Optimum Reaction Temperature Research Articles

Comparison of Plant Genomic DNA Extraction Kits Using the Proofman-LMTIA Amplification Assay.

The extraction of DNA is the basis of molecular biology research. The quality of the extracted DNA is one of the key factors for the success of molecular biology experiments. To select a suitable DNA extraction method for Chinese medicinal herbs and seeds. In this experiment, four commercial DNA extraction kits were used to extract the genomic DNA (gDNA) from the Pinellia ternata (Thunb.) Breit. powder; Arisaema amurense Maxim. powder as well as the seeds of Glycine max (L.) Merr. On the one hand, the concentration and purity of DNA extracted by these four kits were compared. On the other hand, nucleic acid amplification experiments were performed on three samples extracted by each of the four kits by Proofman-LMTIA methods, which is a novel nucleic acid isothermal amplification technique. The concentration and purity of DNA extracted by different kits were used to determine which methods were suitable for the dry powder of Chinese herbal medicines and seeds. The efficiency of the amplification curve to show whether the extracted DNA can be used in nucleic acid amplification experiments. The results showed that the Proofman-LMTIA methods were of high specificity and the optimal reaction temperatures were 63, 59, and 59°C for P. ternata (Thunb.) Makino; A. amurense Maxim. and G. max (L.) Merr., respectively. The concentration and purity of the gDNAs extracted with all kits were within the acceptable ranges; meanwhile, the amplification of the gDNA extracted by Kit II was of the highest efficiency. In this experiment, the principle, concentration, purity, and time taken for extracting DNA with four kits were compared. The automated extraction kit based on the magnetic method is suitable for extracting DNA from Chinese medicinal herbs and seeds. The extracted DNA is suitable for nucleic acid amplification detection.

Exopolygalacturonase Production from the Novel Strain Lichtheimia sp. UV-16 and Enzyme Hydrolysis Properties.

A pectinase-producing strain, Lichtheimia sp. X-8, was isolated from the soil for the first time. Subsequently, Lichtheimia sp. UV-16, with a 1.23-fold increase in pectinase activity, was obtained via UV mutagenesis, and optimization of its liquid fermentation process boosted pectinase activity from 455.6 ± 12.7 to 3202.0 ± 82.1 U/mL. The crude enzyme was purified by salting out and anion exchange resin, with a purification ratio of 2.28-fold and a yield of 36.5%. The optimal reaction temperature for the pure enzyme was 60 °C with an optimal pH of 5.5. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) confirmed that the enzyme was an exopolygalacturonase, achieving over 99% efficiency in pectin hydrolysis. Furthermore, incorporating pure enzymes into juice pulps can substantially enhance the juice yield, which makes this polygalacturonase a promising application in the beverage industry.

An isothermal recombinase polymerase assay coupled with lateral flow dipstick for differentiation of pseudorabies virus wild isolates and gE-deleted vaccine strains.

Pseudorabies virus (PRV) is one of the most important infectious diseases which leads to significant economic losses in the global swine industry. The gE-deleted vaccine is widely used to prevent susceptible pigs from PRV infection. There is no report of the differentiation of PRV wild strain and vaccine strain by recombinase polymerase amplification (RPA) coupled with a lateral flow dipstick (LFD) method. In the present study, the gD and gE gene-targeted primer-probe sets were designed. The RPA-LFD assay could discriminate between the PRV wild strain and the vaccine strain. The RPA reaction conditions were also evaluated. The optimal reaction temperature and reaction time for the RPA-LFD assay were 37℃ and 20 min. The detection limit was 10 genome copies per reaction for PRV wild strain and gE-deleted vaccine strain. The assay did not have cross-reaction with other common swine viral pathogens. The effectiveness of the RPA-LFD assay for detecting the clinical samples was evaluated by testing 80 samples. The result of the assay was compared with that of the conventional PCR. The positive rate of PRV wild strain by the RPA-LFD assay was 20%, whereas the positive rate of PRV wild strain by the PCR assay was 18.8%. The assay therefore provides a novel alternative for differentiation of PRV wild strain and vaccine strain.

