Classification and functional origins of stereocomplementary alcohol dehydrogenases for asymmetric synthesis of chiral secondary alcohols: A review.

Alcohol dehydrogenases (ADHs) mediated biocatalytic asymmetric reduction of ketones have been widely applied in the synthesis of optically active secondary alcohols with highly reactive hydroxyl groups ligated to the stereogenic carbon and divided into (R)- and (S)-configurations. Stereocomplementary ADHs could be applied in the synthesis of both enantiomers and are increasingly accepted as the "first of choice" in green chemistry due to the high atomic economy, low environmental factor, 100 % theoretical yield, and high environmentally friendliness. Due to the equal importance of complementary alcohols, development of stereocomplementary ADHs draws increasing attention. This review is committed to summarize recent advance in discovery of naturally evolved and tailor-made stereocomplementary ADHs, unveil the molecular mechanism of stereoselective catalysis in views of classification and functional basis, and provide guidance for further engineering the stereoselectivity of ADHs for the industrial biosynthesis of chiral secondary alcohol of industrial relevance.

Photocatalytic regeneration of nicotinamide cofactor biomimetics drives biocatalytic reduction by Old Yellow enzymes.

A key approach in developing green chemistry involves converting solar energy into chemical energy of biomolecules through photocatalysis. Photocatalysis can facilitate the regeneration of nicotinamide cofactors during redox processes. Nicotinamide cofactor biomimetics (NCBs) are economical substitutes for natural cofactors. Here, photocatalytic regeneration of NADH and reduced NCBs (NCBsred) using graphitic carbon nitride (g-C3N4) was developed. The process involves g-C3N4 as the photocatalyst, Cp*Rh(bpy)H2O2+ as the electron mediator, and Triethanolamine as the electron donor, facilitating the reduction of NAD+ and various oxidative NCBs (NCBsox) under light irradiation. Notably, the highest reduction yield of 48.32 % was achieved with BANA+, outperforming the natural cofactor NAD+. Electrochemical analysis reveals that the reduction efficiency and capacity of cofactors relies on their redox potentials. Additionally, a coupled photo-enzymatic catalysis system was explored for the reduction of 4-Ketoisophorone by Old Yellow Enzyme XenA. Among all the NCBsox and NAD+, the highest conversion ratio of over 99 % was obtained with BANA+. After recycled for 8 times, g-C3N4 maintained over 93.6 % catalytic efficiency. The photocatalytic cofactor regeneration showcases its outstanding performance with NAD+ as well as NCBsox. This work significantly advances the development of photocatalytic cofactor regeneration for artificial cofactors and its potential application.

Structure-Guided Evolution of Cyclohexanone Monooxygenase Toward Bulky Omeprazole Sulfide: Substrate Migration and Stereoselectivity Inversion.

Structure-guided engineering of a CHMO from Amycolatopsis methanolica (AmCHMO) was performed for asymmetric sulfoxidation activity and stereoselectivity toward omeprazole sulfide. Initially, combinatorial active-site saturation test (CASTing) and iteratively saturation mutagenesis (ISM) were performed on 5 residues at the "bottleneck" of substrate tunnel, and MT3 was successfully obtained with a specific activity of 46.19 U/g and R-stereoselectivity of 99 % toward OPS. Then, 4 key mutations affecting the stereoselectivity were identified through multiple rounds of ISM on residues at the substrate binding pocket region, resulting MT8 with an inversed stereoselectivity from 99 % (R) to 97 % (S). MT8 has a greatly compromised specific activity of 0.08 U/g. By introducing additional beneficial mutations, MT11 was constructed with significantly increased specific activity of 2.29 U/g and stereoselectivity of 97 % (S). Enlarged substrate tunnel is critical to the expanded substrate spectrum of AmCHMO, while reshaping of substrate binding pocket is important for stereoselective inversion. Based on MD simulation, pre-reaction states of MT3-OPSproR, MT8-OPSproS, and MT11-OPSproS were calculated to be 45.56 %, 17.94 %, and 28.65 % respectively, which further confirm the experimental data on activity and stereoselectivity. Our results pave the way for engineering distinct activity and stereoselectivity of BVMOs toward bulky prazole thioethers.

