Patent Description:
A lipase is an enzyme that acts on ester bonds in lipids. Lipases of various origins have been isolated, and are used, for example, for the degradation of fats and oils, food processing, and production of pharmaceuticals. For example, a Candida cylindracea-derived lipase (previously referred to as a Candida rugosa-derived lipase) is expected to be used in fields such as wastewater treatment and food processing (see, for example, PTLs <NUM> to <NUM> and NPL <NUM>).

[D3] <CIT> describes a method for decomposing fats and oils whose action temperature is <NUM>° C or higher using lipase produced by Candida cylindracea U-<NUM> mutant strain producing a lipase with a higher optimum temperature than the conventional strain.

[D4] <CIT> describes nucleic acids that can be used to functionally express heterologous C. rugosa lipase in a common host with the non-universal serine codons (CTG) present in the original gene mutated into universal serine codons (TCT) to allow gene expression in common host cells.

[D8] <CIT> characterizes Cyndida cylindracea derived lipases, their modified versions with the substitutions L428N, G429F, G429M and G429I and their uses in the production of dairy products.

[D10] <CIT> describes methods for improving thermostability of Candida rugosa lipase LIP1 by introducing the F344I/F434Y/F133Y/F121Y substitutions either alone or in combination.

[D1] <NPL> describes G414A, G414M, G414H, or G414W susbstitution mutants of Candida rugosa Lipase <NUM> with improved thermostability and higher activity at high temperatures.

[D2] <NPL> describes the effect of mutations F344I, F434Y, F133Y, F121Y either singular or in combination on thermostability of Candida rugosa Lipase <NUM>.

[D5] <NPL> describes a Lipase B from Candida antarctica with an improved thermostability after introduction of the A162C-K308C substitutions.

[D6] <NPL> compares the Lip1, Lip2 and Lip3 Candida rugosa Lipase isoenzymes in terms of their temperature and pH stability.

[D7] <NPL> characterizes the catalytic performance of recombinant Candida rugosa Lipase <NUM> (Lip5) at varying pH and temperature.

[D9] <NPL> characterizes five Candida rugosa Lipase mutants at Asp457 (Asp457Phe, Asp457Leu, Asp457Met, Asp457Trp, and Asp457Tyr) for protein thermal stability. [D11] <NPL> provides an overview to yeast and fungal lipases, including Candida rugosa lipases Lip1-Lip5.

[D12] <NPL> describes mutagenesis of Candida rugosa Lip1 gene aiming at conversion of the <NUM> non-universal serine codons (CTG) into universal serine codons (TCT) to allow gene expression in Pichia pastoris for further characterization of Lip1.

A Candida cylindracea-derived lipase has high industrial utility. However, there is room for improvement in the stability and reactivity of the lipase at a high temperature. If improvements in its stability/reactivity at a high temperature are made, then it will be possible that it provides increased productivity and expanded use. Therefore, the present invention aims at making improvements in the stability/reactivity of the Candida cylindracea-derived lipase at a high temperature, thereby increasing its usefulness or utility value.

In order to address the above subject, the present inventors undertook to modify a Candida cylindracea-derived lipase. After trial and error, the present inventors were successful in identifying mutation points (amino acid residues) effective for the improvement in the reactivity or stability of the lipase at a high temperature, whereby highly useful variants (modified lipases) were obtained. This result is also important in providing information and means for designing and obtaining variants of a given enzyme that can achieve the purpose of improving the reactivity and stability thereof at a high temperature.

On the other hand, many cases are also encountered in which it is highly likely that for a given enzyme, combinations of two effective mutations result in additive or synergistic effects. In addition, in light of the common technical knowledge that enzymes of the same kind show high levels of similarity in their structures (primary structures and steric structures) and it is highly probable that for these enzymes, a similar mutation leads to similar effects, if a mutations that has been found to be useful in a Candida cylindracea-derived lipase having the amino acid sequence represented by SEQ ID NO: <NUM> is applied to another lipase having a high structural similarity relative to the Candida cylindracea-derived lipase, then there is a high probability that such a lipase has effects comparable to those achieved in the Candida cylindracea-derived lipase. Moreover, one skilled in the art could recognize that this approach is effective.

The present invention described below are based on the above-mentioned results and considerations. The present invention relates to the embodiments as characterized in the claims.

The present invention relates to the embodiments as characterized in the claims. For convenience of description, some of the terms used in relation to the present invention are defined as follows.

The term "modified lipase" refers to an enzyme obtained by modification or mutation of a particular lipase (which is referred to as a "reference lipase" for convenience of description). Typically, the reference lipase is a Candida cylindracea derived lipase having the amino acid sequence of SEQ ID NO:<NUM>. The terms "Candida cylindracea derived lipase" and "Candida rugosa derived lipase" are used interchangeably.

The term "Candida cylindracea derived lipase" is a lipase that is obtained from a strain of Candida cylindracea as the source, and includes lipases produced by Candida cylindracea, lipases expressed, for example, in other microorganism, using the genetic information of such enzyme, or the like.

In the present invention, an "amino acid substitution" is carried out as modification or mutation. Therefore, some amino acid residues are found to be different when a modified lipase and the reference lipase therefor are compared. In the specification, a modified lipase is also referred to as a modified enzyme or as a variant.