Insights into P-doping effect of the activity and anti-SO2/H2O poisoning of V2O5-MoO3/TiO2 catalysts for NH3-SCR

With the increasingly stringent flue gas emission standards, water-sulfur poisoning has become one of the most pressing problems in the SCR process of coal-fired power plants.SO2 not only deactivates the catalyst, but also reacts with water and NH3 to form NH4HSO4, which then corrodes the equipment. Catalysts with water-sulfur tolerance urgently need to be developed. NH4H2PO4-modified V2O5-MoO3/TiO2 SCR catalysts is prepared by an improved wet impregnation method. The effect of P element modification on the denitration performance of the V2O5-MoO3/TiO2 SCR catalyst was studied, and the catalyst was characterized by XRD, BET, SEM, TEM, XPS, TPR, TPD and Raman. When the phosphorus doping amount was 0.6 % (wt%), the P0.6-SCR catalyst could achieve a NOx conversion rate of 95 %, and its optimal reaction temperature was 350 ℃. The characterization results show that the P element made the active components on the catalyst surface further dispersed, delayed the sintering of the catalyst at the calcination stage, and promoted the generation of B-acidic sites, enhances the surface acidity of the catalyst, and promotes the transition of VOx from a monomer state to a polymer state. Meanwhile, the P0.6-SCR catalyst exhibits outstanding sulfur resistance while ensuring high denitration efficiency.

Mechanochemical driven oxidative desulfurization of high-sulfur petroleum coke over [Bpy]PMoVn coupled with amide-based binary deep eutectic solvents

Herein, a novel strategy combining heteropoly acid with deep eutectic solvent was developed to remove the sulfur from HSPC to produce low-sulphur petroleum coke (LSPC) with higher economic value, in which, phosphomolybdenum vanadium pyridine ionic liquid [Bpy]PMoVn (n = 1, 2, 3) and air was used as catalyst and oxidant, and amide-based dibasic deep eutectic solvent (DES) as the extractant and solvent, respectively. The experimental results confirmed that the sulfur content of HSPC can be remarkably decreased from 4.46 wt% to 1.54 wt% under the optimal reaction temperature of 110 °C, reaction time of 8 h, catalyst dosage of 0.20 g, and air flow rate of 100 mL/min, due to the coupling effect between [Bpy]PMoVn and DES. The results of reaction mechanism revealed that peroxyl radicals (O2−) were the key active species in catalyzing the ODS of HSPC in the presence of the [Bpy]PMoV2 catalyst.

Rapid and visual detection of Pyricularia oryzae using coupled recombinase polymerase amplification-lateral flow dipstick assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate and visual detection of P. oryzae is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay to detect P. oryzae. Species-specific RPA primer pairs and probe were designed based on target gene MGG_15975. The optimized reaction temperature and time were set at 37 °C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect P. oryzae isolates from rice, whereas other fungal species or Pyricularia species from grasses were not detected. Additionally, this assay demonstrated highly sensitivity, capable of detecting as low as 10-2 ng/µL of P. oryzae genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, P. oryzae was effectively detected in diseased leaves in rice fields, and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of P. oryzae is rapid, highly sensitive and efficient, which has the potential application for early diagnosis of P. oryzae infection in rice fields.

Improving the catalytic activity and stability of Bacillus alcalophilus serine protease BAPB92 by rational design

The catalytic activity and stability of proteases are essential for their application in the detergent industry. To enhance the catalytic properties of BAPB92, homologous sequence comparison combined with rational design was employed. Six mutants were generated: BAPB92 (A188P), BAPB92 (V262I), BAPB92 (Q239R), BAPB92 (A188P/V262I), BAPB92 (Q239R/V262I), and BAPB92 (Q239R/A188P). Remarkably, the mutant BAPB92 (A188P/V262I) exhibited the most significant improvement, exhibiting a 4.30-fold increase in kcat/Km compared to the wild type, and a 0.75-fold enhancement in thermal stability at 60 °C. The enzymatic activity of BAPB92 (A188P/V262I) reached 6511.81 U/mg, which was 2.95 times higher than that of the wild type BAPB92. Furthermore, the optimal reaction temperature of this mutant increased from 50 °C to 60 °C. The BAPB92 (A188P/V262I) mutant also showed a marked improvement in detergent stability. In sodium tripolyphosphate liquid detergent, its washing efficacy was 17.84 % higher than that of the wild type, and in methyl glycine diacetate liquid detergent, the improvement was 18.51 %. These findings suggested that BAPB92 (A188P/V262I) holds significant potential as a detergent protease in the washing industry. Structural analysis and molecular dynamics simulations further confirmed the enhanced stability of this mutant compared to the wild type. This study provides valuable theoretical insights for the application of the serine protease BAPB92 in detergent formulations.