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher T 50 15 $$ {\mathrm{T}}_{50}^{15} $$ , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

Mining and tailor-made engineering of a novel keto reductase for asymmetric synthesis of structurally hindered γ- and δ-lactones.

A novel carbonyl reductase from Hyphopichia burtoni (HbKR) was discovered by gene mining. HbKR is a NADPH-dependent dual function enzyme with reduction and oxidation activity belonging to SDR superfamily. HbKR strictly follows Prelog priority in the reduction of long-chain aliphatic keto acids/esters containing remote carbonyl groups, such as 4-oxodecanoic acid and 5-oxodecanoic acid, producing (S)-γ-decalactone and (S)-δ-decalactone in >99 % e.e. Tailor-made engineering of HbKR was conducted to improve its catalytic efficiency. Variant F207A/F86M was obtained with specific activity of 8.37 U/mg toward 5-oxodecanoic acid, which was 9.7-fold of its parent. Employing F207A/F86M, 100 mM 5-oxodecanoic acid could be reduced into optically pure (S)-δ-decalactone. Molecular docking analysis indicates that substitution of aromatic Phe with smaller residues renders sufficient space for accommodating substrates in a more stable conformation. This study offers an efficient biocatalyst for the biosynthesis of (S)-lactones, and provides guidance for engineering carbonyl reductases toward structurally hindered substrates.

A comparative study of two aldehyde dehydrogenases from Sphingobium sp.: the substrate spectrum and catalytic mechanism.

Biocatalytic oxidation is one of the most important and indispensable organic reactions for the development of green and sustainable biomanufacturing processes. NAD(P)+-dependent aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Here, two ALDHs, SpALDH1 and SpALDH2, were identified from Sphingobium sp. SYK-6. They belong to different ALDH families and share only 32.30% amino acid identity. Interestingly, SpALDH1 and SpALDH2 exhibit significantly different enzymatic properties and substrate profiles. SpALDH2 has better thermostability than SpALDH1. SpALDH1 is a metalloenzyme and is activated by potassium ions, while SpALDH2 is not metallic-dependent. Compared with SpALDH1, SpALDH2 has a relatively broad substrate spectrum toward aromatic aldehydes. Based on homology modeling and molecular docking analysis, mechanisms underlying the substrate specificity of ALDHs were elucidated. For both ALDHs, hydrophobicity of substrate binding pockets is important for the catalytic properties, especially substrate specificity. Notably, optimization of the flexible loop 444-457 reforms a hydrogen bond between pyridine substrates and SpALDH1, contributing to the high catalytic activity. Finally, a coupling reaction catalyzed by ALDHs and NOX was constructed for efficient production of aromatic carboxylic acids.

Engineering of Methionine Adenosyltransferase Reveals Key Roles of Electrostatic Interactions in Enhanced Catalytic Activity.

As an important dietary supplement, S-adenosylmethionine (SAM) is currently synthesized by methionine adenosyltransferase (MAT) using ATP and methionine as substrates. However, the activity of MAT is severely inhibited by product inhibition, which limits the industrial production of SAM. Here, MAT from Bacteroides fragilis (BfMAT), exhibiting relatively low product inhibition and moderate specific activity, was identified by gene mining. Based on molecular docking, residues within 5 Å of ATP in BfMAT were subjected to mutagenesis for enhanced catalytic activity. Triple variants M3-1 (E42M/E55L/K290I), M3-2 (E42R/E55L/K290I), and M3-3 (E42C/E55L/K290I) with specific activities of 1.83, 1.81, and 1.94 U/mg were obtained, which were 110.5-125.6% higher than that of the wild type (WT). Furthermore, compared with WT, the Km values of M3-1 and M3-3 were decreased by 31.4% and 60.6%, leading to significant improvement in catalytic efficiency (kcat/Km) by 322.5% and 681.1%. All triple variants showed shifted optimal pH from 8.0 to 7.5. Moreover, interaction analysis suggests that the enhanced catalytic efficiency may be attributed to the decreased electrostatic interactions between ATP and the mutation sites (E42, E55, and K290). Based on MD simulation, coulomb energy and binding free energy analysis further reveal the importance of electrostatic interactions for catalytic activity of BfMAT, which could be an efficient strategy for improving catalytic performance of MATs.

Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+ ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA+ . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Effects of obesity on short-term mortality in patients with acute heart failure under different nutritional status.

BACKGROUND: Increased body mass index (BMI) is associated with better survival in patients with acute heart failure (AHF), which is a paradoxical phenomenon. However, it is unclear whether different nutritional status affects this association. METHODS: 1325 patients with AHF from the Medical Information Mart for Intensive Care III database were retrospectively included. Nutritional status was assessed by serum albumin (SA) and prognostic nutritional index (PNI). Patients were divided into High-SA (≥ 3.5 g/dL) and Low-SA groups (< 3.5 g/dL), and they also were divided into High-PNI (≥ 38) and Low-PNI groups (< 38). Propensity-score matching (PSM) was used to control for the effect of baseline confounding factors, multifactor regression model was adopted to assess the association of nutritional status, BMI, and outcomes in AHF patients. RESULTS: Of the 1325 patients (mean age 72.4 ± 13.1 years), 52.1% (n = 690) were male, 13.1% (n = 173) died in hospital and 23.5% (n = 311) died within 90 days. Before PSM, after adjusting for potential confounders, in the High-SA population, compared with the under/normal BMI group, overweight and obesity were negatively correlated with 90-day mortality, with adjusted hazard ratios (HR) of 0.47, 95% confidence interval (CI) (0.30-0.74), P = 0.001; HR 0.45, 95%CI (0.28-0.72), P = 0.001, respectively. However, this correlation was much attenuated in the Low-SA group (overweight BMI: HR 1.06, 95%CI 0.75-1.50, P = 0.744; obese BMI: HR 0.86, 95%CI 0.59-1.24, P = 0.413). After PSM, those who were overweight or obese in the High-SA group had a 50-58% reduction in 90-day risk of death, while the protective effect disappeared in the Low-SA group (HR 1.09, 95% CI 0.70-1.71; HR 1.02, 95%CI 0.66 - 0.59). Similarly, results were similar in analyses using PNI as a nutritional assessment criterion. CONCLUSION: Overweight or Obesity was associated with lower short-term mortality in well-nourished AHF patients, whereas this association was significantly attenuated or even disappeared in malnourished patients. Therefore, further research is needed for weight loss recommendations for malnourished obese patients with AHF.

Efficient production of hyaluronic acid by Streptococcus zooepidemicus using two-stage semi-continuous fermentation.

Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks. To enhance the efficiency of hyaluronic acid production, a semi-continuous fermentation process consisted of two-stage 3-L bioreactors was developed, in which 1.01 g/L/h productivity and 14.60 g/L hyaluronic acid were obtained. To further enhance the titer of hyaluronic acid, recombinant hyaluronidase SzHYal was added into 2nd stage bioreactor at 6 h to reduce the viscosity of broth. The highest hyaluronic acid titer of 29.38 g/L was achieved with a productivity of 1.13 g/L/h at 300 U/L SzHYal after 24 h. This newly developed semi-continuous fermentation process provides a promising strategy for the industrial production of hyaluronic acid and related polysaccharides.

Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu from l-DOPA.

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l-DOPA using tyrosine aminotransferase from Escherichia coli (EcTyrB) and d-isomer-specific 2-hydroxyacid dehydrogenase from Lactobacillus frumenti (LfD2-HDH). Glutamate dehydrogenase from Clostridium difficile (CdgluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH. Under optimal conditions (35 °C, pH 7.0, EcTyrB:LfD2-HDH:CdgluD = 3:2:1, glutamate:NAD+ = 1:1), 98.3% yield (at 20 mM l-DOPA) and space-time yield of 6.61 g L-1 h-1 (at 40 mM l-DOPA) were achieved. Decreased yields of DSS at elevated l-DOPA concentrations (100 mM) could be attributed to an inhibited CdgluD activity caused by NH4+ accumulation. This developed multi-enzyme cascade pathway (including EcTyrB, LfD2-HDH, and CdgluD) provides an efficient and sustainable approach for the production of DSS from l-DOPA.

Engineering the Thermostability of a d-Carbamoylase Based on Ancestral Sequence Reconstruction for the Efficient Synthesis of d-Tryptophan.

