KR102703236B1 - Enantioselectivity Improved Candida antarctica Lipase B Variants - Google Patents

Abstract

Conventional Candida antarctica lipase B has a problem in that it has low stereoselectivity for secondary alcohols having small substituents, thereby lowering the efficiency of producing photochemically pure secondary alcohols, which are building blocks used for synthesizing various compounds such as bioactive compounds. In the present invention, a mutant of Candida antarctica lipase B (CAL-B) was developed, and it was experimentally demonstrated that the mutant has high stereoselectivity for secondary alcohols having small substituents, and thus can efficiently mass-produce photochemically pure secondary alcohols. Therefore, the mutant of Candida antarctica lipase B (CAL-B) of the present invention has an advantage in that it can efficiently produce not only secondary alcohols having large substituents but also pure enantiomers of secondary alcohols having small substituents.

Description

{Enantioselectivity Improved Candida antarctica Lipase B Variants}

The present invention relates to a Candida antarctica lipolytic enzyme B variant having improved stereoselectivity, and more particularly, to a Candida antarctica lipolytic enzyme B variant having improved stereoselectivity for alcohols having small substituents.

Candida antarctica lipase B (CAL-B), also known as Pseudozyma antarctica lipase B or Moesziomyces antarcticus lipase B, is the most widely used biocatalyst for the production of enantiopure sec -alcohols. CAL-B has excellent enantioselectivity, high chemotolerance, and thermostability, and thus has excellent advantages as a biocatalyst.

CAL-B can efficiently distinguish the difference in the size of substituents larger than methyl and ethyl groups, and was confirmed to exhibit high stereoselectivity with an enantiomeric ratio (E) exceeding 200 for a wide range of secondary alcohols. However, CAL-B does not exhibit high stereoselectivity for all secondary alcohols, and in particular, it is known to exhibit low stereoselectivity for secondary alcohols having substituents smaller than a propyl group. For example, the enantiomeric ratio of CAL-B is E > 200 for (±)-pentan-2-ol, whereas the enantiomeric ratios for (±)-butan-2-ol and (±)-but-3-yn-2-ol are E = 7 and E = 4, respectively.

To date, no method has been reported to improve the stereoselectivity of CAL-B for secondary alcohols having small substituents, and improving the stereoselectivity for such secondary alcohols having small substituents still remains a challenging task.

The mirror image but-3-yn-2-ol is used as a useful building block for the synthesis of various bioactive compounds such as the cytotoxic butenolide (-)-akolactone, (-)-Enigmazole A, Fenleuton, Frondosin B, HIV protease inhibitors, iodoalynes (+)-pancratistatin, Meayamycin and tetrahydronaphtyridine. Accordingly, research and development on (±)-but-3-yn-2-ol hydrolyzing enzymes for producing but-3-yn-2-ol, a mirror image of which has very high industrial utility, is actively underway.

Nonetheless, most (±)-but-3-yn-2-ol hydrolases are found to exhibit low stereoselectivity at the level of E=1.0 or intermediate stereoselectivity at the level of E=26, similar to CAL-B.

Bornscheuer et al. used directed evolution and site-directed mutagenesis to improve the stereoselectivity of (±)-but-3-yn-2-ol hydrolase, using Pseudomonas fluorescens esterase (PFE). Bornscheuer et al. produced 7,000 clones derived from the PFE and performed a screening process. As a result, they identified a clone in which the stereoselectivity of PFE was improved from E=3 to E=96, but it did not reach E=200.

Therefore, if a hydrolase with high stereoselectivity for secondary alcohols with small substituents is developed, it is expected that industrial utilization will be improved because it will be possible to efficiently mass-produce pure enantiomeric secondary alcohols, which are building blocks used to synthesize various compounds such as bioactive compounds.

The patent documents and references mentioned in this specification are incorporated by reference into this specification to the same extent as if each document was individually and specifically designated by reference.

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The purpose of the present invention is to provide a variant of Candida antarctica lipase B (CAL-B) having improved stereoselectivity for alcohols having small substituents.

Other objects and technical features of the present invention are more specifically presented in the detailed description of the invention, the claims and the drawings below.

The present invention relates to a mutant of Candida antarctica lipase B (CAL-B), characterized in that the 47th amino acid, serine (S), in the amino acid sequence of SEQ ID NO: 1 (amino acid sequence of Candida antarctica lipase B) is substituted with asparagine (N), glutamine (Q), leucine (L), isoleucine (I), phenylalanine (F), tyrosine (Y), or methionine (M); A mutant of Candida antarctica lipase B (CAL-B), characterized in that the 42nd amino acid, threonine (T), in the amino acid sequence of sequence number 1 is substituted with valine (V), and the 47th amino acid, serine (S), is substituted with asparagine (N), glutamine (Q), leucine (L), isoleucine (I), phenylalanine (F), tyrosine (Y), or methionine (M); A mutant of Candida antarctica lipase B (CAL-B), characterized in that the 42nd amino acid, threonine (T), the 43rd amino acid, proline (P), and the 47th amino acid, serine (S), in the amino acid sequence of sequence number 1 are substituted with valine (V), aspartic acid (D), arginine (R), and asparagine (N), respectively.

