Title:
CREATION OF DIVERSITY IN POLYPEPTIDES
Kind Code:
A1


Abstract:
The inventors realized that the diversity generated by conventional methods may be limited by steric hindrance between amino acid residues in the three-dimensional structures of the resulting polypeptides. The steric hindrance may occur between amino acid residues at widely different positions in the amino acid sequences, e.g. between residues in two different domains of the 3D structure, and resulting polypeptides which include such steric hindrance may never be observed in the conventional recombination methods because they may be ex-pressed in poor yields or may have poor activity or stability. The inventors developed a method to identify and alleviate such steric hindrance in the resulting polypeptides. In an alignment of the three-dimensional structures, steric hindrance is indicated when residues from two different structures are located within a certain distance. Pairs of residues at corresponding positions in the amino acid sequences are not considered, and residues close to the surface (high solvent accessibility) are considered to be less prone to steric hindrance.



Inventors:
Svendsen, Allan (Horsholm, DK)
Beier, Lars (Lyngby, DK)
Application Number:
11/624750
Publication Date:
01/03/2008
Filing Date:
01/19/2007
Assignee:
Novozymes A/S (Bagsvaerd, DK)
Primary Class:
Other Classes:
435/193, 426/549
International Classes:
C12P21/04; A21D10/00; C12N9/00
View Patent Images:



Primary Examiner:
STEADMAN, DAVID J
Attorney, Agent or Firm:
NOVOZYMES NORTH AMERICA, INC. (FRANKLINTON, NC, US)
Claims:
1. 1-13. (canceled)

14. A method of constructing a polypeptide, comprising: a) structurally aligning a three-dimensional structure of a first parent polypeptide and a three-dimensional structure of a second parent polypeptide, b) selecting a first amino acid residue from the structure of the first polypeptide and a second amino acid residue from the structure of the second polypeptide, such that: i) the two selected residues are not aligned with each other in the structural alignment, ii) a non-hydrogen atom of the first amino acid residue and a non-hydrogen atom of the second amino acid residue are located less than 2.7 Å apart, and iii) each of the two residues is not Glycine and has a side chain having less than 30% solvent accessibility, and c) substituting or deleting the first and/or the second residue with a smaller residue, and d) recombining the amino acid sequences after the substitution, and e) preparing a DNA-sequence encoding the polypeptide of step e) and expressing the polypeptide in a transformed host organism.

15. The method of claim 14, wherein the parent polypeptides have an amino acids sequence identity of at least 50% to each other.

16. The method of claim 14, wherein the parent polypeptides have an amino acids sequence identity of at least 80% to each other.

17. The method of claim 14, wherein the parent polypeptides have an amino acids sequence identity of at least 90% to each other.

18. The method of claim 14, wherein the parent polypeptides have an amino acids sequence homology of at least 50% to each other.

19. The method of claim 14, wherein the parent polypeptides have an amino acids sequence homology of at least 80% to each other.

20. The method of claim 14, wherein the parent polypeptides have an amino acids sequence homology of at least 90% to each other.

21. The method of claim 14, further comprising f) superimposing the structures so as to align each non-hydrogen atom located <10 Å of an atom in the first or the second residue, and g) selecting two residues that are less than 1.5 Å apart in the new superimposition.

22. The method of claim 14 wherein the two selected residues after the substitution can form a hydrogen bond, a salt bridge or a cysteine bridge.

23. The method of claim 14 wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.5 Å apart.

24. The method of claim 14 wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.2 Å apart.

25. The method of claim 14 wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.1 Å apart.

26. The method of claim 14 wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.0 Å apart.

27. The method of claim 14 wherein each parent polypeptide is an enzyme having an active site, and the structural alignment is done so as to align each non-hydrogen atom of the amino acid residues of the active sites

28. The method of claim 14 wherein the enzymes belong to glycosyl hydrolase family 13.

29. The method of claim 14 wherein the enzymes are a cyclodextrin glucanotransferase and a maltogenic alpha-amylase.

30. The method of claim 21 which further comprises producing a polypeptide having the recombined amino acid sequence, testing the polypeptide for an enzymatic activity and selecting an enzymatically active polypeptide.

31. A polypeptide which has an amino acid identity of at least 80% to SEQ ID NO: 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25.

32. A polypeptide which: a) has an amino acid sequence which is a hybrid of a maltogenic alpha-amylase and a cyclodextrin glucanotransferase, b) has a smaller residue at a position corresponding to: i) D209, L261, D267, M307, H503, T509, V626, K651 of SEQ ID NO: 6 or ii) K7, Y181, N266, K270, L286, Y574, P592, S676 of SEQ ID NO: 17; and c) has hydrolytic activity on starch.

