AN OMEGA-TRANSAMINASE MUTANT BASED ON ANCESTRAL SEQUENCE RECONSTRUCTION

Abstract

The present invention discloses an -transaminase mutant based on ancestral sequence reconstruction and relates to the technical field of molecular biology. The amino acid sequence of said -transaminase mutant derived from mutation of -transaminase from Aspergillus terreus, is as shown in SEQ ID NO. 4 or SEQ ID NO. 6. Compared with the wild-type enzyme, the half-lives of the -transaminase mutants are all above 24 h, while the half-life of the wild-type is only 6.90 min. The half-inactivation temperature of the mutants are 49.00 C. and 49.03 C., respectively, which are about 11 C. higher than that of the wild-type (37.89 C.), such that the thermal stability is significantly improved.

Claims

1. An -transaminase mutant based on ancestral sequence reconstruction, wherein the -transaminase mutant is derived from mutation of -transaminase from Aspergillus terreus, and the -transaminase mutant has the amino acid sequence as shown in SEQ ID NO. 4 or SEQ ID NO. 6.

2. A method for catalyzing generation of acetophenone from (R)-(+)--methylbenzylamine comprising the step of utilizing the -transaminase mutant as claimed in claim 1.

3. A gene encoding the -transaminase mutant as claimed in claim 1.

4. The gene as claimed in claim 3, wherein the -transaminase mutant has a gene sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 5.

5. A method for catalyzing generation of acetophenone from (R)-(+)--methylbenzylamine comprising the step of utilizing the gene as claimed in claim 3.

6. A recombinant expression plasmid, comprising the gene as claimed in claim 3.

7. A genetically engineered bacterium, comprising the recombinant expression plasmid as claimed in claim 6.

8. A method for in catalyzing generation of acetophenone from (R)-(+)--methylbenzylamine comprising the step of utilizing the genetically engineered bacterium as claimed in claim 7.

9. A method for catalyzing generation of acetophenone from (R)-(+)--methylbenzylamine, wherein acetophenone is generated by a transamination reaction, using (R)-(+)--methylbenzylamine and pyruvic acid as substrates and catalyzed by the -transaminase mutant as claimed in claim 1.

10. A method for catalyzing generation of acetophenone from (R)-(+)--methylbenzylamine, wherein acetophenone is generated by a transamination reaction, using (R)-(+)--methylbenzylamine and pyruvic acid as substrates and catalyzed by the genetically engineered bacterium as claimed in claim 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is the electrophoresis analysis result of SDS-PAGE of wild-type and mutants; in which, each lane is M: protein marker; 1: wild-type enzyme solution (unpurified); 2: wild-type enzyme solution (purified); 3: mutant Ancata-101 enzyme solution (purified); 4: mutant Ancata-124 enzyme solution (purified), respectively.

[0026] FIG. 2 is the result of enzyme activity assay for wild-type -transaminase and -transaminase mutant enzymes; in which, a is specific enzyme activity and b is relative enzyme activity.

[0027] FIG. 3 is the result of stability assay of wild-type -transaminase and -transaminase mutant enzymes; in which, a is T.sub.50.sup.10 between wild-type and Ancata-101 and Ancata-124; b is t.sub.1/2 of wild-type, Ancata-101 and Ancata-124 at 40 C., 45 C. and 47 C.

[0028] FIG. 4 is the result of whole-cell catalytic production of 1-(R)-naphthylethylamine by wild-type -transaminase and -transaminase mutant enzymes.

DESCRIPTION OF THE EMBODIMENTS

Example 1

[0029] The wild-type -transaminase from Aspergillus terreus has an amino acid sequence as shown in SEQ ID NO. 2, and has a gene sequence as shown in SEQ ID NO. 1.

