ABSTRACT
Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.
Subject(s)
Thermomonospora , Actinomycetales , Escherichia coli/genetics , PolyhydroxyalkanoatesABSTRACT
Zearalenone is one of the most widely polluted Fusarium toxins in the world, seriously endangering livestock and human health. Zearalenone hydrolase (ZHD) derived from Clonostachys rosea can effectively degrade zearalenone. However, the high temperature environment in feed processing hampers the application of this enzyme. Structure-based rational design may provide guidance for engineering the thermal stability of enzymes. In this paper, we used the multiple structure alignment (MSTA) to screen the structural flexibility regions of ZHD. Subsequently, a candidate mutation library was constructed by sequence conservation scoring and conformational free energy calculation, from which 9 single point mutations based on residues 136 and 220 were obtained. The experiments showed that the thermal melting temperature (Tm) of the 9 mutants increased by 0.4-5.6 ℃. The S220R and S220W mutants showed the best thermal stability, the Tm of which increased by 5.6 ℃ and 4.0 ℃ compared to that of the wild type. Moreover, the thermal half-inactivation time at 45 ℃ were 15.4 times and 3.1 times longer, and the relative activities were 70.6% and 57.3% of the wild type. Molecular dynamics simulation analysis showed that the interaction force at and around the mutation site was enhanced, contributing to the improved thermal stability of ZHD. The probability of 220-K130 hydrogen bond of the mutants S220R and S220W increased by 37.1% and 19.3%, and the probability of K130-D223 salt bridge increased by 30.1% and 12.5%, respectively. This work demonstrated the feasibility of thermal stability engineering strategy where the structural and sequence alignment as well as free energy calculation of natural enzymes were integrated, and obtained ZHD variants with enhanced thermal stability, which may facilitate the industrial application of ZHD.
Subject(s)
Humans , Hydrolases , Zearalenone , Trichothecenes , Gene Library , Hydrogen BondingABSTRACT
Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (Tm) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.
Subject(s)
Plastics/metabolism , Polyethylene Terephthalates/metabolism , Biodegradation, Environmental , MetagenomeABSTRACT
@#ObjectiveTo identify the sites of thermal stability at Loop structure of xylanase Xyn ASP in Aspergillus saccharolyticus JOP 1030-1 and improve the thermal stability.MethodsThe amino acid sites related to thermal stability of xylanase were predicted,and beneficial mutation sites at Loop structure were screened by Fireprot online server.Singlepoint mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and double-point mutants Xyn(T81Q)and double-point mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(T81Q)retained 37.7%,Xyn(T81Q)retained 37.7%,Xyn(A79Y)and Xyn(A79Y)and Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)at pH 3.0～8.0 showed no significant change compared with the wild type.ConclusionSite-directed mutagenesis(A79Y and T81Q)was carried out at Loop structure,and xylanase mutants with obviously improved thermal stability were obtained,which laid a foundation of the later research on the structure,function and relationship of the enzyme and its industrial application.
ABSTRACT
Objective:To prepare camel-derived nanoantibodies that can bind to the recombinant protein VirB12 antigen with high affinity and lay the foundation for further research.Methods:Xinjiang Bactrian camels were immunized six times with VirB12 recombinant protein, total RNA was extracted from lymphocytes isolated from peripheral blood, and the VHH gene fragment was amplified by nested PCR to construct a phage VHH display library. ELISA solid-phase affinity and enrichment methods were used for screening. After three rounds of affinity screening, the clones enriched in the second and third rounds were randomly picked out, and the binding of a nanoantibody with soluble expression to VirB12 was analyzed by ELISA. After sequence determination and multiple alignment, repetitive sequences were removed, and finally five non-redundant sequences were obtained, which were named D1, E6, H8, H9, and H10. The five identified nanoantibody genes were transformed into the WK6 strain, and the soluble expression of an intercellular substance was carried out at 16 °C. After purified expression of Ni-NTA, the binding ability and thermal stability of nanoantibodies and the antigen VirB12 protein were detected by Western Blot and ELISA.Results:Five strains of nanoantibodies were expressed in WK6 bacteria in soluble form. SDS-PAGE showed that the purity of five anti-VirB12 nanoantibodies was close to 90%, and they had high antigen-binding activity and obvious antigen-antibody concentration dependence. All four strains of nanoantibodies showed high thermal stability, and after being treated at 90 ℃, they could still retain more than 60% binding activity.Conclusions:In the study, a VHH phage display library with a capacity of 2.8×10 8 cfu/ml was constructed from Xinjiang Bactrian camel lymphocytes immunized with VirB12 recombinant protein. Five anti-VirB12 nanoantibodies with high affinity and thermal stability were obtained through solid-phase screening and enrichment and soluble monoclonal ELISA detection. These results laid the foundation for further development of VirB12 nanoantibodies.