33. A dough comprising the polypeptide of claim 32.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation in Part of national application PCT/DK2005/000515 filed Aug. 2, 2005, which claims priority or the benefit under 35 U.S.C. 119 of U.S. provisional application No. 60/598,150 filed Aug. 2, 2004 the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of constructing a hybrid polypeptide from two or more parent polypeptides in order to create diversity. It also relates to hybrid polypeptides constructed by this method.

BACKGROUND OF THE INVENTION

The prior art describes methods of creating diversity by recombination of DNA sequences encoding two or more polypeptides, followed by transformation of a suitable host organism with the recombined DNA sequence and screening of the transformants for enzymatic activity. The recombination may be random or directed. WO 1995022625; U.S. Pat. No. 6,368,805; J. E. Ness et al., Nature Biotechnology, vol. 20, December 2002, pp. 1251-1255; M. C. Saraf et at., 4142-4147, PNAS, Mar. 23, 2004, vol. 101, No. 12.

SUMMARY OF THE INVENTION

The inventors realized that the diversity generated by conventional methods may be limited by steric hindrance between amino acid residues in the three-dimensional structures of the resulting polypeptides. The steric hindrance (also referred to as “structural stop codon”) may occur between amino acid residues at widely different positions in the amino acid sequences, e.g. between residues in two different domains of the 30 structure, and resulting polypeptides which include such steric hindrance may never be observed in the conventional recombination methods because they may be expressed in poor yields or may have poor activity or stability.

The removal of “structural stop codons” can result in improved expression and/or stability of the protein of interest, or in ultimate case expression at all of protein of interest. For example in combining of two or more proteins, i.e. combining multiple hybrids of two or more proteins using various DNA techniques e.g. using shuffling techniques as known in the art (WO9522625, WO9827230 and WO2000482862) the removal of “structural stop codons” from one or more of the included proteins will improve the expression and/or stability of the proteins, and/or create access to a novel diversity not found by other shuffling or hybrid techniques. Combination of protein sequences will often result in accommodation of different sized residues and homologous positions but not always. Sometimes clashes will occur and especially in the core of the protein. The removal of “structural stop codons” results in novel diversity due to allowance of new region combinations not seen because of presence of “structural stop codons”, which otherwise may result in a non functional or non expressed protein.

The inventors developed a method to identify and alleviate such steric hindrance in the resulting polypeptides. In an alignment of the three-dimensional structures, steric hindrance is indicated when residues from two different structures are located within a certain distance. Pairs of residues at corresponding positions in the amino acid sequences are not taken into consideration since only one of the two residues is expected to be present in the recombined polypeptide. Pairs of residues are not taken into consideration if one or both is glycine or if one or both side chains is close to the surface (indicated by a high solvent accessibility) as the residue may be able to reposition to avoid the potential clash.

Accordingly, the invention provides a method of constructing a polypeptide, comprising:

a) selecting at least two parent polypeptides each having an amino acid sequence and a three-dimensional structure,

b) structurally aligning the three-dimensional structures, thereby aligning amino acid residues from different sequences,

c) selecting a first amino acid residue from one structure and a second residue from another structures such that:

    • i) the two residues are not aligned in the superimposition.
    • ii) a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 2.7 Å apart, and
    • iii) each of the two residues is not Glycine and has a side chain having less than 30% solvent accessibility, and

d) substituting or deleting the first and/or the second residue such that the substitution is with a smaller residue, and

e) recombining the amino acid sequences after the substitution, and

f) preparing a DNA-sequence encoding the polypeptide of step e) and expressing the polypeptide in a transformed host organism.

Further the invention relates to a polypeptide which has at least 80%, 85%, 90%, 95% or 98% or 99% identifty to SEQ ID NO: 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25. The invention also relates to a polynucleotide encoding any of the polypeptides.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an alignment of various known CGTase sequences. Details are given below.

FIG. 2 shows the results of a comparison of two 3D structures. The upper sequence is 1qho for the maltogenic alpha-amylase Novamyl (SEQ ID NO: 17), and for the lower sequence is 1a47 for a CGTase (SEQ ID NO: 5). Details are described in Examples 1 and 2.

FIG. 3 and 4 shows hypothetical sequences with “structural stop codons”. Details are described in Examples 6 and 7.

DETAILED DESCRIPTION OF THE INVENTION

Parent Polypeptides

According to the invention, two or more parent polypeptides are selected, each having an amino acid sequence and a three-dimensional structure. The parent polypeptides may in particular be selected so as to be structurally similar, e.g. each pair having a amino acid identity of at least 50%, e.g. at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%. Amino acid identity may be determined as described in U.S. Pat. No. 6,162,628.

