the enzyme using conventional protein engineering techniques, and optionally, e) evaluating the specific activity of the produced variant enzyme.

Detailed description of the invention It has now been shown that selective spatial orientating the relevant enzyme, such that the active site of the enzyme faces the substrate of the relevant enzymatic reaction results in an increased specific activity. Such selective spatial orientation is achieved according to the invention by mutating the wild type enzyme at selected positions thereby constructing variant enzymes having one or more mutations with respect to the wild type of said variant enzyme, wherein said mutations selectively spatially orientate the active region of the variant enzyme towards the substrate, when said variant enzyme is covalently immobilised on a solid support.

The enzymes according to the invention may be present in solution or in immobilized form. Preferably, however, they are present as covalently immobilised enzymes on a solid support. A solid support for covalent immobilization for enzymes could in principle be any polymeric (natural or synthetic) or inorganic material in the form of beads or a surface displaying functional groups for reaction with amino acid residues. The functional groups could be epoxides (oxiranes), cyanate esters or imidocarbonates (e.g. from CNBr activation), activated esters (eg. NHS-esters), activated carboxylic acids (e.g. acid chlorides), vinylsulfones, maleimidos, aldehydes, azides (e.g. for "click chemistry"), thiols, carboxylic acids, primary amines, or any other moiety that will allow formation of a covalent bond to an amino acid.

The covalent linkage might be formed simply by adding enzyme dissolved in a suitable buffer to the solid support, or it might require addition of a coupling agent (e.g. carbodiimides) or a bivalent linker (e.g. dialdehydes).

Preferably, the mutation(s) introduced into the wild type enzyme increases the number of one or more linker amino acid residues selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid, in a selected remote region on the surface of the variant enzyme with respect to the active site and/or decreases the number of said linker amino acid residues on the remaining surface of the variant enzyme.

More preferably the relevant linker amino acid residues are selected from the group consisting of residues of Lysine and Cysteine.

The most preferred linker amino acid residue is a residue of Lysine.

The mutations according to the invention are introduced in spatially selected regions of the wild type enzyme. This means that at least one linker amino acid according to the invention is either introduced or removed at a given selected amino acid position.

In general according to the invention one or more linker amino acid residues may be removed or removed by substitution from any position in the non-remote region of the surface of the enzyme. In one embodiment one or more linker amino acid residues in the active region are removed or substituted, if so present in the wild type enzyme.

Further, linker amino acid residues in the upper region may be removed or removed by sub- stitution, if so present in the wild type enzyme.

Even further, it may be preferred to reduce the number of linker amino acid residues also in the lower part of the enzyme. Thus in one embodiment linker amino acid residues in the lower region are removed or removed by substitution, if so present in the wild type enzyme. In other words the non-remote region wherein the number of linker amino acids may be reduced according to the invention, may consist of regions present in both the upper region well as the lower region of the enzyme surface.

In one embodiment all linker amino acid residues are removed or removed by substitution from the entire surface of the enzyme. Binding to the solid support may then be achieved by binding to the N-terminal amino acid or by inserting one or more linker amino acids in spatially selected remote regions.

In one embodiment according to the invention one or more linker amino acid residues are in- serted or inserted by substitution into on or more selected remote regions of the enzyme. The selected remote regions may be present at both the upper and lower part of the surface of the enzyme.

Linker amino acids present on the upper part of the surface of the variant enzyme result in binding to the solid support such that the active site points downwards or tangentially towards the solid support when immobilised. This embodiment may be preferred when the enzyme substrate is present near or in the boundary region between the solid support and the fluid phase.

Linker amino acids present on the lower part of the surface of the variant enzyme result in binding to the solid support such that the active site points outwards or perpendicular to the solid support when immobilised. This embodiment may be preferred when the enzyme substrate is primarily present in the fluid phase.

The mutations according to the invention are preferably such that the original function of the particular mutated region or position is maintained to the extend possible. This may be achieved by modifying the wild type enzyme as conservatively as possible. Thus, the mutations preferably involve substituting, inserting or deleting amino acid residues having specific n properties in common with the original properties of the mutated region or position. Examples of conservative mutations or modifications are within the group of basic amino acids (ar- ginine, lysine and Histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (Glutamine and Asparagine), hydrophobic amino acids (Leucine, lsoleucine and Valine), aromatic amino acids (Phenylalanine, Tryptophan and Tyrosine), and small amino acids (Glycine, Alanine, Serine, Threonine and Methionine). Amino acid modifications, which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/He, Leu/Val, Ala/Glu, and Asp/Gly as well as the reverse (Taylor, 1986, Journal of Theoretical Biologyl 19:205-218.

