Patent Grant
8883970
The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability pH, /activity profile, pH/stability profile, stability towards oxidation, Ca2+ dependency, specific activity, and solubility in particular under production and application conditions.
Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
In modern and industrial biotechnology high protein concentrations are achieved during fermentation, purification, recovery and in product formulation.
During the fermentation the protein concentration depend on the host cell used. In industrial processes the protein concentration typically lies from above 0.1 g/liter fermentation broth. For high yield recombinant production of alpha-amylases in Bacillus sp. the protein concentration may be as high as 250 g/liter fermentation broth. After purification the protein concentration may reach levels of about 1000 g/liter.
Such high concentrations leads to an undesirable precipitation resulting in lose of active protein. The tendency today is towards products of increasing strength, which make the ability to maintain the enzyme in solution of increasing importance. Often up-concentration of a protein solutions results in protein precipitates, which are difficult to dissolve into active protein.
Thus, it is the object of this application to provide alpha-amylases with altered properties as defined below, in particular increased solubility.
The object of the present invention is to provide Termamyl-like amylases which variants in comparison to the corresponding parent alpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca2+ dependency, specific activity, and solubility, in particular increased solubility under production conditions.
Nomenclature
In the present description and claims, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, alpha-amylase variants of the invention are described by use of the following nomenclature:
According to this nomenclature, for instance the substitution of alanine for asparagine in position 30 is shown as:
A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).
Where a specific alpha-amylase contains a “deletion” in comparison with other alpha-amylases and an insertion is made in such a position this is indicated as:
Multiple mutations are separated by plus signs, i.e.:
When one or more alternative amino acid residues may be inserted in a given position it is indicated as
A30N,E or
A30N or A30E
Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of: R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
Further, “A30X” means any one of the following substitutions: A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short: A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
The object of the present invention is to provide polypeptides, such as enzymes, in particular alpha-amylases, with an alteration in at least one of the following properties relative to said parent polypeptide: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca2+ dependency, specific activity, and solubility, in particular under production conditions. The properties will be defined further below.
Polypeptide
Polypeptides according to the invention include proteins with biological activity, antimicrobial activity, and enzymatic activity.
Contemplated enzyme activities include proteases, amylases, CGTases, mannanases, maltogenic amylases, glucoamylases, carbohydrases, transferases, lyases, oxidoreductases, lipases.
In one preferred embodiment the enzyme is an alpha-amylase, in particular a Bacillus or Aspergillus alpha-amylase. In a preferred embodiment the Bacillus alpha-amylase is a Termamyl-like amylases.
Polypeptides with biological activity include, EPO, TPO, growth hormones, regulatory peptides, blood coagulation factores, antibodies etc.
Polypeptide with Altered Solubility
A protein (polypeptide) crystal is built up of systematic ordered identical units closely packed together in 3 dimensions. The unit cells may contain one or more protein molecules and a significant amount of water. Also ions like Calcium, Sodium, Chloride, Sulphate and larger molecules such as surfactants and substrates—present during crystal growth—can be identified in the tertiary structure. Although the single molecules and the single atoms are very small, the repeating array of identical units facilitates X-ray scattering and thus provide structure information, which can be used for protein engineering purposes.
Relative to crystals, precipitates and aggregates are smaller units and the single molecules are more disordered than in the crystals. Nevertheless some of the intermolecular interactions are the same as is found in a well ordered crystals and thus can the tertiary structure (3D structure) and intermolecular information be used for designing protein engineered alpha-amylase variants with altered properties, in particular with reduced tendency of precipitating, i.e., variant with increased solubility.
Due to the spherical and often irregular surface structures of proteins, large holes in the unit cell exist, which are occupied by more disordered solvents like water. In fact, the large majority of the protein surface is covered with layers of water, which is one reason why structures of proteins solved by X-ray crystallography are the same as those for proteins in solution. Only at a few areas the protein molecules are in direct contact with each other, but also solvent mediated contacts function as the “glue” that binds the crystal together.
In general the solubility of proteins is affected by organic solvents, salts like ammonium sulphate, NaCl and CaCl2, and by pH change altering the surface charge of the molecule. These factors are considered both for enzyme production to keep the enzyme in solution and for crystallography experiment to grow useful crystals. Large symmetric crystals are grown by slow increase in protein concentration or by altering the protein surface thereby enhancing the inter-molecular contacts.
Not all proteins crystallise in a form useful for X-ray crystallography determination methods, but based on existing tertiary structure accurate models can be build if the homology between the template molecule and the molecule of interest is high enough.
When homologous polypeptides, such as enzymes, in particular Termamyl-like alpha-amylases (defined below), are compared on a tertiary structural basis, the large majority of differences are found on the surface of the molecule.
Despite that, the inventors found that surface residues identified to be involved—directly or indirectly through water molecules—in crystal formations for the Termamyl-like amylase (SP722 disclosed in SEQ ID NO: 4) is also playing a key role for crystallisation of other Termamyl-like amylases (defined below).
Below is described one example of modelling a tertialy structure of one Termamyl-like alpha-alpha from another Termamyl-like alpha-amylase structure (SP722 structure disclosed in APPENDIX 1).
It is to be understood that the concept of the invention and the below described modelling method according to the invention can generally be extrapolated to all polypeptides, proteins, in particular enzymes, such as alpha-amylases.
The Tertiary Structure of SP722 and Modelling the Tertiary Structures of Another Termamyl-Like Alpha-Amylase.
Mutants of alpha-amylases of the present invention have been found based on the tertiary structure shown in APPENDIX 1 of SP722 (SEQ ID NO: 4). Mutants of other polypeptides may be found based on other tertiary structures.
Crystals of the alkaline Termamyl-like alpha-amylase (SP722) (shown in SEQ ID NO: 4 and also disclosed in U.S. Pat. No. 5,824,531) were obtained by the hanging drop method (well-known in the art), and the tertiary structure (3D-structure) is disclosed in APPENDIX 1.
The unit cell was found to contain 4 enzyme molecules and each molecule has 8 interacting zones (see
As can be seen from
A model of another alkaline Termamyl-like amylase, AA560 has been build based on the SP722 tertiary structure disclosed in APPENDIX 1. AA560 alpha-amylase is about 87% identical to the template amylase (SP722) and the alignment contains no insertion or deletions. Due to the high homology (identity), the same symmetry and the same crystal interactions, the same interaction zones on the protein surface are involved in crystallization and precipitation at the increasing protein concentrations, which are reached during production, i.e., starting from fermentation stage through the purification stage (see Background section).
Mutations in these interaction zones were constructed, the enzyme expressed and purified, and the protein solubility was measured under different conditions as described in Examples 8 and 9 using the method described in the “Materials and Methods” section.
