Low Thermostability Research Articles

Thermomechanical properties of 3D-printed sand moulds using inorganic binder

Additive Manufacturing of 3D-printed sand cores using the binder jetting process is well-established in prototype manufacturing. Due to the rising focus on sustainability and the fact that printed cores are shifting to serial production, a transition from organic to inorganic binder systems is taking place. To ensure a stable casting process and reduce the scrap rate accurate simulation tools are required. However, a study about the thermomechanical properties and the anisotropy of 3D-printed sand cores has not yet been conducted. In this work the thermomechanical properties of 3D-printed sand cores in three different printing orientations using inorganic binder are given. In contrast to homogeneous materials like metals, the simulation of sand cores requires new material models due to the dependency to hydrostatic pressure. The Drucker-Prager soil plasticity model is used, and the parameters needed for the Drucker-Prager-Cap model until 500°C are analysed using the three-point-bending test, the indirect tensile test and the uniaxial compression test. In addition to these specific parameters, also general parameters required for mechanical simulation like the Young's modulus, the Poisson's ratio, the density and the thermal expansion coefficient are given. In comparison to the reference binder system for shot cores using inorganic binder, the 3D-printed cores showed a higher mechanical strength. In the tensile region due to the higher binder content and in the compressive region due to the higher AFS number. Furthermore, the binder system for printed cores showed a lower thermostability.

A Sewing Approach to the Fabrication of Eco/bioresorbable Electronics.

Eco/bioresorbable electronics represent an emerging class of technology defined by an ability to dissolve or otherwise harmlessly disappear in environmental or biological surroundings after a period of stable operation. The resulting devices provide unique capabilities as temporary biomedical implants, environmental sensors, and related systems. Recent publications report schemes to overcome challenges in fabrication that follow from the low thermostability and/or high chemical reactivity of the eco/bioresorbable constituent materials. Here, this work reports the use of high-speed sewing machines, as the basis for a high-throughput manufacturing technique that addresses many requirements for these applications, without the need for high temperatures or reactive solvents. Results demonstrate that a range of eco/bioresorbable metal wires and polymer threads can be embroidered into complex, user-defined conductive patterns on eco/bioresorbable substrates. Functional electronic components, such as stretchable interconnects and antennas are possible, along with fully integrated systems. Examples of the latter include wirelessly powered light-emitting diodes, radiofrequency identification tags, and temporary cardiac pacemakers. These advances add to a growing range of options in high-throughput, automated fabrication of eco/bioresorbable electronics.

Directed evolution of cholesterol oxidase with improved thermostability using error-prone PCR.

Cholesterol oxidase is industrially important as it is frequently used as a biosensor in food and agriculture industries and measurement of cholesterol. Although, most natural enzymes show low thermostability, which limits their application. Here, we obtained an improved variant of Chromobacterium sp. DS1 cholesterol oxidase (ChOS) with enhanced thermostability by random mutant library applying two forms of error-prone PCR (serial dilution and single step). Wild-type ChOS indicated an optimal temperature and pH of 70 ºC and pH 7.5, respectively. The best mutant ChOS-M acquired three amino acid substitutions (S112T, I240V and A500S) and enhanced thermostability (at 50°C for 5h) by 30%. The optimum temperature and pH in the mutant were not changed. In comparison to wild type, circular dichroism disclosed no significant secondary structural alterations in mutants. These findings show that error-prone PCR is an effective method for enhancing enzyme characteristics and offers a platform for the practical use of ChOS as a thermal-resistance enzyme in industrial fields and clinical diagnosis.