ABSTRACT
Proteus mirabilis lipase (PML) features tolerance to organic solvents and great potential for biodiesel synthesis. However, the thermal stability of the enzyme needs to be improved before it can be used industrially. Various computational design strategies are emerging methods for the modification of enzyme thermal stability. In this paper, the complementary algorithm-based ABACUS, PROSS, and FoldX were employed for positive selection of PML mutations, and their pairwise intersections were further subjected to negative selection by PSSM and GREMLIN to narrow the mutation library. Thereby, 18 potential single-point mutants were screened out. According to experimental verification, 7 mutants had melting temperature (Tm) improved, and the ΔTm of K208G and G206D was the highest, which was 3.75 ℃ and 3.21 ℃, respectively. Five mutants with activity higher than the wild type (WT) were selected for combination by greedy accumulation. Finally, the Tm of the five-point combination mutant M10 increased by 10.63 ℃, and the relative activity was 140% that of the WT. K208G and G206D exhibited certain epistasis during the combination, which made a major contribution to the improvement of the thermal stability of M10. Molecular dynamics simulation indicated that new forces were generated at and around the mutation sites, and the rearrangement of forces near G206D/K208G might stabilize the Ca2+ binding site which played a key role in the stabilization of PML. This study provides an efficient and user-friendly computational design scheme for the thermal stability modification of natural enzymes and lays a foundation for the modification of PML and the expansion of its industrial applications.
Subject(s)
Enzyme Stability , Lipase/chemistry , Molecular Dynamics Simulation , Proteus mirabilis/metabolism , Solvents/chemistryABSTRACT
The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including (NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance (BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLIS_SXAU06. BLIS_SXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLIS_SXAU06 was heterologously expressed in E. coli and the recombinant BLIS_SXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.
Subject(s)
Animals , Anti-Bacterial Agents/pharmacology , Bacillus licheniformis , Bacteriocins/pharmacology , China , Chromatography, Liquid , Escherichia coli/genetics , Listeria monocytogenes , RNA, Ribosomal, 16S , Staphylococcus aureus , Tandem Mass SpectrometryABSTRACT
Nanocapsuling organophosphorus hydrolase (OPH) is a promising strategy, which can improve OPH stability and put it into practical application. Nanocapsule can protect OPH from deactivation, but on the other hand it may block the contact of enzymes and substrates to some extent. Thus, it is worth exploring the influences of nanocapsule density on the enzyme activity and stability. In this study, OPH surface was modified by N-acryloxysuccinimide (NAS), and then the in situ radical polymerization technique was applied to construct a polymer shell around the surface to form the OPH nanocapsule (nOPH). Transmission electron microscope (TEM) and Fourier Transform Infrared (FTIR) was used to characterize the structure of nOPH. The nOPH activity is not influenced by the presence of nanocapsule when the feed ratio of NAS to OPH is below 75. On the contrary, it decreases with the increasing of NAS feed ratio when it is above 75. Furthermore, nOPH activities at high temperatures and in 20% DMSO solutions both first increase and then decrease with the increasing of NAS feed ratio. The results showed that the appropriate density of the nanocapsule could retain the enzyme activity to the maximum extent during the preparation of nOPH nanocapsule, and significantly improve its thermal stability and organic solvent stability. Hence, the results are of great significance to further realize the OPH practical application.