In another preferred embodiment the structurally similar parent potypeptides have a homology of at least 50%, e.g. at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%. Homology may be determined as described in WO 2004067737, i.e. by using the GAP routine of the UWGCG package version 9.1.

The parent polypeptides may be polypeptides having biological activity, structural polypeptides, transport proteins, enzymes, antibodies, carbohydrate binding modules, serum albumin (e.g. human and bovine), insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pituitary hormones. somatomedin, erthropoietin, luteinizing hormone, interleukin, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO) and prolactin.

The enzyme may have an active site, e.g. a catalytic triad, which may consist of Ser, Asp and His. The parent enzymes may be selected so as to have identical residues in the active site.

Three-Dimensional Structure

Three-dimensional structure is meant to be a known crystal structure or a model structure.

The 3D structure of each polypeptide may already be known, or it may be modeled using the known 3D structures of one or more polypeptides with a high sequence homology, using an appropriate modeling program such as Homology, Modeller or Nest. The 3D model may be optimized using molecular dynamics simulation as available, e.g., in Charmm or NAMD. The optimization may particularly be done in a water environment, e.g. a box or sphere.

The Homology, Modeller and Charmm software is available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif. 92121-3752, USA, www accels.com/. The Nest software is distributed free of charge at trantor.bioc.columbia.edu/programs/jackal/index.html. The NAMD software is available at www.ks.uiuc.edu/Research/namd/.

Structural Alignment of 3D Models

The 3D models may be structurally aligned by methods known in the aft. The structural alignment may be done by use of known software. In the structurally aligned models, pairs of residues from different sequences are considered to be aligned when they are located close to each other. The following software may be used.

DALI software, available at www.ebi.ac.uk/dali/

CE software available at ci.sdsc.edu/

STAMP software available at www.compbio.dundee.ac.uk/Software/Stamp/stamp.html

Protein 3Dhome at www-lecb.ncifcrf.gov/˜tsai/

Yale Gernstein Lab—spare parts at bioinfo.mbb.yale.edu/align/

Structural alignment server at www.molmovdb.org/align/

In the case of enzymes having an active site, the structural alignment may be a super-imposition of the structures based on the deviations of heavy atoms (i.e. non-hydrogen atoms) in the active sites, e.g. by minimizing the sum of squares of deviations. Alternatively, the super-imposition may be done so as to keep deviations between corresponding atoms below 0.8 Å, e.g. below 0.6 Å, below 0.4 Å, below 0.3 Å or below 0.2 Å.

Selection of Amino Acid Residues

Steric hindrance (“potential clashes”) between two amino acid residues is indicated if a heavy atoms (i.e. non-hydrogen) of the two residues are located less than 2.7 Å, 2.5 Å or 2.0 Å apart, particularly less than 1.7 Å, 1.5 Å, 1.2 Å, 1.1 Å or 1.0 Å apart, with the following exceptions.

Two residues aligned with each other in the structural alignment (pairs of residues at corresponding positions in the amino acid sequences) are not taken into consideration since only one of the two residues is expected to be present in the recombined polypeptide.

Pairs of residues are not taken into consideration if one or both is glycine.

Pairs of non-glycine residues are not taken into consideration if one or both side chains has more than 20%, 25% or 30% solvent accessibility as a high solvent accessibility is taken as an indication that the residue may be able to reposition to avoid the potential clash. Solvent accessibility can be calculated by use of the DSSP program, available from Centre for Molecular and Biomolecular Informatics, University of Nijmegen, Toernooiveld 1, P.O. Box 9010, 6500 GL Nijmegen, +31 (0)24-3653391, www.cmbi.kun.nl/gv/dssp/. The DSSP program is disclosed in W. Kabsch and C. Sander, BIOPOLYMERS 22 (1983) pp. 2577-2637. The residue total surface areas of the 20 natural amino acids are tabulated in Thomas E. Creighton, PROTEINS; Structure and Molecular Principles, W.H. Freeman and Company, NY, ISBN: 0-7167-1566-X (1984).

To confirm the severity of the potential clash, a local alignment of the two 3D structures may then be made by aligning all residues within a distance of 10 Å.

The steric hindrance may be identified by a comparison of two complete sequences in order, particularly severe clashes (less than 1.2, 1.1 or 1.0 Å apart), to identify potential clashes that may arise no matter how the two sequences are recombined.

Alternatively, the comparison may be made between two partial sequences to be combined in a hybrid and in this case a larger limit may be used for the distance (less than 2.7 Å, 2.5 Å, 2.0 Å, 17 Å or 1.5 Å).