[0030] The protein sequence of Aspergillus terreus -transaminase was uploaded to Fire ProtASR (https://loschmidt.chemi.muni.cz/fireprotasr/, a server for fully automated ancestral sequence reconstruction), and by the fully automated analysis through this website a phylogenetic tree of Aspergillus terreus -transaminase was obtained, then respective nodes on the branch of the phylogenetic tree of evolving to Aspergillus terreus -transaminase were selected, the gene sequences corresponding to these nodes were downloaded from this website, and then the corresponding post-mutation gene sequences were obtained by full gene synthesis. After enzyme expression, purification and thermal stability assay, two mutants with significantly improved thermal stability were finally obtained, named Ancata-101 and Ancata-124, whose mutation position and sequence are as follows:

Ancata-101:

[0031] D5E-A12Q-I17V-S20A-T21S-E22A-T23S-A42H-I77L-T78S-T85S-L87M-R90K-D96E-Q 97E-E104D-T130S-R131K-D134E-I135L-138insN-V143I-D153E-V157T-V162I-V163I-A174S-I175M-V188T-A195S-H210N-Q236E-N245D-A246V-E248R-F250N-F258V-R266Q-T284S-M 288K-G292D-Q294K-I295V-A313P-N322E-E323S-R324A-N325S-325insKKSG, Gene sequence as shown in SEQ ID NO. 3, amino acid sequence as shown in SEQ ID NO. 4;

Ancata-124:

[0032] D5E-A12Q-I17V-S20A-T21S-E22A-T23S-A42H-I77L-T78S-T85A-R90K-D96E-Q97E-E 104D-T130S-R130K-I135L-138insN-V143I-D153E-M154V-V157T-V162I-V163I-A195S-H210 N-Q236E-N245D-A246V-E248R-F250N-F258V-L263M-R266Q-T284S-M288K-G292D-Q294 K-I295V-A313P-N322E-E323S-R324A-N325S-325insKS, Gene sequence as shown in SEQ ID NO. 5, amino acid sequence as shown in SEQ ID NO. 6;

Example 2

(1) Materials and Reagents

[0033] Full genes of (R)--TA and the mutants were synthesized by GENERAL Biosystems (Anhui) Corporation Limited, the vector was pET-28a(+), and the expression host strain was E. coli BL21(DE3); isopropyl--D-thiogalactoside (IPTG), kanamycin sulfate, pyrodoxal-5-phosphate (PLP), and modified Bradford protein concentration assay kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.; protein marker and Ni-NTA chromatography medium were purchased from TransGen Biotech Co., Ltd.; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit was purchased from Beijing CoWin Biotech Co., Ltd.; dimethyl sulfoxide (DMSO), pyruvic acid and (R)--methylbenzylamine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

(2) Enzyme Expression and Purification

[0034] 10 L of wild-type recombinant plasmid bacteria broth and mutant bacteria broth were inoculated into 5 mL of Luria-Bertani liquid medium (LB medium) containing a final concentration of 50 g/mL Kanamycin and incubated for 12 hours at 37 C. and 200 rpm in a shaker. The bacterial broth was transferred to 200 mL of LB liquid medium containing a final concentration of 50 g/mL Kanamycin at 2% inoculum (v/v) and cultured for another 2-3 hours at 37 C. and 200 rpm. When OD.sub.600 reached 0.8, IPTG was added at a final concentration of 0.5 mM and protein expression was induced at 25 C. and 150 rpm. After 20 hours of induction, the bacteria were collected by centrifugation at 6000 rpm, 4 C.

[0035] Bacterial cells were washed once with 50 mM PBS buffer (50 mM sodium dihydrogen phosphate, 50 mM disodium hydrogen phosphate, 300 mM sodium chloride, pH 8.0) to remove residual culture medium and resuspended in the above PBS buffer. The bacterial cells were disrupted by homogenizer under ice bath conditions. The cell disrupting solution was centrifuged at 8000 rpm, 4 C. for 1 hour. The supernatant obtained was collected as crude enzyme solution containing -transaminase. Subsequently, the crude enzyme solution was filtered through a 0.45 m filter membrane and the target protein was purified using a Ni-NTA affinity chromatography column.

[0036] Purification buffers are as follows: [0037] 20 mM imidazole washing buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0; [0038] 50 mM imidazole washing buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 50 mM imidazole, pH 8.0; [0039] 250 mM imidazole eluting buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0.