Employing ancestral sequence reconstruction and consensus sequence analysis, the thermostability of a novel d-carbamoylase derived from Nitratireductor indicus (NiHyuC) was engineered through greedy-oriented iterative combinatorial mutagenesis. A mutant S202P/E208D/R277L (M4Th3) was obtained with significantly elevated thermostability. M4Th3 has a half-life of 36.5 h at 40 °C, about 28.5 times of 1.3 h of its parent M4. For the reaction at 40 °C, M4Th3 can catalyze 10 mM N-carbamoyl-d-tryptophan to produce d-tryptophan with a conversion ratio of 96.4% after 12 h, which is significantly higher than 64.1% of M4. MD simulation reveals that new hydrogen bonds emerging from E208D on the surface can increase the hydrophobicity of the protein, leading to improved stability. More importantly, R277L could contribute to enhanced interface stability of homodimeric M4. This study provides a thermostable d-carbamoylase for the "hydantoinase process", which has potential in the industrial synthesis of optically pure natural and non-natural amino acids.

Engineering of a Baeyer-Villiger monooxygenase reveals key residues for the asymmetric oxidation of omeprazole sulfide.

The structure-guided engineering of a BVMO from Rhodococcus aetherivorans (RaBVMO) was performed for its asymmetric sulfoxidation activity toward omeprazole sulfide. Based on the structural model of RaBVMO, key residues that line the substrate entrance tunnel and the binding pocket were identified, and variants were interrogated with sulfides of varied sizes. The best mutant MT2 (F442A/R337P) was obtained with a specific activity of 2.54 U g-1 and excellent enantioselectivity (≥99%, S) toward omeprazole sulfide, while wild-type RaBVMO exhibited no activity. Further structural analysis reveals that both mutations, F442A and R337P, could render an expanded substrate tunnel and an enlarged substrate binding pocket to enable easier access to the catalytic center for omeprazole sulfide. This work provides valuable guidance for engineering-related BVMOs for improved activity and enantio-preference toward bulky substrates.

Directed reconstruction of a novel ancestral alcohol dehydrogenase featuring shifted pH-profile, enhanced thermostability and expanded substrate spectrum.

Ancestral enzymes are promising for industrial biotechnology due to high stability and catalytic promiscuity. An effective protocol was developed for the directed resurrection of ancestral enzymes. Employing genome mining with diaryl alcohol dehydrogenase KpADH as the probe, descendant enzymes D10 and D11 were firstly identified. Then through ancestral sequence reconstruction, A64 was resurrected with a specific activity of 4.3 U·mg-1. The optimum pH of A64 was 7.5, distinct from 5.5 of D10. The T15 50 and Tm values of A64 were 57.5 °C and 61.7 °C, significantly higher than those of the descendant counterpart. Substrate spectrum of A64 was quantitively characterized with a Shannon-Wiener index of 2.38, more expanded than D10, especially, towards bulky ketones in Group A and B. A64 also exhibited higher enantioselectivity. This study provides an effective protocol for constructing of ancestral enzymes and an efficient ancestral enzyme of industrial relevance for asymmetric synthesis of chiral alcohols.

[Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis].

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

A Novel Scoring System for Predicting the Metastases of Posterior Right Recurrent Laryngeal Nerve Lymph Node Involvement in Patients With Papillary Thyroid Carcinoma by Preoperative Ultrasound.

Objective: Our goal was to investigate the correlation between papillary thyroid carcinoma (PTC) characteristics on ultrasonography and metastases of lymph nodes posterior to the right recurrent laryngeal nerve (LN-prRLN). There is still no good method for clinicians to judge whether a patient needs LN-prRLN resection before surgery, and we also wanted to establish a new scoring system to determine whether patients with papillary thyroid carcinoma require LN-prRLN resection before surgery. Patients and Methods: There were 482 patients with right or bilateral PTC who underwent thyroid gland resection from December 2015 to December 2017 recruited as study subjects. The relationship between the PTC characteristics on ultrasonography and the metastases of LN-prRLN was analyzed by univariate and logistic regression analyses. Based on the risk factors identified in univariate and logistic regression analysis, a nomogram-based LN-prRLN prediction model was established. Result: LN-prRLN were removed from all patients, of which 79 had LN-prRLN metastasis, with a metastasis rate of 16.39%. Multivariate logistic regression analysis revealed that LN-prRLN metastasis was closely related to sex, age, blood supply, larger tumors (> 1 cm) and capsular invasion. A risk prediction model has been established and fully verified. The calibration curve used to evaluate the nomogram shows that the consistency index was 0.75 ± 0.065. Conclusion: Preoperative clinical data, such as sex, age, abundant blood supply, larger tumor (> 1 cm) and capsular invasion, are positively correlated with LN-prRLN metastasis. Our scoring system can help surgeons non-invasively determine which patients should undergo LN-prRLN resection before surgery. We recommend that LN-prRLN resection should be performed when the score is above 103.1.