Agarase cocktail from agarolytic Alteromonas sp. Aga1552 converts homogenized Gelidium amansii into monosaccharide

Marine algae biomass utilization has attracted considerable attention, however, the preparation of monosaccharides from raw algae is still hindered by many technical barriers. In this study, three genes, aga1365, aga1364, and aga1360, encoding key enzymes constituting a complete agar decomposition pathway were expressed and characterized. Recombinant Aga1365, Aga1364, and Aga1360 exhibited high optimal reaction temperatures and excellent thermal stability. Moreover, enzyme cocktail was proved to have higher synergistic effect to prepare monosaccharide from raw seaweed. The enzyme cocktail of Aga1360 (GH117) with Aga1365 (GH16) and enzyme cocktail of Aga1360 with both Aga1365 and 1364 (GH50) were used to synergistically degrade homogenized Gelidium amansii, maximum monosaccharide production of 21.47 mg/g and 39.28 mg/g could be achieved, respectively. This study presents an environment-friendly, time saving and efficient way to prepare monosaccharides from raw seaweed, which also provide a potential strategy to effectively convert algae biomass for biofuel and biochemical production by utilizing the synergistic effects of enzyme cocktail.

Engineering the Non-catalytic Domain to Enhance Catalytic Activity and Thermal Stability of a Nκ2-Producing κ-Carrageenase.

κ-Carrageenases play an important role in achieving the high-value utilization of carrageenan polysaccharides. They can be used in the preparation of even-numbered κ-neocarrageenan oligosaccharides by degrading κ-carrageenan (KC). We previously identified and characterized a κ-carrageenase, CaKC16B, with high specificity for producing a single κ-neocarrabiose. It can produce a single κ-neocarrabiose from degrading KC. However, they also exhibited poor thermal stability and catalytic efficiency. To improve these properties, we introduced noncatalytic domains (nonCDs) from a heat-resistant κ-carrageenase, MtKC16A, into the C-terminus of CaKC16B to construct CaKC16BUN and CaKC16BUNB. Compared to the original CaKC16B, both of them exhibited improved enzymatic properties, including optimal reaction temperature, thermal stability, substrate affinity, and catalytic efficiency. Remarkably, the kcat/Km values of 16BUN and 16BUNB increased by 127 and 290 times, respectively. Importantly, the addition of nonCDs did not alter the final products of degrading KC, retaining the high product specificity of CaKC16B. Interestingly, the addition of nonCDs changed CaKC16B's substrate specificity for hydrolyzing KC and βκ-carrageenan, with mutants exhibiting higher relative activity for βκ-carrageenan. We further observed through biolayer interferometry that the binding and dissociation process between MtUN nonCD and βκ-carrageenan is faster compared to that of KC. This may explain the change in the substrate specificity observed in the mutants of CaKC16B.

Aspergillus flavipes L-methionine γ-lyase-β-cyclodextrin conjugates with improved stability, catalytic efficiency and anticancer activity

Aspergillus flavipes L-methionine γ-lyase (MGL) has been authenticated as a powerful anticancer agent towards various solid tumors, however, the catalytic efficiency and stability of this enzyme remains the main challenge for its further in vivo applications. Thus, the objective of this study was to enhance the catalytic efficiency, structural stability of A. flavipes MGL, in addition to boost their anticancer activity, via conjugation with β-cyclodextrin. The purified A. flavipes MGL was (38.1 μmol/mg/min) was conjugated with β-cyclodextrin, with immobilization yield 80%. The conjugation process of MGL with β-cyclodextrin was verified from the FTIR analysis, molecular docking analysis, ensuring the covalent conjugation process via the hydrogen, and hydrophobic interactions with the cyclodextrin hydroxyl groups and MGL surface amino acids residues. The free and CD-MGL have the same optimum reaction temperature 37 °C, reaction pH 7.5 and pH stability pH 6.5–8.0. The CD-MGL conjugates had a significant stability to proteinase K and trypsin digestion. The affinity of CD-MGL was increased by ~ 2 folds to L-methionine (KM 3.1 mM), compared to the free one (KM 7.2 mM), as well as the catalytic efficiency of MGL was increased by 1.8 folds upon cyclodextrin conjugation. The higher affinity of CD-MGL for L-methionine might be due to re-orientation of the MGL to bind with the substrate by multiple interactions hydrogen, hydrophobic and covalent bonds compared to the free one. The thermal stability of MGL was increased by ~ 2 folds for the tested treatments, upon cyclodextrin conjugation. The in vitro anticancer activity of CD-MGL was enhanced by 2 folds against the HCT-116 (IC50 value 13.9 μmol/mg/min) and MCF7 (IC50 value 9.6 μmol/mg/min), compared to the free MGL (~ 21.4 μmol/mg/min). The enzymes displayed a significant activity against the proliferation of Ehrlich ascites carcinoma in vivo, with an obvious improvement on the liver tissues, as revealed from the histopathological sections

