Candida antarctica lipase B mutant, and methods for making and using the same

Abstract

The present invention relates to the field of bioengineering. It provides a Candida antarctica lipase B mutant and its application. The mutant enzyme overcomes the limit of the parent enzyme that can exhibit high enantioselectivity towards (R)-3-TBDMSO glutaric acid methyl monoester only at temperatures below 5 C. The mutant enzyme successfully increased R-ee value at 5-70 C. The mutant D223V/A281S exhibits high R-ee value (>99%), high conversion rate (80%), and high space-time yield (107.54 g L.sup.1 d.sup.1). The present invention lays a foundation for industrial production of (R)-3-TBDMSO glutaric acid methyl monoester using a biosynthesis approach and provide insights into conformational dynamics-based enzyme design.

Claims

1. A candida antarctica lipase B mutant, wherein said mutant lipase has one of the following amino acid substitutions: D223V, A281S, and D223V/A281S in the parent enzyme of SEQ ID NO:1.

2. The mutant of claim 1, wherein the amino acid sequence of said candida antarctica lipase B mutant is SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.

3. The mutant of claim 1, wherein the nucleotide sequence encoding said mutant lipase is SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

4. A recombinant plasmid vector, wherein said recombinant plasmid vector comprises a nucleotide sequence encoding said mutant lipase of claim 1.

5. A method of producing (R)-3-substituted glutaric acid alkyl monoester compounds, comprising using said mutant lipase of claim 1 as a catalyst in an esterification reaction to make (R)-3-substituted glutaric acid alkyl monoester compounds.

6. The method of claim 5, comprising using said mutant lipase of claim 1 as a catalyst in an esterification reaction to make (R)-3-t-butyl-dimethyl-silyloxy glutaric acid methyl monoester.

7. The method of claim 5, comprising adding said mutant lipase of claim 1 to a substrate, a co-substrate, and an organic solvent to perform a non-aqueous phase esterification reaction, wherein said substrate is 3-substituted glutaric anhydride or 3-substituted glutaric acid and said co-substrate is organic alcohol, and wherein the molar ratio of said substrate to said co-substrate is 1:20-20:1, the mass ratio of said substrate to said mutant lipase is 1:6-6:1, and the molar ratio of said organic solvent to said substrate is 2:1-300:1.

8. The method of claim 7, further comprising adding 0.5% v/v metal ion solution to said esterification reaction, wherein said metal ion solution is MgCl.sub.2, CaCl.sub.2 or KCl.

9. The method of claim 8, wherein concentration of said mutant lipase of claim 1 is 1 to 100 g/L and concentration of said substrate is 10 to 300 g/L, and wherein said esterification reaction is performed at 5-70 C., 200-500 rpm for 2-48 hours.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. The activity pocket of CALB.

(2) FIG. 2. Comparison of the conformational changes between R/S enantiomer-bound CALB EF5 and D223V/A281S mutant. Conformations of the activity pockets are shown in (A) R-enantiomer bound EF5; (B) R-enantiomer bound D223V/A281S mutant; (C) S-enantiomer bound EF5; and (D) S-enantiomer-bound D223V/A281S mutant.

(3) FIG. 3. HPLC analysis of the esterification catalyzed by CALB EF5 and D223V/A281S at 30 C., T.sub.R=6.6 min, Ts=6.9 min. (A) Racemic 3-TBDMSO glutaric acid methyl monoester; (B) EF5-catalyzed esterification (8% R-ee, 82.17% yield); (C) D223V/A281S-catalyzed esterification (>99% R-ee, 80.25% yield).

EXAMPLES

(4) The invention is further illustrated in more detail with reference to the accompanying examples. It is noted that, the following embodiments are only intended for purposes of illustration and are not intended to limit the scope of the invention.

(5) Materials and Methods:

(6) Gene source: the CALB gene was derived from Pseudozyma antarctia JCM 3941, which was purchased from Japan Collection of Microorganisms (JCM). The CALB mutants were obtained by molecular modification, and other chemicals and solvents (analytical grade) were obtained from local suppliers.