The findings of the present invention may be applied on Termamyl-like amylases being at least 60% identical, preferably at least 70% identical, more preferably 80% identical, even more preferably 85% identical, even more preferably 90% identical, even more 95% identical, even more 97% identical, even more 99% identical to the Termamyl-like alpha-amylase shown in SEQ ID NO: 12. In a preferably the findings may be used on alkaline Termamyl-like alpha-amylases, especially alkaline alpha-amylases of the same length, without additional amino residues or gaps in an aligned primary structure in comparison to SP722 (SEQ ID NO: 4 shown as number 1 in the alignment in
Contrary hereto, for instance, Termamyl (shown as sequence number 4 in the alignment in
BAN (shown as sequence number 3 in the alignment in
BSG (shown as sequence number 5 in the alignment in
KSM-K36 and KSM-K38 (EP 1,022,334-A) lack five amino acid residues (positions 1 and 2) and has gaps in positions 174 and 181-182 when aligned with SP722.
AA180, AA20 and Amrk385 (Danish patent application no. PA 2000 00347 or PCT/DK01/00133) have one additional amino acid in position 261 when aligned with SP722.
Below it is described how to model a Termamyl-like alpha-amylase from another alpha-amylase. In Example 4 the modelling of AA560 from SP722 is described. This method can be exprepolated to other polypeptides as for instance the above-mentioned.
Modelling of Termamyl-Like Alpha-Amylases
WO 96/23874 provides the tertiary structure (3D Structure), X-ray crystal structural data for a Termamyl-like alpha-amylase, which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase (BAN™) and amino acids 301-483 of the C-terminal end of the B. licheniformis alpha-amylase (SEQ ID NO: 8). WO 96/23874 further describes methodology for designing (modelling), on the basis of an analysis of the structure of a parent Termamyl-like alpha-amylase, variants of the parent Termamyl-like alpha-amylase which exhibit altered properties relative to the parent.
Other Termamyl-like structures may be modelled in accordance with WO 96/23874, which is hereby incorporated by reference.
In connection with obtaining variant of the present invention the AA560 tertiary structure was designed (modelled) based on the tertiary structure of SP722 (disclosed in APPENDIX 1) as described in Example 1. The structure of other Termamyl-like alpha-amylases (e.g., those disclosed herein) may be built analogously.
Termamyl-Like Alpha-Amylases
A number of alpha-amylases produced by Bacillus spp. are highly homologous (identical) on the amino acid level.
The identity of a number of Bacillus alpha-amylases can be found in the below Table 1:
For instance, the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 8 (commercially available as Termamyl™) has been found to be about 81% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilus alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 6. Further homologous alpha-amylases include SP690 and SP722 disclosed in WO 95/26397 and further depicted in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Other amylases are the AA560 alpha-amylase derived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707 alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 and described by Tsukamoto et al., 1988, Biochemical and Biophysical Research Communications 151: 25-31.
The KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (from KAO Corporation). Also the K38 and K38 alpha-amylases disclosed in EP 1,022,334 are contemplated according to the invention.
Still further homologous alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the alpha-amylases identified in
WO 91/00353 and WO 94/18314. Other commercial Termamyl-like alpha-amylases are comprised in the products sold under the following tradenames: OPTITHERM™ and TAKATHERM™ (available from Solvay); MAXAMYL™ (available from Gist-brocades/Genencor), SPEZYM AA™ and SPEZYME DELTA AA™ (available from Genencor), and KEISTASE™ (available from Daiwa), PURASTAR™ ST 5000E, PURASTAR™ HPAM L (from Genencor Int.).
Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of “Termamyl-like alpha-amylases”.
Accordingly, in the present context, the term “Termamyl-like alpha-amylase” is intended to indicate an alpha-amylase, which, at the amino acid level, exhibits a substantial identity to Termamyl™, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
In other words, all the following alpha-amylases, which has the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 13 are considered to be “Termamyl-like alpha-amylase”. Other Termamyl-like alpha-amylases are alpha-amylases i) which displays at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology with at least one of said amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13, and/or ii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 1, 3, 5, 7, 9 and of the present specification (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and 12, respectively).
In connection with property i), the homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (described above). Thus, Gap GCGv8 may be used with the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, default scoring matrix, for nucleic sequences and 3.0 and 0.1, respectively, from protein sequences. GAP uses the method of Needleman/Wunsch/Sellers to make alignments.
A structural alignment between Termamyl (SEQ ID NO: 8) and another Termamyl-like alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha-amylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., 1987, FEBS Letters 224: 149-155) and reverse threading (Hube et al., 1998, Protein Science 7(1): 142-149.
Hybridization
The oligonucleotide probe used in the characterization of the polypeptide, such as the Termamyl-like alpha-amylase in accordance with property ii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.
Suitable conditions for testing hybridization involve pre-soaking in 5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridisation in the same solution supplemented with 100 mM ATP for 18 hours at ˜40° C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at ˜75° C. (very high stringency). More details about the hybridisation method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
In the present context, “derived from” is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question. The term is also intended to indicate that the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.
Parent Termamyl-Like Alpha-Amylases
According to the invention all Termamy-like alpha-amylases, as defined above, may be used as the parent (i.e., backbone) alpha-amylase. In a preferred embodiment of the invention the parent alpha-amylase is derived from B. licheniformis, e.g., one of those referred to above, such as the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 10. Especially preferred parent alpha-amylases are the SP722 alpha-amylase and the AA560 alpha-amylase. In one embodiment the parent alpha-amyase has one or more of the following mutations/substitutions: Delta (R81-G182); Delta (D183-G184); Delta (D183-G184)+N195F; RR181Q+N445Q+K446N; Delta (D183-G184)+R181Q.
Parent Hybrid Termamyl-Like Alpha-Amylases
The parent alpha-amylase (i.e., backbone alpha-amylase) may also be a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.
The parent hybrid alpha-amylase may be one, which on the basis of amino acid homology (identity) and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like alpha-amylase family. In this case, the hybrid alpha-amylase is typically composed of at least one part of a Termamyl-like alpha-amylase and part(s) of one or more other alpha-amylases selected from Termamyl-like alpha-amylases or non-Termamyl-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin.
Thus, the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial alpha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase. The Termamyl-like alpha-amylase from which a partial amino acid sequence derives, may be any of those specific Termamyl-like alpha-amylase referred to herein.
For instance, the parent alpha-amylase may comprise a C-terminal part of an alpha-amylase derived from a strain of B. licheniformis, and a N-terminal part of an alpha-amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus. For instance, the parent alpha-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis alpha-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase having the amino acid sequence shown in SEQ ID NO: 10 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 10; or b) an amino acid segment corresponding to the 68 N-terminal amino acid residues of the B. stearothermophilus alpha-amylase having the amino acid sequence shown in SEQ ID NO: 6 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
Another suitable parent hybrid alpha-amylase is the one previously described in WO 96/23874 (from Novo Nordisk) constituting the N-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl (amino acids 301-483 of the mature protein).
Altered Properties
The following discusses the relationship between mutations, which are present in variants of the invention, and desirable alterations in properties (relative to those a parent Termamyl-like alpha-amylase), which may result therefrom.
As mentioned above the invention relates to Termamyl-like alpha-amylases with altered properties, in particular under production conditions.
Parent Termamyl-like alpha-amylase specifically contemplated in connection with going through the specifically contemplated altered properties are the above mentioned parent Termamyl-like alpha-amylase and parent hybrid Termamyl-like alpha-amylases. The SP722 alpha-amylase is used as the starting point, but corresponding positions in, e.g., the Termamyl, BSG, BAN, AA560, SP690, AA180, KSM AP1378, and #707, K38, and K36 should be understood as disclosed too.