ABSTRACT
Petite integration frequency 1 (PIF1) helicases are ubiquitous enzymes which play vital roles in nearly all DNA metabolic processes. In recent years, the biochemical activity and three-dimensional structure of several PIF1 helicases have been reported, but there are few reports on the PIF1 helicase of bacteria living in extreme environments. In this paper, a series of biochemical and biophysical techniques were used to study the Thermodesulfovibrio yellowstonii PIF1 (Ty.PIF1) helicase in many aspects. Ty. PIF1 was obtained with a purity of over 90% and good uniformity using the prokaryotic expression and purification system. Ty.PIF1 is a monomer with a calculated molecular weight of 60 kD in solution. Ty. PIF1 has high thermal stability. The secondary structure remains stable when the temperature is below 65 ℃, and the secondary structure changes only when the temperature is above 70 ℃. The optimal unwinding temperature of Ty.PIF1 in vitro is 45 ℃, which is not the optimal temperature for the survival of thermodesulfovibrio yellowstonii. It indicates that when Ty.PIF1 exerts its enzymatic activity in vivo, it may require the participation of other cofactors. Ty.PIF1 can exert unwinding activity in a wide temperature range (20-55 ℃), and the presence of enzyme activity at 55 ℃ indicates that Ty.PIF1 has heat-resistant properties. Ty.PIF1 prefers to bind to substrates containing ssDNA, but there is certain requirement for the length of the ssDNA, which is at least 4 nt in length. Ty.PIF1 can also bind to the G
ABSTRACT
In Sub-Saharan Africa, Lagenaria siceraria is an oleaginous cucurbit widely consumed for its edibleseeds. These seeds were previously categorized as good enzyme sources, including four non-specific acidphosphatases isolated and designated as BLsAP1, BLsAP2, RLsAP1, and RLsAP2. In this study, we investigateon the kinetic and thermodynamic characteristics of these biocatalysts in order to evaluate their thermostability.Thermal inactivation was carried out in the temperature range of 55°C to 80°C from 5 to 60 minutes. The resultsrevealed that thermal inactivation of studied acid phosphatases follows first order kinetics. At their optimumtemperatures, these enzymes showed high half-lives ranging from 169.06 to 495.10 minutes and D valuesfrom 561.71 to 1,645.03 minutes. Moreover, they exhibited high activation energies (from 155.08 to 200.55kJ/mol) and average enthalpy values (from 152.23 to 197.74 kJ.mol−1) suggesting their good thermostability.The comparison of all these values revealed that the two acid phosphatases (RLsAP1 and RLsAP2) from theround-fruited cultivar of L. siceraria showed better thermal stability than those (BLsAP1 and BLsAP2) fromthe blocky-fruited cultivar.
ABSTRACT
Objective: The objective of the present investigation was to develop olive and soybean oil-based oleogels with Span 40 and/or Tween 80 (as gelator and/or surfactant) and determine the critical gelator concentration (CGC), characterise and compare the rheological, thermal properties and drug release profile of the gels formed for topical delivery. Methods: Olive and soybean oil-based Span 40 and Span 40/Tween 80 oleogel formulations were prepared by solid fiber mechanism and subjected to organoleptic evaluation, FT-IR spectroscopy, thermal analysis, rheological study, kinetic modeling of gelation and drug release. Results: The critical gelator (Span 40) concentration was found to be lower for olive oil (12% w/v) and depend on the type of oil. Tween 80 reduced CGC of soybean oleogels only. Soybean oil-based oleogel containing 18% w/v Span 40 was found to form more flexible, less viscous and thermally less stable formulation with better release of paracetamol as evident from lower melt flow index, Tg value, lower β and higher α value compared to olive oil-based oleogel with 12% w/v Span 40 (CGC). Surfactant addition can be assumed to modify the microarchitecture of the oleogels to a great extent to produce more flexible and thermally stable gels with even better drug release profile. Span-Tween based soybean oleogel formed a gel-matrix whereas matrix in olive oil-based oleogels containing Span only became slightly flexible to release the drug in zero-order fashion on the addition of surfactant cogelator. Conclusion: Nature of oil exerts profound influence on the rheological, thermal and release profile of oleogels containing Span 40 as gelator and/or Tween 80 as surfactant cogelator.
ABSTRACT
SUMMARY Some new pyrimidine derivatives have been synthesized and their decomposition characteristics have been studied by thermogravimetric and differential scanning calorimetric analysis. The thermal stability and some kinetics parameters of decomposition were evaluated from thermograms. It is observed that depending upon the structure, substitutions, thermal stability and decomposition kinetics varies in different compounds.
RESUMEN Se sintetizaron algunos nuevos derivados de pirimidina y se estudiaron sus características de descomposición mediante análisis termogravimétrico y calorimétrico diferencial de barrido. La estabilidad térmica y algunos parámetros cinéticos de descomposición se evaluaron a partir de los respectivos termogramas. Se observa que, dependiendo de la estructura y las sustituciones, la estabilidad térmica y la cinética de descomposición varían entre los diferentes compuestos.
ABSTRACT
New hydrogels based on (MAA) and (NVP) copolymers crosslinked with (BAA), were prepared by free radical cross-linkingcopolymerization, with a NVP percent molar composition of 10, 47.5 and 85. These hydrogels have been characterized by(FTIR), (SEM), (TGA /DSC) coupling. The results show four steps of degradation. The degradation rate is inverselyproportional to the mole percent of NVP, and SEM shows that the hydrogels have a pore size between 7.14 to 13.33 μm.