In a particular embodiment the enzyme according to the invention display 0 - 20 linker amino acid residues at spatially selected positions with respect to the active site on the surface of said enzyme.

Preferably the enzyme according to the invention display 1 - 10, more preferably 1 - 3 linker amino acid residues in spatially selected remote regions with respect to the active site on the surface of said enzyme.

More preferably the enzyme according to the invention display 1 - 2 linker amino acid residues at spatially selected remote regions with respect to the active site on the surface of said enzyme.

Most preferably the enzyme according to the invention display 1 linker amino acid residue at spatially selected remote regions with respect to the active site on the surface of said enzyme.

Preferably the enzyme according to the invention display less than 3, more preferably less than 2, and most preferably no linker amino acid residues in selected non-remote regions with respect to the active site on the surface of said enzyme.

Preferably no linker amino acid residues are present in the active region on the surface of the enzyme, unless said linker amino acid residues are crucial to the catalytic function of the en- zyme. In one embodiment according to the inventiun the enzyme only displays linker amino acid residues on the lower surface of the enzyme in a selected remote region opposite to the active site.

Thereby the active region of the enzyme will be directed outwards and towards the substrate.

In another embodiment all linker amino acid residues present on the surface of the enzyme are removed and covalent immobilisation is achieved by linking the N-terminal amino acid residue to the solid support.

The selected remote region may comprise the N-terminal as well as the C-terminal part of the enzyme. Thus linker amino acid residues may be inserted or removed in the N-terminal part of the enzyme. Further linker amino acid residues may be inserted or removed in the C- terminal part of the enzyme.

Enzymes of interest according to the invention are generally hydrolytic enzymes. Hydrolytic enzymes according to the invention comprise lipases, phospholipases, transacylases, proteases, amylases, cutinases, esterases, acylases and amidases. Further enzymes that may benefit from then invention are glucoseisomerases and oxidoreductases.

Lipases and proteases are particularly preferred enzymes according to the invention. In one particular embodiment the enzyme is a lipase. In another embodiment the enzyme is a protease.

Examples of lipases are the Thermomyces lanuginosus lipase (TLL) (SEQ ID NO: 1 ) and the Candida antarctica lipase B (CaI B) (SEQ ID NO:2). An example of a suitable protease is the Nocardiopsis sp. protease called 1 OR (SEQ ID NO:3).

In one embodiment the enzyme according to the invention is provided as part of a composi- tion. Preferably said composition further comprises a solid support, as well as other conventional additives. Such conventional additives could be any organic or inorganic compound that covalently or non-covalently binds to the solid support and/or the enzyme with the purpose of changing hydrophilicity/hydrophobicity, charge/electrostatics, or other physical or chemical properties of the enzyme-solid support complex. Examples include coupling agents (e.g. carbodiimides) and bivalent linkers (e.g. dialdehydes). In one embodiment the invention is a methou of producing a variant enzyme suitable for spatially orientationally selective immobilisation on a solid support, comprising the steps of; (a) identifying the location of the active site using a 3-dimentional structure of said enzyme, (b) selecting the preferred orientation of the active site relative to the solid support, (c) identify- ing the location of linker amino acid residues, (d) increasing the number of linker amino acid residues, in a selected remote region of the surface of the enzyme and/or decreasing the number of said linker amino acid residues on the remaining part of the surface of the enzyme using conventional protein engineering techniques, and (e) optionally, evaluating the specific activity of the produced variant enzyme.

Preferably, the linker amino acid residues are selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid.

More preferably the relevant linker amino acid residues are selected from the group consist- ing of residues of Lysine and Cysteine.

Even more preferably the relevant linker amino acid residue is Lysine.

Examples In describing the various enzyme variants of the present invention, the nomenclature described below is adapted for ease of reference. In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as "T226A". Multiple mutations are separated by comma marks (", "), e.g., "G205R, S41 1 F", representing mutations at positions 205 and 41 1 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position^*. Accordingly, the deletion of glycine at position 195 is designated as "G195^*". Multiple deletions are separated by comma marks (","), e.g., "G195^* + S41 1 ^*". Insertions. For an amino acid insertion, the lollowing nomenclature is used: Original amino acid, position, original amino acid, new inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated "G195GK". Multiple insertions of amino acids are designated Original amino acid, position, original amino acid, new inserted amino acid 5 #1 , new inserted amino acid #2; etc.. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "G195GKA". However in case of inserting amino acids in the N-terminal the following nomenclature is used: Original amino acid, position, new inserted amino acid, original amino acid. Accordingly the insertion of glutamine before glutamic acid at position 1 is designated "E195QE". Multiple insertions of amino acids are des- 10 ignated original amino acid, position, new inserted amino acid #1 , new inserted amino acid #2; etc original amino acid. For example, the insertion of glutamine, glycine and Proline before glutamic acid at position 1 is indicated as "E195QGPE".