In a preferred embodiment the variant of the invention has increased solubility, in particular under washing or cleaning conditions.
In an aspect the invention relates to variant with altered properties as mentioned above.
In the first aspect a variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more positions (using SEQ ID NO: 12 for the amino acid numbering) selected from the group consisting of: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, and N484,
wherein
(a) the alteration(s) are independently
(b) the variant has alpha-amylase activity and
(c) each position corresponds to a position of the amino acid sequence of SEQ ID NO: 12.
In SP722 (SEQ ID NO: 4) such corresponding positions are: R28; N94; L118; N125; Q174; R181; G182; D183; G184; A186; W189; N195, M202, Y298; N299; N302; S303; N306; A310; N314; K320; H324; Q345; F396, T400, W439, Q444; N445, K446, Q449, K458; N471, and K484.
In a preferred embodiment the variant of the invention (using SEQ ID NO: 12 for the numbering) has one or more of the following mutations/substitutions: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N,K; S94K; R118K; N125A,R,K; N174D; R181Q,E,K; G186R; W189R,K; N195F; M202L; Y298H,F; N299A; K302R, S303Q, N306G,D,R,K; R310A,K,Q,E,H,D,N; N314D; R320K; H324K; E345R,D,K,N; Y396F; R400T,K; W439R; R444K; N445K,Q; K446N; Q449E; R458K; N471E; N484Q.
Preferred double, triple and multi-mutations—using SEQ ID NO: 12 as the basis for the numbering—include:
Contemplated variants include the specific ones mentioned in the examples and the above mention mutations in further combination with one or more of the mutations/substitution: Delta (D183-G184); N195F; R181Q; G186R; M202L; V206F,L,M; N193S,T,P
Increased Solubility
All the above-mentioned mutations result in Termamyl-like alpha-amylase variants with altered solubility when used in any alpha-amylase within the group of Termamyl-like alpha-amylase as defined above.
Thus, in a preferred embodiment the variant of the invention is a variant of a parent Termamyl-like alpha-amylase, which has increased solubility (as defined) in comparison to the parent Termemyl-like alpha-amylase, comprising an alteration at one or more positions (using SEQ ID NO: 12 for the amino acid numbering) selected from the group of: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484,
wherein
(a) the alteration(s) are independently
(b) the variant has alpha-amylase activity and
(c) each position corresponds to a position of the amino acid sequence of SEQ ID NO: 12.
In a preferred embodiment a variant of the invention with increased solubility in comparison to the parent alpha-amylase has one or more of the following substitutions: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N,K; S94K; R118K; N125A,R,K; N174D; R181Q,E,K; G186R; W189R,K; N195F; M202L; Y298H,F; N299A; K302R, S303Q, N306G,D,R,K; R310A,K,Q,E,H,D,N; N314D; R320K; H324K; E345R,D,K,N; Y396F; R400T,K; W439R; R444K; N445K,Q; K446N; Q449E; R458K; N471E; N484Q.
In a more preferred embodiment variant of the invention with increased solubility (determined using one of the methods described in the “Materials and Methods” section)—using SEQ ID NO: 12 as the basis for the numbering—include:
Other combinations according to the invention include the specific one mentioned in the examples and one or more of the following substitutions: N195F; R181Q; G186R; M202L; V206F,L,M, N193P.
Stability
In the context of the present invention, mutations (including amino acid substitutions and deletions) of importance with respect to achieving altered stability, in particular improved stability (i.e., higher or lower), at especially high pH (i.e., pH 8-10.5) include any of the mutations listed in the in “Altered properties” section.
Ca2+ Stability
Altered Ca2+ stability means the stability of the enzyme under Ca2+ depletion has been improved, i.e., higher or lower stability. In the context of the present invention, mutations (including amino acid substitutions and deletions) of importance with respect to achieving altered Ca2+ stability, in particular improved Ca2+ stability, i.e., higher or lower stability, at especially high pH (i.e., pH 8-10.5) include any of the mutations listed in the in “Altered properties” section.
Specific Activity
In a further aspect of the present invention, important mutations (including amino acid substitutions and deletions) with respect to obtaining variants exhibiting altered specific activity, in particular increased or decreased specific activity, especially at temperatures from 10-60° C., preferably 20-50° C., especially 30-40° C., include any of the mutations listed in the in “Altered properties” section.
General Mutations in Variants of the Invention
The particularly interesting mutations (including amino acid substitutions and deletions) are those that increase the mobility around the active site of the enzyme. This is accomplished by changes that disrupt stabilizing interaction in the vicinity of the active site, i.e., within preferably 10 Å or 8 Å or 6 Å or 4 Å from any of the residues constituting the active site.
Examples are mutations that reduce the size of side chains, such as
Such mutations are expected to cause increased flexibility in the active site region either by the introduction of cavities or by the structural rearrangements that fill the space left by the mutation.
It may be preferred that a variant of the invention comprises one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more Proline residues present in the part of the alpha-amylase variant which is modified is/are replaced with a non-Proline residue which may be any of the possible, naturally occurring non-Proline residues, and which preferably is an Alanine, Glycine, Serine, Threonine, Valine or Leucine.
Analogously, it may be preferred that one or more Cysteine residues present among the amino acid residues with which the parent alpha-amylase is modified is/are replaced with a non-Cysteine residue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.
Furthermore, a variant of the invention may—either as the only modification or in combination with any of the above outlined modifications—be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 is replaced by an Asn and/or Gln, respectively. Also of interest is the replacement, in the Termamyl-like alpha-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by an Arg.
It will be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.
Furthermore, it may be advantageous to introduce mutations in one or more of the following positions (using SEQ ID NO: 10 (Termamyl™) for the numbering):
In AA560 this corresponds to
Contemplated combinations thereof, include
Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of alpha-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the alpha-amylase-encoding sequence will be discussed.
Cloning a DNA Sequence Encoding an Alpha-Amylase
The DNA sequence encoding a parent alpha-amylase may be isolated from any cell or microorganism producing the alpha-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the alpha-amylase to be studied. Then, if the amino acid sequence of the alpha-amylase is known, homologous, labeled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labeled oligonucleotide probe containing sequences homologous to a known alpha-amylase gene could be used as a probe to identify alpha-amylase-encoding clones, using hybridization and washing conditions of lower stringency.
Yet another method for identifying alpha-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming alpha-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for alpha-amylase, thereby allowing clones expressing the alpha-amylase to be identified.
Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described by Beaucage and Caruthers, 1981, Tetrahedron Letters 22: 1859-1869 or the method described by Matthes et al., 1984, The EMBO J. 3: 801-805. In the phosphoroamidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., 1988, Science 239: 487-491.
Expression of Alpha-Amylase Variants
According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
The recombinant expression vector carrying the DNA sequence encoding an alpha-amylase variant of the invention may be any vector that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.
While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus alpha-amylases mentioned herein comprises a preregion permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
The procedures used to ligate the DNA construct of the invention encoding an alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).
The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.
Examples of suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.
The yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
In a yet further aspect, the present invention relates to a method of producing an alpha-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
The alpha-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
The alpha-amylase variants of this invention possess valuable properties allowing for a variety of industrial applications. In particular, enzyme variants of the invention are applicable as a component in washing, dishwashing and hard surface cleaning detergent compositions. Variant of the invention with altered properties may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP patent publications Nos. 252 730 and 63 909, and WO 99/19467.
Further, variants of the invention are also particularly useful in the production of sweeteners and ethanol from starch, and/or for textile desizing.
Composition
In an aspect the invention relates to a composition comprising a variant of the invention. In an embodiment the composition of the invention comprises one or more other enzymes, including one or more selected from the group of such as a lipase, cutinase, protease, cellulase, mannanase, maltogenic amylase, CGTase, glucoamylase, peroxidase or laccase, and/or another alpha-amylase.
Detergent Compositions
As mentioned above, variants of the invention may suitably be incorporated in detergent compositions. Reference is made, for example, to WO 96/23874 and WO 97/07202 for further details concerning relevant ingredients of detergent compositions (such as laundry or dishwashing detergents), appropriate methods of formulating the variants in such detergent compositions, and for examples of relevant types of detergent compositions.
Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, mannanase, maltogenic amylase, CGTase, glucoamylase, peroxidase or laccase, and/or another alpha-amylase.
Alpha-amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-10 mg (calculated as pure, active enzyme protein) of alpha-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.
The invention also relates to a method of providing alpha-amylases with altered properties relative to the parent alpha-amylase, in particular solubility, especially increased solubility, substrate specificity, substrate binding, substrate cleavage pattern, temperature stability, pH dependence of enzymatic activity, pH dependence of stability, stability towards oxidation, Ca2+-dependency and specific activity, comprising the following steps:
(a) modelling the parent alpha-amylase on the three-dimensional structure of SEQ ID NO: 4 depicted in the Appendix 1 to produce a three-dimensional structure of the parent alpha-amylase;
(b) identifying in the three-dimensional structure obtained in step (a) at least one structural part of the parent wherein an alteration in said structural part is predicted to result in said altered property;
(c) modifying the sequence of a nucleic acid encoding the parent alpha-amylase to produce a nucleic acid encoding a deletion, insertion, or substitution of one or more amino acids at a position corresponding to said structural part; and
(d) expressing the modified nucleic acid in a host cell to produce the variant alpha-amylase,
wherein the variant has alpha-amylase enzymatic activity and has at least one altered property relative to the parent.
In an embodiment the method is a method of constructing a variant of a parent alpha-amylase having an altered property relative to the parent, wherein the parent alpha-amylase has the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 13 or has a sequence at least 60% identity (determined as described above) to said sequence, said method comprising:
(a) modelling the parent alpha-amylase on the three-dimensional structure of SEQ ID NO:4 depicted in the Appendix 1 to produce a three-dimensional structure of the parent alpha-amylase;
(b) comparing the three-dimensional structure obtained in step (a) with a three-dimensional structure of an unrelated alpha-amylase, wherein the unrelated alpha-amylase differs from the parent alpha-amylase in said property;
(c) identifying a structural part of the three-dimensional structure obtained in step (a) which is different from the three-dimensional structure of the unrelated alpha-amylase and which is predicted to be relevant to said property,
(d) modifying the sequence of a nucleic acid encoding the parent alpha-amylase to produce a nucleic acid encoding a deletion, insertion, or substitution of one or more amino acids at a position corresponding to said structural part; and
(e) expressing the modified nucleic acid in a host cell to produce the variant alpha-amylase,
wherein the variant has alpha-amylase activity and has one or more altered properties as compared to the parent alpha-amylase.
The Termamyl-like alpha-amylases contemplated are the ones described above.
Method of Increasing the Solubility of Polypeptides
The present invention also relates to a method of increasing the solubility of polypeptides, such as enzymes, in particular Termamyl-like alpha-amylases.
The method of this invention comprises substitutions, insertions and/or deletion of one or more amino acid residue(s), which residue(s) hold a position close to a neighbour polypeptide molecule located in the protein crystal. In the context of this invention, a polypeptide amino acid residue holding a position close to the neighbour molecule indicates an amino acid residue located within the polypeptide in a way that it is within a potential intermolecular interactive distance from an amino acid residue located at a neighbour enzyme molecule in the crystal or precipitate.
Examples of potential intermolecular interactions include, but are not limited to hydrogen bonding, salt bridge formation, polar interactions, hydrophobic interactions and aromatic interactions.
The term “neighbouring” means in the context of the present invention the shortest distance between two positions in a crystal structure being prepared, e.g., as described in “Protein Crystallization Techniques, Strategies, and Tips”, A Laboratory Manual by Terese M. Bergfors), or as describe on the Internet on the site: hamptonresearch.com.
Thus, in this aspect the invention relates to a method of increasing the solubility of enzyme crystals, wherein one or more amino acid residue(s),
1) located within a distance of 6.0 Å of a neighbouring polypeptide molecule in the tertiary crystal structure, and
2) interacting with said neighbouring polypeptide molecule, are mutated, e.g., by substitution or deletion.
In a preferred embodiment the amino acid residues to be mutated are located with 3.5 Å of a neighbouring polypeptide.
Contemplated polypeptides include antimicrobial polypeptides, polypeptides with biological activity, such as insulin, growth hormone, EPO, TPO, Factor VII, Factor VIII.
Contemplated enzyme activities include protease, amylase, CGTase, carbohydrase, transferase, lyase, oxidoreductase, lipase, cellulase, cutinase, pectate lyase, mannanase, maltogenic amylase, glucoamylase, pectin lyase activity.
In a preferred embodiment the enzyme is an amylase, preferably an alpha-amylase, especially a Termamyl-like alpha-amylase as will be described further below.
An amino acid position “close” to the substrate indicates a distance less than 6 Å (angstrom) corresponding to a protein-protein interaction mediated by a single water molecule. In a preferred embodiment an amino acid position close to the substrate indicates a distance less than 3.5 Å (angstrom) corresponding to a direct protein-protein interaction.
Method of Increasing the Solubility of Termamyl-Like Alpha-Amylases
In this aspect the invention relates to a method of increasing the solubility of Termamyl-like alpha-amylase crystals, wherein one or more amino acid residue(s),
1) located within a distance of 6.0 Å of a neighbouring Termamyl-like alpha-amylase molecule in the tertiary crystal structure, and
2) interacting with said neighbouring Termamyl-like alpha-amylase molecule, are mutated, e.g., by substitution or deletion.
In a preferred embodiment the amino acid residues to be mutated are located with 3.5 Å of a neighbouring Termamyl-like alpha-amylase. The crystal structure of SP722 depicted in APPENDIX 1 may be used as the reference (fix-point) for determining the distance between neighbouring alpha-amylases.
However, it is within the scope of the invention that the reference (fix-point) crystal structure is a structure modelled (as described above or in WO 96/23874) from SP722 depicted in APPENDIX 1.
Preferred Termamyl-like alpha-amylases include Bacillus alpha-amylase selected from the group consisting of alpha-amylase derived from a strain of B. licheniformis, B. amyloliquefaciens, B. stearothermophilus, Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, or DSMZ no. 12649, KSM AP1378.
Termamyl-like alpha-amylase may be any of the alpha-amylases selected from the group depicted in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13, or Termamyl-like alpha-amylase having an amino acid sequence which has a degree of identity to SEQ ID NO: 4 of at least 60%, preferably 70%, more preferably at least 80%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99%, or Termamyl-like alpha-amylase encoded by a nucleic acid sequence, which hybridizes under low, preferably medium, preferred high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 11. The conditions are described in further details above.
Termamyl-like alpha-amylase amino acid residues being less than 6.0 Å from the nearest neighbour amylase molecule are the following (using the SP722 numbering): 19, 20, 21, 22, 25, 28, 29, 53, 76, 84, 87, 90, 93, 94, 124, 125, 126, 128, 142, 144, 156, 157, 158, 159, 160, 161, 170, 171, 172, 173, 174, 175, 183, 184, 185, 186, 187, 188, 189, 190, 193, 195, 196, 197, 209, 212, 226, 229, 256, 257, 258, 259, 280, 281, 298, 299, 300, 302, 303, 304, 305, 306, 310, 311, 314, 319, 320, 321, 322, 341, 345, 405, 406, 408, 444, 447, 448, 449, 463, 464, 465, 466, 467.
Termamyl-like alpha-amylase amino acid residues being less than 3.5 Å from the nearest neighbour amylase molecule are the following (using the SP722 numbering): 22, 25, 28, 76, 94, 125, 128, 158, 160, 171, 173, 174, 184, 189, 209, 226, 229, 298, 299, 302, 306, 310, 314, 320, 345, 405, 447, 466.
The amino acid residues being less than 6.0 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the SP722 amylase: ASN 19, ASP 20, GLY 21, GLN 22, ASN 25, ARG 28, ASP 29, GLN 53, ARG 76, GLN 84, SER 87, HIS 90, LYS 93, ASN 94, ASN 125, ASN 126, ASN 128, LYS 142, ASP 144, LYS 156, TRP 157, ARG 158, TRP 159, TYR 160, HIS 161, PHE 173, GLN 174, ASN 175, ASP 183, GLY 184, LYS 185, ALA 186, TRP 187, ASP 188, TRP 189, GLU 190, SER 193, GLY 196, ASN 197, ASP 209, GLU 212, ASN 226, ASN 229, ALA 256, THR 257, GLY 258, LYS 259, ASN 280, LYS 281, TYR 298, ASN 299, ALA 300, ASN 302, SER 303, GLY 304, GLY 305, ASN 306, ALA 310, ASN 314, GLN 319, LYS 320, HIS 321, PRO 322, GLU 341, GLN 345, PHE 405, ASP 406, HIS 408, GLN 444, ALA 447, GLY 448, GLN 449, THR 463, ILE 464, ASN 465, ALA 466, ASP 467.
The amino acid residues being less than 3.5 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the SP722 amylase: GLN 22, ASN 25, ARG 28, ARG 76, ASN 94, ASN 125, ASN 128, ARG 158, TYR 160, PHE 173, GLN 174, GLY 184, TRP 189, ASP 209, ASN 226, ASN 229, TYR 298, ASN 299, ASN 302, ASN 306, ASN 314, LYS 320, GLN 345, PHE 405, ALA 447, ALA 466.
The amino acid residues being less than 6.0 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the AA560 amylase: ASN 19, ASP 20, ASN 22, ASN 25, ARG 28, SER 29, GLN 53, ARG 76, GLN 84, ALA 87, ASN 90, LYS 93, SER 94, PRO 124, ASN 125, ASN 126, ASN 128, LYS 142, ASP 144, LYS 156, TRP 157, ARG 158, TRP 159, TYR 160, HIS 161, SER 170, ARG 171, LYS 172, LEU 173, ASN 174, ASN 175, ASP 183, GLY 184, LYS 185, GLY 186, TRP 187, ASP 188, TRP 189, GLU 190, THR 193, ASN 195, GLY 196, ASN 197, ASP 209, GLU 212, ASN 226, GLY 229, ALA 256, THR 257, GLY 258, LYS 259, LYS 281, TYR 298, ASN 299, ALA 300, LYS 302, SER 303, GLY 304, ASN 306, ARG 310, GLN 311, ASN 314, GLN 319, ARG 320, HIS 321, PRO 322, GLU 341, GLU 345, LEU 405, HIS 408, ARG 444, ALA 447, GLY 448, GLN 449, THR 463, ILE 464, ASN 465, ALA 466, ASP 467.
The amino acid residues being less than 3.5 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the AA560 amylase: ARG 28, ASN 125, ASN 128, ARG 158, TYR 160, ARG 171, LEU 173, ASN 174, GLY 184, TRP 189, GLY 196, ASP 209, ASN 226, TYR 298, ASN 299, LYS 302, ASN 306, ARG 310, ASN 314, ARG 320, GLU 345, ALA 447, ALA 466.
Substitutions in Alpha-Amylases Leading to Higher Solubility and Thus to Higher Performance
In general, any substitutions disturbing or destroying a protein-protein interaction of any kind will lead to higher solubility and thus to higher performance in a particular application. An amino acid residue participating in protein-protein interaction may thus be changed to a larger, a smaller, more hydrophobic, more hydrophilic, to a charged or to an uncharged amino acid leading to higher solubility.
A larger amino acid residue is capable of making a steric hindrance for the interaction and a smaller amino acid residue makes the distance between the interacting residues to large, weaking the interaction. Polar interaction and in particular saltbridges are destroyed by changing at least one of the involved residues to a hydrophobic residues, by changing at least one of the involved residues to a smaller residue making the distance for a strong interaction to large, or by changing at least one of the involved residues to a larger residue which make sterical hindrance. Hydrophobic interactions are effectively disturbed by changing one of the interacting amino acids to hydrophilic amino acid residues but also smaller or larger hydrophobic residues can destroy this kind of interaction. Aromatic interactions can be prevented by changing to a hydrophilic residue or to a small hydrophobic residue.
Materials and Methods
Enzymes:
SP722: SEQ ID NO: 4, available from Novozymes, and disclosed in WO 95/26397.
AA560: SEQ ID NO: 12; disclosed in WO 00/60060 and available from Novozymes A/S; disclosed in Danish patent application no. PA 1999 00490; deposited on 25 Jan. 1999 at DSMZ and assigned the DSMZ no. 12649.
AA560 was deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig Del.
Bacillus subtilis SHA273: see WO 95/10603
Plasmids
pJE1 contains the gene encoding a variant of SP722 alpha-amylase (SEQ ID NO: 4): viz. deletion of 6 nucleotides corresponding to amino acids D183-G184 in the mature protein. Transcription of the JE1 gene is directed from the amyL promoter. The plasmid further more contains the origin of replication and cat-gene conferring resistance towards kanamycin obtained from plasmid pUB110 (Gryczan et al., 1978, J. Bact. 134:318-329).
Methods:
Modelbuilding
Protein structure databases, such as “The Protein Data Bank (PDB) pdb.bnl.gov/” or “The Brookhaven databank at Brookhaven National Laboratory, US” are search for proteins similar to the molecule in question that a model are to be build of. The amino acid sequences are aligned taking structurally conserved regions into consideration and the coordinates are copied from the reference protein to the subject protein. The coordinates for regions with insertions and deletions are assigned either from other proteins having similar amino acid sequence, or by using the random structure generator function found in most 3D software packages, e.g., in Homology from Biosym, MSI.
When coordinates have been assigned to all amino acids of the subjective protein and the fragments have been linked together, example by the cormands END REPAIR and SPLICE REPAIR, in the Discover program from from Biosym, MSI, the model are to be refined. The energy of the model is minimized first by relaxing the molecule (RELAX command in the Discover program) and second minimised by molecular dynamics.
References can be found in and in the manuals of homology building software, eg. Homology from Biosym, MSI.
Construction of Library Vector pDorK101
The E. coli/Bacillus shuttle vector pDorK101 (described below) can be used to introduce mutations without expression of alpha-amylase in E. coli and then be modified in such way that the alpha-amylase is active in Bacillus. The vector was constructed as follows: The JE1 encoding gene (SP722 with the deletion of D183-G184) was inactivated in pJE1 by gene interruption in the PstI site in the 5′ coding region of SEQ ID NO: 4: SP722 by a 1.2 kb fragment containing an E. coli origin of replication. This fragment was PCR amplified from the pUC19 (GenBank Accession #:X02514) using the forward primer: 5′-gacctgcagtcaggcaacta-3′ (SEQ ID NO: 19) and the reverse primer: 5′-tagagtcgacctgcaggcat-3′ (SEQ ID NO: 20). The PCR amplicon and the pJE1 vector were digested with PstI at 37° C. for 2 hours. The pJE1 vector fragment and the PCR fragment were ligated at room temperature. for 1 hour and transformed in E. coli by electrotransformation. The resulting vector is designated pDorK101.
Filter Screening Assays
The assay can be used to screening of Termamyl-like alpha-amylase variants having an improved stability at high pH compared to the parent enzyme and Termamyl-like alpha-amylase variants having an improved stability at high pH and medium temperatures compared to the parent enzyme depending of the screening temperature setting.
High pH Filter Assay
Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 μg/ml kanamycin at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.
Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10°-60° C.) for 15 min. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in glycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
Low Calcium Filter Assay
The Bacillus library are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.
Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA concentrations (0.001 mM-100 mM). The filters are incubated at room temperature for 1 hour. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH 8.5-10. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours. at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
Method for Obtaining the Regions of Interest:
There are three known 3D structures of bacterial alpha-amylases. Two of B. licheniformis alpha-amylase, Brookhaven database 1BPL (Machius et al., 1995, J. Mol. Biol. 246: 545-559) and 1VJS (Song et al., 1996, Enzymes for Carbohydrate 163 Engineering, Prog. Biotechnol. V 12). These two structures are lacking an important piece of the structure from the so-called B-domain, in the area around the two calcium ions and one sodium ion binding sites. We have therefore used a 3D structure of an alpha-amylase BA2 (WO 96/23874 which is a hybrid between BAN™ (SEQ ID NO: 5) and B. licheniformis alpha-amylase (SEQ ID NO: 4). On basis of the structure a model of B. licheniformis alpha amylase and the SP722 alpha-amylase has been built.
Fermentation and Purification of Alpha-Amylase Variants
Fermentation and purification may be performed by methods well known in the art.
Stability Determination
All stability trials are made using the same set up. The method is as follows:
The enzyme is incubated under the relevant conditions (1-4). Samples are taken at various time points, e.g., after 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) in assay buffer (0.1 M 50 mM Britton buffer pH 7.3) and the activity is measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.
The activity measured before incubation (0 minutes) is used as reference (100%). The decline in percent is calculated as a function of the incubation time. The table shows the residual activity after, e.g., 30 minutes of incubation.
Specific Activity Determination
The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. The manufacturer's instructions are followed (see also below under “Assay for Alpha-Amylase Activity”).
Solubility Determination I
Purified enzyme is dialyzed against a selected buffer overnight. Standard conditions are 0.02 M Tris-HCl, 0.15 M NaCl, pH 7.5, room temperature. A preparation, containing approximately 40 mg enzyme is concentrated on an amicon cell using Millepore ultrafiltration membranes, YM10, NMWL: 10,000. Measuring A280 before and during the process followed concentration of the enzyme. The concentration step is stopped when precipitation started to occur. Measurement of activity in the supernatant is made to ensure that the precipitate actually is amylase. The precipitate is then dissolved again by adding buffer at room temperature and the protein concentration is measured.
Solubility Determination II
Purified enzyme is dialyzed against a selected buffer overnight. Standard conditions are 0.01 M Boric acid, 0.01 M KCl, 0.002 M CaCl2, 0.15 M NaCl pH 7.5. A preparation, containing approximately 40 mg enzyme is concentrated on an amicon cell using Millepore ultrafiltration membranes, YM10, NMWL: 10,000. Measuring A280 before and during the process followed concentration of the enzyme. The concentration step is stopped when precipitation started to occur. Measurement of activity in the supernatant is made to ensure that the precipitate actually is amylase. The precipitate is then dissolved again by adding buffer at room temperature and the protein concentration is measured.
Assays for Alpha-Amylase Activity
1. Phadebas Assay
Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.
For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl2, pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The alpha-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the alpha-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the alpha-amylase activity.
It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
2. Alternative Method
Alpha-amylase activity is determined by a method employing the PNP-G7 substrate. PNP-G7 which is a abbreviation for p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage, the alpha-Glucosidase included in the kit digest the substrate to liberate a free PNP molecule which has a yellow colour and thus can be measured by visible spectophometry at λ=405 nm. (400-420 nm). Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. no. 1054635).
To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309). To prepare the alpha-glucosidase one bottle of alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309). The working solution is made by mixing 5 ml alpha-Glucosidase solution with 0.5 ml substrate.
The assay is performed by transforming 20 μl enzyme solution to a 96 well microtitre plate and incubating at 25° C. 200 μl working solution, 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.
The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.
General Method for Random Mutagenesis by Use of the DOPE Program
The random mutagenesis may be carried out as follows:
Suitable dope algorithms for use in step 6 are well known in the art. One algorithm is described by Tomandl et al., 1997, Journal of Computer-Aided Molecular Design 11: 29-38). Another algorithm, DOPE, is described in the following:
The Dope Program
The “DOPE” program is a computer algorithm useful to optimize the nucleotide composition of a codon triplet in such a way that it encodes an amino acid distribution which resembles most the wanted amino acid distribution. In order to assess which of the possible distributions is the most similar to the wanted amino acid distribution, a scoring function is needed. In the “Dope” program the following function was found to be suited:
where the xi's are the obtained amounts of amino acids and groups of amino acids as calculated by the program, yi's are the wanted amounts of amino acids and groups of amino acids as defined by the user of the program (e.g., specify which of the 20 amino acids or stop codons are wanted to be introduced, e.g., with a certain percentage (e.g., 90% Ala, 3% Ile, 7% Val), and wi's are assigned weight factors as defined by the user of the program (e.g., depending on the importance of having a specific amino acid residue inserted into the position in question). N is 21 plus the number of amino acid groups as defined by the user of the program. For purposes of this function 0° is defined as being 1.
A Monte-Carlo algorithm (one example being the one described by Valleau, J. P. & Whittington, S. G. (1977) A guide to Mont Carlo for statistical mechanics: 1 Highways. In “Stastistical Mechanics, Part A” Equlibrium Techniques ed. B. J. Berne, New York: Plenum) is used for finding the maximum value of this function. In each iteration the following steps are performed:
A cycle normally consists of 1000 iterations as described above in which d is decreasing linearly from 1 to 0. One hundred or more cycles are performed in an optimization process. The nucleotide composition resulting in the highest score is finally presented.
The tertiary structure of the SP722 alpha-amylase was displayed on a UNIX computer station running Insight and all hydrogens and water molecules were deleted. For this minimized amylase structure, all the symetri related amylase molecules were displayed, according to the crystal parameters and the spacegroup determined in the pdb-structure file (Appendix 1). A small computer program was made to calculate the distance from each atom in the mother alpha-amylase to any atom in the symetri related amylase structures. Setting the distance cut-off to 3.5 Å and 6.0 Å, respectively resulted in the lists below:
The amino acid residues being less than 6.0 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the SP722 amylase: ASN 19, ASP 20, GLY 21, GLN 22, ASN 25, ARG 28, ASP 29, GLN 53, ARG 76, GLN 84, SER 87, HIS 90, LYS 93, ASN 94, ASN 125, ASN 126, ASN 128, LYS 142, ASP 144, LYS 156, TRP 157, ARG 158, TRP 159, TYR 160, HIS 161, PHE 173, GLN 174, ASN 175, ASP 183, GLY 184, LYS 185, ALA 186, TRP 187, ASP 188, TRP 189, GLU 190, SER 193, GLY 196, ASN 197, ASP 209, GLU 212, ASN 226, ASN 229, ALA 256, THR 257, GLY 258, LYS 259, ASN 280, LYS 281, TYR 298, ASN 299, ALA 300, ASN 302, SER 303, GLY 304, GLY 305, ASN 306, ALA 310, ASN 314, GLN 319, LYS 320, HIS 321, PRO 322, GLU 341, GLN 345, PHE 405, ASP 406, HIS 408, GLN 444, ALA 447, GLY 448, GLN 449, THR 463, ILE 464, ASN 465, ALA 466, ASP 467.
The amino acid residues being less than 3.5 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the SP722 amylase: GLN 22, ASN 25, ARG 28, ARG 76, ASN 94, ASN 125, ASN 128, ARG 158, TYR 160, PHE 173, GLN 174, GLY 184, TRP 189, ASP 209, ASN 226, ASN 229, TYR 298, ASN 299, ASN 302, ASN 306, ASN 314, LYS 320, GLN 345, PHE 405, ALA 447, ALA 466.
The tertiary structure of the SP722 alpha-amylase was displayed on a UNIX computer station running Insight and all hydrogen and water molecules were deleted. This minimized amylase structure was imported (“getmol”) into the WHAT IF software (Vriend, 1990, J. Mol. Graph. 8: 52-56) and all the symmetry related amylase molecules within 6 angstrom were added to the soup (“symtry, symspg, sympar 6, soushl, soup). A new coordinate file for SP722 including the symmetry molecules was written (makmol, tot 0). The center SP722 amylase was then deleted from the structure file, and a new file including the surrounding molecules was written. A small computer program was made to calculate the distance from each atom in the mother alpha-amylase (original SP72 structure file without water molecules and Hydrogen atoms) to any atom in the symmetry related amylase file. Setting the distance cut-off to 3.5 Å and 6.0 Å, respectively resulted in identification of the position referred to in the description.
What If commands:
The overall homology of the AA560 alpha-amylase (SEQ ID NO: 12) to SP722 (SEQ ID NO: 4) is about 87% as described above. Sequence alignment of AA560 and SP722 shows there to be no insertion or deletions, which can also be seen in
The tertiary structure of the AA560 alpha-amylase was model build on the structure disclosed in Appendix 1 using the method “Modelbuiling” described in the “Materials & Methods”-section.
The structure of SP722 was displayed on a UNIX work station running Insight and Homology software from BIOSYM, MSI. The amino acid sequences were aligned and the Sp722 coordinated assigned to the AA560 amino acids. The coordinates of the first four amino acids in AA560, which are missing in the SP722 structure, were assigned by the “END REPAIR” function.
The AA560 model was refined by first relaxing the amino acid side changes, using the “RELAX” command and then running molecular dynamics to minimise the energy of the 3D model. Default parameters from Insight 95, MSI were chosen for both relaxation molecular dynamics.
Finally the spacegroup and the unit cell dimensions are copied from the SP722 pdb file to the AA560 model file.
The model build structure of AA560 was subjected to the same calculations as was SP722 in Example 1. Because the homology of the two alpha-amylases is as high as about 87% identity, the crystal interactions of AA560 are expected to similar to those of SP722, i.e., AA560 crystallize in the same spacegroup as SP722 and with the similar unity cell parameter.
The tertiary structure of the AA560 alpha-amylase was displayed on a UNIX computer station running Insight and all hydrogens and water molecules were deleted. For this minimized amylase structure, all the symetri related amylase molecules were displayed, according to the crystal parameters and the spacegroup for SP722 (Appendix 1). A small computer program was made to calculate the distance from each atom in the “mother” alpha-amylase to any atom in the symetri related amylase structures. Setting the distance cut-off to 3.5 Å and 6.0 Å, respectively resulted in the lists below:
The amino acid residues being less than 6.0 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the AA560 amylase: ASN 19, ASP 20, ASN 22, ASN 25, ARG 28, SER 29, GLN 53, ARG 76, GLN 84, ALA 87, ASN 90, LYS 93, SER 94, PRO 124, ASN 125, ASN 126, ASN 128, LYS 142, ASP 144, LYS 156, TRP 157, ARG 158, TRP 159, TYR 160, HIS 161, SER 170, ARG 171, LYS 172, LEU 173, ASN 174, ASN 175, ASP 183, GLY 184, LYS 185, GLY 186, TRP 187, ASP 188, TRP 189, GLU 190, THR 193, ASN 195, GLY 196, ASN 197, ASP 209, GLU 212, ASN 226, GLY 229, ALA 256, THR 257, GLY 258, LYS 259, LYS 281, TYR 298, ASN 299, ALA 300, LYS 302, SER 303, GLY 304, ASN 306, ARG 310, GLN 311, ASN 314, GLN 319, ARG 320, HIS 321, PRO 322, GLU 341, GLU 345, LEU 405, HIS 408, ARG 444, ALA 447, GLY 448, GLN 449, THR 463, ILE 464, ASN 465, ALA 466, ASP 467.
The amino acid residues being less than 3.5 Å from the nearest neighboring amylase molecule are the following positions identified in the model structure of the AA560 amylase: ARG 28, ASN 125, ASN 128, ARG 158, TYR 160, ARG 171, LEU 173, ASN 174, GLY 184, TRP 189, GLY 196, ASP 209, ASN 226, TYR 298, ASN 299, LYS 302, ASN 306, ARG 310, ASN 314, ARG 320, GLU 345, ALA 447, ALA 466.
To increase the solubility of the AA560 alpha-amylase random mutagenesis in pre-selected region was performed as described in the following.
The DOPE software (see “Materials and Methods”) was used to determine spiked codons for each suggested change in the SA1 region minimizing the amount of stop codons (see table 1). The exact distribution of nucleotides was calculated in the three positions of the codon to give the suggested population of amino acid changes. The doped regions were doped specifically in the indicated positions to have a high chance of getting the desired residues, but still allow other possibilities.
The resulting doped oligonucleotide strand is shown in table 2 as sense strand: with the wild type nucleotide and amino acid sequences and the distribution of nucleotides for each doped position.
Random Mutagenesis
The spiked oligonucleotides apparent from Table 2 (which by a common term is designated FSA) and reverse primers RSA for the SA1 region and specific SEQ ID NO: 12: AA560 primers covering the SaclI and the DraIII sites are used to generate PCR-library-fragments by the overlap extension method (Horton et al., 1989, Gene 77: 61-68) with an overlap of 21 base pairs. Plasmid pJE1 is template for the Polymerase Chain Reaction. The PCR fragments are cloned in the E. coli/Bacillus shuttle vector pDork101 (see “Materials and Methods” section) enabling mutagenesis in E. coli and immediate expression in Bacillus subtilis preventing lethal accumulation of amylases in E. coli. After establishing the cloned PCR fragments in E. coli, a modified pUC19 fragment is digested out of the plasmid and the promoter and the mutated Termamyl gene is physically connected and expression can take place in Bacillus.
Screening
The library may be screened in the low calcium filter assays described in the “Material and Methods” section above.
The gene encoding the AA560 alpha-amylase shown in SEQ ID NO: 12 is located in a plasmid pTVB223. The amylase is expressed from the amyL promoter in this construct in Bacillus subtilis.
A variant of the invention with delta(D183-G184) mutations was constructed by the mega-primer method as described by Sarkar and Sommer, (1990), BioTechniques 8: 404-407.
Gene specific primer B1 (SEQ ID NO: 16) and mutagenic primer 101458 (SEQ ID NO: 18) were used to amplify by PCR an approximately 645 bp DNA fragment from a pTVB223 plasmid encoding AA560 shown in SEQ ID NO: 12).
The 645 bp fragment was purified from an agarose gel and used as a mega-primer together with primer Y2 (SEQ ID NO: 17) in a second PCR carried out on the same template.
The resulting approximately 1080 bp fragment was digested with restriction enzymes BstEII and AfIIII and the resulting approximately 510 bp DNA fragment was purified and ligated with the pTVB223 plasmid digested with the same enzymes. Competent Bacillus subtilis SHA273 (amylase and protease low) cells were transformed with the ligation and Chlorampenicol resistant transformants and was checked by DNA sequencing to verify the presence of the correct mutations on the plasmid.
The resulting plasmid encoding the AA560 amylase with delta(D183-G184)+N195F was named pTVB232.
The construction of the other variants of the invention was carried out in a similar manner.
AA560 variant was constructed as described above. The solubility was determined as described in the “Material and Methods” section as Solubility Determination I
The concentration step was started with a protein amount of 40 mg and in general, 50% of the amylases were lost during the process. This loss of protein occurred for all enzymes in the initial phase of the process and full recovery of the enzymes were obtained in the terminating phase where the volumes became very limited. Loss of enzymes was due to adsorption to the membrane.
AA560 variants were constructed as described above, using the wild-type AA560 as template. The solubility was determined as described in the “Material and Methods” section under Solubility Determination II
| Number | Date | Country | Kind |
|---|---|---|---|
| 2000 00376 | Mar 2000 | DK | national |
| 2001 00303 | Feb 2001 | DK | national |
This application is a continuation of U.S. patent application Ser. No. 12/204,434 filed Sep. 4, 2008, which is a continuation of U.S. patent application Ser. No. 09/925,576 filed Aug. 9, 2001 (now U.S. Pat. No. 7,432,099), which is a continuation of international application no. PCT/DK01/00144 filed Mar. 7, 2001 (the international application was published under PCT Article 21(2) in English) and claims, under 35 U.S.C. 119, priority or the benefit of Danish application nos. PA 2000 00376 and PA 2001 00303 filed Mar. 8, 2000 and Feb. 23, 2001, respectively, and U.S. provisional application Nos. 60/189,857, and 60/271,382 filed Mar. 15, 2000 and Feb. 26, 2001, respectively, the contents of which are fully incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5824531 | Outtrup | Oct 1998 | A |
| 5856164 | Outtrup | Jan 1999 | A |
| 6093562 | Bisgard-Frantzen | Jul 2000 | A |
| 6187576 | Svendsen | Feb 2001 | B1 |
| 6197565 | Svendsen | Mar 2001 | B1 |
| 6204323 | Wamprecht | Mar 2001 | B1 |
| 6287826 | Norman | Sep 2001 | B1 |
| 6297038 | Bisgard-Frantzen | Oct 2001 | B1 |
| 6361989 | Svendsen et al. | Mar 2002 | B1 |
| 6486113 | Hatada | Nov 2002 | B1 |
| 6528298 | Svendsen | Mar 2003 | B1 |
| 6673589 | Borchert | Jan 2004 | B2 |
| 6867031 | Bisgard-Frantzen | Mar 2005 | B2 |
| 6887986 | Svendsen | May 2005 | B1 |
| Number | Date | Country |
|---|---|---|
| WO 9526397 | Oct 1995 | WO |
| WO 9623874 | Aug 1996 | WO |
| WO 9732961 | Sep 1997 | WO |
| WO 9732985 | Sep 1997 | WO |
| WO 9741213 | Nov 1997 | WO |
| WO 9805748 | Feb 1998 | WO |
| WO 9818323 | May 1998 | WO |
| WO 9919467 | Apr 1999 | WO |
| 9923211 | May 1999 | WO |
| WO 0001712 | Jan 2000 | WO |
| 200060060 | Oct 2000 | WO |
| Entry |
|---|
| WO 1997-032961—EBI Accession No. A65134 (1997). |
| WO 1997-041213—EBI Accession No. AAW31405 (1997). |
| WO 1998-005748—EBI Accession No. AAW48261 (1998). |
| WO 1999-019467—EBI Accession No. AAY07381 (1999). |
| Igarashi et al., Database SPTREMBL, Accession No. 082839 (1998). |
| Igarashi et al., Biochemical and Biophysical Research Communications, vol. 248, No. 2, pp. 372-377 (1998). |
| Seffernick et al., Journal of Bacteriology, vol. 183, No. 8, pp. 2405-2410 (2001). |
| Number | Date | Country | |
|---|---|---|---|
| 20110275136 A1 | Nov 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60271382 | Feb 2001 | US | |
| 60189857 | Mar 2000 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12204434 | Sep 2008 | US |
| Child | 13187014 | US | |
| Parent | 09925576 | Aug 2001 | US |
| Child | 12204434 | US | |
| Parent | PCT/DK01/00144 | Mar 2001 | US |
| Child | 09925576 | US |