In the specification, amino acids are designated according to the common practice, as their single letters as described below:
methionine: M; serine: S; alanine: A; threonine: T; valine: V; tyrosine: Y; leucine: L; asparagine: N; isoleucine: I; glutamine: Q; proline: P; aspartic acid: D; phenylalanine: F; glutamic acid: E; tryptophan: W; lysine: K; cysteine: C; arginine: R; glycine: G; and histidine: H.

In the specification, the positions of amino acids in an amino acid sequence are specified by assigning the numbers from the N-terminus toward the C-terminus of the amino acid sequence, wherein the amino acid residue at the N-termius of a mature protein in which the signal peptide has been removed is assigned to <NUM>, i.e., the first amino acid.

In the specification, an amino acid residue at a mutation site (an amino acid residue to be substituted with another amino acid) is expressed in a combination of the above-described single letter representing the kind of the amino acid residue and the figure representing the position of the amino acid residue. For example, a mutation which substitutes threonine at position <NUM> for cysteine is designated as "T130C".

A first aspect of the present invention relates to a modified lipase (or a modified enzyme) as characterized in the claims. A modified enzyme according to the present invention has an amino acid sequence comprising the amino acid substitution T130C-S153C in the amino acid sequence represented by SEQ ID NO: <NUM>. Due to this feature, the modified lipase has improved reactivity or stability, or improved reactivity and stability, at a high temperature, as compared to a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM>. The amino acid sequence represented by SEQ ID NO: <NUM> corresponds to the mature form (that is, without signal peptide) of Candida cylindracea-derived lipase (referred to herein as "LIP1").

In order to facilitate understanding and judgment/determination of the high-temperature reactivity and stability of modified enzymes, the high temperature at which they are measured for their high-temperature reactivity is set to be "<NUM> to <NUM>" and the high temperature at which they are measured for their high-temperature stability is set to be "<NUM> to <NUM>.

The high-temperature reactivity of modified enzymes can be evaluated based on, for example, the ratio of enzyme activity at <NUM> to that at <NUM> (relative activity at <NUM> with respect to that at <NUM>). A modified enzyme according to the present invention has improved high-temperature reactivity as compared to a reference lipase, that is, a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM>, and thus the relative activity of the modified enzyme will be higher than that of the reference lipase. The relative activity of the modified enzymes is, for example, <NUM> to <NUM> times that of the reference lipase. Preferably, the relative activity of the modified enzymes is <NUM> to <NUM> times that of the reference lipase. Here, the relative activity is calculated as follows.

The high-temperature stability of modified enzymes can be evaluated based on, for example, the residual activity after treatment at <NUM> for <NUM> minutes. A modified enzyme according to the present invention has improved high-temperature stability as compared to a reference lipase, that is, a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM>, and thus the residual activity percent of the modified enzyme will be higher than that of the reference lipase. The residual activity percent of the modified enzymes is, for example, <NUM> to <NUM> times that of the reference lipase. Preferably, the residual activity percent of the modified enzymes is <NUM> to <NUM> times that of the reference lipase. Here, the residual activity percent is calculated as follows.

As used herein, "comprising an amino acid substitution" means that the substituted amino acid is located at the mutation point, that is, the position of the amino acid residue at which a specified amino acid substitution occurs. Therefore, when an amino acid sequence comprising an amino acid substitution, i.e., a mutated amino-acid sequence, is compared with that represented by SEQ ID NO: <NUM> (reference amino-acid sequence) having no amino acid substitution, the mutated amino-acid sequence will be found to have a different amino acid residue at the position at which the amino acid substitution has occurred.

T130C-S153C denotes a mutation in which the amino acid at position <NUM> (threonine) in the amino acid sequence represented by SEQ ID NO: <NUM> is substituted with cysteine and the amino acid at position <NUM> (serine) is substituted with cysteine. A249P denotes a mutation in which the amino acid at position <NUM> (alanine) in the amino acid sequence represented by SEQ ID NO: <NUM> is substituted with proline. F259Y denotes a mutation in which the amino acid at position <NUM> (phenylalanine) in the amino acid sequence represented by SEQ ID NO: <NUM> is substituted with tyrosine. S282P denotes a mutation in which the amino acid at position <NUM> (serine) in the amino acid sequence represented by SEQ ID NO: <NUM> is substituted with proline. S283Y denotes a mutation in which the amino acid at position <NUM> (serine) in the amino acid sequence represented by SEQ ID NO: <NUM> is substituted with tyrosine. S300P denotes a mutation in which the amino acid at position <NUM> (serine) in the amino acid sequence represented by SEQ ID NO: <NUM> is substituted with proline.

Specific examples of a modified enzyme according to the present invention includes a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM>, corresponding to variant <NUM> (T130C-S153C). Variants <NUM> (A249P), <NUM> (F259Y), <NUM> (S282P), <NUM> (S283Y), and <NUM> (S300P) do not belong to the present invention and are included only as examples herein. As shown in Examples described below, a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM> (variant <NUM> (T130C-S153C)) and a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM> (variant <NUM> (S283Y)) have been ascertained to show particularly improved reactivity at the high temperature. On the other hand, a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM> (variant <NUM> (A249P)), a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM> (variant <NUM> (S283Y)), and a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM> (variant <NUM> (S300P)) have been ascertained to show particularly improved stability at the high temperature.