Multiple mutations. Variants comprising multiple modifications are separated by comma 15 marks (","), e.g., "FM 7OY, G195E" representing modifications at positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.

Example 1. Thermomyces lanuginosus lipase (TLL)

Thermomyces lanuginosus lipase (TLL) was analysed according to the invention. The PDB id 20 chosen was 1 TIB. The geometrical centre was obtained with the coordinates of the C_α atoms present (269). The coordinates of the geometrical centre of the catalytically relevant atoms were obtained using the C_α atoms of residues serine 146, Histidine 258 and Aspartate 201.

"Upper" region amino acid residues present at the surface of the enzyme were determined as 25 residues number:

1 ,3,4,1 1 ,37,38,39,61 ,62,83,84,85,86,87,88,89,90,91 ,92,93,94,95,96, 179,200,205,206,208,209,210,21 1 ,212,221 ,223,226,227,228,229,231 , 232,233,234,236,237,239,240,241 ,244,245,248,249,250,251 ,252,253, 30 254,255,256,259,260,264,267,269.

"Lower" region amino acid residues present at the surface of the enzyme were determined as residues number:

35 5,8,9,24,27,29,30,31 ,32,33,35,43,45,46,48,52,53,56,57,58,59,60,71 ,72, 73,74,98,99,101 ,102,105,106,108,1 15,1 18,1 19,122,123,125,126,127, 129,130,133,134,135,136,137,139,158,160,161 ,162,163,164,165,180, 183,184,186,187,188,189,192,194. ¹³

Wild type TLL contains 7 lysine residues at positions 24, 46, 74, 98, 127, 223 and 237. Further wild type TLL has Cysteine at positions 22, 36, 41 , 104, 107 and 268, Histidine at 1 10, 5 135, 145, 198, 215 and 258, Tyrosine at 16, 21 , 53, 138, 164, 171 , 194, 213, 220, 261.

TLL-variants were constructed having the following mutations: Variant 1 (TLL1 ): K24R, K74R, K98R, K127R, R175K, K223R, K237R Variant 2 (TLL2): K24R, K46R, K74R, K98R, K127R, D137K, K223R, K237R.

10 Variant 3 (TLL3): K24R,K46R,K74R,K98R,K127R,K223R,K237R,T267K

Variant 4 (TLL4): K24R,N33Q,K46R,K74R,K98R,K127R,R175K,K223R,K237R

Variant 5 (TLL5): K24R,N33Q,K46R,K74R,K98R,K127R,R205K,K223R,K237R

Variant 6 (TLL6): K24R,K46R,K74R,K98R,K127R,K223R,K237R,T267K

Variant 7 (TLL7): K24R,K46R,K74R,K98R,K127R,K223R,K237R (no lysine residues on sur-

15 face. Binds the support through the N-terminal amino acid residue).

Example 2. Candida antarctica lipase B (CaI B)

Candida antarctica lipase B (CaI B) was analysed according to the invention. The PDB id chosen was 1TCA. The geometrical centre was obtained with the coordinates of the C_α at- 20 oms present (317). The coordinates of the geometrical centre of the catalytically relevant atoms were obtained using the C_α atoms of residues serine 105, Histidine 224 and Aspartate 187.

43,45,46,49,71 ,136,139,140,143,144,146,147,148,149,155,159,160,186, 188,189,191 ,192,194,195,196,198,199,203,205,208,214,215,216,218, 219,221 ,223,238,249,250,251 ,252,255,256,257,258,259,260,261 ,264, 30 265,267,268,269,270,271 ,272,273,275,276,278,279,280,282,283,285, 286,287,289,290,291 ,292,293.

"Lower" region amino acid residues present at the surface of the enzyme were determined as residues number: 35

1 ,3,4,5,6,8,9,10,1 1 ,12,13,14,15,17,18,21 ,23,24,25,26,27,28,29,31 ,32, 57,58,60,67,69,70,74,88,91 ,92,95,96,97,98,120,122,123,124,126,127, 168,169,171 ,174,175,176,178,206,242,244,^5,247,296,298,299,300, 302,303,304,306,307,308,309,31 1 ,312,314,316,317.