The above variant of Candida antarctica lipase B (CAL-B) is characterized in that the enantioselectivity (E) for (±)-butan-2-ol is improved 2 to 5 times compared to Candida antarctica lipase B, and the enantioselectivity (E) for (±)-but-3-yn-2-ol is improved 35 times or more compared to Candida antarctica lipase B.

The present invention provides a polynucleotide encoding a variant of the Candida antarctica lipase B (CAL-B); a vector comprising the polynucleotide; and a microorganism producing a variant of Candida antarctica lipase B (CAL-B), comprising at least one of the variant of the Candida antarctica lipase B (CAL-B), the polynucleotide, and the vector.

In addition, the present invention provides a method for producing a variant of Candida antarctica lipase B (CAL-B), comprising: a polynucleotide encoding a variant of Candida antarctica lipase B (CAL-B); a vector comprising the polynucleotide; a step of culturing a microorganism producing a variant of Candida antarctica lipase B (CAL-B), the microorganism including at least one of the variant of Candida antarctica lipase B (CAL-B), the polynucleotide, and the vector in a medium; and a step of recovering the variant of Candida antarctica lipase B (CAL-B) from the cultured medium or microorganism.

Conventional Candida antarctica lipase B has a low stereoselectivity for secondary alcohols with small substituents, which results in a low production efficiency of pure enantiomeric secondary alcohols, which are building blocks used to synthesize various compounds such as bioactive compounds.

In the present invention, a mutant of Candida antarctica lipase B (CAL-B) was developed, and it was experimentally demonstrated that the mutant has high stereoselectivity for secondary alcohols having small substituents, enabling efficient mass production of pure enantiomeric secondary alcohols.

Therefore, the variant of Candida antarctica lipase B (CAL-B) of the present invention has the advantage of being able to efficiently produce pure enantiomers of secondary alcohols having small substituents.

Figure 1 shows the purification process of CAL-B and CAL-B variant of the present invention by SDS-PAGE. M represents a marker for confirming protein size, S represents a soluble fraction obtained after destroying microorganisms, I represents an insoluble fraction obtained after destroying microorganisms, F represents a fraction that passed through an affinity column, W, W ₁ , and W ₂ represent fractions that washed the affinity column, and E represents a fraction that eluted the affinity column.
Figure 2 shows the results of analyzing the stereoselectivity of CAL-B and CAL-B variants using gas chromatography. Panel a) shows the gas chromatography analysis result of 5D-CAL-B, and the upper graph of the panel shows 1% result and the lower graph shows 40% result. Panel b) shows the gas chromatography analysis result of CAL-B-S47N, and the upper graph of the panel shows 1% result and the lower graph shows 40% result. The retention time of the fast enantiomer was 3.2 minutes; the retention time of the slow enantiomer was 3.4 minutes; the retention time of vinyl butyrate was 3.7 minutes; the retention time of ( R )-but-3-yn-2-yl butyrate was 10.3 minutes; and the retention time of ( S )-but-3-yn-2-yl butyrate was 10.8 minutes. The retention time of butanoic acid was 18 minutes.
Figure 3 shows the results of a three-dimensional structural analysis of the amino acid local sequence and binding pocket of CAL-B of the present invention. Panel a) shows the results of a comparative analysis of the local sequences of CAL-B and PBL, and panel b) shows the results of an analysis of the key amino acid side chains of the middle binding pocket.
Figure 4 shows the model structures of CAL-B and CAL-B-S47N of the present invention bound to (S)-but-3-yn-2-ol as a substrate. Panel a) shows a binding structure model of CAL-B and (S)-but-3-yn-2-ol, and panel b) shows a binding structure model of CAL-B-S47N and (S)-but-3-yn-2-ol. The red solid line indicates the distance between the O _γ atom of serine or the O _δ1 atom of asparagine and the terminal carbon atom of the acetylene group.

The present invention relates to a variant of Candida antarctica lipase B (CAL-B) having high stereoselectivity for secondary alcohols having small substituents. The variant means one or more amino acid sequences are substituted with a different type of amino acid by a change in the genetic sequence, and is used interchangeably with the term mutant in the present specification.

The present invention provides a variant of Candida antarctica lipase B (CAL-B, Blast access code: P41365), characterized in that serine (S), the 47th amino acid in the amino acid sequence (SEQ ID NO: 1) of Candida antarctica lipase B, is substituted with asparagine (N), glutamine (Q), leucine (L), isoleucine (I), phenylalanine (F), tyrosine (Y), or methionine (M); Provided is a mutant of Candida antarctica lipase B (CAL-B), characterized in that the 42nd amino acid, threonine (T), is substituted with valine (V), and the 47th amino acid, serine (S), is substituted with asparagine (N), glutamine (Q), leucine (L), isoleucine (I), phenylalanine (F), tyrosine (Y), or methionine (M); A mutant of Candida antarctica lipase B (CAL-B) is provided, characterized in that the 42nd amino acid, threonine (T), the 43rd amino acid, proline (P), and the 47th amino acid, serine (S), in the amino acid sequence are substituted with valine (V), aspartic acid (D), arginine (R), and asparagine (N), respectively. Glutamine (Q), Leucine (L), Isoleucine (I), Phenylalanine (F), Tyrosine (Y), or Methionine (M), which replace the 47th amino acid Serine (S), have the characteristic that the residue size is larger than Asparagine (N). Among the amino acids that replace the 47th amino acid Serine (S), Asparagine (N) is preferable.