Total turnover number prediction of an aggregating biocatalyst: Amino ester hydrolase (AEH)

Amino ester hydrolase (AEH) from Xanthomonas campestris is a promising candidate for β-lactam synthesis but suffers from low thermostability and rapid deactivation. Wild-type AEH was found to deactivate with an apparent order that varies with temperature from n = 2 at 30 °C (close to the temperature of optimum activity Topt) to n = 1.5 at 25 °C. AEH features two transitional temperatures: (1) from native ‘N’ to partially unfolded, inactive intermediate ‘I’, and (2) from partially unfolded to fully unfolded entity ‘U’ at the melting temperature Tm. CD and light scattering data suggest aggregation near Topt. To determine the total turnover number TTN, AEH was deactivated by imposing a temperature gradient and recording instantaneous rates of cephalexin hydrolysis. The TTN increased ∼ 5-fold from the wild-type (WT) for the quadruple variant N186D/A275P/E143H/V622I (QVH) at 25 °C and 10 nM AEH but varied significantly with AEH concentration and temperature.

Simultaneous enhancement of thermostability and catalytic activity of κ-carrageenase from Pseudoalteromonas tetraodonis by rational design

κ-Carrageenase provides an attractive enzymatic approach to preparation of κ-carrageenan oligosaccharides. Pseudoalteromonas tetraodonis κ-carrageenase is active at the alkaline conditions but displays low thermostability. To further improve its enzymatic performance, two mutants of Q42V and I51H exhibiting both improved thermostability and enzyme activity were screened by the PoPMuSiC algorithm. Compared with the wild-type κ-carrageenase (WT), Q42V and I51H increased the enzyme activity by 20.9% and 25.4%, respectively. After treatment at 50 ℃ for 40 min, Q42V and I51H enhanced the residual activity by 31.1% and 25.9%, respectively. The Tm values of Q42V, I51H, and WT determined by differential scanning calorimetry were 58.2 ℃, 54.8 ℃, and 51.2 ℃, respectively. Compared with untreated and HCl-treated κ-carrageenans, Q42V-treated κ-carrageenan exhibited higher pancreatic lipase inhibitory activity. Molecular docking analysis indicated that the additional pi-sigma force and hydrophobic interaction in the enzyme-substrate complex could account for the increased catalytic activity of Q42V and I51H, respectively. Molecular dynamics analysis indicated that the improved thermostability of mutants Q42V and I51H could be attributed to the less structural deviation and the flexible changes of enzyme conformation at high temperature. This study provides new insight into κ-carrageenase performance improvement and identifies good candidates for their industrial applications.

Extraction Optimization and Physical Modification of Fish Gelatin from Thunnus tonggol Using Ultraviolet (UV) Irradiation

ABSTRACT In this study, the yield of gelatin extraction from fish waste was optimized using response surface method. The highest yield was obtained with 0.8% citric acid concentration at 72.95°C for 58 minutes. Then, fish gelatin was modified using ultraviolet radiation to overcome fish gelatin defects including unsuitable rheological properties and low thermo-stability. UV irradiation can create crosslinks via alkyl or phenyl groups of the proteins. The modified sample also showed the highest melting point due to the creation of cross-links and improved emulsification and foaming properties of fish gelatin through structural modification and increased surface hydrophobicity of gelatin.

Three Molecular Modification Strategies to Improve the Thermostability of Xylanase XynA from Streptomyces rameus L2001

Glycoside hydrolase family 11 (GH11) xylanases are the preferred candidates for the production of functional oligosaccharides. However, the low thermostability of natural GH11 xylanases limits their industrial applications. In this study, we investigated the following three strategies to modify the thermostability of xylanase XynA from Streptomyces rameus L2001 mutation to reduce surface entropy, intramolecular disulfide bond construction, and molecular cyclization. Changes in the thermostability of XynA mutants were analyzed using molecular simulations. All mutants showed improved thermostability and catalytic efficiency compared with XynA, except for molecular cyclization. The residual activities of high-entropy amino acid-replacement mutants Q24A and K104A increased from 18.70% to more than 41.23% when kept at 65 °C for 30 min. The catalytic efficiencies of Q24A and K143A increased to 129.99 and 92.26 mL/s/mg, respectively, compared with XynA (62.97 mL/s/mg) when using beechwood xylan as the substrate. The mutant enzyme with disulfide bonds formed between Val3 and Thr30 increased the t1/260 °C by 13.33-fold and the catalytic efficiency by 1.80-fold compared with the wild-type XynA. The high thermostabilities and hydrolytic activities of XynA mutants will be useful for enzymatic production of functional xylo-oligosaccharides.

