Enhancing Thermostability Research Articles

Enhancing thermostability of tryptophan hydroxylase via protein engineering and its application in 5-hydroxytryptophan production

5-Hydroxytryptophan (5-HTP), as the precursor of serotonin and melatonin in animals, can regulate mood, sleep, and behavior, which is widely used in pharmaceutical and health products industry. The enzymatic production of 5-hydroxytryptophan (5-HTP) from L-tryptophan (L-Trp) using tryptophan hydroxylase (TPH) show huge potential in application due to its advantages, such as mild reaction conditions, avoidance of protection/deprotection processes, excellent regioselectivity and considerable catalytic efficiency, compared with chemical synthesis and natural extraction. However, the low thermostability of TPH restricted its hydroxylation efficiency toward L-Trp. In this study, we aimed to improve the thermostability of TPH via semi-rational design guided by (folding free energy) ΔΔG fold calculation. After two rounds of evolution, two beneficial mutants M1 (S422V) and M30 (V275L/I412K) were obtained. Thermostability evaluation showed that M1 and M30 possessed 5.66-fold and 6.32-fold half-lives (t1/2) at 37 °C, and 4.2 °C and 6.0 °C higher melting temperature (Tm) than the WT, respectively. The mechanism behind thermostability improvement was elucidated with molecular dynamics simulation. Furthermore, biotransformation of 5-HTP from L-Trp was performed, M1 and M30 displayed 1.80-fold and 2.30-fold than that of WT, respectively. This work provides important insights into the thermostability enhancement of TPH and generate key mutants that could be robust candidates for practical production of 5-HTP.

Enhancing thermostability of alkaline κ-carrageenase from Pseudoalteromonas tetraodonis by rational design of disulfide bonds

The industrial applications of the κ-carrageenases have been restricted by their poor thermostability. In this study, based on the structural information of alkaline κ-carrageenase from Pseudoalteromonas tetraodonis (WT) and with the aid of Disulfide by Design2 computational tool, two disulfide bond mutants of K44C-A119C and T120C-Q250C were identified with improved thermostability and enzymatic activity. K44C-A119C and T120C-Q250C increased the enzyme activity by 19.0% and 7.3%, respectively. After treatment at 50 °C for 30 min, the residual activities of K44C-A119C, T120C-Q250C, and WT were 80.1%, 78.6%, and 45.4%, respectively. The Tm values of K44C-A119C, T120C-Q250C, and WT determined by differential scanning calorimetry were 56.0 °C, 54.8 °C, and 51.2 °C, respectively. The increasement of disulfide bond and its adjacent non-covalent interactions would account for the increased thermostability of the mutants. Molecular dynamics simulation analysis revealed that the improved thermostability and catalytic activities of K44C-A119C and T120C-Q250C could be attributed to the increased flexibility of finger F6 and the less structural deviation in some loops and β-sheets. In addition, the κ-carrageenan hydrolysates by K44C-A119C exhibited higher tyrosinase inhibitory activity than the untreated κ-carrageenan. These findings provide a useful strategy to improve κ-carrageenases’ thermostability and enzyme activity for their better industrial applications.

Intra-Ligand H···F Interactions: Non-negligible Forces for Enhancing Thermostability of Cobalt Complexes in 1,3-Butadiene Polymerization

Intra-Ligand H···F Interactions: Non-negligible Forces for Enhancing Thermostability of Cobalt Complexes in 1,3-Butadiene Polymerization

Enhancing thermostability and activity of sucrose phosphorylase for high-level production of 2-O-α-d-glucosylglycerol

Sucrose phosphorylase (SPase) can transfer the glucosyl group of sucrose to different compounds and has been widely used in industry. To overcome the low thermostability of the sucrose phosphorylase from Leuconostoc mesenteroides ATCC 12291 (LmSP), a method named PROSS was used to construct mutants with increased thermostability. All variants were screened by measuring their residual activities after heating at 50°C. Then, a single point mutant and a combined mutant with improved thermostability and activity were obtained. The half-lives of mutants at 50°C were approximately twice as high as those of the wild type. In addition, 2-O-α-d-glucosylglycerol (αGG) was synthesized by the wild type and the two improved variants, and the reaction conditions were optimized. Under the conditions of glycerol concentration of 3.2 mol/L, sucrose concentration of 1.2 mol/L, and enzyme concentration of 40 U/mL at 37°C for 60 h, the yield of αGG reached the maximum, and the sucrose conversion rate of the wild type, the mutant V23L and the combined mutant V23L/S424R were 62.3%, 70.7% and 76.3%, respectively. In this study, SPase mutants with higher activity and stability were obtained, and achieved high-level production of αGG.

Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.

Enhancing thermostability of a psychrophilic alpha-amylase by the structural energy optimization in the trajectories of molecular dynamics simulations

The cold-adapted alpha-amylase (PHA) from Pseudoalteromonas haloplanktis is a psychrophilic enzyme which demonstrates high activity at low temperatures, but poor thermostability. Most of the method only employed the crystal structure to design the target protein. However, the trajectory of protein molecular dynamics (MD) simulation contained clues about the protein stability. In this study, we combined MD simulation and energy optimization methods to design mutations located at non-conserved residues. Two single point mutants (S255K, S340P) and one integrated mutant (S255K/S340P) enhanced thermostability without affecting the optimal catalytic activity. After incubation at 40 °C for 80 min, the residual activities of mutants S255K, S340P and S255K/S340P were 1.6-, 2.4-, and 2.6-fold greater than that of the wild type (WT). Additionally, the catalytic efficiency values (kcat/Km) of S255K, S340P, and S255K/S340P also increased 1.9-, 2.0-, and 2.7-fold when compared to WT.

Enhancing thermostability by modifying flexible surface loops in an evolved high‐redox potential laccase

AbstractHigh‐redox potential laccases (HRPLs) from white‐rot fungi are versatile biocatalysts whose practical use is highly dependent on their thermostability. In this work, an evolved HRPL variant was subjected to structure‐guided evolution to improve its thermostability. We first selected several surface flexible loops in the laccase structure by inspecting them through molecular dynamics and an analysis of B‐factors. The resulting segments were grouped into three MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo Grouping) blocks, which were constructed in Saccharomyces cerevisiae and explored at high temperatures. This evolution process gave rise to a double mutant that showed a half‐life at 70°C enhanced by 31 min with an optimum temperature for activity of 75°C and similar kinetic parameters. The Ser264Lys and Ser356Asn mutations modified the contacts established between these residues and those that surround them, altering the surface loops and thereby the enzyme properties.

Enhancing thermostability of Yarrowia lipolytica lipase 2 through engineering multiple disulfide bonds and mitigating reduced lipase production associated with disulfide bonds

The limited thermostability of Yarrowia lipolytica lipase 2 (Lip2) hampers its industrial application. To improve its thermostability, we combined single disulfide bonds which our group identified previously. In this study, combining different regional disulfide bonds had greater effect than combining same regional disulfide bonds. Furthermore, mutants with 4, 5, and 6 disulfide bonds exhibited dramatically enhanced thermostability. Compared with the wild-type, sextuple mutant 6s displayed a 22.53 and 31.23 ℃ increase in the melting temperature (Tm) and the half loss temperature at 15 min (T15 50), respectively, with greater pH stability and a wider reaction pH range. Molecular dynamics simulation revealed that multiple disulfide bonds resulted in more rigid structures of mutants 4s, 5s and 6s, and prolonged enzyme unfolding times. Moreover, secretions of mutants 5s and 6s were significantly increased by 60% and 80% by co-expressing with the chaperone protein disulfide isomerase (PDI), which mitigated the reduced production issue caused by multiple disulfide bonds. Results of this study indicated that enhanced heat endurance giving more potential for industrial application.

Enhancing thermostability of iron ethylene polymerization catalysts through N,N,N-chelation of doubly fused α,α′-bis(arylimino)-2,3:5,6-bis(hexamethylene)pyridines

At temperatures up to 80 °C, high catalytic activity and high molecular weight PE was achievable using the Fe-catalyst depicted.

Enhancing thermostability and removing hemin inhibition of Rhodopseudomonas palustris 5-aminolevulinic acid synthase by computer-aided rational design.

To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3°C and 6.0°C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1h incubation at 37°C was obtained for H29R (2.0U/mg) and H15K (3.1U/mg) compared to wild-type (0.3U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10μM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.

Engineering Clostridium absonum 7α-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and ΔΔG Prediction.