The above-mentioned Antartica lipolytic enzyme B has high stereoselectivity for secondary alcohols and is the most widely used biocatalyst for the production of enantiopure secondary alcohols.

Antartica lipolytic enzyme B (CAL-B) of the present invention has an amino acid sequence disclosed in SEQ ID NO: 1, and its homolog Pseudozyma brasiliensis lipase GHG001 (PBL, GHG001, Blast access code: EST06015 or XP_016291004) has an amino acid sequence disclosed in SEQ ID NO: 2. The present invention experimentally demonstrated that a position-directed mutation at residues 42 to 47 corresponding to the binding pocket site or a substitution at residues 42 to 47 between homologs enhances the stereoselectivity of CAL-B by conducting a mass screening for position-directed mutations between homologs.

The inventors of the present invention have searched for homologous enzymes with more than 60% identical amino acid sequences through previous studies and confirmed hydrolysis reactivity using 4-nitrophenyl acetate and 4-nitrophenyl butyrate as reaction substrates (Seongsoon Park, 2015 and Young-Hyun Kim et al., 2017). In the case of homologs with more than 60% amino acid sequence identity, all of them, except for PBL, had serine (S) located at position 47, and it was confirmed that the selectivity was similar to that of CAL-B. Therefore, it is judged that even if the amino acid structurally corresponding to the 47th amino acid of Candida antarctica lipase B (CAL-B) in the homologous enzymes having an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence identical by more than 60% is substituted with Asparagine (N), Glutamine (Q), Leucine (L), Isoleucine (I), Phenylalanine (F), Tyrosine (Y), or Methionine (M), the same effect as the CAL-B variant of the present invention will be exhibited. The homologous enzymes having an amino acid sequence identical by more than 60% are as shown in Table 1 below.

According to an embodiment of the present invention, the variant of Candida antarctica lipase B (CAL-B) of the present invention has an enantioselectivity (E) for the substrate (±)-butan-2-ol that is improved by 2 to 5 times compared to Candida antarctica lipase B, and has an enantioselectivity (E) for the substrate (±)-but-3-yn-2-ol that is improved by 35 times or more compared to Candida antarctica lipase B. Therefore, the use of the mutant of Candida antarctica lipase B (CAL-B) of the present invention has improved stereoselectivity compared to conventional CAL-B, and thus enables efficient production of photochemically pure secondary alcohols used as starting materials for various organic synthetic products.

The present invention provides a polynucleotide encoding a variant of the Candida antarctica lipase B (CAL-B) described above. The polynucleotide is a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, and is a DNA or RNA strand of a certain length or longer, and more specifically, refers to a polynucleotide fragment encoding the variant.

The present invention provides a vector comprising a polynucleotide encoding a variant of the Candida antarctica lipase B (CAL-B) described above. The vector refers to a DNA preparation containing a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable regulatory sequence so as to enable expression of the target polypeptide in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. The vector may be replicated or function independently of the host genome after being transformed into a suitable host cell, and may be integrated into the genome itself. The vector used in the present invention is not particularly limited, and any vector known in the art may be used.

The present invention provides a microorganism producing a variant of Candida antarctica lipase B (CAL-B), comprising at least one of the variants of Candida antarctica lipase B (CAL-B), a polynucleotide thereof, and a vector thereof as described above.

The microorganism producing the above mutant refers to a microorganism in which the ability to produce a CAL-B mutant is imparted to a parent strain that has no or weak ability to produce a CAL-B mutant. Specifically, the microorganism may be a microorganism expressing a mutant of Candida antarctica lipase B (CAL-B) of the present invention. The type of the microorganism producing the CAL-B mutant is not particularly limited as long as it can produce CAL-B or a mutant of CAL-B, but may be a recombinant microorganism widely used in recombinant protein production, such as E. coli, and in case of yeast, specifically, may be Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces , Neurospora crassa , etc.

The present invention provides a method for producing a variant of Candida antarctica lipase B (CAL-B), comprising the steps of: culturing a microorganism producing a variant of Candida antarctica lipase B (CAL-B) in a medium, wherein the microorganism comprises at least one of a variant of Candida antarctica lipase B (CAL-B), a polynucleotide thereof, and a vector comprising the polynucleotide; and recovering the variant of Candida antarctica lipase B (CAL-B) from the cultured medium or microorganism.

The above cultivation means growing the microorganism under appropriately controlled environmental conditions, and can be performed according to appropriate media and cultivation conditions known in the art. The cultivation process can be easily adjusted and used by those skilled in the art according to the selected strain.

The above medium refers to a material containing nutrients necessary for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water essential for survival and growth. The medium can be used to culture microorganisms in a general medium containing an appropriate carbon source, a nitrate source, a phosphorous source, inorganic compounds, amino acids, and/or vitamins, while controlling temperature, pH, etc. under aerobic conditions. The carbon source may include carbohydrates such as glucose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) can be used. As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, and the like; amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, and the like can be used. The phosphorous source can include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and in addition, amino acids, vitamins, and/or appropriate precursors, etc., can be included. During the cultivation of microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium, and an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble production. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain anaerobic and microaerophilic states, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected. The temperature of the medium can be 20°C to 50°C, specifically 30°C to 37°C, and the cultivation period can be continued until the desired amount of useful substances produced is obtained.