Trends in in-silico guided engineering of efficient polyethylene terephthalate (PET) hydrolyzing enzymes to enable bio-recycling and upcycling of PET

Polyethylene terephthalate (PET) is the largest produced polyester globally, and less than 30% of all the PET produced globally (∼6 billion pounds annually) is currently recycled into lower-quality products. The major drawbacks in current recycling methods (mechanical and chemical), have inspired the exploration of potentially efficient and sustainable PET depolymerization using biological approaches. Researchers have discovered efficient PET hydrolyzing enzymes in the plastisphere and have demonstrated the selective degradation of PET to original monomers thus enabling biological recycling or upcycling. However, several significant hurdles such as the less efficiency of the hydrolytic reaction, low thermostability of the enzymes, and the inability of the enzyme to depolymerize crystalline PET must be addressed in order to establish techno-economically feasible commercial-scale biological PET recycling or upcycling processes. Researchers leverage a synthetic biology-based design; build, test, and learn (DBTL) methodology to develop commercially applicable efficient PET hydrolyzing enzymes through 1) high-throughput metagenomic and proteomic approaches to discover new PET hydrolyzing enzymes with superior properties: and, 2) enzyme engineering approaches to modify and optimize PET hydrolyzing properties. Recently, in-silico platforms including molecular mechanics and machine learning concepts are emerging as innovative tools for the development of more efficient and effective PET recycling through the exploration of novel mutations in PET hydrolyzing enzymes. In-silico-guided PET hydrolyzing enzyme engineering with DBTL cycles enables the rapid development of efficient variants of enzymes over tedious conventional enzyme engineering methods such as random or directed evolution. This review highlights the potential of in-silico-guided PET degrading enzyme engineering to create more efficient variants, including Ideonella sakaiensis PETase (IsPETase) and leaf-branch compost cutinases (LCC). Furthermore, future research prospects are discussed to enable a sustainable circular economy through the bioconversion of PET to original or high-value platform chemicals.

Light-up split aptamers: binding thermodynamics and kinetics for sensing.

Due to their programmable structures, many aptamers can be readily split into two halves while still retaining their target binding function. While split aptamers are prevalent in the biosensor field, fundamental studies of their binding are still lacking. In this work, we took advantage of the fluorescence enhancement property of a new aptamer named OTC5 that can bind to tetracycline antibiotics to compare various split aptamers with the full-length aptamer. The split aptamers were designed to have different stem lengths. Longer stem length aptamers showed similar dissociation constants (Kd) to the full-length aptamer, while a shorter stem construct showed an 85-fold increase in Kd. Temperature-dependent fluorescence measurements confirmed the lower thermostability of split aptamers. Isothermal titration calorimetry indicated that split aptamer binding can release more heat but have an even larger entropy loss. Finally, a colorimetric biosensor using gold nanoparticles was designed by pre-assembling two thiolated aptamer halves, which can then link gold nanoparticles to give a red-to-blue color change.

Gold Nanozymes: Smart Hybrids with Outstanding Applications

Nanozymes are nanostructured artificial enzymes that have attracted great attention among researchers because of their ability to mimic relevant biological reactions carried out by their natural counterparts, but with the capability to overcome natural enzymes’ drawbacks such as low thermostability or narrow substrate scope. The promising enzyme-like properties of these systems make nanozymes excellent candidates for innovative solutions in different scientific fields such as analytical chemistry, catalysis or medicine. Thus, nanozymes with different type of activities are of special interest owing to their versatility since they can reproduce several biological reactions according to the substrates and the environmental conditions. In this context, gold-based nanozymes are a representative example of multifunctional structures that can perform a great number of enzyme-like activities. In addition, the combination of gold-based materials with structures of organic and inorganic chemical nature yields even more powerful hybrid nanozymes, which enhance their activity by providing improved features. This review will carry out a deep insight into gold-based nanozymes, revisiting not only the different type of biological enzymatic reactions that can be achieved with these kinds of systems, but also structural features of some of the most relevant hybrid gold-based nanozymes described in the literature. This literature review will also provide a representative picture of the potential of these structures to solve future technological challenges.