Sustainable one-pot chemo-enzymatic synthesis of chiral furan amino acid from biomass via magnetic solid acid and threonine aldolase.

Sustainable synthesis of valuable noncanonical amino acids from renewable feedstocks is of great importance. Here, a feasible chemo-enzymatic procedure was developed for the synthesis of chiral ß-(2-furyl)serine from biomass catalyzed by a solid acid catalyst and immobilized E. coli whole-cell harboring l-threonine aldolase. A novel magnetic solid acid catalyst Fe3O4@MCM-41/SO42- was successfully synthesized for conversion of corncob into furfural in an aqueous system. Under the optimum conditions, furfural yield of 63.6% was achieved in 40 min at 180 ℃ with 2.0% catalyst (w/w). Furthermore, biomass-derived furfural was converted into an aldol-addition product ß-(2-furyl)serine with 73.6% yield, 99% ee and 20% de by immobilized cells in 6 h. The magnetic solid acid and biocatalyst can be readily recovered and efficiently reused for five consecutive cycles without significant loss on product yields. This chemo-enzymatic route can be attractive for producing noncanonical amino acids from biomass.

Kinetic Resolution of Nearly Symmetric 3-Cyclohexene-1-carboxylate Esters Using a Bacterial Carboxylesterase Identified by Genome Mining.

A new bacterial carboxylesterase (CarEst3) was identified by genome mining and found to efficiently hydrolyze racemic methyl 3-cyclohexene-1-carboxylate (rac-CHCM) with a nearly symmetric structure for the synthesis of (S)-CHCM. CarEst3 displayed a high substrate tolerance and a stable catalytic performance. The enantioselective hydrolysis of 4.0 M (560 g·L-1) rac-CHCM was accomplished, yielding (S)-CHCM with a >99% ee, a substrate to catalyst ratio of 1400 g·g-1, and a space-time yield of 538 g·L-1·d-1.

Co-immobilized Alcohol Dehydrogenase and Glucose Dehydrogenase with Resin Extraction for Continuous Production of Chiral Diaryl Alcohol.

Ni2+-functionalized porous ceramic/agarose composite beads (Ni-NTA Cerose) can be used as carrier materials to immobilize enzymes harboring a metal affinity tag. Here, a 6×His-tag fusion alcohol dehydrogenase Mu-S5 and glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-immobilized on Ni-NTA Cerose to construct a packed bed reactor (PBR) for the continuous synthesis of the chiral intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl) methanol ((S)-CPMA) NADPH recycling, and in situ product adsorption was achieved simultaneously by assembling a D101 macroporous resin column after the PBR. Using an optimum enzyme activity ratio of 2:1 (Mu-S5: BmGDH) and hydroxypropyl-ß-cyclodextrin as co-solvent, a space-time yield of 1560 g/(L·d) could be achieved in the first three days at a flow rate of 5 mL/min and substrate concentration of 10 mM. With simplified selective adsorption and extraction procedures, (S)-CPMA was obtained in 84% isolated yield.

Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation.

Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that ß-subunit played a key role in adjusting the flexibility of α-subunit and the different conformations of linker in "α5-helix-coil ring" supersecondary structure of ß-subunit can affect the interaction between ß-subunit and α-subunit. Mutant NHCTA1-α6 C with a random coil linker and mutant NHCTA1-αßγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-α6 C and NHCTA1-αßγ (Tmα6C = 74.4°C, Tmαßγ = 65.6°C) verified that the flexibility of α-subunit adjusted by ß-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes.