Enhancing the imidase activity of BpIH toward 3-isobutyl glutarimide via semi-rational design.

(R)-3-Isobutylglutarate monoamide (R-IBM) is a key intermediate in the synthesis of the analgesic drug pregabalin. Recently, the imidase BpIH derived from Burkholderia phytofirmans was identified as a promising catalyst for the industrial production of R-IBM. Notably, this catalyst has the distinct advantage of achieving a 100% theoretical yield from 3-isobutyl glutarimide (IBI). In this study, homology modeling and structure alignment techniques were used to determine the substrate binding pocket of BpIH. Semi-rational design was used to analyze the amino acid residue conservation in the binding pocket region of BpIH. Interestingly, mutations of several low-conserved amino acid located 6-9 Å from the substrate significantly enhanced the catalytic activity of BpIH. Among them, the triple mutant Y37FH133NS226I (YHS-I) showed approximately a fivefold increase in enzyme activity and a significantly improved catalytic efficiency (kcat/Km). Under the same reaction time and conditions, YHS-I successfully converted IBI into R-IBM with a conversion rate of 88.87%, with an enantiomeric excess (ee) of the product exceeding 99.9%. In comparison, wild-type BpIH had a conversion rate of only 38.15%. Molecular dynamics and docking results indicated that YHS-I had higher rigidity around the mutation sites. The synergistic substitutions of Y37F, H133N, and S226I altered the interaction network within the mutation site, enhancing the protein's affinity for the substrate and improving catalytic efficiency. KEY POINTS: • 100% theoretical yield of R-IBM by BpIH compared with 50% by resolution • Semi-rational design of BpIH based on conservativity with homologous enzymes • Mutant with enzyme activity of sixfold and product ee value of 99.9.

Channel Engineering of a Glutamate Exporter.

Mechanosensitive channel MscCG2 is involved in glutamate excretion in most C. glutamicum strains. Improving the excretion efficiency of MscCG2 is beneficial to the production of glutamate. In this study, structure-based rational design was carried out to obtain an improved efflux ability of exporter MscCG2 and its mechanistic advance via two strategies: widening the channel entrance for smoother entry of glutamate and reducing the electronegativity at the entrance of the channels to minimize the rejection of negatively charged glutamate entry. The designed variants were found to enhance glutamate excretion by 2 to 3.3-fold in the early phase and 1.1-fold to 1.5-fold in the late phase of fermentation. The enhanced glutamate excretion was further confirmed by using glutamate toxic analog 4-fluoroglutamate (4-FG) and Glu-Glu peptide uptake and glutamate export assay. Molecular dynamic (MD) simulations revealed that the amino acid substitutions indeed enlarged the channel entrance and reduced the repulsion of glutamate when entering the channel. The finding of this study is important for understanding the underlying structure-function relationship and the mechanism of glutamate secretion to improve glutamate efflux efficiency of glutamate exporter.

Enhancing the thermal stability and activity of zearalenone lactone hydrolase to promote zearalenone degradation via semi-rational design.

Zearalenone (ZEN) is a fungal toxin produced by Fusarium exospore, which poses a significant threat to both animal and human health due to its reproductive toxicity. Removing ZEN through ZEN lactonase is currently the most effective method reported, however, all published ZEN lactonases suffer from the poor thermal stability, losing almost all activity after 10 min of treatment at 55℃. In this study, we heterologously expressed ZHD11A from Phialophora macrospora and engineered it via semi-rational design. A mutant I160Y-G242S that can retain about 40 % residual activity at 55℃ for 10 min was obtained, which is the most heat-tolerant ZEN hydrolase reported to date. Moreover, the specific activity of the I160Y-G242S was also elevated 2-fold compared to ZHD11A from 220 U/mg to 450 U/mg, which is one of the most active ZEN lactonses reported. Dynamics analysis revealed that the decreased flexibility of the main-chain carbons contributes to increased thermal stability and the improved substrate binding affinity and catalytic turnover contribute to enhanced activity of variant I160Y-G242S. In all, the mutant I160Y-G242S is an excellent candidate for the industrial application of ZEN degradation.

Semi-rational engineering of ω-transaminase for enhanced enzymatic activity to 2-ketobutyrate.

Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus (ppTA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type ppTA (WT). Furthermore, the mutant enzyme ppTA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines.

Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

Thermostability and activity improvement in l-threonine aldolase through targeted mutations in V-shaped subunit.

l-threonine aldolase (LTA) catalyzes the synthesis of ß-hydroxy-α-amino acids, which are important chiral intermediates widely used in the fields of pharmaceuticals and pesticides. However, the limited thermostability of LTA hinders its industrial application. Furthermore, the trade-off between thermostability and activity presents a challenge in the thermostability engineering of this enzyme. This study proposes a strategy to regulate the rigidity of LTA's V-shaped subunit by modifying its opening and hinge regions, distant from the active center, aiming to mitigate the trade-off. With LTA from Bacillus nealsonii as targeted enzyme, a total of 25 residues in these two regions were investigated by directed evolution. Finally, mutant G85A/M207L/A12C was obtained, showing significantly enhanced thermostability with a 20 °C increase in T5060 to 66 °C, and specific activity elevated by 34 % at the optimum temperature. Molecular dynamics simulations showed that the newly formed hydrophobicity and hydrogen bonds improved the thermostability and boosted proton transfer efficiency. This work enhances the thermostability of LTA while preventing the loss of activity. It opens new avenues for the thermostability engineering of other industrially relevant enzymes with active center located at the interface of subunits or domains.

Switching engineered Vitreoscilla hemoglobin into carbene transferase for enantioselective SH insertion.

An attractive strategy for efficiently forming CS bonds is through the use of diazo compounds SH insertion. However, achieving good enantioselective control in this reaction within a biocatalytic system has proven to be challenging. This study aimed to enhance the activity and enantioselectivity of to enable asymmetric SH insertion. The researchers conducted site-saturation mutagenesis (SSM) on 5 amino acid residues located around the iron carbenoid intermediate within a distance of 5 Å, followed by iterative saturation mutagenesis (ISM) of beneficial mutants. Through this process, the beneficial variant VHbSH(P54R/V98W) was identified through screening with 4-(methylmercapto) phenol as the substrate. This variant exhibited up to 4-fold higher catalytic efficiency and 6-fold higher enantioselectivity compared to the wild-type VHb. Computational studies were also conducted to elucidate the detailed mechanism of this asymmetric SH insertion, explaining how active-site residues accelerate this transformation and provide stereocontrol.

Semi-rational design based on the interaction between SmFMO and FAD isoalloxazine ring to enhance the enzyme activity.

Flavin monooxygenases (FMOs) have been widely used in the biosynthesis of natural compounds due to their excellent stereoselectivity, regioselectivity and chemoselectivity. Stenotrophomonas maltophilia flavin monooxygenase (SmFMO) has been reported to catalyze the oxidation of various thiols to corresponding sulfoxides, but its activity is relatively low. Herein, we obtained a mutant SmFMOF52G which showed 4.35-fold increase in kcat/Km (4.96 mM-1s-1) and 6.84-fold increase in enzyme activity (81.76 U/g) compared to the SmFMOWT (1.14 mM-1s-1 and 11.95 U/g) through semi-rational design guided by structural analysis and catalytic mechanism combined with high-throughput screening. By forming hydrogen bond with O4 atom of FAD isoalloxazine ring and reducing steric hindrance, the conformation of FAD isoalloxazine ring in SmFMOF52G is more stable, and NADPH and substrate are closer to FAD isoalloxazine ring, shortening the distances of hydrogen transfer and substrate oxygenation, thereby increasing the rate of reduction and oxidation reactions and enhancing enzyme activity. Additionally, the overall structural stability and substrate binding capacity of the SmFMOF52G have significant improved than that of SmFMOWT. The strategy used in this study to improve the enzyme activity of FMOs may have generality, providing important references for the rational and semi-rational engineering of FMOs.

Engineering UDP-Glycosyltransferase UGTPg29 for the Efficient Synthesis of Ginsenoside Rg3 from Protopanaxadiol.

Rare ginsenosides Rg3 and Rh2, which exhibit diverse pharmacological effects, are derivatives of protopanaxadiol (PPD). UDP-glycosyltransferases, such as the M315F variant of Bs-YjiC (Bs-YjiCm) from Bacillus subtilis and UGTPg29 from Panax ginseng, can efficiently convert PPD into Rh2 and Rh2 into Rg3, respectively. In the present study, the N178I mutation of Bs-YjiCm was introduced, resulting in an increase in Rh2 production. UDP-glycosyltransferase UGTPg29 was then engineered to improve its robustness through semi-rational design. The variant R91M/D184M/A287V/A342L, which indicated desirable stability and activity, was utilized in coupling with the N178I variant of Bs-YjiCm and sucrose synthase AtSuSy from Arabidopsis thaliana to set up a "one-pot" three-enzyme reaction for the biosynthesis of Rg3. The influential factors, including the ratio and concentration of UDP-glycosyltransferases, pH, and the concentrations of UDP, sucrose, and DMSO, were optimized. On this basis, a fed-batch strategy was adopted to achieve a Rg3 yield as high as 12.38 mM (9.72 g/L) with a final yield of 68.78% within 24 h. This work may provide promising UDP-glycosyltransferase candidates for ginsenoside biosynthesis.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays.

The thermal instability of γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays has imposed limitations on its industrial applications, affecting both stability and activity at reaction temperatures. In this study, disulfide bridges were introduced through a combination of directed evolution and rational design to enhance GMAS stability. Among the variants that we generated, M12 exhibited a 1.46-fold improvement in relative enzyme activity and a 6.23-fold increase in half-life at 40℃ compared to the wild-type GMAS. Employing variant M12 under optimal conditions, we achieved the production of 645.7 mM (112.49 g/L) L-theanine with a productivity of 29.3 mM/h, from 800 mM substrate in an ATP regeneration system. Our strategy significantly enhances the biosynthesis efficiency of L-theanine by preserving the structural stability of the enzyme during the catalysis process.

Efficient production of a highly active lysozyme from European flat oyster Ostrea edulis.

Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZdm) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZdm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 103 U mL-1 to 2.11 × 104 U mL-1 in shake flask culture, and eventually reaching 2.05 × 105 U mL-1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.

Semi-rational design of nitroarene dioxygenase for catalytic ability toward 2,4-dichloronitrobenzene.

Rieske non-heme dioxygenase family enzymes play an important role in the aerobic biodegradation of nitroaromatic pollutants, but no active dioxygenases are available in nature for initial reactions in the degradation of many refractory pollutants like 2,4-dichloronitrobenzene (24DCNB). Here, we report the engineering of hotspots in 2,3-dichloronitrobenzene dioxygenase from Diaphorobacter sp. strain JS3051, achieved through molecular dynamic simulation analysis and site-directed mutagenesis, with the aim of enhancing its catalytic activity toward 24DCNB. The computationally predicted activity scores were largely consistent with the detected activities in wet experiments. Among them, the two most beneficial mutations (E204M and M248I) were obtained, and the combined mutant reached up to a 62-fold increase in activity toward 24DCNB, generating a single product, 3,5-dichlorocatechol, which is a naturally occurring compound. In silico analysis confirmed that residue 204 affected the substrate preference for meta-substituted nitroarenes, while residue 248 may influence substrate preference by interaction with residue 295. Overall, this study provides a framework for manipulating nitroarene dioxygenases using computational methods to address various nitroarene contamination problems.IMPORTANCEAs a result of human activities, various nitroaromatic pollutants continue to enter the biosphere with poor degradability, and dioxygenation is an important kickoff step to remove toxic nitro-groups and convert them into degradable products. The biodegradation of many nitroarenes has been reported over the decades; however, many others still lack corresponding enzymes to initiate their degradation. Although rieske non-heme dioxygenase family enzymes play extraordinarily important roles in the aerobic biodegradation of various nitroaromatic pollutants, prediction of their substrate specificity is difficult. This work greatly improved the catalytic activity of dioxygenase against 2,4-dichloronitrobenzene by computer-aided semi-rational design, paving a new way for the evolution strategy of nitroarene dioxygenase. This study highlights the potential for using enzyme structure-function information with computational pre-screening methods to rapidly tailor the catalytic functions of enzymes toward poorly biodegradable contaminants.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Semi-rational design for enhancing thermostability of Culex pipiens acetylcholinesterase and sensitivity analysis of acephate.

Acetylcholinesterase (AChE) has emerged as a significant biological recognition element in the biosensor field, particularly for the detection of insecticides. Nevertheless, the weak thermostability of AChE restricts its utilization due to the complexities associated with production, storage, and application environments. By evaluating the binding affinity between representative AChE and insecticides, an AChE from Culex pipiens was screened out, which displayed a broad-spectrum and high sensitivity to insecticides. The C. pipiens AChE (CpA) was subsequently expressed in Escherichia coli (E. coli) as a soluble active protein. Furthermore, a three-point mutant, M4 (A340P/D390E/S581P), was obtained using a semi-rational design strategy that combined molecular dynamics (MD) simulation and computer-aided design, which exhibited a four-fold increase in half-life at 40 °C compared to the wild-type (WT) enzyme. The mutant M4 also demonstrated an optimal temperature of 50 °C and a melting temperature (Tm) of 51.2 °C. Additionally, the sensitivity of WT and M4 to acephate was examined, revealing a 50-fold decrease in the IC50 value of M4. The mechanism underlying the improvement in thermal performance was elucidated through secondary structure analysis and MD simulations, indicating an increase in the proportion of protein helices and local structural rigidity. MD analysis of the protein-ligand complexes suggested that the enhanced sensitivity of M4 could be attributed to frequent specific contacts between the organophosphorus (OP) group of acephate and the key active site residue Ser327. These findings have expanded the possibilities for the development of more reliable and effective industrial enzyme preparations and biosensors.