Generally, when the amino acid sequence of a given protein is partially changed by mutation, the protein obtained after the mutation may have the same function as that before the mutation. In other words, it is sometimes observed that a mutation in the amino acid sequence of a given protein does not substantially affect the function of the resulting protein and in these proteins, the function is maintained before and after the mutation. In addition, it is highly probable that two proteins exhibit equivalent properties when sharing high identity in their amino acid sequences. In light of common technical knowledge of these things, a modified enzyme can be considered to be an enzyme that is substantially identical to the above-described modified enzyme (or can be referred to as a substantially identical lipase), with the proviso that such a modified enzyme has an amino acid sequence that is not completely identical (i.e., does not have a sequence identity of <NUM>%) to that of any of the above-described modified enzymes, that is, an "amino acid sequence comprising an amino acid substitution of T130C-S153C in the amino acid sequence represented by SEQ ID NO: <NUM> (specific examples of which amino acid sequence is this represented by SEQ ID NO: <NUM>)" and displays a high level of sequence identity therewith, and has improved reactivity and/or stability at a high temperature. Such a high level of sequence identity is preferably <NUM>% or more, more preferably <NUM>% or more, even more preferably <NUM>% or more, and most preferably <NUM>% or more. If a substantially identical lipase is compared with the above-described modified enzyme, then the lipase will be found to have a slightly different amino acid sequence. Note that a slight difference in the amino acid sequence of a substantially identical lipase is to be generated at a position or positions other than that at which the above-mentioned amino acid substitution is made. Accordingly, a slight difference in the amino acid sequence of a substantially identical lipase will be generated at a position or positions other than those corresponding to cysteine at positions <NUM> and <NUM> in the amino acid sequence represented by SEQ ID NO: <NUM>, when the respective amino acid sequences are used as a reference sequence to determine the sequence identity. In other words, in amino acid sequences having a sequence identity of <NUM>% or more with that represented by SEQ ID NO: <NUM>, both the amino acids at the positions corresponding to positions <NUM> and <NUM> therein are cysteine.

Here, a "slightly different amino acid sequence" results from amino acid deletion, substitution, addition, insertion, or a combination thereof. This means that in typical cases, a given amino acid sequence is mutated (or changed) by deletion or substitution of one to several (for example, up to three, five, seven, ten) amino acids constituting the amino acid sequence, or addition or insertion of one to several (for example, up to three, five, seven, ten) amino acids, or a combination thereof. A "slightly different amino acid sequence" preferably results from a conservative amino acid substitution. Here, by "conservative amino acid substitution" is meant that a certain amino acid residue is substituted with an amino acid residue of which the side chain is similar in properties. Amino acid residues are classified into several families, on the basis of their side chains, such as families of amino acids having basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (for example, threonine, valine, isoleucine), and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). The conservative amino acid substitution preferably is a substitution between amino acid residues within the same family. In connection with this, it is known that the amino acid residues constituting the active center of Candida cylindracea-derived lipase (LIP1) (SEQ ID NO: <NUM>) are glutamic acid at position <NUM>, histidine at position <NUM>, and serine at position <NUM>, and thus mutation should be made so that there is no influence on these amino acid residues. An example of an amino acid sequence in which a slightly different amino acid sequence does not substantially affect the function of the resulting protein and the function is maintained before and after the mutation is one in which the amino acid at position <NUM> in the amino acid sequence represented by SEQ ID NO: <NUM> (phenylalanine) is changed to tyrosine. A lipase having this mutated amino acid sequence was found equivalent to a lipase having the amino acid sequence represented by SEQ ID NO: <NUM> when specific activities and high-temperature reactivities were compared.

The identity (%) between two amino acid sequences or two nucleic acid sequences (hereinafter, the term "two sequences" are used for representing either of two sequences) can be determined by the following procedure. Firstly, two sequences are aligned for optimum comparison of the two sequences (for example, a gap may be introduced into the first sequence so as to optimize the alignment with respect to the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence and a molecule in the corresponding position in the second sequence are the same as each other, the molecules in the positions are defined as being identical. The identity between two sequences is a function of the number of identical positions shared by the two sequences (i.e., identity (%) = number of identical positions / total number of positions × <NUM>). Preferably, the number and size of the gaps, which are required to optimize the alignment of the two sequences, are taken into consideration.

The comparison and determination of the identity between two sequences can be carried out by using a mathematical algorithm. A specific example of the mathematical algorithm that can be used for comparing the sequences includes an algorithm described in <NPL> and modified by <NPL>. However, the algorithm is not necessarily limited to this. Such an algorithm is incorporated in NBLAST program and XBLAST program (version <NUM>) described in <NPL>. In order to obtain an equivalent nucleic acid sequence, for example, BLAST nucleotide search with score = <NUM> and word length = <NUM> may be carried out by the NBLAST program. In order to obtain an equivalent amino acid sequence, for example, BLAST polypeptide search with score = <NUM> and word length = <NUM> may be carried out by the XBLAST program. In order to obtain gapped alignments for comparison, Gapped BLAST described in <NPL> can be utilized. In using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. In detail, see http://www. Another example of the mathematical algorithm that can be used for comparing sequences includes an algorithm described in <NPL>. Such programs are incorporated into the ALIGN program that can be used for, for example, GENESTREAM network server (IGH Montpellier, France) or ISREC server. When the ALIGN program is used for comparison of the amino acid sequences, for example, PAM120 weight residue table can be used in which a gap length penalty is <NUM> and a gap penalty is <NUM>.