[0040] Specific purification steps: [0041] 1) Equilibrating the Ni-NTA affinity chromatography column: washing the column for 3 column volumes with 20% (v/v) ethanol aqueous solution, deionized water and 20 mM imidazole washing buffer in sequence; [0042] 2) Sample loading: the crude enzyme solution was taken by syringe and filtered through 0.45 m filter membrane, and the target protein with a tag of 6 histidine could bind to the packing material. [0043] 3) Washing: washing the column with the 20 mM imidazole washing buffer and 50 mM imidazole washing buffer for 3 column volumes each, and using Bradford's solution to test if the protein impurities were washed out; [0044] 4) Elution: rinsing the column with the 250 mM imidazole eluting buffer and collecting 5 mL filtrate. [0045] 5) Preserving the column: washing the column with 20 mM imidazole washing buffer, deionized water and 20% (v/v) ethanol aqueous solution in sequence for 3 column volumes each, and finally preserving the column in 20% (v/v) ethanol aqueous solution.

(3) Determination of Protein Content.

[0046] A modified Bradford protein concentration determination kit was used to establish a protein content standard curve to determine the concentration of the pure enzyme obtained from step (2) in Example 2, and the preparation steps of the protein standard curve were carried out with reference to the instructions. The molecular weight and purity of the purified protein were identified by SDS-PAGE method. Specific steps were as follows:

[0047] Preparation of the gel: 12% separation gel, 5% concentration gel. The formulation is as shown in Table 1.

TABLE-US-00001 TABLE 1 Formulation of separation gel and concentration gel for SDS-PAGE protein electrophoresis 12% separation 5% concentration mass fraction gel gel Volume (mL) 5.00 2.00 Double distilled water (mL) 1.70 0.67 30% Acr-Bis (29:1) (mL) 2.00 0.33 Separation/concentration gel buffer 1.25 1.00 (mL) 10% Ammonium persulfate (mL) 0.05 0.02 TEMED (mL) 0.003 0.002 Note: Acr-Bis: Acrylamide-Bisacrylamide; TEMED: N,N,N,N-Tetramethylethylenediamine.

[0048] Sample processing: 40 L of enzyme solution and 10 L of 5 protein spiking buffer were mixed and kept in boiling water bath for 10 min.

[0049] Loading: protein marker 10 L, sample 15 L.

[0050] Electrophoresis conditions: electrophoresis runs at 120 V for about 90 min; stop the electrophoresis when bromophenol blue indicator is moved to about 1 cm from the lower edge of the gel.

[0051] Staining: The gel was covered by the staining solution, heated in microwave oven for 1 min and stained in shaker for 25 min.

[0052] Decolorization: The staining solution was recovered and replaced with decolorization solution, which was changed every hour until the protein bands were clear.

[0053] Protein content determination: The target protein was diluted to the linear range of the BSA standard curve, and the diluted protein concentration was obtained by measuring the A595 value by a microplate reader.

[0054] The SDS-PAGE electrophoresis profiles of the wild type and mutants are shown in FIG. 1. The electrophoretic bands of wild type and mutants were located at the same position and were consistent with the theoretical molecular weight of 36.1 kDa, which laid the foundation for subsequent experiments.

(4) Determination of Enzyme Activity

1) Determination of Enzyme Activity

[0055] 20 L of pure enzyme was reacted with 180 L of substrate solution (10 mM PLP, 2.5 mM (R)--MBA ((R)-(+)--methylbenzylamine), 2.5 mM pyruvic acid, 0.25% DMSO, 50 mM PBS, pH 8.0) for 3 min at 25 C., and the production of OD.sub.245 acetophenone was determined by reference to the literature (Rapid and sensitive kinetic assay for characterization of -transaminases. Anal Chem, 2009, 81: 8244-8248). Enzyme activity (U) was defined as the amount of enzyme required for transaminase to catalyze transamination reaction of the substrate pyruvic acid and (R)--MBA to produce 1 moL of acetophenone per minute under certain conditions.

[0056] The enzyme activities of the wild type and two mutants are shown in FIG. 2(a). Compared with the enzyme activities of the wild type -transaminase, the enzyme activities of the two mutants were significantly higher, being 1.67 and 1.46 times of that of the wild type, respectively.

2) Determination of Residual Enzyme Activity

[0057] The purified wild-type and mutants were incubated at 40 C. for 10 min, and immediately cooled on ice for 10 min after incubation. Then, 20 L of the heat-treated enzyme solution was reacted with 180 L substrate solution (10 mM PLP, 2.5 mM (R)--MBA, 2.5 mM pyruvic acid, 0.25% DMSO, 50 mM PBS, pH 8.0) at 25 C. for 3 min. The residual activity of the wild type and mutants was determined. The experiments were performed in triplicates, and the enzyme activity of the wild type without incubation at 40 C. was set as 100%, and the mutants with higher relative enzyme activity than the wild type were screened. The residual activity of the wild type and the two mutants after heat treatment at 40 C. for 10 min is shown in FIG. 2(b). The activity of mutants Ancata-101 and Ancata-124 was basically undiminished, while that of the wild type decreased by about 60%.

