Abstract
Background: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. So, improving the tolerance of enzymes in organic solvents is essential.
Main methods and results: Herein, the method of regional random mutation combined with combinatorial mutation was used to improve the resistance of transaminase from Aspergillus terreus(At ATA) in organic solvents, and the best mutant T23I/T200K/P260S (M3) was acquired. In different concentrations (ranging from 25% to 45%, v/v ) of dimethyl sulfoxide (DMSO), the catalytic efficiency (k cat/K m) toward 1-acetylnaphthalene and the stability (half-lifet 1/2) were higher than the wild-type (WT) ofAt ATA. M3 also showed enhanced stability against six organic solvents with different oil-water partition coefficients (log P values). The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Intra- and intermolecular interaction analysis indicated that the increased hydrogen bonds and hydrophobic interactions within monomers or at the interface of two monomers also strengthened the stability of the overall structure against organic solvents. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R )-(+)-1(1-naphthyl)-ethylamine ((R )-NEA), which was a resolving agent for producing L-menthol by resolution of monomenthyl phthalate, and an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, 3~10 mM 1-acetylnaphthalene can be converted to (R )-NEA with 94.2~38.9% yield and a strictR -stereoselectivity (enantiomeric excess (e.e. ) value >99.5%) within 10 h under 25% DMSO, which was higher than WT and expected to be a potential biocatalyst for industrial application.
Conclusion: The beneficial mutation sites were identified to tailor At ATA’s organic solvents stability via regional random mutation. The “best” mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R )-NEA.
Keywords: transaminase, organic solvent stability, regional random mutation, MD simulations, (R )-(+)-1(1-naphthyl)-ethylamine
1. Introduction
(R )-1-(1-Naphthyl)-ethylamine ((R )-NEA) is a critical chiral aromatic amine compound, which is widely used in pharmaceutical industry, chemical industry, materials and other fields.[1] For example, (R )-NEA can be used as a resolving agent for acid/ester enantiomers by exploiting the weakly basic chemical properties of amino group.[2] Dudas et al. reported (R )-NEA was a resolving agent for producing L-menthol by resolution of monomenthyl phthalate.[2] Morever, (R )-NEA can be used to prepare the intermediate of Cinacalcet Hydrochloride from 1-acetylnaphthalene, which is a kind of pharmaceutical for the treatment of secondary hyperthyroidism and hypercalcemia.[3]The synthesis of (R )-NEA via biocatalysts, such as amine oxidases, imine reductase, amine dehydrogenases, and transaminases,[4] has attracted extensive attention of chemists due to its advantages of high selectivity, mild reaction conditions, and environmental friendliness.[5] The ω-transaminases are capable of catalyzing the asymmetric amination from prochiral ketones to chiral amines with strict stereoselectivity and 100% theoretical yield,[6] indicating that they are promising biocatalysts for the production of chiral amines. However, due to the low solubility of organic substrates in the aqueous phase, it is difficult for enzyme to convert substrates towards the desired product.
Biocatalysis in organic solvents is high-efficient for converting organic substrates. Organic solvents can dissolve more nonpolar substrates[7] and restrain microbial growth and pollution.[8] Biocatalysis in organic phase is helpful for the separation and purification of products due to the low boiling points of organic solvents.[9] Benefiting from these advantages, biocatalysis in organic solvents has been employed to produce various high value-added products.[10] However, the stability of enzymes is poor, and the activity is decreased or even inactivated in high concentration organic solvents. Therefore, improving the enzyme resistance and clarifying the mechanism of enzyme stability in organic solvents are needed.[7]
The method for improving the stability of enzymes in organic solvents can be achieved by rational (semi-rational) design and random mutation.[11,12] Tian et al. reported a semi-rational method for improving methanol tolerance ofThermomyces lanuginosus lipase,[13] which aimed for the high B-factor residues[14] to perform iterative saturation mutagenesis (ISM). In the directed evolution campaign of metalloprotease PT121 via random mutation, eleven variants with enhanced stability against 25% (v/v ) acetonitrile were obtained.[15] The beneficial “wing‐type gate” for improving organic solvents resistance of ω-ATA fromArthrobacter cumminsii ZJUT212 was identified via a semi‐rational design and two beneficial variants were obtained.[16] Strengthening protein surface hydration via surface charge engineering combined with molecular dynamics (MD) simulation is an efficient rational strategy for tailoring enzyme stability in organic solvents.[17] Meng et al. obtained two optimal ω-transaminase mutants from Pseudomonas jessenii with improved activity and high concentrations of isopropylamine and co-solvent tolerance by computational interface design.[18] Generally, the design principle is still a considerable challenge for constructing a “small and smart” mutation library. Combining the advantages of rational design and random mutation will be helpful in obtaining more positive mutants quickly and efficiently.[19,20]
In our previous work, wild type (WT) of amine transaminase fromAspergillus terreus (At ATA) has been cloned and expressed in Escherichia coli , and At ATA showed excellent catalytic efficiency and high R -enantioselectivity toward various ketones.[21,22] Herein, the substrate 1-acetylnaphthalene could be catalyzed to produce (R )-NEA byAt ATA. However, engineering of At ATA to develop a robust biocatalyst for green biomanufacturing of (R )-NEA is in huge demand due to the poor stability in organic solvents. In this study, we identified the critical hot spots by a strategy of regional random mutation, which analyzed the structure of At ATA, performed the error-prone PCR (epPCR) and combinatorial mutation, to evolve the organic solvents resistance of At ATA. For the “best” variant, its enzymatic properties towards 1-acetylnaphthalene in different organic solvents were performed in detail. Moreover, the underlying molecular mechanism was clarified by docking analysis and MD simulations.