Creation of Highly Dispersed Supported Polyoxometalate Cesium Salts via Cation Exchange from Alkylammonium Salts on Supports

AbstractWe have reported several precisely designed metal‐oxo clusters on lacunary polyoxometalate (POM) tetra‐n‐butylammonium (TBA) salts in organic solvents. However, owing to their low thermostability and/or low effective surface area, POM TBA salts are not amenable to heterogeneous catalysts for gas‐phase reactions at high temperature. To avoid this problem, we developed a new preparation method that obtains highly dispersed supported‐POM Cs salts (Cs−POM/support) via cation exchange from the corresponding TBA salts on supports. In this study, a model Keggin‐type POM called TBA4PVMo11O40 (TBA−PVMo11) is supported on oxides (TBA−PVMo11/support) using an incipient wetness method in acetonitrile. This TBA−PVMo11/support was cation‐exchanged with cesium trifluoromethanesulfonate in ethanol to obtain supported‐POM Cs salts (Cs−PVMo11/support). Various characterizations revealed that the TBA−PVMo11/support maintained the original Keggin structure during cation exchange and that Cs−PVMo11 was highly dispersed on the supports. Furthermore, Cs−PVMo11/support maintained the Keggin structure even after calcination at 573 K under an aerobic condition.

Loop optimization of Trichoderma reesei endoglucanases for balancing the activity–stability trade‐off through cross‐strategy between machine learning and the B‐factor analysis

AbstractTrichoderma reesei endoglucanases (EGs) have limited industrial applications due to its low thermostability and activity. Here, we aimed to improve the thermostability of EGs from T. reesei without reducing its activity counteracting the activity–stability trade‐off. A cross‐strategy combination of machine learning and B‐factor analysis was used to predict beneficial amino acid substitution in EG loop optimization. Experimental validation showed single‐site mutated EG concomitantly improved enzymatic activity and thermal properties by 17.21%–18.06% and 49.85%–62.90%, respectively, compared with wild‐type EGs. Furthermore, the mechanism explained mutant variants had lower root mean square deviation values and a more stable overall structure than the wild type. According to this study, EGs loop optimization is crucial for balancing the activity–stability trade‐off, which may provide new insights into how loop region function interacts with enzymatic characteristics. Moreover, the cross‐strategy between machine learning and B‐factor analysis improved superior enzyme activity–stability performance, which integrated structure‐dependent and sequence‐dependent information.

Analysis of lytic polysaccharide monooxygenase activity in thermophilic fungi by high-performance liquid chromatography-refractive index detector.

Most current methods for analysing the activity of LPMO are based on the quantification of H2O2, a side product of LPMO; however, these methods cannot assay the LPMO activity of thermophilic fungi because of the low thermostability of H2O2. Therefore, we present a high-performance liquid chromatography-refractive index detector (HPLC-RID) method to assay the LPMO activity of the thermophilic fungus Thermoascus aurantiacus. According to the established method, the specific activities of nTaAA9A C1 and C4 oxidation were successfully analysed and were 0.646 and 0.574 U/mg, respectively. By using these methods, we analyzed the C1 and C4 oxidation activities of the recombinant TaAA9A (rTaAA9A) and mutated rTaAA9A (Y24A, F43A, and Y212A) expressed in Pichia pastoris. The specific activities of rTaAA9A C1 and C4 oxidation were 0.155 and 0.153 U/mg, respectively. The specific activities of Y24A, F43A, and Y212A C1 and C4 oxidation were 0.128 and 0.125 U/mg, 0.194 and 0.192 U/mg, and 0.097 and 0.146 U/mg, respectively. In conclusion, the method can assay the LPMO activity of thermophilic fungi and directly target C1 and C4 oxidation, which provides an effective activity assay method for LPMOs of thermophilic fungi.