ABSTRACT
Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
Subject(s)
Escherichia coli , Green Fluorescent Proteins , Hydrophobic and Hydrophilic Interactions , Peptides , Pichia , Recombinant Fusion Proteins , MetabolismABSTRACT
OBJECTIVE: Thermal stability of Curcuma longa L. was studied by GC-MS- analysis. The affect to Curcuma longa L.by seven antioxidants were explored. METHODS: Volatile oil of Curcuma longa L. were extracted by steam extraction and heated at different temperature. The compounds of Curcuma longa L. were analysis by GC-MS. Volatile oil were heated in different temperature and the temperature which make volatile oil most obvious changing were selected. The volatile oil was heated at 100 ℃ in different time and the time which make volatile oil most obvious changing were selected. The affect to Curcuma longa L.by seven antioxidants were explored and the antioxidant which improved thermal stability of Curcuma longa L. was selected. RESULTS: The compounds of volatile oil from Curcuma longa L. was changed most significant by heated 8 h and heated at 20 ℃.The upward trend of α-turmerone was appropriate inhibited by thymol. The downward trend of ar-curcumen was better inhibited by tocopherol. CONCLUSION: The volatile oil from Curcuma longa L. shown obvious thermal instability. The volatility compounds from volatile oil of Curcuma longa L. was changed most by heated. The thermal stability of volatile oil from Curcuma longa L. can improve by antioxidants.
ABSTRACT
Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. T he molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and ßmercaptoethanol, but it was reduced by Cu+2 and Pb+2 . The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.
Subject(s)
Penicillium chrysogenum/metabolism , Polysaccharides/metabolism , Endo-1,4-beta Xylanases/biosynthesis , Temperature , Enzyme Stability , Biomass , Endo-1,4-beta Xylanases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , HydrolysisABSTRACT
Abstract Morchella esculenta (L) Pers. is a highly valued edible and medicinal fungus that remains underutilized. For this study, the effects of glycation treatment on antioxidant activity and characteristics of the M. esculenta protein isolate (MPI) were investigated via the Maillard reaction. Conjugation between MPI and xylose was proven via UV-vis, FT-IR, intrinsic fluorescence analysis, and SDS-PAGE. Amino acid analysis revealed involvement of lysine, arginine and tyrosine in MPI, forming a covalent cross-link with xylose. Differential scanning calorimetry (DSC) results showed that glycated MPI (MPIG) possesses a more favorable thermal stability compared to native MPI (MPIN), heated MPI (MPIH) and an unheated mixture of MPI and xylose (MPI-XM). MPIG exhibited significantly enhanced antioxidant activity compared to MPIN, MPIH, and MPI-XM. These results indicate MPIG can serve as a promising novel source of nutraceutical and functional ingredients that exert antioxidant activity.
ABSTRACT
Thalidomide was indicated as a sedative and antiemetic and prescribed for pregnant women. Its tragic teratogenic effects culminated in withdrawal from the market. Since the discovery of its anti-angiogenic and anti-inflammatory actions, thalidomide has been used in the treatment of leprosy and multiple myeloma, which justify studies of its stability. We investigated the effects of irradiation of thalidomide up to 100 kGy (fourfold the usual sterilizing dose for pharmaceutics). The β polymorph of thalidomide was obtained in an isothermal experiment at 270 °C. All samples underwent gamma irradiation for specific times. At different doses, decomposition of the pharmaceutical was not observed up to 100 kGy. The observed effect was angle turning between the phthalimide and glutarimide rings modulated by repulsion towards the carbonyl group, leading to a stable energetic configuration, as measured by the equilibrium in the torsion angle after irra-diation. The thalidomide molecule has a center of symmetry, so a full turn starting from 57.3° will lead to an identical molecule. Further irradiation will start the process again. Samples irradiated at 30 and 100 kGy have more compact unit cells and a lower volume, which leads to an increase in the intermolecular hydrogen interaction within the unit cel , resulting in higher thermal stability for polymorph α.
