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 polypeptides 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 , erythropoietin , 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 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 . accelrys . 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 /. the 3d models may be structurally aligned by methods known in the art . 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 : in the case of enzymes having an active site , the structural alignment may be a superimposition 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 superimposition 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 . 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 , 1 . 7 å or 1 . 5 å ). 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 ): the substitution may be such that the two residues after the substitution can form a hydrogen bond , a salt bridge or a cysteine bridge . 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 , e . g ., as described in wo 1995022625 . in the case of the parent polypeptides being enzymes , the transformants can be screened for enzymatic activity . 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 subfamily 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 . 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 ( 1cdg ), 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 . fig1 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 : 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 : 12 . cdgt_bacsp . gcg bacillus sp . ( strain 17 - 1 , wo 2003068976 ) ( seq id no : 4 ) 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 fig2 . other sequences may be aligned by conventional methods , e . g . by use the software gap from uwgcg version 8 . 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 . 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 . 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 fig2 aligned residues shown vertically above each other , with gaps inserted to separate non - aligned residues . the two structures were analyzed , and the following unaligned residues were identified as having a side chain with less than 30 % solvent accessibility 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 a : the above residues are marked by asterisks in fig2 . to design hypothetical hybrids , residues in a partial sequence of novamyl ( seq id no : 17 ) were compared with residues in the complementary part 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 fig2 . 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 clashes . for comparison , similar variants were constructed without substitutions . novamyl residues substitutions novamyl 1 - 494 + cgtase 495 - 683 k7s + y181a cgtase 1 - 494 + novamyl 495 - 686 r545s novamyl 1 - 499 + cgtase 500 - 683 k7s + y181a cgtase 1 - 499 + novamyl 500 - 686 r545s novamyl 1 - 410 + cgtase 410 - 683 k7s + y181a novamyl 1 - 378 + cgtase 378 - 683 k7s + y181a novamyl 1 - 204 + cgtase 207 - 683 k7s , w107f cgtase 1 - 139 + novamyl 131 - 204 + y181a cgtase 207 - 683 novamyl 1 - 204 + cgtase 207 - 683 k7s , w107f , y181a novamyl 1 - 204 + cgtase 207 - 683 k7s , y181a the first eight of the above hybrids are found in seq id no : 18 to seq id no : 25 . 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 . 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 . the two hybrids improve the softness of breadcrumb as compared to cgtase . bahy2 improves the mobility of free water as compared to cgtase , whereas bahy1 has the same effect as cgtase . this example illustrates the possible outcome of a hybridization between two proteins having the sequences seqa and seqb ( fig3 ): 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 clashes 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 for 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 . ito 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 & lt ; a = s = c & lt ; v = t & lt ; p & lt ; l = i = n = d = m & lt ; e = q & lt ; k & lt ; h & lt ; r & lt ; f & lt ; y & lt ; 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 2 20 = 1048576 and for the clashes it is 2 18 = 262144 . 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 ( fig4 ). 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 : where d is diversity without “ structural stop codons ”, n the number of proteins , k the number segments , and p the number of pairs ( ie . 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 ”. 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 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 . 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 . 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 : 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 1 gt6 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 . 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 . 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 : 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 : 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 1xnb ( 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 : 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 :