Enhancing thermostability of the 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) is beneficial to its industrial application broadly. For protein engineering to enhance thermostability the nonrational strategy, directed evolution, has been applied in obtaining more stable proteins through error-prone PCR or DNA rearrangement generating random mutations. However, the successful application of directed evolution needs to build a large mutant library. Site-directed mutations of CA 7α-HSDH had been performed to probe the relationship between the compactness increasing and thermostability enhancing. Although most of the mutations in β-sheet core predicted by MAESTRO became more stable than wild type, unfortunately, all the mutations suffered dramatic activity loss. The main objective of this study was to verify effects of the mutations in helices selected from the predicting results through MAESTRO on thermostability improving of CA 7α-HSDH. Seven mutants, S22L, P124L, A125L, N171L, A195Q, L197E and Y259E were synthesized and verified through DNA sequencing in Sangon Biotech (Sangon, Shanghai, China). The two mutants, A104P and G105P were prepared by over-lapping PCR. The GST-fusion expression vector, pGEX-6p-1 (GE Healthcare), was used for protein expression with restriction sites BamH I and Not I. Thermostability was measured by circular dichroism (CD) spectrometer (MOS-450, BioLogic Inc). All the enzymes were diluted in PBS (pH 7.3, 10 mM) to OD222 value between 0.8 and 1, and temperature varied from 20°C to 95°C. Activity of enzyme was assayed by measuring the production of NADPH by UV-visible spectrophotometer at 340 nm. The activity assay was performed in 2 mL reaction mixture which contained PBS (pH 7.3, 10 mM), NADP+ (0.5 mM) and taurocholic acid (TCA) at 25°C. Based on unfolding free energy changes (ΔΔG) prediction seven mutations of Clostridium absonum (CA) 7α-HSDH were selected and experimentally verified, and these mutants fitted three-state denaturation model well, among which S22L located in the αA possessed the greatest Tm N→I increase (> 8°C). Mutants P124L, L197E, N171L and Y259E also became more stable than wild type CA 7α-HSDH with different ranges. Meanwhile, thermostability of the two mutants, A104P and G105P (in the coil between βD and αD) resulting from the proline substitution method decreased significantly. Enzyme activity assays indicated that mutant L197E located in αF maximally maintained 28.7% of catalytic efficiency, and activity of the five mutants, P124L, A125L, N171L, A104P and G105P cannot be detected. Although all the mutants' activities decreased, the mutant L197E with the maximum activity retain suggested that the loop structure (residues 194 to 211) may be the favored candidate sites to enhance thermostability. In addition, CA 7α-HSDH may suffer structural destruction resulting from the proline substitution in A104 and G105.

Enhancing thermostability of β-amylase from Bacillus flexus CCTCC 2015368 based on in vitro molecular evolution

We used in vitro molecular evolution technology by error-prone PCR and high-throughput screening to improve thermostability of Bacillus flexus CCTCC 2015368 β-amylase. Mutant D476N with significant thermostability increase was selected by LB agar starch plate colorimetric assay and 96-well plate enzyme activity assay. The optimum pH was 6.5 for the mutant D476N, compared to 7.0 of the wild type. The optimal temperature was 55 ℃ for both mutant D476N and the wild type. The T₅₀ value of the mutant D476N was 4 ℃ higher than that of the wild type. The half-life of mutant D476N at 55 ℃ was 35 min, 95% higher than that of the wild type. The Km of the mutant D476N was 97.98 μmol/L, 1.14 times of that of the wild type (85.86 μmol/L). The thermostability of the mutant D476N was slightly lower than that of the wild type. The three-dimensional structure of wild type and mutant D476N was simulated by SWISS-MODEL and analyzed by PyMol software. The mutated amino acid residue Asn476 was located on the loop of protein surface. The molecular free energy(ΔG) of D476N was calculated by MOE software was 106.0 kcal/mol, reduced by 10.3% compared to the wild enzyme. These results were consistent with the theory that the protein molecular free energy and thermostability were negatively correlated.

The β-Sheet Core is the Favored Candidate of Engineering SDR for Enhancing Thermostability but not for Activity.