The identity between two amino acid sequences can be determined by using the GAP program in the GCG software package, using Blossom <NUM> matrix or PAM250 matrix with the gap weight of <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, and the gap length weight of <NUM>, <NUM>, or <NUM>. The identity between two nucleic acid sequences can be determined by using the GAP program in the GCG software package (available at http://www. com), with the gap weight of <NUM>, and the gap length weight of <NUM>.

Typically, a modified enzyme according to the present invention is produced by subjecting a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM>, that is, Candida cylindracea-derived lipase, to mutation (the above-described T130C-S153C). A substantially identical lipase as mentioned above can be obtained by subjecting a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM> to mutation (the above-described T130C-S153C,), followed by further mutation, or by applying an equivalent mutation to a lipase consisting of an amino acid sequence having a high sequence identity with that represented by SEQ ID NO: <NUM>, such as a lipase derived from a strain of the same genera and species as a strain of Candida cylindracea that produces a lipase consisting of the amino acid sequence represented by SEQ ID NO: <NUM>, or subjecting a variant resulting therefrom to additional mutation. The "equivalent mutation" in this case will lead to a substitution being made at an amino acid residue corresponding to that at any of the mutation points disclosed in the present invention (position <NUM>, <NUM> in the amino acid sequence represented by SEQ ID NO: <NUM>), in an amino acid sequence having a high sequence identity with that represented by SEQ ID NO: <NUM>. Examples of a lipase consisting of an amino acid sequence having a high sequence identity with that represented by SEQ ID NO: <NUM> can be, by way of illustration, isozymes of LIP1, i.e., LIP2 (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>%), LIP3 (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>%), LIP4 (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>%), and LIP5 (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>%), andlipases derived from Diutina rugosa (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>%), Candida cylindracea (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>% ), and Candida sp. AC-IITM (SEQ ID NO: <NUM>, showing a sequence identity of <NUM>%).

Herein, the term "corresponding" when used for an amino acid residue in the present specification means contributing equally to exhibition of functions among proteins (enzymes)being compared. For example, when an amino acid sequence for comparison to the base amino acid sequence (that is, the amino acid sequence set forth in SEQ ID NO: <NUM>) is aligned while considering partial homology of the primary structure (that is, an amino acid sequence) so that the most appropriate comparison can be achieved (in this event, the alignment may be optimized by introducing gaps if necessary), an amino acid located at a position corresponding to a specific amino acid in the base amino acid sequence can be specified as a "corresponding amino acid". The "corresponding amino acid" can also be specified by comparison between conformations (three-dimensional structures) in place of or in addition to the comparison between primary structures. Utilization of conformational information can give highly credible comparison results. In this case, a technique of performing an alignment with comparing atomic coordinates of conformations of a plurality of enzymes can be adopted. Conformational information of an enzyme to be mutated is available from, for example, the Protein Data Bank (http://www. org/index_j.

One example of a method for determination of a protein conformation by the X-ray crystal structure analysis will be shown below.

The second aspect of the present invention provides a nucleic acid relating to the modified enzyme of the invention. That is, provided is a gene coding for the modified enzyme as characterized in the claims.

The gene coding for a modified enzyme is typically used in preparation of the modified enzyme. According to a genetic engineering procedure using the gene coding for a modified enzyme, a modified enzyme in a more homogeneous state can be obtained. Further, the method can be a preferable method also in the case of preparing a large amount of a modified enzyme. Note that uses of the gene coding for a modified enzyme are not limited to preparation of a modified enzyme. For example, the nucleic acid can also be used as a tool for an experiment intended for clarification of action mechanisms of a modified enzyme or a tool for designing or preparing a further modified form of an enzyme.

The "gene coding for a modified enzyme" herein refers to a nucleic acid capable of obtaining the modified enzyme when it is expressed, and includes, as a matter of course of a nucleic acid having a base sequence corresponding to the amino acid sequence of the modified enzyme, also a nucleic acid obtained by adding a sequence that does not code for an amino acid sequence to such a nucleic acid. Degeneracy of a codon is also considered.

Example of the (base) sequence of the gene encoding a modified enzyme are represented in SEQ ID NO: <NUM> to <NUM>. These sequences encode variants described in the Examples section which follows, as indicated below.

In Candida cylindracea, the CTG codon encodes serine. If a gene is recombinantly expressed using other yeasts and the like as a host, then it is necessary that depending on the host to be used, the CTG codon is changed to another codon encoding serine (TCT, TCC, TCA, ATG, or AGC).