(7) The analysis of conformational dynamics of CALB: performing MD simulations and calculating the RMSF of -carbons to analyze changes of the conformational dynamics. The MD simulations were performed with the GROMACS 4.5.5 and the AMBER03 force field following three main steps of energy minimization, system equilibration and production protocols.

(8) The analysis of enzyme structure and its interaction with substrate: structural analysis was performed with Pymol. Molecular docking was performed with Autodock.

(9) Determination of R-ee value and conversion rate: the concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselectivity were determined by HPLC. The mobile phase consisted of 96% hexane and 4% iso-propanol with 0.02% (v/v) trifluoroacetic acid, filtered through a 0.22 m membrane before use. Analysis was performed by injecting a 20 L sample into the chromatograph, with detection temperature of 25 C. and 1 mL/min flow rate; sample detection time was 15 min. The R-ee value was defined as follows: R-ee=(RS)/(R+S)*100%, wherein R and S represent the concentrations of R and S enantiomer, respectively.

Example 1: Selection of Mutation Sites

(10) Structural analysis: CALB EF possesses the catalytic triad Asp187-His224-Ser105 which lay between the two binding pockets (the acyl binding pocket and the alcohol binding pocket). The acyl binding pocket is mainly composed of A141, L144, V149, D134, T138 and Q157, and the alcohol binding pocket is mainly composed of T42, K47, W104, L278, A281 and A282. Residues A281, A282, and 1285 point towards alcohol moiety of substrates and limit the size of the channel.

(11) Selection of mutation sites: six residues (D134, A148, V149, 1189, V190 and Q157) on acyl binding pocket and five residues (T42, T43, W104, A281 and A282) on alcohol binding pocket and the entrance of the channel were selected. Besides, D223 and T186, which are in front of the catalytic residues His224 and Asp187, were also selected. The residues are shown in FIG. 1.

Example 2: The Effects of Candidate Residues on Enantioselectivity

(12) Mutant libraries of residues D134, A148, V149, 1189, V190, Q157, T42, T43, W104, A281, A282, D223 and T186, which were chosen based on the structural analysis, were constructed, and the effects of the mutants on the enantioselectivity were examined through high throughput screening. Nine combination libraries were constructed, including library 1 (A148/V149), library 2 (I189/V190), library 3 (Q157), library 4 (T42/T43), library 5 (W104), library 6 (A281/A282), library 7 (D223), library 8 (T186), library 9 (D134). Out of the 7000 mutants that were screened, only D223V, A281S and D223V/A281S mutants exhibited significant change in the R-ee value as compared to that of the parent enzyme (CALB EF5). In addition, A282S, W104A and Q157N mutants were also selected further experimental evaluation.

Example 3: Construction of CALB Mutants

(13) Six mutants, D223V, A281S, A282S, W104A, Q157N and D223V/A281S, were successfully constructed by site directed mutagenesis using PCR. The PCR primers used for site-directed mutagenesis were shown in SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14. The DNA template is a plasmid comprising a CALB EF5 parent gene. 1 L of Dpn I (10 U L.sup.1) was added to 25 L of the PCR reaction mixture and incubated 3 hr at 37 C. to eliminate the template plasmid. The digested PCR product was inserted into pGAOZA plasmid and transformed into Escherichia coli JM109 for plasmid amplification. The plasmid pGAPZA-mutants were obtained from E. coli JM109 and linearized by AvrII, and were then purified and transformed into P. pastoris GS115. Recombinant P. pastoris GS115 were inoculated into yeast extract peptone dextrose (YPD) medium (10 g L.sup.1 of yeast extract, 20 g L.sup.1 of peptone, and 20 g L.sup.1 of glucose) and grown at 30 C. on a rotary shaker (200 rpm) for 2 days.

Example 4: Measurement of the Initial Generation Rates of R/S Enantiomers in Esterification Reactions Catalyzed by CALB Mutants

(14) For the esterification reaction, 1.23 mM 3-TBDMSO glutaric anhydride and 1.23 mM methanol were dissolved in acetonitrile (5 mL), followed by ultrasonic dispersion. Enzyme (400 mg) was then added to the reaction system. The mixed system was incubated at 30 C., 200 rpm. At appropriate times, samples were collected and analyzed by high-performance liquid chromatography (HPLC). Several data points were collected to determine the initial generation rate of each enantiomer. The activities of the immobilized mutant enzymes were maintained at the same level.