The CAL-B variant of the present invention produced by the above-mentioned culture may be released into the medium or may remain in the cell without being released. The method for producing the CAL-B variant may include a step of recovering the CAL-B variant from the cultured microorganism or medium. The method for recovering the CAL-B variant produced in the above-mentioned culturing step of the present invention may utilize a suitable method known in the art depending on the culturing method, and for example, the CAL-B variant may be recovered from the medium or microorganism by using a suitable method known in the art such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC.

Additionally, the recovery step may include a purification process and may be performed using a suitable method known in the art, such as filtration using a membrane.

For reference, the variant of CAL-B produced according to the method of the present invention can be directly used in the reaction as an enzyme itself, but can be manufactured in the form of an immobilized enzyme by physically or chemically immobilizing it on an insoluble carrier (matrix) and using it. The immobilized enzyme has the advantage of being able to be used repeatedly since the reactants and enzymes can be easily separated after the enzymatic reaction is completed, and the stability of the enzyme with respect to reaction conditions can be increased. The technology for immobilizing a lipase enzyme such as the CAL-B of the present invention on a carrier such as an acrylic resin (acrylic resin), a fabric membrane, polypropylene, celite, diatomaceous earth, etc. is widely known, and the method for immobilizing the variant of CAL-B of the present invention and the selection of the carrier are not particularly limited, but it is preferable to use the immobilized enzyme by pulverizing it into a fine powder having a size of nanometers (nm) to micrometers (㎛) in order to increase the enzyme reaction speed and thus the conversion rate in a short period of time.

The present invention is described in detail below through examples.

Example

1. Experimental method

1) Experimental materials

Reagents used in the experiment were purchased from TCI or Thermo Fisher Scientific. Vector (pBADgⅢa) was purchased from Invitrogen Korea (Seoul, Korea). Poly-Prep chromatography column and protein assay solution were purchased from Bio-Rad. Ni-NTA agarose resin was purchased from QIAGEN. CIRCLEGROW® medium was purchased from MO Biomedicals. DNA sequencing was performed by Solgent Co. (Daejeon, Korea), and gas chromatography was performed using an Agilent 6890N equipped with a chiral capillary column (Cyclosil-B 30 m × 0.25 mm).

2) Position-directed mutation

Overlap-extension PCR was performed using the CAL-B gene (5D-CAL-B) as a template and primers for mutation. The primers for mutation are as shown in Table 2 below.

In the above Table 2, the F1 refers to a forward primer and the R1 refers to a backward primer. In addition, the above 5DCALB_T42V refers to a mutation in which threonine (T) at residue 42 of CAL-B is substituted with valine (V), the above 5DCALB_S47N refers to a mutation in which serine (S) at residue 47 of CAL-B is substituted with asparagine (N), the above 5DCALB_T42V/S47N refers to a mutation in which threonine (T) at residue 42 of CAL-B is substituted with valine (V) and serine (S) at residue 47 is substituted with asparagine (N), and the above 5DCALB_42-47 refers to a mutation in which residues 42 to 47 are substituted with Pseudozyma brasiliensis lipase GHG001 (Pseudozyma brasiliensis lipase). brasiliensis lipase, PBL, GHG001) refers to a mutation in which residues 42 to 47 are substituted.

The above mutant gene was synthesized, digested with restriction enzymes NcoI and SalI, and inserted into the pBADgⅢa vector treated with the same restriction enzymes, followed by ligation to produce a plasmid. The constant plasmid was transformed into an E. coli strain (Top 10).

3) Expression and purification of CAL-B and mutant CAL-B

E. coli transformed with the plasmid was cultured overnight at 37°C and 180 rpm in 15 ml of CIRCLEGROW® medium containing Amplcilin (15 μl, 100 mg/ml). The cultured medium was transferred to 500 ml of the CIRCLEGROW® medium and further cultured under the same conditions until OD ₆₀₀ = 0.5. L-(+)-arabinose (500 μl, 2% w/v) was added to the CIRCLEGROW® medium cultured until OD ₆₀₀ = 0.5 to initiate protein expression, and then the medium was further cultured at 20°C for 20 hours. The medium cultured at 20°C was centrifuged at 10,000 rpm, 4°C for 20 minutes to obtain an E. coli cell pellet. The obtained cell pellet was resuspended in BES buffer (5 ml per 1 g of cells, 5 mM, pH 7.2). The resuspended cell pellet was sonicated, then frozen and thawed. Then, syringe pumping was performed to disrupt the cells, and centrifugation was performed at 10,000 rpm and 4°C for 10 minutes to separate the soluble fraction and the insoluble fraction. The soluble fraction was mixed with N-NTA resin for 1 hour and then purified using a Poly-Prep chromatography column. The column was washed three times with dissolution buffer (5 ml, 50 mM NaH ₂ PO ₄ ; 300 mM NaCl; 10 mM imidazole, pH 8.0) and then eluted with elution buffer (5 ml, 50 mM NaH ₂ PO ₄ ; 300 mM NaCl; 250 mM imidazole, pH 8.0). The buffer of the eluted fraction was exchanged with the BES buffer using a centrifugal concentrator, and the purified enzyme protein was confirmed through SDS-PAGE (see Fig. 1).

3) Measuring protein concentration

The concentration of the purified enzyme protein was measured using the Bradford assay. A bovine serum albumin (BSA) standard solution (1 mg/mL) was prepared and then diluted to a range of 0.05 to 0.5 mg/mL. 10 μl of the prepared BSA standard solution or 10 μl of an appropriately diluted enzyme solution was mixed with 200 μl of the assay solution, and the absorbance was measured at 595 nm after 5 minutes to derive a BSA calibration curve and calculate the enzyme concentration.

4) Measurement of hydrolysis activity

Five microliters of enzyme solution was mixed with 100 microliters of assay solution (200 mM p-nitrophenyl acetate or p-nitrophenyl butyrate, 20 μl; acetonitrile, 870 μl; 5 mM BES buffer, 11,110 μl), and the absorbance change was measured at 404 nm at 5 or 7 s intervals.

5) Fixation of Cellite 545

A protein solution (1 mL, 10 mg/mL) containing 60 mg of enzyme protein was mixed with 1 g of Celite 545, shaken for 15 minutes, the suspension was spread onto a weighing dish, covered with perforated aluminum foil, and dried overnight in a fume hood.

6) Measurement of mirror image ratio for ester exchange reaction

18.3 ㎕ of 0.2 mmol butan-2-ol or 15.7 ㎕ of 0.2 mmol but-3-yn-2-ol as a substrate, and 55.4 ㎕ of 0.6 mmol vinyl acetate or 76.2 ㎕ of 0.6 mmol vinyl butyrate as an acyl donor were dissolved in 2 ㎖ of t-butyl methyl ether (MTBE) containing immobilized enzyme protein (CAL-B 400 ㎍, CAL-B-T42V 400 ㎍, CAL-B-S47N 400 ㎍, CAL-B-42-47 400 ㎍, or CAL-B-T42V/S47N 400 ㎍). A reaction mixture was prepared. The reaction mixture was stirred at 25°C and 500 rpm and the reaction was stopped at a level showing about 40% conversion. The enantiomeric ratio was calculated using the method of Chen et al., which uses both the starting alcohol and the ester product in enantiomeric excess. The amounts of the substrate (starting alcohol) and the product (ester product) were analyzed using gas chromatography (see Table 3).

7 ) Analysis of specific activity

The specific activities of CAL-B and CAL-B-S47 were compared and analyzed. To this end, the substrate, the acyl donor (3 equal parts), and the immobilized enzyme were mixed with the MTBE to prepare a reaction mixture, which was stirred and reacted at 500 rpm at 25°C. The reaction mixture was reacted for 160 minutes and recovered in small amounts at 20-minute intervals. Considering that the reaction rates of the enzyme proteins are different, the reaction was performed by increasing the reaction amount or the amount of the enzyme protein. For the measurement of the initial rate of the fast enantiomer, (R)-(+)-but-3-yn-2-ol, vinyl butyrate (3 equal parts), and hexadecane (0.1 equal part) as an internal standard were mixed with MTBE and reacted at 25°C. Gas chromatography was performed under the same conditions as above to measure the stereoselectivity.

8) Results of stereoselectivity measurement using gas chromatography

As the ratio of enantiomers increased in the experiments, the peak differences of the products could be distinguished more clearly through gas chromatography analysis. Figure 2 shows the results of gas chromatography analysis of the transesterification reaction for but-3-yn-2-ol under conditions containing vinyl butyrate. Panel (a) of Figure 2 shows the result for 5D-CAL-B, panel (b) for CAL-B-T42V, panel (c) for CAL-B-S47N, panel (d) for CAL-B-42-47, and panel (e) for CAL-B-T42V/S47N. The retention times for the substrate were 3.2 min for the fast enantiomer and 3.4 min for the slow enantiomer. Vinyl butyrate was detected at 3.7 min. The product was identified in 10.3 min for the fast enantiomer and in 10.8 min for the slow enantiomer.

2. Experimental results and discussion

1) Local sequence comparison analysis of CAL-B and PBL

The present invention shows the improved stereoselectivity for (±)-but-3-yn-2-ol through mutation of CAL-B residues. Through previous studies, the inventors of the present invention have confirmed that Pseudozyma brasiliensis lipase (PBL, GHG001), a homolog of CAL-B, shows lower catalytic activity than the CAL-B of the present invention, but has better stereoselectivities for (±)-butan-2-ol and (±)-but-3-yn-2-ol than CAL-B, with E (enantioselectivity) >200 and E = 17, respectively.

The above PBL has 72% sequence identity with the CAL-B of the present invention. Therefore, the PBL and CAL-B are expected to share common structure, function, and stereoselectivity. Therefore, it is judged that by performing a local sequence comparison analysis of PBL and CAL-B, a clue can be obtained to improve the stereoselectivity of CAL-B for (±)-butan-2-ol and (±)-but-3-in-2.

2) Stereoselectivity of CAL-B

In general, the enantioselectivity of lipase follows the Kazlauskas rule, which can be used to determine the enantioselectivity of lipase toward chiral secondary alcohols. The Kazlauskas rule is based on the structural features of lipases related to the substrate binding pocket, and CAL-B also shows (R)-selectivity according to the rule.