[0058] In sum, using the ancestral sequence reconstruction method, two mutants with significantly enhanced thermal stability were screened. Ancata-101 (D5E-A12Q-I17V-S20A-T21S-E22A-T23S-A42H-I77L-T78S-T85S-L87M-R90K-D96E-Q97 E-E104D-T130S-R131K-D134E-I135L-138insN-V143I-D153E-V157T-V162I-V163I-A174S-I175M-V188T-A195S-H210N-Q236E-N245D-A246V-E248R-F250N-F258V-R266Q-T284S-M288K-G292D-Q294K-I295V-A313P-N322E-E323S-R324A-N325S-325insKKSG, Gene sequence as shown in SEQ ID NO. 3, amino acid sequence as shown in SEQ ID NO. 4);

Ancata-124

[0059] (D5E-A12Q-I17V-S20A-T21S-E22A-T23S-A42H-I77L-T78S-T85A-R90K-D96E-Q97E-E104 D-T130S-R130K-I135L-138insN-V143I-D153E-M154V-V157T-V162I-V163I-A195S-H210N-Q236E-N245D-A246V-E248R-F250N-F258V-L263M-R266Q-T284S-M288K-G292D-Q294K-I 295V-A313P-N322E-E323S-R324A-N325S-325insKS, Gene sequence as shown in SEQ ID NO. 5, amino acid sequence as shown in SEQ ID NO. 6.

(5) Determination of Enzyme Kinetic Parameters

[0060] Different concentrations of 0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM (R)--MBA and pyruvic acid substrate solutions were respectively prepared with PBS buffer (50 mM, pH 8.0) containing 0.01 mM PLP. The enzyme activity of wild type and mutant -transaminase at different concentrations was determined by enzyme activity assay. The reaction rates V for different substrates as well as different substrate concentrations [S] were brought into the Michaelis-Menten equation V=V.sub.max[S]/(K.sub.m+[S]), nonlinear curve fit was performed using Origin 8.0 software, and the enzyme kinetic parameters K.sub.m and V.sub.max were calculated for wild type and mutants; the conversion number k.sub.cat and catalytic efficiency k.sub.cat/K.sub.m were calculated for wild type and mutants from the equation k.sub.cat=V.sub.max/[E], where [E] was the molar concentration of the enzyme. The results are shown in Table 2. The enhancement of conversion number of pyruvic acid by the two mutant enzymes was lower than the enhancement of affinity, and the catalytic efficiency was lower than that of the wild type for pyruvic acid. The K.sub.m.sup.-MBA values of both mutant enzymes Ancata-101 and Ancata-124 were 0.28 mM, which were slightly higher than that of the wild type, but the k.sub.cat.sup.-MBA value of Ancata-101 was 1.67 times higher than that of the wild type, while the k.sub.cat.sup.-MBA value of Ancata-124 was slightly lower than that of the wild type. k.sub.cat/K.sub.m.sup.-MBA was calculated for Ancata-101 and Ancata-124, which were 3.81 s.sup.1.Math.mM.sup.1 and 2.07 s.sup.1.Math.mM.sup.1, respectively, and the catalytic efficiency of Ancata-101 for -MBA was 1.35 times of the wild type, which was slightly enhanced, while the catalytic efficiency of Ancata-124 was lower than that of the wild type. Taken together, the catalytic efficiencies of the two mutants were not significantly enhanced.

TABLE-US-00002 TABLE 2 Kinetic parameters of wild type and mutants Name WT-AT Ancata-101 Ancata-124 k.sub.cat.sup.pyruvate(s.sup.1) 0.50 0.01 1.50 0.03 0.74 0.03 K.sub.m.sup.pyruvate(mM) 0.23 0.02 1.15 0.02 0.93 0.01 k.sub.cat/K.sub.m.sup.pyruvate(L/(s .Math. mmol)) 2.22 1.30 0.79 k.sub.cat.sup.-MBA(s.sup.1) 0.64 0.01 1.07 0.02 0.60 0.01 K.sub.m.sup.-MBA(mM) 0.23 0.03 0.28 0.03 0.28 0.01 k.sub.cat/K.sub.m.sup.-MBA(L/(s .Math. mmol)) 2.82 3.81 2.07

(6) Determination of Thermal Stability.