Trehalose-6-phosphate phosphatase expression and enzymatic properties of Fusariumgraminearum

This study presents an exhaustive characterization of the enzymatic attributes and structural properties of trehalose-6-phosphate phosphatase (TPP) derived from Fusarium graminearum. Enzyme activity was evaluated through a meticulously designed enzymatic assay. The findings indicate that the molecular weight of the enzyme is approximately 99.8 kDa, with an optimal reaction temperature and pH of 40 °C and 6.5, respectively. Magnesium ions (Mg2+) markedly enhance the enzymatic activity, resulting in a specific activity of 1.795 U/μg. Kinetic analysis revealed a Km value of 0.96 μmol/L and a Vmax of 15.79 μmol/L/min. Subsequent computational analysis elucidated the three-dimensional architecture of the enzyme and identified the binding site for the substrate trehalose-6-phosphate (T6P). T6P was found to form hydrogen bonds with TPP at residues Lys754, Arg720, His665, Glu758, and Asn756. Additionally, hydrophobic interactions were observed between T6P and residues Phe802, Ile610, Asp801, Pro752, and Gly753. The binding energy calculated for the T6P-TPP complex stood at −5.7 kcal/mol.

Near‐Unity Photothermal CO2 Hydrogenation to Methanol based on a Molecule/Nanocarbon Hybrid Catalyst

Solar‐driven CO2‐to‐methanol conversion provides an intriguing route for both solar energy storage and CO2 mitigation. For scalable applications, near‐unity methanol selectivity is highly desirable to reduce the energy and cost endowed by low‐value byproducts and complex separation processes, but so far has not been achieved. Here we demonstrate a molecule/nanocarbon hybrid catalyst composed of carbon nanotube‐supported molecularly dispersed cobalt phthalocyanine (CoPc/CNT), which synergistically integrates high photothermal conversion capability for affording an optimal reaction temperature with homogeneous and intrinsically‐efficient active sites, to achieve a catalytic activity of 2.4 mmol gcat‐1 h‐1 and selectivity of ~99% in direct photothermal CO2 hydrogenation to methanol reaction. Both theoretical calculations and operando characterizations consistently confirm that the unique electronic structure of CoPc and appropriate reaction temperature cooperatively enable a thermodynamic favorable reaction pathway for highly selective methanol production. This work represents an important milestone towards the development of advanced photothermal catalysts for scalable and cost‐effective CO2 hydrogenation.

Near-Unity Photothermal CO2 Hydrogenation to Methanol Based on a Molecule/Nanocarbon Hybrid Catalyst.

Solar-driven CO2-to-methanol conversion provides an intriguing route for both solar energy storage and CO2 mitigation. For scalable applications, near-unity methanol selectivity is highly desirable to reduce the energy and cost endowed by low-value byproducts and complex separation processes, but so far has not been achieved. Here we demonstrate a molecule/nanocarbon hybrid catalyst composed of carbon nanotube-supported molecularly dispersed cobalt phthalocyanine (CoPc/CNT), which synergistically integrates high photothermal conversion capability for affording an optimal reaction temperature with homogeneous and intrinsically-efficient active sites, to achieve a catalytic activity of 2.4 mmol gcat -1 h-1 and selectivity of ~99 % in direct photothermal CO2 hydrogenation to methanol reaction. Both theoretical calculations and operando characterizations consistently confirm that the unique electronic structure of CoPc and appropriate reaction temperature cooperatively enable a thermodynamic favorable reaction pathway for highly selective methanol production. This work represents an important milestone towards the development of advanced photothermal catalysts for scalable and cost-effective CO2 hydrogenation.

Transgenic rice with microbial high-temperature-resistant β-glucosidase gene significantly improved 2-acetyl-1-pyrroline content and edible quality.