CAL-B has two binding pockets that accommodate two different substituents, a large substituent and a small substituent, on the chiral carbon atom of the secondary alcohol. One of them is the large binding pocket where the substrate binds, and the other is the middle binding pocket located deep inside CAL-B. Kazlauskas rule states that the middle binding pocket accommodates smaller substituents than large substituents. Previous experiments have shown that CAL-B accommodates substituents smaller than a propyl group, which means that CAL-B cannot distinguish the size difference between a methyl group, an ethyl group, or an acetylene group. Therefore, it is thought that analyzing the differences in the amino acid sequences of the middle binding pockets of PBL and CAL-B may explain the difference in stereoselectivity between PBL and CAL-B.

3) Mutant CAL-B

The middle binding pocket of CAL-B is composed of T40, T42, S47, and W104, and the corresponding amino acid residues in PBL are identified as T40, V42, N47, and W104. The W represents tryptophan (W), and the W104 corresponds to the same residue in both enzymes.

The amino acid sequences of the two enzymes were compared for the remaining residues 39 to 48 (see panel a of Fig. 3). As a result, residues 39 (Glycine, G), 40 (T), 41 (G), 44 (G), 46 (Glutetamine, Q), and 48 (Phenylalanine, F) were confirmed to be the same in both enzymes, while residues 42, 43, 45, and 47 were confirmed to be different in the two enzymes. Among the different residues, the side chains of residues 42 and 47 are located toward the active site.

A CAL-B mutant, CAL-B-42-47, was prepared in which residues 42 to 47 of CAL-B are entirely replaced with the corresponding residues of PBL. To this end, genes corresponding to residues 42 to 47 of the PBL were synthesized and introduced into a CAL-B template (original CAL-B without mutation, referred to as 5D-CAL-B hereinafter) to modify the CAL-B gene. The CAL-B-42-47 protein was expressed as a protein from the CAL-B-42-47 mutant gene vector injected into E. coli (Top10) and was optimized to exhibit the same stereoselectivity and expression level as the original CAL-B protein.

4) Stereoselectivity analysis of CAL-B-42-47

In order to analyze the stereoselectivity of the above CAL-B-42-47, the above CAL-B-42-47 was fixed on celite, and then the transesterification reaction of (±)-butan-2-ol and (±)-but-3-yn-2-ol was performed using vinyl acetate or vinyl butyrate as an acyl donor at 25°C. The analysis results showed that the above CAL-B-42-47 showed high stereoselectivity for both substrates, (±)-butan-2-ol and (±)-but-3-yn-2-ol, compared to 5D-CAL-B. The stereoselectivity for (±)-butan-2-ol of the above CAL-B-42-47 was confirmed to be enhanced by 3 to 4 times (E=13 or 26) compared to 5D-CAL-B, respectively, and the stereoselectivity for (±)-but-3-yn-2-ol was confirmed to be enhanced by 40 to 50 times (E>200; >99% ee for (R)-4b and (R)-5b (E=6-7 and 4-5) compared to 5D-CAL-B, respectively) compared to 5D-CAL-B (see reaction scheme and Table 2).

The reaction conditions of the following reaction scheme 1 are as follows: lipase enzyme 20 mg (1%, immobilized on Celite); rac-1a or rac-1b, 0.1 mmol; acyl donor (vinyl acetate 2 or vinyl butanoate 3), 0.3 mmol, MTBE, 1 mL; and 25°C. E ^b in Table 4 below describes the stereoselectivity described in Sih et al., wherein rac-1a means (±)-butan-2-ol, rac-1b means (±)-but-3-yn-2-ol, acyl donor 2 means vinyl acetate, acyl donor 3 means vinyl butanoate, and MTBE means t-butyl methyl ether.

[Reaction Formula 1]

The stereoselectivity of CAL-B-42-47 was confirmed to be similar to the stereoselectivity of PBL toward (±)-butan-2-ol (E = 13 or 27) and the stereoselectivity of PBL toward (±)-but-3-yn-2-ol (E > 200). The above results imply that the stereoselectivity of CAL-B was affected by the change in the stereospecific pocket. In particular, it is noteworthy that the stereoselectivity of CAL-B-42-47 toward (±)-but-3-yn-2-ol was improved to the level of E > 200 by the present invention, which is a dramatic change that could not be achieved until now.

5) Stereoselectivity analysis of CAL-B-T42V or CAL-B-S47N mutations

In the present invention, it was confirmed which residues are essential for the improved stereoselectivity of CAL-B. It was confirmed that the residues of PBL corresponding to T42 and S47 of CAL-B are different as V42 and N47 (see panel a of Fig. 3). Therefore, it was confirmed what effect the difference in the residues has on the improvement of stereoselectivity of the two enzymes.

The side chains of residues 42 and 47 above are confirmed to be directed toward the active site in both enzymes. Therefore, it is judged that the residues above will contribute to forming the stereospecific pocket of the enzyme, and if the residues above are substituted with other residues and the side chains change, the degree of substrate binding is also expected to change. In order to confirm this, in the present invention, a mutant CAL-B-T42V/S47N in which T42 is substituted with V42 and S47 is substituted with N47 was prepared, and the stereoselectivity for (±)-butan-2-ol and (±)-but-3-yn-2-ol was measured using the same method as above (see Table 4).