Chemoenzymatic Synthesis of Selegiline: An Imine Reductase-Catalyzed Approach.

(R)-Homobenzylic amines are key structural motifs present in (R)-selegiline, a drug indicated for the treatment of early-stage Parkinson's disease. Herein, we report a new short chemoenzymatic approach (in 2 steps) towards the synthesis of (R)-selegiline via stereoselective biocatalytic reductive amination as the key step. The imine reductase IR36-M5 mutant showed high conversion (97%) and stereoselectivity (97%) toward the phenylacetone and propargyl amine substrates, offering valuable biocatalysts for synthesizing alkylated homobenzylic amines.

[Site-specific mutation of ω-transaminase and the biocatalytic preparation of (S)-1-(2-fluorophenyl) ethylamine].

(S)-1-(2-fluorophenyl) ethylamine plays a crucial role as a chiral building block in pharmaceutical synthesis. ω-transaminases are widely recognized as environmentally friendly and efficient catalysts for the preparation of chiral amines. In this study, we isolated a novel ω-transaminase, PfTA, from Pseudogulbenkiania ferrooxidans through gene mining in the NCBI database. By employing semi-rational design, we obtained a Y168R/R416Q variant with enhanced enzyme activity. This variant exhibited the ability to catalyze the synthesis of (S)-1-(2-fluorophenyl) ethylamine from 2-fluorophenone, achieving a yield of 83.58% and an enantioselectivity exceeding 99% after a 10 h reaction. Compared to the wild type, the specific enzyme activity of the Y168R/R416Q variant reached 47.04 U/mg, which represents an increase of 11.65 times. Additionally, the catalytic efficiency, as measured by kcat/Km, was increased by 20.9 times. Molecular docking and structural simulation analysis revealed that the primary factor contributing to the improved catalytic efficiency is the expansion of the enzyme's active pocket and the alleviation of steric hindrance.

Engineering non-conservative substrate recognition sites of extradiol dioxygenase: Computation guided design to diversify and accelerate degradation of aromatic compounds.

Extradiol dioxygenases (EDOs) catalyzing meta-cleavage of catecholic compounds promise an effective way to detoxify aromatic pollutants. This work reported a novel scenario to engineer our recently identified Type I EDO from Tcu3516 for a broader substrate scope and enhanced activity, which was based on 2,3-dihydroxybiphenyl (2,3-DHB)-liganded molecular docking of Tcu3516 and multiple sequence alignment with other 22 Type I EDOs. 11 non-conservative residues of Tcu3516 within 6 Å distance to the 2,3-DHB ligand center were selected as potential hotspots and subjected to semi-rational design using 6 catecholic analogues as substrates; the mutants V186L and V212N returned with progressive evolution in substrate scope and catalytic activity. Both mutants were combined with D285A for construction of double mutants and final triple mutant V186L/V212N/D285A. Except for 2,3-DHB (the mutant V186L/D285A gave the best catalytic performance), the triple mutant prevailed all other 5 catecholic compounds for their degradation; affording the catalytic efficiency kcat/Km value increase by 10-30 folds, protein Tm (structural rigidity) increase by 15 °C and the half-life time enhancement by 10 times compared to the wild type Tcu3516. The molecular dynamic simulation suggested that a stabler core and a more flexible entrance are likely accounting for enhanced catalytic activity and stability of enzymes.

Improvement of the catalytic performance of chitosanase Csn-PD from Paenibacillus dendritiformis by semi-rational design.

Chitooligosaccharides (COS) possess versatile functional properties that have found extensive applications across various fields. Chitosanase can specifically hydrolyze ß-1,4 glycosidic bonds in chitosan to produce COS. In this study, Csn-PD, a glycoside hydrolase family 46 chitosanase from Paenibacillus dendritiformis, which produces (GlcN)2 as its main product, was rationally redesigned aiming to improve its catalytic performance. Based on the results of molecular docking analysis and multiple sequence alignment, four amino acid residues in Csn-PD (I101, T120, T220, and Y259) were pinpointed for targeted mutations. Beneficial mutations in terms of enhanced catalytic activity were then combined by site-directed mutagenesis. Notably, the most promising variant, Csn-PDT6 (Csn-PD I101M/T120E/T220G), exhibited an impressive eight-fold surge in activity compared to the wild-type Csn-PD. This heightened enzymatic activity was complemented by an enhanced pH stability profile. A compelling feature of Csn-PDT6 is its preservation of the hydrolytic product profile observed in Csn-PD. This characteristic further accentuates its candidacy for the targeted production of (GlcN)2. The success of our strategic approach is vividly illustrated by the significant improvements achieved in the catalytic performance of the chitosanase, encompassing both its activity and stability. These developments offer a valuable model that may have implications for the semi-rational design of other enzymes.

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.