When a gene according to the present invention is to be expressed in a host, the gene will usually be inserted into the host in the form of a gene construct in which the above-described sequence has a signal peptide-coding sequence (a signal sequence) added thereto at the <NUM>' end of the above sequence (of SEQ ID NO:<NUM>). The signal sequence of wild-type LIP1 is represented in SEQ ID NO: <NUM>. The amino acid sequence encoded by this signal sequence (that is, the signal peptide) is represented in SEQ ID NO: <NUM>. The signal sequence may be selected depending on the host to be used. Any signal sequence that can express a variant of interest can be used in the present invention. Examples of the signal sequence that can be used in the present invention can be illustrated by the following: a sequence encoding the signal peptide of the α-factor (<NPL>), a sequence encoding the signal peptide of the α-factor receptor, a sequence encoding the signal peptide of the SUC2 protein, a sequence encoding the signal peptide of the PHO5 protein, a sequence encoding the signal peptide of the BGL2 protein, a sequence encoding the signal peptide of the AGA2 protein, a sequence encoding the signal peptide of TorA (trimethylamine N-oxidoreductase), a sequence encoding the signal peptide of Bacillus subtilis derived PhoD (phosphoesterase), a sequence encoding the signal peptide of Bacillus subtilis derived LipA (lipase), a sequence encoding the signal peptide of Aspergillus oryzae derived Taka-amylase (<CIT>), a sequence encoding the signal peptide of Bacillus amyloliquefaciens derived α-amylase (<NPL>)), a sequence encoding the signal peptide of Bacillus subtilis derived neutral protease (<NPL>), and a sequence encoding the signal peptide of Bacillus derived cellulase (<CIT>).

The nucleic acid of the present invention can be prepared in an isolated state by use of a standard genetic engineering technique, molecular biological technique, biochemical technique, chemical synthesis and the like in reference to the present specification or the sequence information disclosed in the appended sequence listing.

Another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (the gene coding for a modified enzyme). The recombinant DNA of the invention is provided in, for example, a form of a vector. The term "vector" in the present specification refers to a nucleic acid molecule that can transfer a nucleic acid inserted in the vector to a target such as a cell.

A suitable vector is selected according to its intended use (cloning, expression of a protein) and in consideration of a kind of a host cell. Examples include a M13 phage or an altered form thereof, a λ phage or an altered form thereof, and pBR322 or an altered form thereof (e.g., pB325, pAT153, pUC8), etc. as a vector having Escherichia coli as a host, pYepSec1, pMFa, pYES2 and pPIC3. <NUM> as a vector having a yeast as a host, pAc, pVL, etc. as a vector having an insect cell as a host, and pCDM8, pMT2PC, etc. as a vector having a mammal cell as a host.

The vector of the present invention is preferably an expression vector. The "expression vector" refers to a vector capable of introducing a nucleic acid inserted in the expression vector into a target cell (host cell) and expressing it in the cell. The expression vector generally contains a promoter sequence necessary for expression of a nucleic acid inserted, an enhancer sequence for promoting expression, and the like. An expression vector containing a selective marker can also be used. When such an expression vector is used, presence or absence (and its degree) of introduction of the expression vector can be confirmed using a selective marker.

Insertion of the nucleic acid of the present invention into the vector, insertion of a selective marker gene (if necessary), insertion of a promoter (if necessary), and the like can be performed in a standard recombinant DNA technique (for example, a known method of using a restriction enzyme and a DNA ligase, which can be referred in <NPL>).

As host cells, there can be employed, for example, microbial cells of koji mold (for example, Aspergillus oryzae), bacilli (for example, Bacillus subtilis), Escherichia coli, and Saccharomyces cerevisiae, in terms of easy handling; however, any host cell in which a recombinant DNA can be replicated and a gene encoding a modified enzyme can be expressed can be utilized. Preferably, Escherichia coli and Saccharomyces cerevisiae can be employed as a host organism. Candida yeasts such as Candida cylindracea can also be used as a host organism. In addition, Pichia yeasts such as Pichia pastoris can also be used as a host organism. Strains of Escherichia coli can be Escherichia coli strain BL21(DE3)pLysS in cases of using a T7-based promoter, and Escherichia coli strain JM109 in other cases. Strains of Saccharomyces cerevisiae can be Saccharomyces cerevisiae strain SHY2, AH22, or INVSc1 (Invitrogen).

Another aspect of the present invention relates to a microorganism having the recombinant DNA of the invention (that is, a transformant). The microorganism of the invention can be obtained by transfection or transformation using the vector of the invention described above. The transfection or transformation can be performed in, for example, the calcium chloride method (<NPL>)), the Hanahan method (<NPL>)), the SEM method (<NPL>)), a method by <NPL>)), the calcium phosphate coprecipitation method, electroporation (<NPL>)), and lipofectin (<NPL>)). The microorganism of the present invention can be used for producing the modified enzyme of the invention.

The modified enzyme of the present invention is provided, for example, in the form of an enzyme preparetion. The enzyme preparetion may contain an excipient, a buffer agent, a suspending agent, a stabilizer, a preservative, an antiseptic, saline and the like besides the active ingredient (the modified enzyme of the present invention). As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white soft sugar, glycerol and the like can be used. As the buffer agent, phosphates, citrates, acetates and the like can be used. As the stabilizer, propylene glycol, ascorbic acid and the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the antiseptic, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

