Molecular evolution of candida antarctica lipase B and its application in catalytic synthesis of lactone compounds

Abstract

The present application provides Candida antarctica lipase B and mutants, and methods of making and using the same. Specifically, the present application relates to in vitro directed evolution of Candida antarctica lipase B and catalytic synthesis of lactone compounds. The lipases of the present application are Candida antarctica lipase B (CalB) and mutants thereof, including mutants of lipase B that have significantly improved activity for catalyzing the synthesis of lactone compounds. The present application also provides in vitro cyclization condensation reaction methods, including the step of converting a particular substrate into a lactone compound by cyclization condensation reaction in the presence of Candida antarctica lipase B (CalB) or a mutant thereof. The enzymes and mutants of the present application can be used for in vitro enzymatic synthesis of lactone compounds.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biocatalysis, specifically relating to the molecular evolution of Candida antarcticis lipase B and its efficient catalytic synthesis of lactone compounds. Background Technology

[0002] Lactones are an important class of high-value compounds and intermediates with wide applications in the food flavoring industry, pharmaceuticals, and medical devices. Based on the position of their hydroxyl condensation, aliphatic lactones are classified into different types, including macrocyclic lactones and α-, β-, γ-, and δ-lactones. Among them, small-ring δ-lactones have high commercial value in the food and cosmetics industries due to their rich fruity aroma, unique fragrance, and stable physicochemical properties. In particular, δ-dodecyl lactone, with its creamy flavor characteristics, is one of the most important δ-lactones and is widely used as a chemical flavoring agent in food.

[0003] δ-lactones can be produced in a variety of ways, including direct extraction from plants and through chemical and biosynthetic processes. The most common extraction method is from plants; however, the cost of extracting δ-lactones from plants is high due to their low natural concentrations and the complexity of the separation and purification processes. Currently, there are two main chemical synthetic processes for the production of δ-lactones; the first is the esterification of δ-hydroxy acids to form δ-lactones. The second, and more popular, chemical route involves the synthesis of the important intermediate 2-alkylcyclopentanone, followed by the oxidation of the δ-lactone via the Baeyer-Villiger process. These processes require toxic catalysts and environmentally unfriendly reagents. As an effective alternative to the synthesis of δ-lactones, biosynthesis has emerged as an important environmentally friendly and sustainable method, allowing for mild reaction conditions and high selectivity. Recently, an enzyme system has been developed that uses Waltomyceslipofer to synthesize δ-lactones from corresponding hydroxy acids via whole-cell catalysis.

[0004] As a multifunctional enzyme, CalB can catalyze various types of reactions involving ester bond formation and cleavage, and has been used to catalyze the synthesis of lactones. Robinson et al. reported a biosynthetic route for obtaining hexadecanoic acid from 16-hydroxyhexadecanoic acid using an immobilized lipase from Antarctica, but the conversion rate was only 57.4%, which is insufficient for production requirements. This indicates that while CalB has great potential for the large-scale production of δ-lactones, there is an urgent need in the field to develop highly efficient catalytic reactions. Summary of the Invention

[0005] The purpose of this invention is to provide molecular evolution of Candida antarcticis lipase B and its efficient catalytic synthesis of lactone compounds.

[0006] In a first aspect of the present invention, an in vitro cyclization condensation method is provided, comprising the steps of:

[0007] In the presence of Candida antarcticis lipase B or its mutant, compound (I) undergoes a cyclization condensation reaction to form compound (II):

[0008]

[0009] Wherein, R1 is H or methyl;

[0010] R2 is H, Na, or K;

[0011] n = 2 or 3.

[0012] When n = 2, the product is compound γ-decanolide of formula (II)a:

[0013]

[0014] In another preferred embodiment, the mutant has a mutation at a site selected from the group consisting of: position 188, position 189, or a combination thereof, wherein the site is based on the wild-type Candida antarcticis lipase B sequence shown in SEQ ID No:1.

[0015] In another preferred embodiment, the mutant has a 188-position mutation selected from the group consisting of: E188Q, E188D.

[0016] In another preferred embodiment, the mutant has the following mutation: I189F.

[0017] In another preferred embodiment, the mutant has mutations selected from the group consisting of: E188Q / I189F, E188D / I189F.

[0018] When n = 3, the product is compound δ-dodecanolide of formula (II)b:

[0019]

[0020] In another preferred embodiment, the mutant has a mutation at a site selected from the group consisting of: position 188, position 189, or a combination thereof, wherein the site is based on the wild-type Candida antarcticis lipase B sequence shown in SEQ ID No:1.

[0021] In another preferred embodiment, the mutant has a 188-position mutation selected from the group consisting of: E188Q, E188D, and E188R.

[0022] In another preferred embodiment, the mutant has the following mutations: I189F, I189Y, and I189M.

[0023] In another preferred embodiment, the mutant has mutations selected from the group consisting of: E188Q / I189F, E188D / I189Y, E188D / I189F, E188D / I189M, and E188R / I189F.