(15) As illustrated in Table 1, the V.sub.S values of the mutants A281S, D223V, and D223V/A281S significantly decreased from 79.993.18 in the parent CALB-EF5 to 3.140.04, 2.000.01, and 0.460.01 mol h.sup.1, respectively. However, compared with that of the parent CALB-EF5, the V.sub.R values of the mutant lipases only decreased slightly (Table 1). As a result, the values of V.sub.R/V.sub.S increased from 1.17 (the parent enzyme) to 29.78 (A281S), 46.71 (D223V), and 200.11 (D223V/A281S). Overall, it indicated that the decreases in the dynamics of the pocket and channel resulting from mutations at sites 223 and 281 led to a sharp decline in the initial generation rate of the S-enantiomer (V.sub.S) and thereby increased R-enantioselectivity at 30 C. The mutant A282S exhibited similar initial generation rate in comparison to that of the parent enzyme.

(16) TABLE-US-00001 TABLE 1 The formation rate of R and S enantiomers catalyzed by CALB mutants at 30 C. Initial formation rate [mol h.sup.1] Mutants V.sub.R V.sub.S V.sub.R/V.sub.S EF5 93.90 4.35 79.99 3.18 1.17 A282S 93.85 5.54 78.98 5.31 1.19 A281S 93.52 8.02 3.14 0.04 29.78 D223V 93.42 2.35 2.00 0.01 46.71 D223V/A281S 92.05 4.82 0.46 0.01 200.11 W104A 93.75 5.35 75.23 5.94 1.25 Q157N 94.00 6.54 93.85 5.54 1.00

Example 5: Measurement of Kinetic Parameters of the CALB Mutants

(17) The kinetic parameters of the R/S-enantiomers, including K.sub.m and k.sub.cat, were calculated by measuring the initial rates of product formation at different concentrations of R/S-enantiomers (1-20 mM) at 30 C. Samples were withdrawn, extracted, and analyzed by HPLC. All assays were carried out at least three times. The data were plotted, and K.sub.m and k.sub.cat values were obtained by the double reciprocal method.

(18) The kinetic parameters of the CALB mutants were determined with optically pure R- and S-enantiomers as substrates, and the results were listed in Table 2. For the mutant D223V/A281S, the k.sub.cat, K.sub.m, and k.sub.cat/K.sub.m, values towards the R-enantiomer were 5.6% higher, 34% lower, and 58.2% higher, respectively, than those of the parent EF5 enzyme. The k.sub.cat, K.sub.m, and k.sub.cat/K.sub.m values towards the S-enantiomer exhibited a 16.2% decrease, 88.1-fold increase, and 100-fold decrease, respectively, compared with the corresponding values in the parent EF5 enzyme.

(19) TABLE-US-00002 TABLE 2 Kinetic parameters of CALB mutants R-enantiomer S-enantiomer k.sub.cat/K.sub.m k.sub.cat/K.sub.m k.sub.cat K.sub.m [mM.sup.1 k.sub.cat K.sub.m [mM.sup.1 Mutant [s.sup.1] [mM] s.sup.1] Fold [s.sup.1] [mM] s.sup.1] Fold EF5 4.96 0.60 8.29 1.00 4.59 0.64 7.12 1.00 A282S 4.94 0.61 8.10 0.98 4.60 0.65 7.11 1.00 A281S 5.02 0.55 9.12 1.10 4.52 15.40 0.30 0.04 D223V 5.13 0.42 12.21 1.47 4.01 15.42 0.26 0.04 D223V/ 5.24 0.40 13.10 1.58 3.95 56.43 0.07 0.01 A281S W104A 5.01 0.55 9.10 1.10 4.58 0.60 7.63 1.07 Q157N 4.98 0.58 8.59 1.04 4.61 0.52 8.87 1.25

Example 6: Determination of R-Ee Value and Conversion Rate of the CALB Mutants

(20) The esterification reaction was carried out by immobilized EF5 or new mutants (80 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 12 hr at 37 C. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselectivity were determined by HPLC.