A further aspect of the present invention relates to uses of modified enzymes and enzyme preparations according to the present invention, thereby providing different types of reaction (hydrolysis, synthesis, transformation) using the modified enzymes or enzyme preparations as characterized in the claims. Specifically, modified enzymes or enzyme preparations according to the present invention can be used, for example, for the degradation of fats and oils contained in fat-and-oil-containing wastewater and within grease traps, food processing (for example, production of milk flavor, degradation of fats and oils, production of FPA/DHA, bread making, egg white treatment), and organic synthesis reactions (for example, optical resolution of racemates, asymmetrization of symmetric compounds, regioselective acylation of hydroxyl groups). The modified enzymes according to the present invention have excellent reactivity and stability at a high temperature. These properties enhance the reactivity especially toward fats and oils that solidify at room temperature (such as oils and fats containing saturated fatty acids, and animal fats), and allow the modified enzymes to work advantageously in applications as described above. There is now described, as a specific example of these applications, a method for degradation of fats and oils in wastewater and the like, in which it is desired that the reaction with the lipase is carried out at a high temperature. Fats and oils present in wastewater and within grease straps often contain those that have high melting/freezing points and are thus difficult to be hydrolyzed by the lipase at room temperature (<NUM> ± <NUM>), for example, animal fats and oils such as lard and tallow. The method for degradation of fats and oils according to the present invention is useful for efficient degradation of such fats and oils. Therefore, as a preferred embodiment, the method for degradation of fats and oils according to the present invention is intended to degrade a material containing fats and oils with high melting/freezing points, which may be referred to hereinafter as a fat/oil containing material. Examples of the fat/oil containing material include wastewater from restaurants and eating places, hospitals, hotels, and the like; household wastewater; industrial wastewater discharged from food-processing and fat/oil-processing plants, and the like; and wastewater and deposits within grease traps placed at kitchens, and the like. The "grease trap" is a device for separating and collecting oils in wastewater, and typically includes three compartments. The first compartment is equipped with a basket to capture pieces of foodstuffs, garbage, and others. The second compartment is for separating the oils from the water. The wastewater from which the oils have been separated is sent to the third compartment to remove sedimentary debris and others. Grease traps are compulsorily required to be placed at kitchens for business use in restaurants and eating places, hospitals, hotels, and the like.

In the method for degradation of fats and oils according to the present invention, a modified enzyme according to the present invention is allowed to act on a fat/oil containing material as described above. For example, a modified enzyme or enzyme preparation according to the present invention is added to a solution including a fat/oil containing material to form a state where the modified enzyme and the fat/oil-containing material are in contact with each other, thereby to perform an enzyme reaction. In order to take advantage of characteristics of the present invention and at the same time, achieve efficient hydrolysis, the enzyme reaction is preferably carried out at a high temperature. Here, the high temperature refers to <NUM> to <NUM>, preferably <NUM> to <NUM>, further preferably <NUM> to <NUM>. The reaction time can be set so as to obtain a desired rate of degradation, taking into consideration the type and amount of the fat/oil containing material to be treated, the amount of the enzyme used, and others. The reaction time is, by way of example, from <NUM> hour to <NUM> days.

A further aspect of the present invention relates to a method for preparing a modified enzyme according to the present invention as characterized in the claims. In the method for preparation according to the present invention, modified enzymes that have been successfully obtained by the present inventors are prepared by genetic engineering procedures. In this embodiment, a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: <NUM> to <NUM> is prepared (step (I)). Here, a "nucleic acid encoding a particular amino acid sequence" gives, upon its expression, a polypeptide having the amino acid sequence encoded thereby, and includes not only a nucleic acid consisting of a base sequence corresponding to the amino acid sequence, but also a nucleic acid that may have an additional sequence added thereto, which may or may not encode an amino acid sequence. The degeneracy of codons is also taken into consideration. A "nucleic acid encoding the amino acid sequence represented by SEQ ID NO: <NUM>" can be prepared in an isolated state, for example, by standard genetic engineering, molecular biological, and biochemical procedures, with reference to the sequence information disclosed in the specification and accompanying sequence listing. As mentioned above the amino acid sequence represented by SEQ ID NO: <NUM> results from a particular mutation of the amino acid sequence of the Candida cylindracea-derived lipase. Therefore, a nucleic acid (gene) encoding the amino acid sequence represented by SEQ ID NO: <NUM> can also be obtained by applying a required mutation to the gene encoding the Candida cylindracea-derived lipase. A large number of methods for site-specific base sequence substitution are known in the art (see, for example, <NPL>), and from among these, suitable methods can be selected and used. As a site-specific mutagenesis method, site-specific saturation mutagenesis of amino acids can be employed. The site-specific saturation mutagenesis of amino acids is a "Semi-rational, semi-random" method in which the position involved in the desired function of a given protein is estimated on the basis of the three-dimensional structure of the protein, which is then subjected to amino acid saturation mutagenesis (<NPL>)). For example, site-specific saturation mutagenesis of amino acids can be performed by using kits, such as QuickChange® (Stratagene), and overlap extention PCR (<NPL>)). As the DNA polymerase for use in the PCR, for example, Taq polymerase can be employed. Preferably, use is made of high-accuracy DNA polymerases such as KOD-PLUS- (Toyobo Co. ) and Pfu turbo (Stratagene).

Following the step (I), the prepared nucleic acid is expressed (step (II)). For example, firstly, an expression vector inserted with the above described nucleic acid is prepared and a host cell is transformed using this constructed vector.

Then, a transformant is cultured under the condition of producing a modified enzyme that is an expressed product. Culture of the transformant may follow a general method. An assimilable carbon compound may be used as a carbon source used for a medium, and examples such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid are used. An available nitrogen compound may be used as a nitrogen source, and examples such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean bran alkali extract are used. Other than those substances, phosphate, carbonate, sulfate, salts of magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins, and the like are used according to necessity.