[0024] In another preferred embodiment, the mutant catalyzes sodium 5-hydroxydodecanoate to directly generate the corresponding product δ-dodecanolate.

[0025] In another preferred embodiment, the mutant catalyzes sodium 4-hydroxydecanoate to directly generate the corresponding product γ-decanolide.

[0026] In another preferred embodiment, the substrate concentration is 0.5-2 mM substrate.

[0027] In another preferred embodiment, the reaction system is a mixed organic solvent system.

[0028] In another preferred embodiment, the type of organic solvent is not particularly limited, as long as it does not affect the substrate and product of the reaction.

[0029] In another preferred embodiment, the final concentration of the organic solvent in the mixed reaction system is 80%-95%.

[0030] In another preferred embodiment, the temperature is 37°C-52°C.

[0031] In another preferred embodiment, the reaction time is 11-15 hours.

[0032] A second aspect of the invention provides an isolated or purified Candida antarctic lipase B mutant, said mutant having a mutation at a site selected from the group consisting of: position 188, position 189, or a combination thereof, wherein said site is based on the wild-type Candida antarctic lipase B sequence shown in SEQ ID No: 1.

[0033] In another preferred embodiment, the mutant has a 188-position mutation selected from the group consisting of: E188Q, E188D, and E188R.

[0034] In another preferred embodiment, the mutant has the following mutations: I189F, I189Y, and I189M.

[0035] In another preferred embodiment, the mutant has mutations selected from the group consisting of: E188Q / I189F, E188D / I189F, E188Q / I189F, E188D / I189Y, E188D / I189F, E188D / I189M, and E188R / I189F.

[0036] Preferably, the mutant is E188Q / I189F.

[0037] In another preferred embodiment, the Candida antarcticis lipase B mutant has the following activity: catalyzing the cyclization and condensation reaction of sodium 5-hydroxydodecanoate substrate to form δ-dodecyl lactone product.

[0038] In another preferred embodiment, the Candida antarcticis lipase B mutant has the following activity: catalyzing the cyclization and condensation reaction of sodium 4-hydroxydecanoate substrate to form γ-decanoic acid lactone product.

[0039] In a third aspect, the present invention provides a codon-optimized polynucleotide encoding wild-type Candida antarctica lipase B, the sequence of which is shown in SEQ ID NO:2.

[0040] In a fourth aspect, the present invention provides a carrier containing the polynucleotide described in the third aspect of the present invention.

[0041] In a fifth aspect, the present invention provides a genetically engineered host cell containing the vector described in the fourth aspect of the present invention, or having the polynucleotides described in the third aspect of the present invention integrated into its genome.

[0042] In another preferred embodiment, suitable host cells include Gram-positive bacteria such as Bacillus subtilis, Gram-negative bacteria such as Escherichia coli, actinomycetes such as Streptomyces, yeasts such as Saccharomyces cerevisiae, and fungi such as Aspergillus, whose cells are all commonly used host cells for recombinant vectors.

[0043] In a sixth aspect of the invention, the use of the Antarctic Candida lipase B and mutant described in the second aspect of the invention is provided, for use in catalyzing cyclization condensation reactions, or for use in preparing catalysts for catalyzing cyclization condensation reactions.

[0044] A seventh aspect of the present invention provides a method for the in vitro catalytic synthesis of lactones from Candida antarctica lipase B and its mutants, comprising the steps of: performing a cyclization condensation reaction on a compound of formula (I) catalyzed by Candida antarctica lipase B and its mutants as described in the second aspect of the present invention, thereby forming a lactone product.

[0045] An eighth aspect of the present invention provides a reaction system for carrying out a cyclization condensation reaction, said reaction system comprising:

[0046] (S0) The Antarctic Candida lipase B or a mutant thereof as described in the second aspect of the present invention;

[0047] (S1) Formula (Ⅰ) compound:

[0048]

[0049] Wherein, R1 is H or methyl;

[0050] R2 is H, Na, or K;

[0051] n = 2 or 3; and

[0052] (S2) Organic solvent / Tris-HCl two-phase solvent system.

[0053] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0054] Figure 1 The results of SDS-PAGE electrophoresis analysis of purified Candida antarcticis lipase B are shown.

[0055] Figure 2 The results of the screening of mutant libraries for the efficient synthesis of δ-dodecanoate from sodium 5-hydroxydodecanoate catalyzed by Candida antarctica lipase B and mutants are shown.

[0056] Figure 3 The GC-MS results of the synthesis of δ-dodecyl lactone catalyzed by the lipase mutant E188Q / I189F are shown.

[0057] Figure 4 The results of screening a mutant library for efficient synthesis of γ-decanolide catalyzed by Candida antarctica lipase B and mutants are shown.

[0058] Figure 5 The figure shows the effect of solvent on the synthesis of δ-dodecyl lactone catalyzed by the lipase mutant E188Q / I189F.