1) Determination of T.sub.50.sup.10

[0061] T.sub.50.sup.10 is the temperature when the residual enzyme activity is reduced to 50% after incubation of the pure enzyme at 4-60C for 10 min. The purified wild enzyme and its mutants were incubated at 4 C., 25 C., 30 C., 35 C., 40 C., 45 C., 47 C., 49 C., 50 C. and 55 C. for 10 min, then placed on ice to cool down for 10 min immediately after incubation, then the residual activity of the wild type and its mutants were determined. The T.sub.50.sup.10 of the wild type and the mutants were calculated by using the temperature as the horizontal coordinate and the ratio of enzyme activity between heat-treated enzyme and untreated enzyme as the vertical coordinate and plotting using Origin 8.0 software.

2) Determination of t.sub.1/2

[0062] t.sub.1/2 is the time when the residual enzyme activity decreases to 50% after incubation of the pure enzyme at 40 C. for different times. The purified wild type and its mutants were incubated at 40 C. for 0-24 hours. After incubation, the wild type and its mutants were immediately cooled on ice for 10 min, and the residual activity of the wild type and its mutants were measured. t.sub.1/2 of the wild type and the mutants at 40 C. were calculated using time as horizontal coordinate and the ratio of enzyme activity between heat-treated enzyme and untreated enzyme as the vertical coordinate and plotting using Origin 8.0 software.

TABLE-US-00003 TABLE 3 Stability parameters of wild type and mutants Name WT-AT Ancata-101 Ancata-124 T.sub.50.sup.10 ( C.) 37.89 0.5 49.00 0.4 49.03 0.5 Increased temperature of 11.11 11.14 T.sub.50.sup.10 ( C.) t.sub.1/2 (min) 6.90 0.6 >1440 >1440 Increase fold of t.sub.1/2 207 207

[0063] The stability measurements of mutants Ancata-101 and Ancata-124 are shown in FIG. 3 and Table 3. The T.sub.50.sup.10 of the wild type was 37.89 C., and the T.sub.50.sup.10 of mutants Ancata-101 and Ancata-124 were 49.00 C. and 49.03 C., respectively, which were 11.11 C. and 11.14 C. higher than that of the wild type, respectively. t.sub.1/2 of mutants Ancata-101 and Ancata-124 were both greater than 24 hours (1440 min), while the wild-type t.sub.1/2 was only 6.90 min. At 45 C., the residual activity of the wild-type enzyme decreased to 50% after incubation for less than one minute, while the residual activity of the two mutants was still greater than 50% after over 6 h of incubation.

(7) Determination of Substrate Spectrum

[0064] The catalytic reaction was performed with (R)--MBA as amine group donor and benzaldehyde, acetophenone, 4-fluoroacetophenone, 4-chloroacetophenone, 4-bromoacetophenone, 4-methoxyacetophenone, 4-methylacetophenone, 4-trifluoromethylacetophenone, 4-nitroacetophenone, 1-acetylnaphthalene, 2-acetylnaphthalene, -tetralone as amino group acceptors. 2 mL reaction system comprised 10 mmol/L (R)--MBA, 10 mmol/L each ketone substrate, 0.1 mmol/L PLP, 50 mmol/L PBS (pH 8.0), and 0.15 mg/mL pure enzyme solution. The amount of each product produced was determined by HPLC after 24 h of reaction at 30 C. and 40 C. and 180 r/min, and yield and e.e. values of the product were calculated.

[0065] HPLC detection method: The reaction solution was filtered through a 0.22 m filter membrane and then detected by HPLC on Agilent InfinityLab Poroshell 120 EC-C18 column (4.6150 mm, 4.0 m) with mobile phase being acetonitrile:water=40:60 (v/v) at a flow rate of 1.0 mL/min. The UV detection wavelength was 210 nm.