Amino Acid Substitution

When a potential clash between two residues has been identified, one or both residues is substituted with a smaller residue. In this connection, the residues are ranked as follows from smallest to largest: (an equal sign indicates residues with sizes that are practically indistinguishable):

G<A=S=C<V=T<P<L=I=N=D=M<E=Q<K<H<R<F<Y<W

The substitution may be such that the two residues after the substitution can form a hydrogen bond, a salt bridge or a cysteine bridge.

Recombination of Amino Acid Sequences

After making amino acid substitutions to alleviate potential clashes, the substituted amino acid sequences are recombined. The recombination may be done by designing hybrids or by gene shuffling.

Hybrids may be constructed by switching from one sequence to another between aligned residues. Once constructed, the hybrids can be produced by conventional methods by preparing a DNA sequence encoding it and expressing it in a transformed host organism.

Alternatively, genes can be prepared encoding each substituted amino acid sequence, by shuffling the genes by known methods, transforming a suitable host organism with the shuffled genes. The shuffling can be done, I e.g., as described in WO 1995022625.

In the case of the parent polypeptides being enzymes, the transformants can be screened for enzymatic activity.

Enzymes

The parent enzymes may have hydrolase, oxidoreductase or transferase activities, e.g. activities such as protease, lipolytic enzyme, glycosyl hydrolase, laccase, oxidoreductases with oxygen as acceptor (e.g. glucose oxidase, hexose oxidase or galactose oxidase), glycosyl transferase, esterase, cellulase, xylanase, amylase, isoamylase, pullulanase, branching enzyme, pectate hydrolase, cyclodextrin glucanotransferase, or maltogenic alpha-amylase activity. One or more of the parent enzymes may have a carbohydrate-binding domain.

The method may particularly be applied to two or more structurally similar enzymes, e.g. belonging to the same family in a structural classification of enzymes. Thus, they may belong to the same structural family for glycosyl hydrolases and glycosyl transferases as described, e.g., in the following literature. The enzymes may be of family 13 and may particularly include a maltogenic alpha-amylase and a cyclodextrin glucanotransferase.

    • Henrissat B., A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 280:309-316 (1991).
    • Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 293:781-788 (1993).
    • Henrissat B., Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316:695-696 (1996).
    • Davies G., Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure 3:853-859 (1995).

The parent enzymes may be lipolytic enzymes belonging to the same homologous family as described at www.led.uni-stuttgart.de/families.html. The 3D structures of the lipolytic enzymes may all include a so-called “lid” in open or closed form.

The enzymes may be proteases or peptidases belonging to the same family or sub-family as described by MEROPS in “The Peptidase Database”, available at merops.sanger.ac.uk/. The proteases may be subtilases, e.g. belonging to the same sub-group as described by Siezen R J and Leunissen J A M, 1997, Protein Science, 6, 501-523; one of these sub-groups is the Subtilisin family.

CGTase

The cyclodextrin glucanotransferase (CGTase) may have an amino acid sequence as shown in SEQ ID NOS: 1-16 and may have a three-dimensional structure found under the following identifier in the Protein Data Bank (rcsb.org): B. circulans (1COG), alkalophilic Bacillus (1PAM), B. stearothermophilus (1CYG) or Thermoanaerobacterium thermosulfurigenes (1CIU, 1A47). 3D structures for other CGTases may be constructed as described in Example 1 of WO 9623874.

FIG. 1 shows an alignment of the following known CGTase sequences, each identified by accession number in the GeneSeqP database and by source organism. Some sequences include a propeptide, but only the mature peptide is relevant for this invention.

SEQ ID NO: 1. aab71493.gcg B. agaradherens

SEQ ID NO: 2. aau76326.gcg Bacillus agaradhaerans

SEQ ID NO: 3. cdg1_paema.gcg Paenibacillus macerans (Bacillus macerans).

SEQ ID NO: 4. cdg2_paema.gcg Paenibacillus macerans (Bacillus macerans).

SEQ ID NO: 5. cdgt_thetu.gcg Thermoanaerobacter thermosulfurogenes (Clostridium thermosulfurogenes) (SEQ ID NO 2:)

SEQ ID NO: 6. aaw06772.gcg Thermoanaerobacter thermosulphurigenes sp. ATCC 53627 (SEQ ID NO: 3)

SEQ ID NO: 7. cdgt_bacci.gcg Bacillus circulans

SEQ ID NO: 8. cdgt_bacli.gcg Bacillus sp. (strain 38-2)

SEQ ID NO: 9. cdgt_bacs0.gcg Bacillus sp. (strain 1011)

SEQ ID NO: 10. cdgt_bacs3.gcg Bacillus sp. (strain 38-2)

SEQ ID NO: 11. cdgu_bacci.gcg Bacillus circulans

SEQ ID NO: 12. cdgt_bacsp.gcg Bacillus sp. (strain 17-1, WO 2003068976) (SEQ ID NO: 4)