Fully rosin-based epoxy vitrimers with high mechanical and thermostability properties, thermo-healing and closed-loop recycling

Bio-massed epoxy vitrimers are significant for sustainable industrial development, however, most of them show low mechanical properties and thermostability. The rosin derivates with rigid hydrophenanthrene framework have been synthesized as epoxy monomer and curing agent, for preparation of fully rosin-based epoxy vitrimer networks with dynamic β-hydroxy ester bonds. Meanwhile, to further improve mechanical properties and thermostability of the networks, zinc acetylacetonate was coordinated with the network by interaction of zinc metal with carbonyl group in the networks. The obtained fully rosin-based epoxy vitrimers have been characterized by FTIR, SEM, and DMA measurements. The obtained networks possess the higher mechanical properties (tensile strength = 40.8 MPa, toughness = 10.77 MJ/m3) and thermostability (T5d = ∼270 °C) than these of vegetable oil-based epoxy vitrimers. Moreover, the performances of thermo-healing, welding, shape memory, physical and chemical recycling have been investigated. Present results demonstrate obtained epoxy vitrimers with good functionalities. In addition, fully rosin-based epoxy vitrimers display an excellent degradability under ethylene glycol solvent and can be applied as environmentally friendly materials.

A single-phase white-emitting La(BO3,PO4):Dy3+ phosphor with high thermostability

Developing single-phase white-emitting phosphor has increasingly become an effective strategy to solve the emerging challenges of multiple phosphors-converted white light-emitting diodes such as unavoidable reabsorption, high production cost, and low thermostability. In this work, a single-phase white-emitting La(BO3,PO4):Dy3+ phosphor was synthesized. The photoluminescence performance characterizes the two strong visible emissions of Dy3+: blue (470–500 nm, 4F9/2 → 6H15/2) and yellow lights (570–600 nm, 4F9/2 → 6H13/2). The content quenching of Dy3+ presents at x = 0.03 mol, which is controlled by the dipole–dipole interaction. The optimized product La0.97Dy0.03(BO3,PO4) has a high thermostability. It has also the comparable color coordinates with the ideal white light due to the proper yellow-blue intensity ratio. The above studies confirm that the Dy3+-activated La(BO3,PO4) phosphor could be a prospective white-emitting candidate in lighting application.

Characterization and Preliminary Application of a Novel Lipoxygenase from Enterovibrio norvegicus.

Lipoxygenases have proven to be a potential biocatalyst for various industrial applications. However, low catalytic activity, low thermostability, and narrow range of pH stability largely limit its application. Here, a lipoxygenase (LOX) gene from Enterovibrio norvegicus DSM 15893 (EnLOX) was cloned and expressed in Escherichia coli BL21 (DE3). EnLOX showed the catalytic activity of 40.34 U mg−1 at 50 °C, pH 8.0. Notably, the enzyme showed superior thermostability, and wide pH range stability. EnLOX remained above 50% of its initial activity after heat treatment below 50 °C for 6 h, and its melting point temperature reached 78.7 °C. More than 70% of its activity was maintained after incubation at pH 5.0–9.5 and 4 °C for 10 h. In addition, EnLOX exhibited high substrate specificity towards linoleic acid, and its kinetic parameters of Vmax, Km, and Kcat values were 12.42 mmol min−1 mg−1, 3.49 μmol L−1, and 16.86 s−1, respectively. LC-MS/MS analysis indicated that EnLOX can be classified as 13-LOX, due to its ability to catalyze C18 polyunsaturated fatty acid to form 13-hydroxy fatty acid. Additionally, EnLOX could improve the farinograph characteristics and rheological properties of wheat dough. These results reveal the potential applications of EnLOX in the food industry.