[0066] To verify the catalytic performance of the ancestral enzymes Ancata-101 and Ancata-124 for different substrates, the enzyme-catalyzed reactions of WT, Ancata-101 and Ancata-124 were carried out at 10 mmol/L of each substrate concentration, and the results are shown in Tables 4 and 5. After 24 hours of reaction, except for benzylamine generated from benzaldehyde, Ancata-101 and Ancata-124 showed elevated yields compared to WT for the remaining nine substrates catalyzed, and all the products produced were in strict R-configuration (e.e. >99.5%) except for benzaldehyde which produced the corresponding product. In contrast to WT, which catalyzed various substrates (except benzaldehyde and 4-nitroacetophenone) in lower yields at 40 C. than at 30 C., both ancestral enzymes obtained higher catalytic yields at 40 C. than at 30 C. Overall the ancestral enzymes Ancata-101 and Ancata-124 showed better catalytic activity for aromatic ketone substrates than WT.

TABLE-US-00004 TABLE 4 Parameters of substrate profiles of wild type and mutants at 30 C. WT Ancata-101 Ancata-124 Substrate Yield (%) Yield (%) Yield (%) Benzaldehyde 69.4 57.8 61.6 4-Fluoroacetophenone 20.6 4.2 4.4 4-Chloroacetophenone 30.8 30.1 26.6 4-Bromoacetophenone 29.6 29.4 29.6 4-Methylacetophenone 15.5 2.6 7.2 4-Methoxyacetophenone 6.1 6.8 1.7 4-Trifluoromethylacetophenone 36.0 30.8 33.8 4-Nitroacetophenone 42.9 45.9 42.5 1-Acetyl Naphthalene 38.5 42.2 42.6 2-Acetyl Naphthalene 24.5 24.1 26.0

TABLE-US-00005 TABLE 5 Parameters of substrate profiles of wild type and mutants at 40 C. WT Ancata-101 Ancata-124 Substrate Yield (%) Yield (%) Yield (%) Benzaldehyde 79.1 77.3 73.5 4-Fluoroacetophenone 13.9 28.4 29.0 4-Chloroacetophenone 16.0 37.5 34.9 4-Bromoacetophenone 16.4 33.7 32.6 4-Methylacetophenone 5.7 22.9 24.3 4-Methoxyacetophenone 2.9 8.3 12.3 4-Trifluoromethylacetophenone 32.6 39.9 38.4 4-Nitroacetophenone 47.7 52.2 36.3 1-Acetyl Naphthalene 16.0 44.5 44.6 2-Acetyl Naphthalene 22.0 28.2 29.5

(8) Whole-Cell Catalytic System Construction

[0067] The catalytic reaction was carried out using 1-(R)-phenylethylamine as amine group donor and 1-acetylnaphthalene as amino group acceptor. 10 mL of the reaction system comprised 10 mmol/L 1-(R)-phenylethylamine, 10 mmol/L each ketone substrate, 0.1 mmol/L PLP, 50 mmol/L PBS (pH 8.0), and 10 g/L -transaminase wet bacteria. After reaction for 15 hours at 40 C. and 180 r/min, the amount of each product produced was determined by HPLC and the yield and e.e. values of the products were calculated.

[0068] HPLC detection method: The reaction solution was filtered through a 0.22 m filter membrane and then detected by HPLC on Agilent InfinityLab Poroshell 120 EC-C18 column (4.6150 mm, 4.0 m) with mobile phase being acetonitrile:water=40:60 (v/v) at a flow rate of 1.0 mL/min. The UV detection wavelength was 210 nm.

[0069] As shown in the whole-cell catalytic results in FIG. 4, the transaminase mutants Ancata-101 and Ancata-124 could catalyze 1-acetylnaphthalene at 40 C. for more than 15 hours, which was significantly longer than the wild-type catalyzing time of 1 hour. Among them, the ancestral enzyme Ancata-101 was able to achieve more than 60% conversion at a catalytic temperature of 40 C. Overall, whole-cell catalytic experiments could show that the ancestor enzymes with greatly improved thermal stability could greatly extend the catalytic time and increase the catalytic efficiency.

[0070] Applicant hereby electronically submits the amended Sequence Listing in XML (CRF format) with the file name of USSN18287879_SEQ_LIST.XML, created on Sep. 6, 2024 and with the size of 10,229 bytes. The replacement Sequence Listing XML does not include new matter.