SEQ ID NO: 13. cdgt_bacoh.gcg Bacillus ohbensis

SEQ ID NO: 14. cdgt_bacs2.gcg Bacillus sp. (strain 1-1)

SEQ ID NO: 15. cdgt_bacst.gcg Bacillus stearothermophilus

SEQ ID NO: 16. cdgt_klepn.gcg Klebsiella pneumoniae

To develop variants of a CGTase without a known 3D structure, the sequence may be aligned with a CGTase having a known 3D structure. An alignment for a number of CGTase sequences is shown in FIG. 2. Other sequences may be aligned by conventional methods, e.g. by use the software GAP from UWGCG Version 8.

Maltogenic Alpha-Amylase

The maltogenic alpha-amylase (EC 3.2.1.133) may have the amino acid sequence shown in SEQ ID NO: 17 (in the following referred to as Novamyl), having the 3D structure described in U.S. Pat. No. 6,162,628 and found in the Protein Data Bank with the identifier 1QHO. Alternatively, the maltogenic alpha-amylase may be a Novamyl variant described in U.S. Pat. No. 6,162,628. A 3D structure of such a variant may be developed from the Novamyl structure by known methods, e.g. as described in T. L. Blundell et al., Nature, vol. 326, p. 347 ff (26 Mar. 1987); J. Greer, Proteins: Structure, Function and Genetics, 7:317-334 (1990), or Example 1 of WO 9623874.

Use of Hybrid Polypeptide

The hybrids may be useful for the same purpose as the parent enzymes.

Thus, a hybrid of a maltogenic alpha-amylase and a cyclodextrin glucanotransferase may form linear oligosaccharides as an initial product by starch hydrolysis and a reduced amount of cyclodextrin and may be useful for anti-staling in baked products.

A hybrid of laccases and/or other enzymes belonging to EC 1.10.3 may be useful for e.g. hair dyeing or reduction of malodor.

EXAMPLES

Example 1

Comparison of Complete Sequences

Superimposition of Parent Enzymes

Two glycosyl hydrolases of family 13 were selected. One was a maltogenic amylase (Novamyl) having the amino acid sequence shown in SEQ ID NO: 17 and having a 3D structure published under number 1 QHO. The other was a CGTase having the amino acid sequence shown in SEQ ID NO: 5 and the 3D structure 1A47, and this was also taken to represent the structure of the highly homologous CGTase having the sequence SEQ ID NO: 6. The two 3D structures were superimposed so as to align the active sites, and the alignment of residues of the two sequences is shown in FIG. 2 Aligned residues shown vertically above each other, with gaps inserted to separate non-aligned residues.

Identification of Potential Clashes

The two structures were analyzed, and the following unaligned residues were identified as having a side chain with less than 30% solvent accessibitity and with a heavy atom less than 1.5 Å (or less than 1.0 Å) apart from a heavy atom of a residue in the other structure. The following pairs of residues were found to come within 1.0 Å, The potential clashes are shown as CGTase residue and atom, Novamyl residue and atom, and distance in Å:

D209OD2A676CB0.89
L261CD1K270NZ0.93
D267CGN266O0.94
D267OD1N266O0.48
M307CEL286CD10.77
H503CD2K7NZ0.97
T509OG1Y574CZ0.65
V626CBY181CZ0.41
V626CG1Y181OH0.99
V626CG2Y181CD20.76
K651NZP592CG0.35

The above residues are marked by asterisks in FIG. 2.

Example 2

Comparison of Complementary Sequences

To design hypothetical hybrids, residues in a partial sequence of Novamyl (SEQ ID NO: 17) were compared with residues in the complementary pan of the CGTase sequence (SEQ ID NO: 6), and residues with heavy atoms located less than 1.7 Å apart were identified. The potential clashes are shown as in Example 1. The identified residues are marked with asterisks in FIG. 2.

Novamyl 1-494+CGTase 495-683

H503CD2K7NZ0.97
N575OY317OH1.68
V626CBY181CZ0.41

CGTase 1-494+Novamyl 495-686

D3CR545NH21.36
D209OD2A676CB0.89

Novamyl 1-499+CGTase 500-683

H503CD2K7NZ0.97
N575OY317OH1.68
V626CBY181CZ0.41

CGTase 1-499+Novamyl 500-686

D3CR545NH21.36
D209OD2A676CB0.89

Novamyl 1-410+CGTase 410-683

H503CD2K7NZ0.97
N575OY317OH1.68
V626CBY181CZ0.41

Novamyl 1-378+CGTase 378-683

N409OE1R354N1.63
H503CD2K7NZ0.97
N575OY317OH1.68
V626CBY181CZ0.41

Novamyl Residues 1-204+CGTase Residues 207-683

W219CZ2L75CD21.66
H503CD2K7NZ0.97
V626CBY181CZ0.41

CGTase Residues 1-139 and 207-683+Novamyl Residues 131-204

V626CBY181CZ0.41

Example 3

Construction of Hybrids

Hybrids were constructed with the following combinations of Novamyl residues and CGTase residues (SEQ ID NO: 6) and with substitutions of Novamyl residues as indicated to alleviate potential crashes. For comparison, similar variants were constructed without substitutions.