The content of 2-acetyl-1-pyrroline (2-AP) directly affects the aroma and taste of rice. Δ1-Pyrroline and methylglyoxal are the precursors of 2-AP synthesis, and β-glucosidase plays an important role in the synthesis of methylglyoxal. In this study, β-glucosidase gene cloned from Pyrococcus furiosus was molecularly modified to obtain the high-temperature-resistant β-glucosidase gene 371-β-glucosidase (T371A), which was transformed into kitaake varieties (Oryza sativa L. subsp. japonica) by Agrobacterium-mediated transformation method, and transgenic rice with heterologous expression of T371A was obtained. Experiments were conducted in transgenic rice to investigate whether this gene had an effect on the synthesis of 2-AP. Under the optimum reaction temperature of 50°C and cooking temperature of 100°C, the enzyme activity of β-glucosidase in transgenic rice seeds was prominently increased by 260-280% and 419-426% over that of the control, respectively. The content of 2-AP in transgenic rice seeds significantly increased by 75-105% under normal temperature and high-temperature cooking conditions compared with the control. It was also found that transgenic rice increased the content of methylglyoxal and decreased the expression of betaine aldehyde dehydrogenase (BADH2). The high-temperature-tolerant β-glucosidase gene obtained in this study provides an innovative technical strategy for molecular breeding of high-edible aroma crops and has wide application potential. © 2024 Society of Chemical Industry.

Assisting denitrification and strengthening combustion by using ammonia/coal binary fuel gasification-combustion: Effects of injecting position and air distribution

The direct co-firing of ammonia (NH3) with coal is an effective approach for reducing carbon emissions from coal-fired plants. However, the limitations of NH3 combustion, such as its high ignition energy requirement and high NOx emissions, restrict its large-scale co-combustion in coal-fired units. In this study, a self-sustaining gasification–combustion experimental system was employed, and its denitrification performance and combustion strengthening capability for NH3/coal binary fuel under different NH3 injection positions and air distribution methods were evaluated. The results of co-firing 20 % NH3 in the gasifier revealed that its operating temperature can be flexibly controlled within the optimal reaction temperature window of SNCR by adjusting the primary air ratio (λ1). Increasing λ1 was beneficial for NH3 and coal conversion and denitrification in the gasifier; at λ1 = 0.53, the conversion rate of NH3 and N2 reached 86.37 % and 83.59 %, respectively, with no NOx being detected at the gasifier outlet. Furthermore, increasing λ1 promoted the development of gasified char pore structure, thereby improving reactivity. After entering the down-fired combustor (DFC), increasing λ1 promoted NH3 and coal burnout and effectively controlled NOx emissions, reaching a level similar with that of pure coal under λ1 = 0.53. Co-firing 20 % NH3 in the DFC also demonstrated that the introduction of inner secondary air was conducive to char burnout, however, increased NH3 slip. The outer secondary air promoted NH3 combustion, however, exacerbated NOx emissions and inhibited gasified char combustion. The uniform air distribution method balanced the combustion of NH3 and gasified char, and effectively controlled NOx emissions. When the same air distribution method was used, co-firing NH3 in the gasifier was more favourable for NOx control, whereas co-firing NH3 in the DFC benefited coal burnout. This study provides innovative ideas and serves as a reference for developing enhanced combustion and low NOx emission technologies for large-scale NH3 co-firing in coal-fired units.

In situ thermal-assisted photocatalytic decarboxylation of high-concentration biomass-derived fatty acids to alkanes

Heating is a straightforward method to increase reaction rates to meet industrial production, yet photocatalysis seldom incorporates it because their intrinsic driving force depends on the separation of photogenerated carriers, which is rarely affected by temperature. Here, we report a temperature-dependent photocatalytic decarboxylation strategy for producing long-chain n-alkanes from biomass-derived fatty acids using only the photothermal effect of the SnS catalyst. Under concentrated light irradiation without any external heating, and by employing high boiling-point solvents to reach optimal reaction temperatures (∼254 °C), Cn-1 n-alkane allows a very high concentration output (∼0.5 M for stearic acid to n-heptadecane) in a single operation, which is far beyond the capacity limit of conventional photocatalysis, typically no more than the mM levels. Mechanistic studies suggest that the in-situ heat brings the standing C-chain at low temperature down onto the SnS surface, increasing strain on the C-COO- bonds and facilitating reaction with photo-induced hole-electron pairs. We demonstrate that the majority of the incident light energy, when converted to heat, accelerates the photocatalytic decarboxylation.