On the other hand, a culture temperature can be set within the range from <NUM> to <NUM> (preferably at around <NUM>). A culture time can be set by considering growing characteristics of a transformant to be cultured and production characteristics of a modified enzyme. A pH of a medium is set within the range wherein a transformant grows and an enzyme is produced. The pH of a medium is preferably set at about <NUM> to <NUM> (preferably at around pH <NUM>).

Subsequently, the expressed product (modified enzyme) is collected (step (III)). A culture liquid containing fungas bodies after culture may be used as an enzyme solution directly or after undergoing condensation, removal of impurities, or the like, but the expressed product is generally once collected from the culture liquid or fungas bodies. When the expressed product is a secretion type protein, it can be collected from the culture liquid, and in other cases, the expressed product can be collected from cells. In the case of collecting from the culture liquid, for example, an undissolved substance is removed by filtration and centrifugation on a culture supernatant, and then, a purified product of a modified enzyme can be obtained by separation and purification in combination of vacuum concentration, membrane concentration, salting out using ammonium sulfate or sodium sulfate, fractional precipitation by methanol, ethanol, or acetone, dialysis, heating treatment, isoelectric treatment, various kinds of chromatography such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography (for example, gel filtration with Sephadex® gel (GE Healthcare Life Sciences), etc., DEAE sepharose® CL-6B (GE Healthcare Life Sciences), octyl sepharose® CL-6B (GE Healthcare Life Sciences), CM sepharose® CL-6B (GE Healthcare Life Sciences)). On the other hand, in the case of collecting the expressed product from cells, a culture liquid is subjected to filtration, centrifugation, or the like, to thus obtain the cells, then the cells are crushed by a mechanical method such as a pressure treatment and an ultrasonic treatment, or an enzymatic method with a lysozyme or the like, thereafter carrying out separation and purification in the same manner as described above, and a purified product of a modified enzyme can be thus obtained.

The purified enzyme obtained as described above can be provided after being powdered, for example, by freeze dry, vacuum dry, or spray dry. In this time, the purified enzyme may be previously dissolved in a phosphoric acid buffer solution, a triethanol amine buffer solution, a tris-hydrochloric acid buffer solution, or a GOOD buffer solution. Preferably, a phosphoric acid buffer solution and a triethanol amine buffer solution can be used. Note that, for the GOOD buffer solution herein, PIPES, MES or MOPS is exemplified.

Generally, genetic expression and collection of the expressed product (modified enzyme) are carried our using an appropriate host-vector system as described above, but a cell-free synthesis system may also be employed. Herein, the "cell-free synthesis system (cell-free transcription system, cell-free transcription/translation system)" refers to in vitro synthesis of mRNA or a protein from a nucleic acid (DNA or mRNA) being a template, which codes for the mRNA or the protein, using a ribosome, a transcription/translation factor derived from living cells (alternately, obtained in a genetic engineering technique) or the like, not using living cells. In the cell-free synthesis system, a cell extraction obtained from a cell disruptor that is purified according to necessity is generally used. The cell extraction generally includes ribosome necessary for protein synthesis and various factors such as an initiation factor, and various enzymes such as tRNA. When a protein is synthesized, this cell extraction is added with other substances necessary for protein synthesis, such as various amino acids, energy sources (e.g., ATP and GTP), and creatine phosphate. As a matter of course, ribosome and various factors and/or various enzymes, and the like, which are separately prepared, may be supplemented if necessary in the protein synthesis.

Development of a transcription/translation system reconstructing various molecules (factors) necessary for protein synthesis has also been reported (<NPL>). In this synthesis system, a gene of <NUM> kinds of factors composed of <NUM> kinds of initiation factors constituting a protein synthesis system of bacteria, <NUM> kinds of elongation factors, <NUM> kinds of factors associated with termination, <NUM> kinds of aminoacyl tRNA synthesis enzymes that make each amino acid combine with tRNA, and a methionyl tRNA formyl transfer enzyme is amplified from an Escherichia coli genome, and a protein synthesis system is reconstructed in vitro using them. Such a reconstructed synthesis system may be used in the present invention.

The term "cell-free transcription/translation system" is interchangeably used with a cell-free protein synthesis system, an in vitro translation system or an in vitro transcription/translation system. In the in vitro translation system, RNA is used as a template to synthesize a protein. Any of RNA, mRNA, an in vitro transcribed product, or the like is used as the template RNA. On the other hand, in the in vitro transcription/translation system, DNA is used as a template. The template DNA should include in a ribosome bonding region, and preferably contains a suitable terminator sequence. In addition, in the in vitro transcription/translation system, a condition of adding factors necessary for each reaction is established so that a transcription reaction and a translation reaction proceed sequentially.

With the aim of improving high-temperature reactivity or stability of Candida cylindracea-derived lipase (SEQ ID NO: <NUM>), mutation points (amino acid residues for which substitution was to be made) were selected from the viewpoints of formation of disulfide bonds, loop stabilization (Pro introduction), enhancement of hydrogen bonding, and enhancement of hydrophobicity, whereby <NUM> variants (modified lipases) were designed. The respective variants designed were produced by the method described below, and evaluated for their properties. Here, the amino acid sequence represented by SEQ ID NO: <NUM> represents the mature form (without signal peptide) of Candida cylindracea-derived lipase; the amino acid sequence comprising the signal peptide sequence thereof, and the gene sequence encoding it are set forth in SEQ ID NOs: <NUM> and <NUM>, respectively.