ResiduesNovamyl substitutions
Novamyl 1-494 + CGTase 495-683K7S + Y181A
CGTase 1-494 + Novamyl 495-686R545S
Novamyl 1-499 + CGTase 500-683K7S + Y181A
CGTase 1-499 + Novamyl 500-686R545S
Novamyl 1-410 + CGTase 410-683K7S + Y181A
Novamyl 1-378 + CGTase 378-683K7S + Y181A
Novamyl 1-204 + CGTase 207-683K7S, W107F
CGTase 1-139 + Novamyl 131-204 + CGTaseY181A
207-683
Novamyl 1-204 + CGTase 207-683K7S, W107F,
Y181A
Novamyl 1-204 + CGTase 207-683K7S, Y181A

The first eight of the above hybrids are found in SEQ ID NO: 18 to SEQ ID NO: 25.

Example 4

Screening of Hybrids for Amylase Activity

Four hybrids of the previous example were produced by preparing a DNA-sequence encoding the hybrid and expressing the hybrid in a transformed organism cultivating a transformant, and the amylase activity was assayed by letting the culture broth act on Phadebas (dye-labelled substrate, available from Pharmacia) and measuring the absorbance at 650 nm. The amylase assay was made at pH 5.5 at two different temperatures: 50° C. and 60° C. Reference hybrids without substitutions were included for comparison.

ABSABS
(650 nm)(650 nm)
NovamylpH 5.5,pH 5.5,
Residuessubstitutions60° C.50° C.
Novamyl 1-410 + CGTase 410-6830.010.01
Novamyl 1-410 + CGTase 410-683K7S, Y181A0.491.66
Novamyl 1-378 + CGTase 378-6830.010.01
Novamyl 1-378 + CGTase 378-683K7S, Y181A0.160.37
CGTase 1-139 + Novamyl0.060.02
131-204 + CGTase 207-683
CGTase 1-139 + NovamylY181A0.210.07
131-204 + CGTase 207-683

Example 5

Baking with Hybrids

Further two hybrids were produced by cultivating a transformant and tested for baking. The two hybrids are:

    • BaHy1: CGTase (SEQ ID NO: 6) residue 1-139+Novamyl (SEQ ID NO: 17) residue 131-204+CGTase (SEQ ID NO: 6) residue 207-683; and
    • BaHy2: Novamyl (SEQ ID NO: 17) residue 1-577+CGTase (SEQ ID NO: 6) residue 580-683+Y181A mutation in Novamyl.

The effect of the two hybrids in straight dough was compared to that of CGTase with respect to a number of parameters: Softness of breadcrumb, elasticity, and mobility of free water.

Approximately 1 mg/kg of flour was dosed.

The two hybrids improve the softness of breadcrumb as compared to CGTase.

The two hybrids improve the elasticity as compared to CGTase.

BaHy2 improves the mobility of free water as compared to CGTase, whereas BaHy1 has the same effect as CGTase.

Example 6

Structural Stop Codons—Impact on Diversity

This example illustrates the possible outcome of a hybridization between two proteins having the sequences SeqA and SeqB (FIG. 3):

If combination sites (marked with |) comprises a “structural stop codon” (marked with X), the resulting protein not be expressed properly or maybe even not at all. Segment 14 in SeqA and segment 7 in SeqB indicates such potential crashes due to the presence of “structural stop codons”. The result will be a lowering of the diversity, as combinations containing these two segments most likely not will be able to accommodate the clashes and therefore not be present in the diversity of protein molecules,

If X in SeqA and/or SeqB is made smaller the accommodation might result in a functional protein. Accommodation may also be obtained by changing the shape or charge of the residue e.g. I to L and D to N. The “structural stop codon” can also be removed by inserting the proper match of residues by mutating the particular residues and/or mutating the surrounding residues around the clashing residues thus creating accommodation. Smaller residues can be found in the list; G<A=S=C<V=T<P<L=I=N=D=M<E=Q<K<H<R<F<Y<W.

If the “structural stop codon” gives 100% non-functional protein—the lowering of diversity is 25% for one “structural stop codon” residue pair—compared to the situation without any “structural stop codons”. That is the diversity for the segments are 220=1048576 and for the clashes it is 218=262144.

Example 7

Structural Stop Codons—Impact on Diversity when Combining More than Two Proteins

In this example we have three proteins illustrated by SEQ1, SEQ2 and SEQ3 (FIG. 4). SEQ1 has a “structural stop codon” with SEQ2 called X. SEQ1 has a “structural stop codon” with SEQ3 called Z, and SEQ 2 has a structural stop codon” with SEQ3 called Y. The diversity will hereby be lowered dramatically as exemplified above. We will have the common equation for the number of non-“structural stop codon” containing proteins termed D for diversity in the cases where the “structural stop codons” pairs are found in separate segments not containing other “structural stop codons” and the number of segments are higher or equal to the number of pairs:
D=NK−P*N(K−2) Equation I:
where D is diversity without “structural stop codons”, N the number of proteins, K the number segments, and P the number of pairs (i.e. X, Y and Z).

For other situations e.g. with “structural stop codons” in the same segment or other situations other equations can be derived.

Using equation I we get D to be ⅔ for the numbers shown in present example and for the numbers in shown in the above example we get 0.75. Consequently the diversity may be increased significantly by removing “structural stop codons”.

Example 8

Structural Stop Codons—Impact on Extending Combination Possibilities for Proteins with Low Homology to a Better Result

One important aspect is the possibility of combining more distant related proteins by hybridisation or shuffling techniques and not only closely related proteins. The combination by hybridisation or shuffling techniques may go below the 90. or the 80, or the 70, or the 60, or the 50 percent homology level. At the upper level of homology, around 70-90 percent homology, the amount of diversity—meaning the number of active clones coming out of a hybridisation or shuffling experiment—or at the lower level around 50-80 percent homology creation of active clones at all might be the outcome.

Example 9

Example on Finding “Structural Stop Codons” for Combining Proteins e.g. Shuffling or Hybrid Formation

The set of parent sequences are analyzed using the 3D structures. The 3D structures can be based on existing known structures or obtained by X-ray crystallography, NMR methods or modeled using appropriate modeling programs like NEST, MODELLER or HOMOLOGY. The two structures are superimposed by optimizing the RMSD of the C-alpha atom distances using a appropriate program as listed in the description. The superimposed structures are analyzed for possible clashes between residues. For each type of atoms (a,b). where atom a is in structure A and atom b is in structure B the distance d(a,b) between the atoms is calculated as the standard Euclidian distance. All atom pairs with distance smaller than a given predefined threshold are potentially structural clashes. A set of rules is imposed to filter out atom pairs with distance smaller than the threshold which are not to be considered as clashes. The rules are:

    • i. Atom pairs that form part of the residue that are aligned in the alignment based on the superimposition are filtered out.
    • ii. Atom pairs that form part of residues that are adjacent to aligned residue are filtered out.
    • iii. Atom pairs where both atoms are backbone atoms are filtered out.
    • iv. Atom pairs that form part of residues that are both surface exposed are filtered out.
      Surface exposed can be computed based on the “solvent exposed surface area” computed by the DSSP-program by division by the standard accessibilities in the following list; A=62, C=92, D=69, E=156, F=123, G=50, H=130, I=84, K=174, L=97, M=103, N=85, P=67, Q=127, R=211, S=64, T=80, V=81, W=126 and Y=104. The threshold fro interatomic distances can be 3 Å, or 2.7 Å, or 2.5 Å or 2.3 Å, or 2.1 Å or 2 Å. The minimal relative surface exposed area for filtering out an atom pair is 20% or preferably 30% for each residue. The found clashes are visualized and inspected in a graphic display program.

Example 10

“Structural Stop Codons” for Combining Protease—Subtilisin S8A

After the superimposition of the two X-ray structures of BPN′ (1SBT—also disclosing the amino acid sequence) and Savinase (1SVN—also disclosing the amino acid sequence) using a suitable display software like INSIGHT II from Accellrys inc. a “structural stop codon” can be found i.e. a clash between to residues with distance lower than a certain threshold here 2.5 Å. The residues giving a clash can be seen are located in the core of the two proteins and having the following residues below 2.5 Å apart to I198 from Savinase structure 1SVN and I268 BPN′ 3D structure 1SBT. Mutation of either 1SVN to I198V or A or G or T, or the SBT sequence to I268V or A or G or T will remove the interaction.

So for example making the hybrid construction 1SVN sequence A1-G219 and 1SBT sequence N218-Q275 should include the mutations suggested above to obtain the best result regarding expression.