Online in-situ modification of biochar for the efficient removal of elemental mercury and co-benefit of SO2/NO removal

In this study, non-thermal plasma discharge was proposed for online in-situ modification of biochar injected into coal-biomass co-combustion flue gas, aiming to achieve the combined removal of elemental mercury (Hg0), NO, and SO2 from flue gas. After plasma discharge, the pore structure of biochar was slightly improved whereas the surface morphology of biochar was almost unchanged. After plasma discharge under O2, SO2, and HCl atmospheres, the oxygen, sulfur, and chlorine contents of biochar were obviously increased. More exactly, additional functional groups were formed on the modified biochar surface, such as CO, C(O)OC, CS, and CCl. Compared with raw biochar, the biochar modified under HCl, SO2, O2, H2O and CO2 atmospheres exhibited higher Hg0 removal efficiency, and longer modification time resulted in better Hg0 removal performance. The optimal modification atmosphere and reaction temperature were the simulated flue gas (SO2 + HCl + CO2 + O2 + H2O + NO + N2) and 20 ℃-100 ℃, respectively. The modified biochar exhibited excellent resistance to SO2 and H2O. The addition of HCl, CO2, NO, and O2 contributed to Hg0 removal. The removal efficiency of NO and SO2 attained a maximum of 100 %. The Hg0 removal mechanism was further revealed, where CO, C(O)OC, CS, and CCl served as active components.

Developing PVDF hollow fiber membrane enhanced by braided tube and modified by bionic coating for treating aldehyde-containing wastewater

This study originates from the difficulties encountered in the wastewater treatment from a large-scale methionine production line. Despite the superior performance of the polytetrafluoroethylene (PTFE) membrane, significant issues still persist. The PTFE layer tend to peer off leading to substandard effluent quality, and membrane fouling rate is relatively rapid. To overcome the peeling issue, braided tubes were used to enhance the mechanical strength. To improve the interface bonding between the PVDF layer and braided tubes, two modifiers are introduced to modify the surfaces of braided tubes. Then, the embedded braided tube enhanced PVDF membrane is prepared. On this basis, the one-step bionic coating PVDF (OSBC-PVDF) membrane was prepared by utilizing the polydopamine coating, polyethylene glycol and Cu nanoparticles. It was found that the embedded structure resembling “teeth and gums” formed, significantly improving the mechanical properties to prevent the peeling off of the separation layer. The optimal reaction temperature, reaction time and modifier dosage for preparing the OSBC-PVDF membrane are separately 25 °C, 2 h and 20 g. More importantly, the OSBC-PVDF membrane has withstood the test of 300 days of continuous operation. The stable water quality of the effluent demonstrated that the above issues had been overcome.

Random mutagenesis and disulfide bond formation improved thermostability in microbial transglutaminase

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T50 of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (kcat/Km) at 37.0 °C was increased from 3.3 × 102 M−1 s−1 for the wild type to 5.9 × 102 M−1 s−1. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency.Key points• Improved heat resistance is essential to broaden the application of MTG.• The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability.• X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.

Bisphenol F Synthesis from Formaldehyde and Phenol over Zeolite Y Extrudate Catalysts in a Catalyst Basket Reactor and a Fixed-Bed Reactor

The objective of this study was to evaluate the applicability of zeolite Y as a catalyst for producing bisphenol F (BPF) from phenol and formaldehyde. Catalyst extrudates were prepared by extrusion after adding pseudoboehmite sol (PS) and Ludox (Lu) as alumina and silica binders, respectively. The compressive strength of the catalyst extrudates increased with the addition of Ludox. However, the formaldehyde conversion decreased as more Ludox was used as a binder, resulting in a decrease in the yield of BPF. This decrease is attributed to the reduction in the total amount of acid sites caused by the addition of Ludox. In this study, the Y_PS5_Lu5 catalyst was selected as the most suitable for BPF synthesis. In the BPF synthesis over the Y_PS5_Lu5 catalyst in a catalyst basket reactor, the optimum reaction temperature was determined to be 110 °C. The effect of stirring speed on the yield of BPF was found to be negligible in the range of 200 rpm to 350 rpm. The spent catalyst was able to recover a specific surface area and reaction activity similar to those of a fresh catalyst through regeneration in an air atmosphere at 500 °C. When the Y_PS5_Lu5 extruded catalyst was used in a continuous reaction in a fixed-bed reactor, there was no noticeable deactivation of the catalyst at low space velocities of the reactants. However, when the space velocity was increased to 18.0 h−1, catalyst deactivation was clearly observed. This suggests that periodic regeneration of the catalyst is inevitable in a continuous reaction using the Y_PS5_Lu5 extruded catalyst.