A Pichia pastoris host expression system (Pichia Expression Kit, available from Invitrogen) was used. As a plasmid, use was made of pPIC3. The gene of Candida cylindracea-derived LIP1 as a template was codon-optimized for use in budding yeast (Saccharomyces cerevisiae). Mutations were made by an inverse PCR method (using Takara Bio Primestar mutagenesis kit), thereby to prepare genes encoding variants with amino acid substitutions at the selected mutation points (SEQ ID NOs: <NUM> to <NUM>). A plasmid containing a variant of the LIP1 gene was transformed into E. Subsequently, the plasmid was extracted from the transformed E. coli cells.

The plasmid containing the variant of the LIP1 gene was transformed into Pichia pastoris GS115 (using Pichia Expression Kit, available from Invitrogen). Pichia pastoris transformants obtained were cultured, and the cultured supernatants were collected. The culturing can be performed by the method described in the instruction manual of the kit. For example, a Pichia pastoris transformant was inoculated in BMGY medium (<NUM>% Peptone; <NUM>% Yeast extract; <NUM> Potassium phosphate, pH <NUM>; <NUM>% Yeast Nitrogen Base (without Amino Acids); <NUM>µg/mL Biotin; <NUM>% Glycerol) and cultured with shaking in a test tube at <NUM> for <NUM> days. Then, the cultured solution was inoculated into a baffled flask containing <NUM> of BMMY medium (<NUM>% Peptone; <NUM>% Yeast extract; <NUM> Potassium phosphate, pH <NUM>; <NUM>% Yeast Nitrogen Base (without Amino Acids); <NUM>µg/mL Biotin; <NUM>% Methanol) and incubated at <NUM>, at <NUM> rpm for <NUM> day. The cells were harvested, and then suspended in <NUM> of BMMY medium and subjected to culturing in a baffled flask under the same conditions. Methanol was added about every <NUM> hours to a final concentration of <NUM>% for the induction of enzyme expression for <NUM> days. For the respective transformants, the cultured supernatant (crude enzyme solution) was collected and evaluated for their properties, i.e., high-temperature reactivity and stability, of the variant enzyme. By comparison with the wild-type enzyme, eight variants were selected that were found to have improvements in these properties.

For the selected variants, the cultured supernatant was desalted and concentrated, and replaced into <NUM> Mcllvaine buffer (pH <NUM>). The solution after the replacement was applied to a column of SP-sepharose® (GE healthcare) equilibrated with the same buffer. The column was eluted with a linear gradient of <NUM> to <NUM> mol/L NaCl, and fractions with lipase activity were collected to obtain a purified enzyme.

Each of the purified enzymes was measured for enzyme activity at <NUM> and at <NUM> with the lipase kit S (DS Biopharma Medical Co. ) to ascertain whether the enzyme had improved reactivity at the high temperature. The high-temperature reactivity of the enzyme was evaluated in terms of the relative activity thereof, which is a ratio of enzyme activity at <NUM> to that at <NUM>, calculated by the following formula.

The evaluation results are shown in <FIG>. Six variants were observed to have improvements in the reactivity at the high temperature. Variants <NUM> (T130C-S153C) and <NUM> (S283Y) have particularly high reactivity at the high temperature.

Each of the purified enzyme solutions was diluted with phosphate buffer (pH <NUM>) so that the absorbance (A280) of the resulting enzyme solution at <NUM> was equal. The diluted solution was heated at <NUM> for <NUM> minutes, and then cooled on ice, followed by centrifugation. For the respective samples, the enzyme activity of the centrifuged supernatant was measured with the lipase kit S (DS Biopharma Medical Co. The residual activity percent of the enzyme was calculated as described below, from this measurement result, that is, the enzyme activity after heat treatment, and that before heat treatment, to evaluate the high-temperature stability thereof.

The evaluation results are shown in <FIG>. Six variants were observed to have improvements in the stability at the high temperature. Variants <NUM> (A249P), <NUM> (S283Y), and <NUM> (S300P) have particularly high stability at the high temperature.

As described above, <NUM> variants having excellent reactivity or stability at the high temperature were successfully obtained. The amino acid sequences of these variants are shown below.

The modified lipases of the present invention have excellent reactivity or stability at a high temperature. Therefore, the modified lipases are of high utility value in various applications in which enzyme reactions at a high temperature are desired.

Claim 1:
A modified lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of an amino acid sequence represented by SEQ ID NO: <NUM>, the modified lipase having an amino acid sequence of SEQ ID NO: <NUM> with an amino acid substitution in the amino acid sequence which is T130C-S153C, or an amino acid sequence (i) having <NUM>% or more sequence identity with the amino acid sequence of SEQ ID NO: <NUM>, and (ii) with the amino acid substitution T130C-S153C, wherein the high temperature for measuring the high-temperature reactivity is <NUM> to <NUM> and the high temperature for measuring the high-temperature stability is <NUM> to <NUM>.