7α-Hydroxysteroid dehydrogenases (7α-HSDHs) can stereoselectively catalyze steroids, aromatic α-ketoesters, and benzaldehyde analogues playing a critical role in the biotransformation and poor thermostability that hinders their biomedical and industrial applications. This study was to investigate how to enhance the thermostability of 7α-HSDH from Clostridium absonum (CA 7α-HSDH). Based on the three-dimensional structure of CA 7α-HSDH, recently reported program MAESTRO was used to compute the ΔΔG and predict the single-point mutants that could enhance its thermostability. The selected mutants were verified experimentally. The results from the circular dichroism spectrum indicated that three of the mutants, N89L, N184I, and A185I, fitted a three-state model and the values for Tm N→I and Tm I→D increased with different ranges. In particular, the Tm N→I for the N184I mutant increased maximally by 9.93°C. Meanwhile, the denaturation process of the G189I mutant fitted the two-state model and it was more stable than the wild type, judging from the denaturation curves. Nevertheless, the enzyme catalytic activity analysis suggested that only the N89L mutant held a 2.28% catalytic efficiency, compared to the wild type, CA 7α-HSDH, and the activities of the other three mutants could not be detected. Molecular dynamics (MD) simulations were performed to determine the structural changes that occurred in the mutations and the results indicated that β-sheet structures in the mutants without detectable activity had changed significantly. Judging from the locations of the mutated sites, residues in the β-sheet core were considered as the favored candidates for SDR engineering to enhance the thermostability but not for activity holding.

Single Amino Acid Substitution in the Pullulanase of Klebsiella variicola for Enhancing Thermostability and Catalytic Efficiency.

Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of V max and k cat/K m, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently.

Enhancing thermo-stability to ethylene polymerization: synthesis, characterization and the catalytic behavior of 1-(2,4-dibenzhydryl-6-chlorophenylimino)-2-aryliminoacenaphthylnickel halides

The title complexes exhibited high activities up to 1.09 × 107 g of PE/(mol of Ni per h) in ethylene polymerization with better thermo-stability activated with ethylaluminium sesquichloride.

Computational approach for designing thermostable Candida antarctica lipase B by molecular dynamics simulation

Candida antarctica lipase B (CalB) is one of the most useful enzyme for various reactions and bioconversions. Enhancing thermostability of CalB is required for industrial applications. In this study, we propose a computational design strategy to improve the thermostability of CalB. Molecular dynamics simulations at various temperatures were used to investigate the common fluctuation sites in CalB, which are considered to be thermally weak points. The RosettaDesign algorithm was used to design the selected residues. The redesigned CalB was simulated to verify both the enhancement of intramolecular interactions and the lowering of the overall root-mean-square deviation (RMSD) values. The A251E mutant designed using this strategy showed a 2.5-fold higher thermostability than the wild-type CalB. This strategy could apply to other industry applicable enzymes.

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-β-D-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

Enhancing Thermostability and Organic Solvent Tolerance of ω‐Transaminase through Global Incorporation of Fluorotyrosine

AbstractHere, we have utilized the incorporation of non‐canonical amino acids as a tool kit to improve enzyme properties for organic synthesis applications. The global incorporation of 3‐fluorotyrosine (FY) into ω‐transaminase (ω‐TA) to give ω‐TA[FY] enhanced the thermostability and organic solvent tolerance without altering substrate specificity and enantioselectivity. Moreover, ω‐TA[FY] was able to completely convert 25 mM of acetophenone into (S)‐1‐phenylethylamine (ee>99%) in the presence of 20% DMSO (v/v) which is ∼2‐fold higher when compared to wild‐type ω‐TA.magnified image

Enhancing thermostability of the biocatalysts beyond their natural function via protein engineering

Majority of the naturally occurring enzymes lacks essential features required during the harsh conditions of the industrial processes, because of their less stability. Protein engineering tool offers excellent opportunity to improve the biochemical properties of these biocatalysts. These techniques further help in understanding the structure and function of the proteins. Most common methods employed in protein engineering are directed evolution and rational mutagenesis. Several research groups have utilized these methods for engineering the stability/activity of diverse class of enzymes. In silico tools further plays an important role in designing better experimental strategy to engineer these proteins. The availability of vast majority of data on protein thermostability will enable one to envisage the possible factors that may contribute significantly in maintaining the protein structure and function during various physical conditions. This review discusses the common method employed in protein engineering along with various molecular/computational approaches that are being utilized for altering protein activity, along with important factors associated with these processes. Key words: Computational database, rational, directed evolution, thermostability, hydrophobicity, three dimensional structure, configuration, biocatalysts.

Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface

To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60 degrees C for 5 h and increased synthetic activity in organic solvent at 60 degrees C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.