[0059] Figure 6 The results show the time curves and temperature effects of the synthesis of δ-dodecyl lactone catalyzed by the lipase mutant E188Q / I189F.

[0060] Figure 7 The figure shows the effect of enzyme amount on the synthesis of δ-dodecyl lactone catalyzed by the lipase mutant E188Q / I189F. Detailed Implementation

[0061] Through extensive and in-depth research, the inventors have developed applications for Candida antarcticus lipase B and its mutants. Specifically, the inventors developed lipase B from Candida antarcticus and performed directed evolutionary modification to obtain mutants with improved lipase activity, such as the mutant E188Q / I189F, which significantly enhances the activity of 5-hydroxydodecanoic acid or its salts. The Candida antarcticus lipase B and its mutants of this invention can be used for the in vitro enzymatic synthesis of various useful compounds (including δ-dodecanoic acid and γ-decanoic acid). This invention is based on this foundation.

[0062] Specifically, the inventors studied the sequence and structure of *Candida antarcticus* lipase B, and screened it using co-evolutionary analysis based on principles such as protein structural similarity, conserved site analysis, and diverse host origins. Subsequently, the original and evolved genes were functionally expressed in an *E. coli* expression system, and purified *Candida antarcticus* lipase B and its mutants were obtained. Experiments showed that the obtained important paired residue sites (E188 / I189) resulted in a catalytic efficiency increase of approximately 1.0 to 3.9 times. The mutant E188Q / I189F showed significant effects in the synthesis of δ-dodecanolide and γ-decanolide, directly catalyzing the cyclization and condensation reactions of specific substrates to form lactone products. After optimizing the conditions for the synthesis of δ-dodecanolide catalyzed by the mutant E188Q / I189F, with an enzyme loading of 3 mg, esterification of 1 mM substrate in toluene / Tris-HCl solvent (9:1, v / v) at 42°C for 13 h achieved a conversion rate of 99.53%.

[0063] Protein of the present invention

[0064] As used herein, the terms “Candida antarcticis lipase B (CalB)”, “enzyme of the present invention”, “Candida antarcticis lipase B (CalB) of the present invention and its mutants” or “lipase of the present invention” are used interchangeably and all refer to Candida antarcticis lipase B (CalB). It should be understood that the term includes wild-type and mutant Candida antarcticis lipase B (CalB) (e.g., derivative polypeptides derived from Candida antarcticis lipase B (CalB)).

[0065] The present invention provides *Candida antarcticis* lipase B (CalB) and its encoding gene, which are derived from *Candida antarcticis*. The wild-type amino acid sequence of the protein is SEQ ID NO:1, and an optimized nucleotide sequence is SEQ ID NO:2.

[0066] In this invention, CalB can also be a derived protein that has the same function (i.e., cyclization condensation function) as the protein shown in SEQ ID NO:1, by substitution, deletion or addition of one or more amino acids.

[0067] The enzymes of the present invention also include mutants of *Candida antarcticis* lipase B (CalB). Typically, these mutants are obtained by substituting one or more amino acid residues at positions 188 and 189 of the wild-type *Candida antarcticis* lipase B (CalB) with another amino acid residue; the preferred substitution positions are positions 188 and 189 of the amino acid sequence of *Candida antarcticis* lipase B (CalB) represented by SEQ ID NO:1. The mutant *Candida antarcticis* lipase B (CalB) of the present invention catalyzes the cyclization and condensation reaction of 5-hydroxydodecanoic acid or its salt to form a δ-dodecanoic acid product, exhibiting significantly enhanced activity compared to the wild type.

[0068] Furthermore, the *Candida antarcticis* lipase B (CalB) mutant is obtained by replacing one or more amino acid residues at positions of *Candida antarcticis* lipase B (CalB) with an amino acid sequence that exhibits at least 90% homology with the wild-type *Candida antarcticis* lipase B (CalB) using another amino acid residue; the preferred substitution positions are positions 188 and 189 of the amino acid sequence of *Candida antarcticis* lipase B (CalB) represented by SEQ ID NO:1. The mutant can catalyze the cyclization and condensation reaction of 5-hydroxydodecanoic acid or its salt to form a δ-dodecyl lactone product, with significantly improved activity compared to the wild type.

[0069] Furthermore, the other amino acid residue used to replace the original amino acid residue is preferably glutamine (amino acid abbreviation Q) or phenylalanine (amino acid abbreviation F).

[0070] In this invention, the nucleotide encoding corresponding to the mutation site of Candida antarcticis lipase B (CalB) should be understood as the nucleotide encoding of the "other amino acid residue" described in this invention.

[0071] In this invention, some preferred Candida antarcticis lipase B (CalB) mutants and their encoded genes are also provided, wherein the gene sequence of the starting Candida antarcticis lipase B (CalB) is SEQ ID NO:2 in the sequence listing, and the starting amino acid sequence is SEQ ID NO:1 in the sequence listing.