Potential Lysine-positions in matured lipase B from Candida antarctica lipase (CaIB, 5 CAA83122) with the N-terminal Ipsgsdpafsq are 13, 32, 98, 124, 136, 208, 271 , 290 and 308. The most relevant single lysine variants are thus

Variant 1 (CaM ): K13R K32R K124R K136R K208R K271 R K290R K308R Variant 2 (CaI 2): K13R K98R K124R K136R K208R K271 R K290R K308R Variant 3 (CaI 3): K13R K32R K98R K136R K208R K271 R K290R K308R 10

Potential Cysteine-positions in matured lipase B from Candida antarctica lipase (CaIB, CAA83122) with the N-terminal Ipsgsdpafsq are 22, 64, 216, 258, 293 and 31 1 , Histidine is 224 and tyrosine are 61 , 82, 91 , 135, 183, 203, 234, 253 and 300.:

15 Example 3. Nocardiopsis sp. protease 10R (10R)

Nocardiopsis sp. protease 10R (10R) was analysed as described above. The PDB file chosen is disclosed in WO 2005/035747. The geometrical centre was obtained with the coordinates of the C_α atoms present (188). The coordinates of the geometrical centre of the cata- lytically relevant atoms were obtained using the C_α atoms of residues Aspartate 61 , Histidine

20 35 and serine 143. The regions as from the above definition are:

"Upper" region amino acid residues present at the surface of the enzyme were determined as residues number:

25 1 1 ,12,13,14,42,44,49,66,68,1 15,135,139,140,163,165,166,167,169.

30 24,25,27,69,89,92,93,95,96,97,1 13,180,185,188.

Wild type Nocardiopsis sp. protease 10R (10R) does not contain Lysine-positions on the surface. Potential Cysteine-positions in matured Nocardiopsis sp. protease 10R (10R) with the N-terminal I adiigglayt are 15, 36, 101 , 1 1 1 , 137 and 164, Histidine 35, 91 and 1 10, tyrosine 35 9, 80, 85, 123, 147 and 172.

10R-variants were constructed having the following mutations: Variant 1 (1 OR 1 ): N92K ¹⁵

Variant 2 (10R 2): T151 K Variant 3 (10R 3): S99K

Example 4. Activity of immobilised TLL-variants

Two TLL-variants TLL 1 and TLL 2 were used in the assay. The wt TLL and the two TLL variants were immobilised on two different solid support types, Eupergit and Accurel, according to the experimental protocol below.

Immobilisation on Eupergit:

Enzyme was transferred to a 10 ml phosphate buffer, pH 8 using Amicon Ultra Centrifugal Filter Devices, MWCO 1 OkDa. The enzyme concentration was determined from absorption at 280 nm. Eupergit (100 to 250 mg) was weighed into a 10 ml PP syringe fitted with a PE frit and closing valve and enzyme solution was added in a volume corresponding to 20 mg en- zyme per g Eupergit. Buffer was added to reach a final volume of approx. 7 ml. The syringe was placed in an orbital shaker over night. The immobilised product was washed with buffer (5 X 7 ml) and acetone (2 X 7ml), and dried in vacuum over night.

Immobilisation on Accurel: The enzyme concentration was determined from absorption at 280 nm. Accurel (100 to 250 mg) was weighed into a 10 ml PP syringe fitted with a PE frit and closing valve. Ethanol (7 ml) was added and the mixture was shaken for 30 min. Solvent was drained off and the Accurel was washed with buffer (10 mM phosphate, pH 8). Enzyme solution was added in a volume corresponding to 20 mg enzyme per g Accurel, followed by buffer to reach a final volume of 7 ml. The syringe was placed in an orbital shaker over night. The immobilised product was washed with buffer (5 X 7 ml) and acetone (2 X 7ml), and dried in vacuum over night.

Eupergit covalently immobilise the enzymes through the lysine residues by reaction with the epoxides on Eupergit. Accurel immobilize the enzyme by non-residue-specific adsorption.

Amino acid analysis was used to determine the enzyme loading. Immobilised product (10 mg) was hydrolyzed with 18.5% HCI, 0.1 % phenol at 1 10 °C for 16 hours, filtered, and analyzed on an Biochrom 20 Plus amino acid analyzer. Enzyme loading was calculated as mg protein per g total weight. The activity of the enzymes (assayed as conversion in %) was determined for the reaction of 1 -propanol with lauric acid. A reaction mixture was made from 1 -propanol (50.4 g, 63 ml), water (6.72 g), and lauric acid (168.2 g). The mixture was heated to 60⁹C for 30 min. A portion (10.73 g) was transferred to a 100 ml conical flask closed with a screw cap. The flask was heated to 60 °C and enzyme (30 mg) was added. After 4 hours reaction at 60 °C and shaking at 200 rpm, the conversion or lauric acid to propyl laurate is determined by ¹H NMR (10 μl_ reaction mixture in 0.7 ml CDCI₃).