Example 11

“Structural Stop Codons” for Combining Protease TY145 and Savinase

After the superimposition of the two X-ray structures of TY145 (see patent application WO2004067737 A3, also disclosing the amino acid sequence (SEQ ID NO: 1)) and Savinase (1SVN—also disclosing the amino acid sequence) using a suitable display software like INSIGHT II from Accellrys inc. a “structural stop codon” can be found i.e. a clash between to residues with distance lower than a certain threshold here 2.1 Å:

  • TY145 P308 clashes with Savinase I198
  • TY145 W101 clashes with Savinase M119
  • TY145 103 clashes with Savinase W113
  • Savinase Y263 clashes with TY145 Mainchain

Example 12

“Structural Stop Codons” for Combining Lipases

Two hybrid enzymes consisting of the N-terminal from Thermomyces lanuginosus lipase (TLL, SEQ ID NO: 26) and the C-terminal from Fusarium sp. lipase (KVL, SEQ ID NO: 27) have been constructed (Construct 1 and Construct 2). The point of crossover resides within conserved regions within the two enzymes. A study of the three-dimensional structure of Thermomyces lanuginosus lipase 1GT6 and a model of the Fusarium sp. lipase build based on the 1GT6 structure reveals two places of residue clashes when making the two hybrid constructs.

In general the following “structural stop codons” can be found:

  • TLL F142 clashes with KVL F136
  • TLL T64 clashes with KVL F24
  • TLL I222 clashes with KVL Y226
  • TLL F80 clashes with KVL 160
  • TLL F55 clashes with KVL A62

The structural problem has been alleviated by introduction of the following mutations T64G and T64G/I222L into the two hybrid enzymes Construct 1 and Construct 2, respectively.

The constructs for two specific hybrids are (the numbers are taken for KVL and TLL protein sequences):

    • Construct 1. KVL 1-28 and TLL 29-269
    • Construct 2. KVL 1-28 and TLL 29-227 and KVL 225-267
    • Construct 3. KVL 1-28 and TLL 29-269 and TLL T64G
    • Construct 4. KVL 1-28 and TLL 29-227 and KVL 225-267 and TLL T64G and TLL I222L
      The 3D structures of the KVL lipase was build using the Accelrys software HOMOLOGY program other suitable software like NEST could also be used.

Example 13

“Structural Stop Codons” for Combining Laccases

Analyzing the three dimensional structure of the Coprinus cinerius laccase (CLL, SEQ ID NO:28) and the three dimensional structure model of Myceliophthora thermophila laccase (MTL, SEQ ID NO: 29) build using the NEST software based on the Melanocarpus albomyces laccase structure (1GWO—also disclosing the amino acid sequence), it can be found that several “structural stop codons” can be found. Focusing on the core “structural stop codons” the following residues can found to be important to mutate. There are the following important “structural stop codons” that has to be removed before attempting shuffling of the two laccases of CCL and MTL:

MTL M301A and/or CCL F124L

CCL E239A or D

CCL E453A

MTL W464L

MTL W420F

There are besides the mentioned changes other important issues concerning the cystin bridges MTL C301/C267 and CCL C135/C222. Securing of no overlaps in theses regions are of great importance. To avoid the problems the following are a plausible way to go further:

MTL C379S/C345S and CCL C135G/C222V

Alternatively “transfer” CCL cystinbridge to MTL: MTL G193C/V281C.

Example 14

“Structural Stop Codons” for Combining Xylanases

Analysing the three dimensional structure of the Bacillus agaradherens xylanase (BAX), having the X-ray structure 1QH7 (also disclosing the amino acid sequence), and the three dimensional structure of Bacillus halodurans xylanase (BHX) having the X-ray structure 1XNS (also disclosing the amino acid sequence) it can be found that several “structural stop codons” can be found. Focusing on the core “structural stop codons” the following residues can found to be important to mutate:

  • BAX R49 clashes with BHX Y165
  • BAX K53 clashes with BHX Y5
  • BAX K136+E56 clashes with BHX R73
  • BAX F163 clashes with BHX F145
  • BAX L199 clashes with BHX W42
  • BAX M28 clashes with BHX W6
    Analysing the three dimensional structure of the Bacillus agaradherens xylanase (BAX), having the X-ray structure 1QH7, and the three dimensional structure model of Paenibacillus sp. xylanase (PSX) having the X-ray structure 1BVV (also disclosing the amino acid sequence), it can be found that several “structural stop codons” can be found. Focusing mostly on the core “structural stop codons” the following residues can found to be important to mutate:
  • BAX R49 clashes with PSX Y166+Q7
  • BAX K53 clashes with PSX Y5
  • BAX L199 clashes with PSX A42
  • BAX F163 clashes with PSX F146
  • BAX M28 clashes with PSX W6
  • BAX Y195 clashes with PSX N54.