(21) The R-ee of A281S/D223V was the highest, reaching 99%. The R-ee of A281S and D223V were 93.5% and 95.8%, respectively. The enantioselectivity of other mutants were shown in the Table 3 below.

(22) TABLE-US-00003 TABLE 3 Production of R-enantiomers by CALB mutants Temperature Time R-ee Conv. space-time yield Mutants [ C.] [h] [%] [%] [g .Math. L.sup.1 .Math. d.sup.1] EF5 5 60 98.50 70.49 18.89 EF5 37 12 8 82.17 110.11 A282S 37 12 8.32 82.10 110.01 A281S 37 12 93.50 81.23 108.85 D223V 37 12 95.80 79.75 106.87 D223V/ 37 12 >99 80.25 107.54 A281S W104A 37 12 10% 78.33 104.96 Q157N 37 12 1% 76.25 102.18

Example 7: Effects of Metal Ions on Enantioselectivity

(23) The esterification reaction was carried out by immobilized EF5 or new mutants (80 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 12 hr at 30 C. 0.5% v/v metal ion solution (2.4 M, MgCl.sub.2, CaCl.sub.2 LiCl, NaCl, BaCl.sub.2 or KCl) was added to the system. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselecitivity were determined by HPLC. The results indicated that MgCl.sub.2 showed the greatest effect on R-ee, increased to 99%, and CaCl.sub.2 and KCl showed slight effect, while LiCl, NaCl and BaCl.sub.2 showed no effect.

Example 8: The Preparation of (R)-3-Substituted Glutaric Acid Monoesters

(24) The esterification reaction was carried out by immobilized EF5 or new mutants (60 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 12 hr at 37 C. 0.5% v/v metal ion solutions (MgCl.sub.2 2.4 M) were added to the system. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselecitivity were determined by HPLC. The results indicated that the R-ee of A281S, D223V and A281S/D223V mutants reached 99%.

Example 9: The Preparation of (R)-3-Substituted Glutaric Acid Monoesters

(25) The reaction was carried out by immobilized EF5 and new mutants (60 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 30 hr at 20 C. 0.5% v/v metal ion solutions (MgCl.sub.2 2.4 M) were added to the system. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselecitivity were determined by HPLC. The results indicated that the R-ee of A281S, D223V and A281S/D223V mutants reached 99%.

Example 10: The Preparation of (R)-3-Substituted Glutaric Acid Monoesters

(26) The reaction was carried out by immobilized EF5 and new mutants (60 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 8 hr at 55 C. 0.5% v/v metal ion solutions (MgCl.sub.2 2.4 M) were added to the system. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselecitivity were determined by HPLC. The results indicated that the R-ee of A281S, D223V and A281S/D223V mutants reached 98.5%.

Example 11: The Preparation of (R)-3-Substituted Glutaric Acid Monoesters

(27) The reaction was carried out by immobilized EF5 and new mutants (60 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 2 hr at 70 C. 0.5% v/v metal ion solutions (MgCl.sub.2 2.4 M) were added to the system. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselecitivity were determined by HPLC. The results indicated that the R-ee of A281S, D223V and A281S/D223V mutants reached 98%.

Example 12: The Preparation of (R)-3-Substituted Glutaric Acid Monoesters

(28) The reaction was carried out by immobilized EF5 and new mutants (80 g/L) in acetonitrile, containing 60 g/L 3-TBDMSO glutaric anhydride with methanol for 48 hr at 5 C. 0.5% v/v metal ion solutions (MgCl.sub.2 2.4 M) were added to the system. The activity of immobilized mutant enzymes was maintained at the same level. The concentrations of (R)-3-TBDMSO glutaric acid methyl monoester and enantioselecitivity were determined by HPLC. The results indicated that the R-ee of A281S, D223V and A281S/D223V mutants reached 99%.

(29) The above preferred embodiments are described for illustration only, and are not intended to limit the scope of the invention. It should be understood, for a person skilled in the art, that various improvements or variations can be made therein without departing from the spirit and scope of the invention, and these improvements or variations should be covered within the protecting scope of the invention.