The immobilised enzymes were assayed for specific activity. Results are shown in table 1 .

As can be seen from the results the orientationally controlled immobilisation of TLL 2 on Eupergit increase the activity compared to the activity of the wt TLL from 1.37 times to 2.24 times. The same holds true for the orientationally controlled immobilisation of TLL 1 on Eu- pergit were the increase in activity compared to the activity of the wt TLL was from 0.65 times to 1.39 times.

Claims

1. A variant enzyme having one or more mutations with respect to the wild type of said vari- ant enzyme, wherein said mutations selectively spatially orientate the active region of the variant enzyme towards the substrate, when said variant enzyme is covalently immobilised on a solid support.

2. The enzyme according to claim 1 , covalently immobilised on a solid support.

3. The enzyme according to claim 1 or 2 wherein said mutation(s) has increased the number of one or more linker amino acid residues selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid, in a selected remote region on the surface of the variant enzyme with respect to the active site and/or decreased the number of said linker amino acid residues on the remaining surface of the variant enzyme.

4. The enzyme according to claim 3 wherein said linker amino acid residues are selected from the group comprising Lysine and Cysteine.

5. The enzyme according to claim 4 wherein said linker amino acid residue is Lysine.

6. The enzyme according to any of claims 3 - 5 displaying 0 - 20 of said linker amino acid residues on the surface of said enzyme.

7. The enzyme according to any of claims 3 - 5 displaying 0 - 20 of said linker amino acid residues in the non-active areas on the surface of said enzyme.

8. The enzyme according to any of claims 7, wherein the number of said linker amino acid residues displayed in a selected remote region with respect to the active site on the surface of said enzyme is from 1 to 3.

9. The enzyme according to claim 8 wherein the number of said linker amino acid residues displayed in the selected remote region with respect to the active site on the surface of said enzyme is from 1 to 2.

10. The enzyme according to claim 9 wherein the number of said linker amino acid residues displayed in the selected remote region with respect to the active site on the surface of said enzyme is 1.

5 1 1 . The enzyme according to any of claims 3 - 5 displaying 0 - 3 of said linker amino acid residues in the selected non-remote regions with respect to the active site on the surface of said enzyme.

12. The enzyme according to claims 1 1 , wherein the number of said linker amino acid resi- 10 dues displayed at spatially selected non-remote regions with respect to the active site on the surface of said enzyme is from 0 - 2.

13. The enzyme according to claim 12, wherein the number of said linker amino acid residues displayed at spatially selected non-remote regions with respect to the active site on

15 the surface of said enzyme is from 0-1.

14. The enzyme according to claim 13, wherein the number of said linker amino acid residues displayed at spatially selected non-remote regions with respect to the active site on the surface of said enzyme is 0.

20

15. The enzyme according to claim 14, wherein the immobilisation to the solid support is achieved through the N-terminal of the enzyme.

16. The enzyme according to any of the above claims wherein said enzyme is a hydrolytic 25 enzyme.

17. The enzyme according to claim 16, wherein said enzyme is selected among the group consisting of lipases, phospholipases, transacylases, proteases, amylases, cutinases, esterases, acylases and amidases.

30

18. The enzyme according to claim 16 or 17 wherein said enzyme is a lipase.

19. The enzyme according to claim 18 wherein said lipase is CaI B or TLL.

35 20. The enzyme according to claim 16 or 17 wherein said enzyme is a protease.

21 . The enzyme according to claim 20 wherein said protease is 10R.

22. A method of producing a variant enzyme suitable for spatially orientationally selective immobilisation on a solid support, comprising the steps of; a. identifying the location of the active site using a 3-dimentional structure of said enzyme, 5 b. selecting the preferred orientation of the active site relative to the solid support, c. identifying the location of linker amino acid residues selected from the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and As- partic acid on the surface of the enzyme, d. increasing the number of one or more linker amino acid residues selected from 10 the group consisting of residues of Lysine, Cysteine, Histidine, Tyrosine, Glutamic acid and Aspartic acid, in a selected remote region of the surface of the enzyme and/or decreasing the number of said linker amino acid residues on the remaining part of the surface of the enzyme using conventional protein engineering techniques, 15 e. optionally, evaluating the specific activity of the produced variant enzyme,

23. The method according to claim 22 wherein said linker amino acid residues in step d. are selected from the group consisting of Lysine and Cysteine.

20 24. The method according to claim 22 wherein said linker amino acid residue is Lysine.