[0072] Some preferred mutation types are selected from the following group: they are mutants in which glutamic acid at position 188 is replaced by glutamine, and mutants in which isoleucine at position 189 is replaced by phenylalanine.

[0073] As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide will produce a single master band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using its amino acid sequence.

[0074] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptides of the present invention can be dehydrated or non-dehydrated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0075] The present invention also includes fragments, derivatives, and analogs of the said polypeptide. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the said polypeptide.

[0076] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituents or groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify the polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0077] Modifications (typically without altering the primary structure) include: chemically derived forms of peptides, either in vivo or in vitro, such as acetylation or carboxylation. Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). They also include peptides modified to improve their resistance to proteolysis or optimize their solubility.

[0078] The preferred sequence of the polypeptide is the polypeptide shown in SEQ ID NO:1. This term also includes variants and derived polypeptides having the same function as the polypeptide shown in SEQ ID NO:1. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10); and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus.

[0079] For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of a protein. Similarly, adding one or more amino acids to the C-terminus and / or N-terminus typically does not change the function of a protein. This term also includes the active fragment and active derivatives of the *Candida antarcticis* lipase B (CalB) of the present invention. The present invention also provides analogues of the said polypeptide.

[0080] These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0081] The amino or carboxyl terminus of the protein of this invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in this invention. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly–His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0082] To enable the translated protein to be expressed secretively (e.g., secreted extracellularly), a signal peptide sequence, such as the pelB signal peptide, can be added to the amino terminus of the *Candida antarcticis* lipase B (CalB). The signal peptide can be cleaved during the secretion of the polypeptide from the cell.

[0083] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding the protein having SEQ ID NO:1 but differing from the coding region sequence shown in SEQ ID NO:2.

[0084] The polynucleotide encoding the mature polypeptide of SEQ ID NO:1 includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0085] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0086] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring isoforms or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0087] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:1.

[0088] This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used herein, a “nucleic acid fragment” is at least 15 nucleotides long, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding Candida antarctica lipase B (CalB) protein.

[0089] The polypeptides and polynucleotides in this invention are preferably provided in isolated form, and more preferably purified to homogenization. The full-length nucleotide sequence or fragments of the *Candida antarcticis* lipase B (CalB) of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0090] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0091] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0092] Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis. The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0093] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or the Candida antarcticis lipase B (CalB) protein coding sequence, and methods for generating the polypeptides of the present invention via recombinant technology.

[0094] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant Candida antarcticis lipase B (CalB) polypeptide. Generally, the following steps are involved:

[0095] Transformation or transduction of suitable host cells using the polynucleotide (or variant) encoding the Candida antarcticis lipase B (CalB) polypeptide of the present invention, or using a recombinant expression vector containing the polynucleotide; host cells cultured in a suitable culture medium; isolation and purification of the protein from the culture medium or cells.

[0096] In this invention, the polynucleotide sequence of *Candida antarcticis* lipase B (CalB) can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0097] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding Candida antarctic lipase B (CalB) and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of E. coli; the PL promoter of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0098] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0099] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0100] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; plant cells; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0101] In another preferred embodiment, suitable host cells include Gram-positive bacteria such as Bacillus subtilis, Gram-negative bacteria such as Escherichia coli, actinomycetes such as Streptomyces, yeasts such as Saccharomyces cerevisiae, and fungi such as Aspergillus, whose cells are all commonly used host cells for recombinant vectors.

[0102] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0103] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0104] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0105] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0106] The recombinant peptides described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0107] application

[0108] This invention also provides applications of the *Candida antarcticis* lipase B (CalB), particularly in catalyzing the cyclization and condensation reaction of compounds of formula (I) to form lactone products. Typically, the *Candida antarcticis* lipase B (CalB) of this invention can be used to prepare compounds such as δ-dodecyl lactone and γ-decyl lactone.

[0109] δ-dodecanolide, catalyzed by Candida antarcticis lipase B (CalB), is a fragrance with a distinctive aroma. The Candida antarcticis lipase B (CalB) of this invention can catalyze the production of δ-dodecanolide.

[0110] Because certain mutant Candida antarcticis lipase B (CalB) of this invention has significantly enhanced catalytic activity for sodium 4-hydroxydecanoate and sodium 5-hydroxydodecanoate (e.g., E188Q / I189F), it can be used for in vitro enzymatic synthesis of γ-decanolide and δ-dodecanolide.

[0111] Typically, this invention provides a method for synthesizing corresponding lactones in vitro using *Candida antarcticus* lipase B (CalB) and mutants of this invention. In this method, sodium 5-hydroxydodecanoate can be used as a starting material to directly generate the corresponding product δ-dodecanoate under the catalysis of *Candida antarcticus* lipase B (CalB). Sodium 4-hydroxydecanoate can be used as a starting material to directly generate the corresponding product γ-decanoate under the catalysis of *Candida antarcticus* lipase B (CalB).

[0112] Preferably, the *Candida antarcticis* lipase B (CalB) or the *Candida antarcticis* lipase B (CalB) mutant provided in the second aspect above. Under optimized conditions, the *Candida antarcticis* lipase B (CalB) mutant catalyzes a 99.53% conversion rate in the reaction of sodium 5-hydroxydodecanoate to δ-dodecyl lactone.

[0113] Preferably, the substrate concentration is 0.5-2 mM substrate.

[0114] Preferably, the reaction system is a mixed organic solvent system.

[0115] Preferably, there are no particular restrictions on the type of organic solvent, as long as it does not affect the substrate and product of the reaction.

[0116] Preferably, the final concentration of the organic solvent in the mixed reaction system is 80%-95%.

[0117] Preferably, the temperature is 37℃-52℃.

[0118] Preferably, the reaction time is 11-15 hours.

[0119] Methods for synthesizing lactone compounds

[0120] In this invention, a method for synthesizing lactone compounds in vitro using the Antarctic Candida lipase B (CalB) and mutants of this invention is also provided.

[0121] Typically, this method includes: using sodium 5-hydroxydodecanoate as a raw material, δ-dodecanoate can be directly generated under the catalysis of Candida antarcticis lipase B (CalB); using sodium 4-hydroxydecanoate as a raw material, γ-decanoate can be directly generated under the catalysis of Candida antarcticis lipase B (CalB).

[0122] Experimental results show that the enzyme of this invention is particularly suitable for the in vitro enzymatic synthesis of δ-dodecanoate and γ-decanoate. Under unoptimized experimental conditions, the conversion rate of δ-dodecanoate to δ-dodecanoate after 19 hours of catalysis by the *Candida antarcticum* lipase B (CalB) mutant E188Q / I189F was preferably 75.4%, which is 2.75 times that of the wild type (WT). The conversion rate of γ-decanoate to δ-decanoate after 19 hours of catalysis by the *Candida antarcticum* lipase B (CalB) mutant E188Q / I189F was 41.3%, which is 1.79 times that of the wild type (WT). Under optimized conditions, the conversion rate of the reaction of sodium 5-hydroxydodecanoate to δ-dodecanoate catalyzed by the *Candida antarcticum* lipase B (CalB) mutant was 99.53%.

[0123] Preferably, the substrate concentration is 0.5-2 mM substrate.

[0124] Preferably, the reaction system is a mixed organic solvent system.

[0125] Preferably, there are no particular restrictions on the type of organic solvent, as long as it does not affect the substrate and product of the reaction.

[0126] Preferably, the final concentration of the organic solvent in the mixed reaction system is 80%-95%.

[0127] Preferably, the temperature is 37℃-52℃.

[0128] Preferably, the reaction time is 11-15 hours.

[0129] The main advantages of this invention include:

[0130] (a) The catalytic action of Candida antarcticis lipase B (CalB) and its mutants described in this invention has successfully enabled the in vitro enzymatic synthesis of lactone compounds such as δ-dodecyl lactone and γ-decyl lactone.

[0131] (b) The conversion rate of δ-dodecanoate to sodium 5-hydroxydodecanoate by the Candida antarcticis lipase B (CalB) mutant E188Q / I189F after catalyzing the substrate was 75.4% after 19 h, which is 2.75 times that of the wild type (WT).

[0132] (c) The conversion rate of γ-decanoic acid lactone synthesized from sodium 4-hydroxydecanoate by the Candida antarcticis lipase B (CalB) mutant E188Q / I189F after catalyzing the substrate for 19 h was 41.3%, which is 1.79 times that of the wild type (WT).

[0133] (d) Using the Candida antarcticis lipase B (CalB) mutant described in this invention and the optimized reaction system, the corresponding δ-dodecyl lactone was synthesized in vitro by enzymatic method, with a conversion rate of up to 99.53%.

[0134] The present invention will be further described in detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0135] Materials and reagents

[0136] General description of the source of the biological materials described in this invention:

[0137] Primer synthesis: All primers used in this invention were synthesized and prepared by BGI Genomics Co., Ltd.

[0138] The PrimeSTARMax DNA polymerase used in the experiment was purchased from TakaRa; the DNA gel extraction kit and plasmid miniprep kit were purchased from Axygen.

[0139] Example 1: Construction of Candida antarcticis lipase B (CalB)

[0140] Specifically, in this embodiment, the present invention is based on Candida antarcticis lipase B (CalB) and its coding sequence. Its amino acid sequence is shown in SEQ ID No:1: LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP (SEQ ID No:1)

[0141] The CalB gene underwent codon optimization to facilitate expression in E. coli. The optimized coding sequence is shown in SEQ ID No:2:

[0142] CTACCTTCCGGTTCGGACCCTGCCTTTTCGCAGCCCAAGTCGGTGCTCGATGCGGGTCTGACCTGCCAGGGTGCTTCGCCATCCTCGGTCTCCAAACCCATCCTTCTCGTCCCCGGAACCGGCACCACAGGTCCACAGTCGTTCGACTCGAACTGGATTCCCCTCTCAACGCAGTTGGGTTACACACCCTGCTGGATCTCACCCCCGCCGTTCATGCTCAACGACACCCAGGTCAACACGGAGTACATGGTCAACGCCATCACCGCGCTCTACGCTGGTTCGGGCAACAACAAACTTCCCGTGCTTACCTGGTCCCAGGGTGGTCTGGTTGCACAGTGGGGTCTGACCTTCTTCCCCAGTATCAGGTCCAAGGTCGATCGACTTATGGCCTTTGCGCCCGACTACAAGGGCACCGTCCTCGCCGGCCCTCTCGATGCACTCGCGGTTAGTGCACCCTCCGTATGGCAGCAAACCACCGGTTCGGCACTCACCACCGCACTCCGAAACGCAGGTGGTCTGACCCAGATCGTGCCCACCACCAACCTCTACTCGGCGACCGACGAGATCGTTCAGCCTCAGGTGTCCAACTCGCCACTCGACTCATCCTACCTCTTCAACGGAAAGAACGTCCAGGCACAGGCCGTGTGTGGGCCGCTGTTCGTCATCGACCATGCAGGCTCGCTCACCTCGCAGTTCTCCTACGTCGTCGGTCGATCCGCCCTGCGCTCCACCACGGGCCAGGCTCGTAGTGCAGACTATGGCATTACGGACTGCAACCCTCTTCCCGCCAATGATCTGACTCCCGAGCAAAAGGTCGCCGCGGCTGCGCTCCTGGCGCCGGCAGCTGCAGCCATCGTGGCGGGTCCAAAGCAGAACTGCGAGCCCGACCTCATGCCCTACGCCCGCCCCTTTGCAGTAGGCAAAAGGACCTGCTCCGGCATCGTCACCCCCTGA(SEQ ID NO:2)

[0143] The sequence shown in SEQ ID No:2 was synthesized using a whole-genome synthesis method, with NcoⅠ and XhoⅠ restriction sites added to both ends. This full-length sequence was then cloned into the NcoⅠ and XhoⅠ restriction sites of the commercially available pET-22b(+) plasmid.

[0144] Example 2: Expression and purification of Candida antarcticis lipase B

[0145] The recombinant expression plasmids containing the selected genes were heat-shock transformed into *E. coli* Rosetta(DE3) competent cells for gene expression and protein purification. The recombinant bacteria were cultured to OD200. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.01mM and induce culture overnight at a low temperature of 15℃ and 220rpm.

[0146] The bacterial cells were collected by centrifugation and resuspended in 20 mM Tris-HCl buffer (pH 8.0, 500 mM NaCl, 20 mM imidazole). 250 mL of cultured cells were finally resuspended in 40 mL of buffer and disrupted using a high-pressure cell disruptor (4–6 °C, 700 Pa). The cell disruption buffer was then centrifuged at 12,000 rpm for 30 min (4 °C), and the supernatant was collected. This centrifugation at 12,000 rpm for 30 min (4 °C) was repeated. The supernatant was then used to purify the protein using a Ni-NTA column affinity chromatography. Impurities were eluted with 20 mM Tris-HCl buffer (pH 8.0, 500 mM NaCl, 50 mM imidazole), and the target protein was eluted with 200 mM Tris-HCl buffer (pH 8.0, 500 mM NaCl, 200 mM imidazole). The eluted protein was then concentrated and desalted to obtain the purified protein.

[0147] The purified protein was stored in 50 mM Tris-HCl buffer (pH 8.0), and the purified protein was detected by electrophoresis using 12% SDS-PAGE. The protein concentration was determined using the Bradford Protein Assay Kit (Shanghai Sangon Biotech).

[0148] The results are as follows Figure 1 As shown in the figure. The results indicate that a clear band was obtained at approximately 35 kDa, suggesting that the target protein CalB has been purified.

[0149] Example 3: Construction of the Antarctic Candida lipase B mutant and determination of its enzyme activity during the catalytic synthesis of δ-dodecanolide.

[0150] In this embodiment, based on the activity characteristic of Candida antarcticis lipase B in catalyzing the synthesis of δ-dodecanolide, the enzyme was subjected to directed evolutionary modification using the SCA.SIM co-evolutionary analysis method and rationally designed selection sites to obtain a mutant that can efficiently catalyze the synthesis of δ-dodecanolide.

[0151] Using recombinant plasmid pET-22b(-)-CalB as a template, and a pair of complementary oligonucleotides with a mutation site as a degenerate base (NNK) as primers, the whole plasmid was amplified by Primestar high-fidelity enzyme to obtain a recombinant plasmid with a specific mutation site.

[0152] The primer sequences are as follows: Mutants corresponding to the substitution of glutamic acid at position 188 and isoleucine at position 189 in SEQ NO:2 with 19 other amino acids: E188 / I189-FTCGGCGACCGACNNKNNKGTTCAGCCTCAG (SEQ ID No:4). E188 / I189-RGGCTGAACMNNMNNGTCGGTCGCCGAGTAG (SEQ ID No:5)

[0153] The amplification system consisted of 20 ng of recombinant plasmid template, 1 μL each of primers (10 μM), 25 μL of PrimeSTAR Max DNA polymerase, and double-distilled water to a final volume of 50 μL. The amplification conditions were: 98℃ pre-denaturation for 3 minutes, 98℃ denaturation for 15 seconds, 72℃ extension for 2 minutes, for a total of 25 cycles, followed by a final extension at 72℃ for 10 minutes. After the reaction, the amplified products were detected by 1% agarose gel electrophoresis. The products were purified and recovered using a PCR product purification kit, and digested with DpnI enzyme (NEB) at 37℃ for 2 hours to degrade the initial template. The digested products were transformed into E. coli Rosetta (DE3) competent cells, plated on LB agar plates containing 100 μg / mL ampicillin and 340 μg / mL chloramphenicol, and incubated overnight at 37℃. Positive clones were screened and sequenced for verification. A saturated mutant recombinant strain of *Candida antarcticis* with a specified site for lipase B was obtained.

[0154] The purified protein of the saturated mutant of Candida antarcticis lipase B at a specified site was obtained according to the method in Example 2.

[0155] The mutant enzyme activity assay was performed using a 1000 μL reaction system, comprising 1.5 mg / mL of purified mutant enzyme and 1 mM sodium 5-hydroxydodecanoate, in toluene / Tris-HCl (9:1, v / v). The reaction was incubated at 37 °C with shaking at 220 rpm for 19 h. The enzyme was then dried by rotary evaporation and analyzed by gas chromatography (GC).

[0156] The treated reaction product was redissolved in n-hexane and centrifuged at 12,000 rpm for 40 min. Then, 1 μL of sample was injected into an HP-5 capillary column (30 m × 0.32 mm × 0.25 μm). Typical GC operating conditions were as follows: injection port and detector temperatures of 250 °C and 300 °C, respectively; nitrogen as carrier gas at a flow rate of 1 mL·min⁻¹; and an initial oven temperature of 150 °C held for 5 min. The oven temperature was then increased at 3 °C·min⁻¹. -1 The rate was increased to 180°C, and then at 5°C·min. -1 The reaction rate was increased until the final temperature reached 240°C, and then maintained for 5 minutes. To calculate the conversion of the reaction, δ-dodecanolide was used as a standard reference to quantify the reaction products.

[0157] The results are shown in Table 1. Figure 2 As shown in the figure. The results indicated that among the saturated mutants at sites 188 and 189, several mutants exhibited higher conversion rates than the wild type. Among them, the E188Q / I189F mutant achieved a 75.4% conversion rate of δ-dodecanolide after 19 hours of catalysis, which was 2.75 times that of the wild type (WT). This site is a key site affecting the catalytic activity of CalB.

[0158] Table 1. Conversion rates of dodecalactone synthesis in the presence of wild-type CalB or its different mutants.

[0159]

[0160] like Figure 3 As shown, the gas chromatography-mass spectrometry (GC-MS) results indicate that the product is δ-dodecanolide, consistent with previous reports.

[0161] Example 4: Enzyme activity determination during the synthesis of γ-decanolide by the Antarctic Candida lipase B mutant.

[0162]

[0163] The mutant enzyme activity assay was performed using a 1000 μL reaction system, comprising 1.5 mg / mL of purified mutant enzyme and 1 mM sodium 4-hydroxydecanoate, in toluene / Tris-HCl (9:1, v / v). The reaction was incubated at 37 °C with shaking at 220 rpm for 19 h. The enzyme was then dried by rotary evaporation and analyzed by gas chromatography (GC).

[0164] The treated reaction product was redissolved in n-hexane and centrifuged at 12,000 rpm for 40 min. Then, 1 μL of sample was injected into an HP-5 capillary column (30 m × 0.32 mm × 0.25 μm). Typical GC operating conditions were as follows: injection port and detector temperatures of 250 °C and 300 °C, respectively; nitrogen as carrier gas at a flow rate of 1 mL·min⁻¹; and an initial oven temperature of 150 °C held for 5 min. The oven temperature was then increased to 180 °C at a rate of 3 °C·min⁻¹, followed by increases at a rate of 5 °C·min⁻¹ until a final temperature of 240 °C was reached, which was then held for 5 min. To calculate the conversion of the reaction, γ-decanolide was used as a standard reference to quantify the reaction product.

[0165] The results are shown in Table 2. Figure 4 As shown in the figure, among the saturated mutants at positions 188 and 189, several mutants exhibited higher conversion rates than the wild type. Specifically, the E188Q / I189F mutant achieved a 41.3% conversion rate of γ-decanolide after 19 hours of catalysis, which was 1.79 times that of the wild type (WT). This site is a key site affecting the catalytic activity of CalB.

[0166] Table 2. Conversion rates of γ-decanolide synthesis in the presence of wild-type CalB or its different mutants.

[0167]

[0168] Example 4: Optimization of conditions for the synthesis of δ-dodecanolide by Candida antarcticis lipase B mutant

[0169] The reaction conditions for the synthesis of δ-dodecanoate were optimized. Sodium 5-hydroxydodecanoate was used at a concentration of 1 mM. Reactions were carried out at different temperatures (25℃, 30℃, 37℃, 42℃, and 51℃), different solvents (toluene, benzene, chloroform, and dichloromethane), and different enzyme loadings (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL), with incubation at 220 rpm for different times (4 h, 8 h, 13 h, 19 h, 24 h, and 37 h). The resulting products were then dried by rotary evaporation and analyzed by gas chromatography (GC).

[0170] The results are as follows Figure 5 As shown in the figure. The results indicate that the highest conversion rate, reaching 75.4%, was achieved with a sodium 5-hydroxydodecanoate concentration of 1 mM, an enzyme loading of 1.5 mg / mL, and toluene as the organic solvent.

[0171] like Figure 6As shown, the concentration of sodium 5-hydroxydodecanoate was 1 mM, the organic solvent was toluene, and the enzyme loading was 1.5 mg / mL. With changes in temperature and time, the conversion rate increased at a slow rate until it reached 99.51% at 37 hours.

[0172] like Figure 7 As shown, with an enzyme loading of 3 mg / mL, a high conversion rate of 99.53% can be obtained after 13 hours of reaction.

[0173] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0174]

Claims

1. An in vitro cyclization condensation method, characterized in that, Including the following steps: In the presence of the Candida antarcticis lipase B mutant, compound (I) undergoes a cyclization condensation reaction to form compound (II): Wherein, R1 is H or methyl; R2 is H, Na, or K; n = 2 or 3; The mutation sites of the mutants are: E188Q / I189F, E188D / I189F, E188D / I189Y, E188D / I189M, or E188R / I189F, which are based on the wild-type Candida antarcticis lipase B sequence shown in SEQ ID No:

1.

2. The method as described in claim 1, characterized in that, When n=2, the product is compound γ-decanolide of formula (Ⅱa): The mutation sites of the mutants are E188Q / I189F or E188D / I189F.

3. The method as described in claim 1, characterized in that, When n=3, the product is compound δ-dodecanolide of formula (Ⅱb): 。 4. The method as described in claim 1, characterized in that, The substrate concentration was 0.5-2 mM.

5. The method as described in claim 4, characterized in that, The reaction system is a mixed organic solvent system.

6. The method as described in claim 4, characterized in that, The final concentration of the organic solvent in the mixed reaction system is 80%-95%.

7. The method as described in claim 4, characterized in that, The reaction time is 11-15 hours.

8. An isolated or purified Candida antarcticis lipase B mutant, characterized in that, The mutation sites of the mutants are: E188Q / I189F, E188D / I189F, E188D / I189Y, E188D / I189M, or E188R / I189F; wherein the sites are based on the wild-type Candida antarcticis lipase B sequence shown in SEQ ID No:

1.

9. The mutant as described in claim 8, characterized in that, The mutation sites of the mutants are selected from the following groups: E188Q / I189F, E188D / I189F.

10. The mutant as described in claim 8, characterized in that, The aforementioned Candida antarcticis lipase B mutant exhibits the following activity: catalyzing the cyclization and condensation reaction of sodium 5-hydroxydodecanoate substrate to form δ-dodecyl lactone product.

11. The mutant as described in claim 9, characterized in that, The aforementioned Candida antarcticis lipase B mutant exhibits the following activity: catalyzing the cyclization and condensation reaction of sodium 4-hydroxydecanoate substrate to form γ-decanoic acid lactone product.

12. An isolated polynucleotide, characterized in that, The polynucleotide encodes the Antarctic Candida lipase B mutant of claim 8.

13. A carrier, characterized in that, The carrier contains the polynucleotide of claim 12.

14. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 13, or the polynucleotide of claim 12 is integrated into its genome.

15. The use of the *Candida antarcticis* lipase B mutant according to claim 8, characterized in that, It is used to catalyze cyclization condensation reactions, or to prepare catalysts for cyclization condensation reactions.

16. A reaction system for carrying out a cyclization condensation reaction, characterized in that, The reaction system includes: (S0) The Antarctic Candida lipase B mutant according to claim 8; (S1) Formula (Ⅰ) compound: Wherein, R1 is H or methyl; R2 is H, Na, or K; n=2 or 3; and (S2) Organic solvent / Tris-HCl two-phase solvent system.

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