2 Materials and methods
2.1 Materials
E. coli BL21(DE3) used for cloning and recombinant protein expression was purchased from TransGen Biotech Co. Ltd. (Beijing, China). The strain E. coli BL21(DE3)/pET28a(+)-At ATA was constructed in previous work and preserved in our laboratory.[22] Prime STAR® Max DNA polymerase was purchased from Takara Biotechnology (Dalian, China). The restriction enzyme Dpn I was purchased from Thermo Scientific (Shanghai, China). All polymerase chain reaction (PCR) primers were synthesized by Tsingke Biological Technology (Hangzhou, China) and listed in Table S1. Other chemicals were of analytical grade and obtained from standard commercial sources.
2.2 Methods
2.2.1 Selection of organic solvent
According to the log P values, seven organic solvents containing acetonitrile (ACN), acetone (AC), methanol (MeOH), dimethyl sulfoxide (DMSO), N,N -dimethylformamide (DMF), isopropanol (IPA), and ethanol (EtOH) were selected for co-solvent of At ATA in this study. The reaction mixture contained 1-(R )-phenylethylamine (1-(R )-PEA) (5 mM), 1-acetylnaphthalene (5 mM), PLP (0.1 mM), the purified enzyme ofAt ATA-WT, and different organic solvents (the concentration ranging from 5~50%, v/v ). The reactions were performed at 30 °C, 500 rpm for 30 minutes and the product (R )-NEA was analyzed by high performance liquid chromatography (HPLC). One unit of enzyme activity (U) was defined as the amount of enzyme required for 1 μmol of (R )-NEA formed per minute under optimum conditions. The specific activity was expressed as the units of activity per gram purified enzyme (U/g). All experiments were conducted in triplicates.
2.2.2 Mutagenesis and screening
Three regions of amino acid sequence fragments were selected to construct mutagenesis libraries via in situ epPCR based on WT as template. MnCl2 (0.1 mM) was added to the PCR mixture to control the mutagenesis rate (1 to 2 mutation sites per gene). The PCR product was digested by Eco R I and Xho I, and ligated to the vector pET-28a. The PCR reaction conditions were supplemented in Table S2 to Table S5, and the template was digested by Dpn I at 37 °C for 0.5 h (refer to Table S6 for details). The PCR products were transformed into E. coli BL21 (DE3) competent cells for enzyme expression.
Colonies were picked up in 96 deep-well plates containing 1 mL of LB medium with 50 μg/mL kanamycin and cultured for 8 h at 37 °C. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to 96 deep-well plates with a final concentration of 0.1 mM to induce protein expression. After incubation for 20 h at 25oC, the cells were harvested by centrifugation for 15 min at 3900 × g. The cells in 96 deep-well plates were resuspended in 250 μL PBS buffer (100 mM, pH 8.0) with 5 mg/mL lysozyme at 37 °C for 30 min. The crude enzyme solution was centrifuged for 30 min at 3500 × g. Next, 50 μL crude enzyme solution and 50 μL of 80% (v/v ) DMSO was added to a new 96-well plate and incubated at 30 °C for 30 min.
A chromogenic reaction-based screening method was developed. In this section, 10 mM 1-(R )-PEA, 40% (v/v ) DMSO, 25 mM 4-nitrophenylethylamine, 50 mM sodium phosphate (pH 8.0), 0.1 mM PLP, and enzymes incubating by 40% (v/v ) DMSO, were added to 96-well plate at 30 °C, 600 rpm for 30 min.[23] The absorbance values of the reaction solution were measured in 96-well microtiter plates at 500 nm using SpectraMax 190 Microplate Reader (Molecular Devices, USA), and Optical Density at 500 nm (OD500) was calculated.
2.2.3 Protein expression and purification
All enzymes that we constructed were expressed and purified as described in our previous work.[24] The mutants and WT with His6-Tag were purified by nickel affinity chromatography and analyzed by SDS-PAGE.
2.2.4 Determination of kinetic parameters and half-lives in different concentration DMSO
The purified At ATAs were incubated in different concentrations of DMSO (the concentration ranging from 15~45%,v/v ) for 15 min, and the temperature was maintained at 30 °C. Then, enzyme activity assay was performed. Each sample of 1 mL mixture was centrifuged at 13800 ×g for 2 min, and the supernatant was subjected to microfiltration with 0.22 μm PTFE organic membranes. The product and remaining substrate were analyzed by HPLC to measure the 1-acetylnaphthalene conversion, (R )-NEA yield, and enantiomeric excess (e.e .). All the assays were conducted in triplicate.
The half-lives (t 1/2) of At ATAs were determined by incubating each purified enzyme (1 mg/mL) for appropriate times in different concentrations of DMSO (25%, 35%, and 45%,v/v ) at 30 °C, respectively, followed by measuring the residual activity. The half-lives were calculated according to the first order deactivation equation 1 and 2:
ln(A/A0 )=-k dt(equation 1)
t 1/2=ln2/k d (equation 2)
where A 0 is the initial activity, A is the residual activity at time t during the thermal deactivation,k d is the deactivation rate constant (min-1)
2.2.5 Enzyme kinetics characterization under different concentrations of DMSO
The reaction solution was added to DMSO (the final concentration of DMSO to 25%, 35%, or 45%, v/v ). The initial rates were measured at various concentrations of 1-acetylnaphthalene in a range of from 0.1 to 15 mM with a fixed 1-(R )-PEA concentration (5 mM) and PLP (0.1 mM). For 1-(R )-PEA, 1-acetylnaphthalene (5 mM) and 1-(R )-PEA in the range of 0.1 to 1.5 mM were used for the enzyme kinetic assays. The kinetic parameters were obtained by nonlinear fitting the experimental data to Michaelis-Menten equation.[25,26]
2.2.6 MD simulation
The starting model was generated from the crystallography structure (PDB ID: 4CE5).[22] The models of the variants were generated by FoldX software. MD simulations of At ATAs were performed via software YASARA at 308 K for 20 ns under AMBER14 force field.[24] A cubic box was constructed that filled with 15~45% (v/v) DMSO. The simulation system was constructed with 15818~36056 water molecules, the number of which varied depending on the DMSO concentration. Counterions Na+ and Cl were used to neutralize the total net charge of the systems, and all resulting systems had a net charge of zero. During the simulation, coordinates, energies, and velocities were stored per 0.5 ns for further analysis.
2.2.7 Asymmetric synthesis of (R )-NEA by WT and mutant M3
The bio-asymmetric ammoniation 1-acetylnaphthalene to produce (R )-NEA was performed in a 20-mL scale reaction including 6.0 gdry cell weight/L recombinant E.coli whole cells expressing WT or M3, 3~20 mM 1-(R )-PEA, 3~20 mM 1-acetylnaphthalene, and 25% (v/v ) DMSO at 30 °C, 600 rpm. The yield and e.e . value of (R )-NEA were assayed via HPLC.
2.2.8 Analytical methods
The e.e. value of the product was determined after derivatization with Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alaninamide, FDAA) as follows: 50 μL of the reaction solution was mixed with 100 μL of 1% (m/v ) FDAA solution in acetone and 20 μL of NaHCO3 solution. After incubation at 40 °C for 2 h, 20 μL of 2 M HCl was added to terminate the reaction. The samples were extracted with 3 times volume of dichloromethane, and evaporated at room temperature. Then, samples were dissolved in 50% (v/v ) acetonitrile aqueous solution for HPLC analysis. An HPLC 1220 Infinity II system (Agilent Technologies) with an EC-C18 column (4.6 × 150 mm, 4 μm) was used at 30 °C. Detector wavelength was set at 340 nm. The mobile phase was composed of acetonitrile and ultra-pure water at a volumetric ratio of 60: 40 (v/v ), and ran at a flow rate of 1 mL/min for 8 min.
The contents of 1-acetylnaphthalene and (R )-NEA were quantitatively analyzed by using an HPLC 1220 Infinity II system (Agilent Technologies) with a C-18 column (4.6 × 150 mm, 4 μm). Detector wavelength was set at 210 nm. The mobile phase was composed of 0.15% ethanolamine, acetonitrile and ultra-pure water at a volumetric ratio of 1 : 39 : 60 (v/v/v ), and ran at a flow rate of 1.0 mL/min. The column temperature was maintained at 30 °C. Each measurement was conducted at least 3 times with a standard deviation of less than 5%.
3. Results and discussion
3.1 Determination of enzyme activity under different organic solvents
ACN, AC, MeOH, DMSO, DMF, IPA, and EtOH are the representatively and commonly used organic co-solvents with different log P values. The enzyme activities of At ATA in different concentrations of organic solvents were shown in Figure 1. All seven kinds of organic solvents had good solubility for 1-acetylnaphthalene (5 mM), and the activity ofAt ATA in DMSO was higher than other six organic solvents. Enzyme in 25% (v/v ) DMSO displayed the highest activity, which the specific activity reached 8.86 U/g and the activity showed a bell-shape trend with the DMSO concentration increasing. Instead, the enzyme activities were decreased when the concentrations of other six organic solvents increased. Many reports indicated that DMSO was extensively used in the fields of organic synthesis and biocatalysis due to its favorable properties (amphiphilicity, dissolving ability, low chemical reactivity).[27,28] In the following experiments, DMSO was selected as the organic solvent in this study.