Versatile Copolymer for Stretchable and Self-healable Liquid-free Ionic Conductive Elastomers.

To facilitate the practical use of ionic conductive materials for flexible electronics, the issues existing in hydrogels and ionogels, such as low thermostability and possible solvent leakage, need to be resolved but are inevitable. Liquid-free ionic elastomers (ICEs) as an alternative option are free of such concerns but have been facing the drawbacks of low conductivity and less satisfying mechanical properties. Here, a versatile copolymer with π-π stacking and cation-π interactions for high-performance ICE is proposed. The ICEs presented tunable mechanical and electrical properties by varying the feed ratio of the ternary monomers. The optimized ICE possessed high stretchability and strength, fast shape-recovery, self-healing, decent conductivity, and desirable stability against heat and under ambient conditions. The use of virgin and self-healed ICEs as the conductors for dielectric elastomer actuators (DEA) is demonstrated and exhibits comparable actuating performance to the reported DEA employing organogels and ionogels. The work provides a facile approach for fabricating ICEs with versatile properties that can be used for flexible electronics.

Synthesis and properties of photochromic polymer contain spiro-oxazine induced by ultraviolet light

ABSTRACT The special photochromic mechanism of spiro-oxazine (SO) refers to low thermostability and weak fatigue resistance. High light transmittance and biocompatibility are the two advantages of polymethyl methacrylate (PMMA), but it does not perform well in terms of tensile strength. Here, a new type of SO (1-(4'methacryloyloxybutyl)- 3,3-dimethyl-9'-hydroxy- 3H-spiro-naphthyl oxazine) was synthesized and introduced as a photochromic monomer into methyl methacrylate (MMA) system, which is an environment-friendly method. Compared with other photochromic polymers that have been reported, the advantages of this material mainly perform in the following areas: (1) The active group was introduced into the N-alkyl chain of SO and copolymerized with PMMA, and the fading rate of the photochromic polymer was dropped by 76.3% compared to SO. (2) Physical and chemical methods were used to improve the flexibility of PMMA-SO. The tensile strength of polymers has been reduced to 3.92 MPa. It also has a high elongation of 269.84% at a break. (3) PMMA-SO still has a significant fatigue resistance after 25 cycles of UV and visible light irradiation. In the future, it has potential applications in the fields of smart glass, data storage, biomimetic materials, and so on.

Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags.

1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.

Engineering and linker-mediated co-immobilization of carotenoid cleavage oxygenase with phenolic acid decarboxylase for efficiently converting ferulic acid into vanillin

The artificial cascade system with a coenzyme-independent decarboxylase and oxygenase was a very attractive approach for bioconversion of ferulic acid into vanillin. However, its potential was impeded by the low activity and thermostability of oxygenase. In this study, a combined strategy including the protein engineering of a carotenoid cleavage oxygenase from Thielavia terrestris (TtCCO) and its linker-mediated co-immobilization with a decarboxylase from Bacillus atrophaeus (BaPAD) was explored with aims to improve the catalytic efficiency and robustness of this artificial cascade system. We proved that engineering the enzyme by inserting an extra short peptide between E450 to E476 greatly affected TtCCO substrate specificity, catalytic activity, and thermostability. Among three variants, TtCCO2 displayed better thermostability and had 1.4-, 55- and 1.3-fold higher activity than TtCCO on 4-vinylguaiacol, resveratrol, and isoeugenol, respectively. The excellent protein and enzyme activity yield of immobilization was achieved by linker-mediated immobilization of 4lp-TtCCO2 @ mesoporous Y zeolite. A 100% conversion rate of ferulic acid and a 55% of the molar conversion yield of vanillin were obtained by co-immobilized 4lp-TtCCO2/4lp-BaPAD@mesoporous Y zeolite. This work demonstrated an effective engineering strategy for more efficient and robust oxygenases and the potential of this bi-enzyme cascade system for converting ferulic acid into vanillin.