The measurement results showed that CAL-B-T42V/S47N showed high stereoselectivity similar to CAL-B-42-47. The results imply that T42 and S47 of CAL-B play an important role in stereoselectivity simultaneously or individually.

To further confirm the results above, CAL-B-T42V, in which only T42 was substituted with V42, and CAL-B-S47N, in which only S47 was substituted with N47, were prepared and expressed, and their stereoselectivities were measured according to the above method. The results showed that CAL-B-T42V and CAL-B-S47N showed different stereoselectivities (see Table 3). The stereoselectivities of CAL-B-T42V for (±)-butan-2-ol and (±)-but-3-yn-2-ol were E=7 to 8 and E=3 to 4, respectively, whereas the stereoselectivities of the mutant CAL-B-S47N for (±)-butan-2-ol and (±)-but-3-yn-2-ol were E=14 to 30 and E>200, respectively. The results show that the T42 and S47 residues have a significant effect on the stereoselectivity of CAL-B. In particular, substituting S47 of CAL-B with N was found to enhance the stereoselectivity toward (±)-but-3-yn-2-ol.

The improvement in stereoselectivity of the mutant enzyme can be interpreted as the possibility that the reaction rate for the fast enantiomer became faster and the reaction rate for the slow enantiomer became much slower compared to the original enzyme. This can be confirmed by measuring the kinetic parameters of the two enantiomers for the enzyme. However, the kinetic parameter for the slow enantiomer has a limitation in that it is difficult to measure because of the very slow reaction rate of the slow enantiomer. Accordingly, in the present invention, an analysis was performed by comparing the specific activities for the fast enantiomer and the slow enantiomer (see Table 5). The specific activity reaction can be expressed by the following reaction scheme 2 and was calculated based on the amount of protein used instead of the total amount of immobilized enzyme. The specific activity reaction was as follows: 5D-CAL-B, 10 mg (1%, immobilized on Celite); (R)- or (S)-but-3-yn-2-ol, 0.1 mmol; vinyl butanoate, 0.3 mmol; MTBE (methyl tert-butylether), 4 mL, and the reaction conditions were 25°C. In the case of CAL-B-S47N, CAL-B-S47N, 40 mg (1%, immobilized on Celite); (R)- or (S)-but-3-yn-2-ol, 0.1 mmol; vinyl butanoate, 0.3 mmol, MTBE, 4 mL, and the reaction conditions were 25°C. The above reactions were performed at least three times, and the average values were analyzed, and the standard deviation was used to obtain the error.

The analysis results showed that CAL-B-S47N had lower activity than 5D-CAL-B (see Table 4). Therefore, a larger amount of CAL-B-S47N was used to obtain a reliable reaction rate. The reaction mixture was recovered at time intervals, and the reaction progress was analyzed by gas chromatography (GC).

[Reaction Formula 2]

The experimental results showed that in the case of 5D-CAL-B, the specific activity was 50% lower in the slow enantiomer (S)-but-3-yn-2-ol than in the fast enantiomer (R)-but-3-yn-2-ol, whereas in the case of mutant CAL-B-S47N, the specific activity was 120 times lower in the slow enantiomer (S)-but-3-yn-2-ol than in the fast enantiomer (R)-but-3-yn-2-ol, confirming the difference clearly. The above results show that when S47 is substituted with N47, the reaction toward the slow enantiomer becomes much slower. Therefore, it is judged that the binding of mutant CAL-B-S47N to the slow enantiomer in the transition state is less favorable than the binding of the original CAL-B.

6) Molecular modeling analysis

Through molecular modeling, we investigated how the substitution of residue 47 affects the improved stereoselectivity of CAL-B-S47N. To this end, we created a molecular model by minimizing the energy of the tetrahedral intermediate of CAL-B and mutant CAL-B-S47N for the slow enantiomer. Unfortunately, it was determined that the molecular modeling analysis above could not sufficiently explain the improved stereoselectivity of CAL-B-S47N.

For reference, the energy-minimized structures of CAL-B and CAL-B-S47N were compared and analyzed, and it was confirmed that they are not significantly different except for the distance between the terminal carbon atom of the acetylene group and the O _γ atom of S47 or the O _δ1 atom of N47. Although the distance between the O _δ1 atom of N47 and the terminal carbon of the acetylene group (4.8Å) was confirmed to be shorter than the distance between the O _γ atom of S47 and the terminal carbon atom (5.7Å), both distances were analyzed to be outside the optimal distance for van der Waals interaction. The above results imply that since the side chain of N47 is still far from the slow enantiomer, N47 itself may not significantly affect the binding of the slow enantiomer. However, a possible explanation for the improved stereoselectivity is that water molecules hydrogen bond to the side chain of N47 and hinder the binding of the slow enantiomer in the transition state.

The specific embodiments described in this specification are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

<110> Sungshin R&D Foundation <120> Enantioselectivity Improved Candida antarctica Lipase B Variants <130> MP21-0079 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 317 <212> PRT <213> Candida antarctica <400 > 1 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu 1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Pro Ser Ser Val Ser Lys 20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe 35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Thr Gln Leu Gly Tyr Thr Pro Cys 50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asp Thr Gln Val Asn Thr 65 70 75 80 Glu Tyr Met Val Asn Ala Ile Thr Ala Leu Tyr Ala Gly Ser Gly Asn 85 90 95 Asn Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln 100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu 115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu 130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile 165 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro 180 185 190 Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys 195 200 205 Asn Val Gln Ala Gln Ala Val Cys Gly Pro Leu Phe Val Ile Asp His 210 215 220 Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val 260 265 270 Ala Ala Ala Ala Leu Leu Ala Pro Ala Ala Ala Ala Ile Val Ala Gly 275 280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe 290 295 300 Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315 <210> 2 <211> 317 <212> PRT <213> Unknown <220> <223> Kalmanozyma brasiliensis GHG001, Taxonomy ID: 1365824 (for references in articles please use NCBI:txid1365824) <400> 2 Leu Pro Ser Gly Ser Asp Pro Ala Leu Ser Thr Pro Gln Ala Val Leu 1 5 10 15 Asp Ala Gly Leu Phe Cys Gln Asn Gly Ser Pro Ser Ala Gln Asn Lys 20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Val Asp Gly Arg Gln Asn Phe 35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Thr Met Leu Gly Tyr Ser Pro Cys 50 55 60 Trp Val Ser Pro Pro Pro Phe Met Leu Asn Asp Ser Gln Ile Asn Ala 65 70 75 80 Glu Tyr Val Val Asn Ala Val Arg Arg Leu Tyr Ala Gly Ser Gly Ser 85 90 95 Thr Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Thr Gln 100 105 110 Trp Ala Leu Thr Phe Phe Pro Ser Thr Arg Asn Gln Ile Asp Arg Leu 115 120 125 Val Ala Phe Val Pro Asp Tyr Lys Gly Thr Val Asn Ala Tyr Pro Leu 130 135 140 Thr Ala Thr Asp Thr Ala Ser Pro Ser Ile Trp Gln Gln Arg Ala Gly 145 150 155 160 Ser Ala Phe Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Asn Lys Ile 165 170 175 Val Pro Thr Thr Asn Thr Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro 180 185 190 Gln Ile Thr Asn Ser Pro Ile Asp Ser Ser Phe Leu Phe Asn Ala Lys 195 200 205 Asn Ile Gln Ser Gln Ser Val Cys Gly Pro Ala Phe Val Ala Asp His 210 215 220 Ala Gln Ser Leu Val Asn Gln Phe Ala Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Ser Thr Gly Gln Ala Gln Ser Ser Asp Tyr Ser Ile Ala 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asp Pro Leu Thr Pro Gln Gln Lys Ala 260 265 270 Glu Ser Ser Thr Leu Leu Leu Val Ala Ala Ala Asn Val Ala Ala Gly 275 280 285 Pro Lys Thr Asn Cys Glu Pro Asp Leu Lys Pro Tyr Ala Arg Pro Phe 290 295 300 Ala Pro Gly Ser Gln Thr Cys Ser Gly Phe Thr Thr Phe 305 310 315

Claims (9)

A mutant of Candida antarctica lipase B (CAL-B), characterized in that the 47th amino acid, serine (S), in the amino acid sequence of sequence number 1 is substituted with asparagine (N).
A mutant of Candida antarctica lipase B (CAL-B), characterized in that the 42nd amino acid, threonine (T), in the amino acid sequence of sequence number 1 is substituted with valine (V), and the 47th amino acid, serine (S), is substituted with asparagine (N).
A mutant of Candida antarctica lipase B (CAL-B), characterized in that the 42nd amino acid, threonine (T), the 43rd amino acid, proline (P), and the 47th amino acid, serine (S), in the amino acid sequence of sequence number 1 are substituted with valine (V), aspartic acid (D), arginine (R), and asparagine (N), respectively.
In the third paragraph, the 42nd to 47th amino acid sequence (VDGRQN) of the Candida antarctica lipase B (CAL-B) mutant is characterized in that it is identical to the 42nd to 47th amino acid sequence of Pseudozyma brasiliensis lipase (PBL, GHG001).
A variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4, characterized in that the enantioselectivity (E) for (±)-butan-2-ol is improved by 2 to 5 times compared to Candida antarctica lipase B, and the enantioselectivity (E) for (±)-but-3-yn-2-ol is improved by 35 times or more compared to Candida antarctica lipase B.
A polynucleotide encoding a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4.
A vector comprising a polynucleotide of claim 6.
A microorganism producing a variant of Candida antarctica lipase B (CAL-B), comprising at least one of: a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4; a polynucleotide encoding a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4; and a vector comprising a polynucleotide encoding a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4.
A step of culturing a microorganism producing a variant of Candida antarctica lipase B (CAL-B) in a medium, comprising at least one of: a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4; a polynucleotide encoding a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4; and a vector comprising a polynucleotide encoding a variant of Candida antarctica lipase B (CAL-B) according to any one of claims 1 to 4; and
A method for producing a mutant of Candida antarctica lipase B (CAL-B), comprising a step of recovering a mutant of Candida antarctica lipase B (CAL-B) from a medium in which the microorganism is cultured or from the cultured microorganism.