ABSTRACT
Glucose oxidase catalyzes the oxidation of β-D-glucose to gluconic acid and its derivatives, thus shows a great potential in the development of antibiotic-free feed. However, its production and processing still have the problem of poor thermal stability of enzyme activity. In this study, fusion of amphiphilic peptide technology was used to improve the stability of glucose oxidase. Herein, eight self-assembling peptides with different amino acid lengths and Linkers were fused to the N terminus of the glucose oxidase, yielding eight chimeric fusions SAP1-GS-GOD, SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD, SAP4-PT-GOD, SAP5-PT-GOD, SAP6-PT-GOD and SAP7-PT-GOD. Then, the 8 recombinant proteins were expressed in P. pastoris GS115. After separation and purification, the stability of glucose oxidase at 60 ℃was determined. The relative enzyme activities of the PT Linker-linked fusion enzyme incubated at 60 ℃ for 60 min were higher than those of the original enzyme, and the relative activity of SAP5-PT-GOD was 67% at 60 ℃ for 30 min, which was 10.9 times higher than that of the initial enzyme with the same treatment. Among them, the Kcat/Km value of SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD and SAP5-PT-GOD of the fusion enzyme was further improved than that of the initial enzyme. Through the analysis of the intramolecular force of the fusion enzyme, the increase of the thermal stability of the fusion enzyme is mainly due to the increase of the hydrogen bond. In summary, the study indicates that translational fusion of self-assembling peptides with PT Linker was able to augment the thermo-stability of glucose oxidase, which has certain potential in the production and application of glucose oxidase. The glucose oxidase with improved thermostability obtained in the above study and the related mechanism will play an important role in improving the activity of related enzymes in the proceeding of processing and application.
ABSTRACT
Se realizó un estudio in vitro donde se evaluaron once sistemas adhesivos, para establecer si las condiciones de trabajo clínico favorecen su eficiencia. Se plantea que los sistemas adhesivos autoacondicionantes presentan menor estabilidad térmica que los convencionales. Determinar el comportamiento térmico de once sistemas adhesivos sometidos a diferentes rangos de temperatura en ambientes con 100 % de humedad, agua acidulada, humedad ambiental, saliva artificial y la correlación con su composición. Se utilizaron adhesivos autograbantes y convencionales, se prepararon muestras en cápsulas de aluminio pequeñas y se fotopolimerizaron con una lámpara LED. Se les realizó el estudio de espectroscopía infrarrojo con transformada de Fourier (FT-IR), asignando las bandas de absorción de los grupos funcionales orgánicos de cada compuesto, correspondientes a los grupos químicos que poseen. Posteriormente fueron sometidas a examen termogravimétrico entre temperatura ambiente y 500 °C para establecer un patrón de comportamiento térmico en ambiente inerte y luego de permanecer en agua acidulada, 100 % humedad, humedad ambiental y saliva artificial. Los termogramas informaron las temperaturas de descomposición y los porcentajes de pérdida de masa. Se correlacionaron los resultados del estudio de FT-IR infrarrojo con el comportamiento térmico de los sistemas adhesivos. Se observó pérdida de masa, eficiencia de la polimerización y cantidad de masa residual. Se observaron dinámicas de degradación diferentes por el diseño de las curvas y por los cambios en la línea base. Conclusión: Los sistemas adhesivos de ambos grupos analizados, presentaron elevado grado de polimerización. Sin embargo, el efecto producido por las condiciones a las cuales fueron sometidos, depende del tipo de adhesivo, siendo que, los adhesivos convencionales, a diferencia de los autoacondicionantes, se degradan en menor extensión, reflejado por su mayor estabilidad térmica.
An in vitro study of eleven adhesive systems was carried out to establish if clinical work conditions could improve their efficiency. It has been observed that self-etch adhesive systems have less thermal stability than conventional ones. As a consequence, early replacement of unsatisfactory aesthetic restorations is needed. The objectives of this work were to determine the thermal behavior of eleven polymeric adhesive systems under different rates of temperatures and a variety of conditions such as 100 % humidity, presence of acidulated water (pH 3), environmental relative humidity and artificial saliva. Another aim was to establish if these factors are correlated with the adhesive systems composition. For each type of adhesive system, samples were prepared in small aluminum caps and polymerized with a LED lamp. All samples were analyzed with the Fourier-Transform Infrared Spectroscopy (FT-IR) method, which assigned absorption bands to organic functional groups of each compound corresponding to their chemical type. Subsequently, a thermogravimetric analysis was performed in a range temperature from room temperature to 500 °C in order to establish thermal behavior in an inert environment and after staying in acidulated water, 100 % humidity, environmental humidity and artificial saliva. Thermograms were obtained to collect data about decomposition temperatures and loss of mass percentages. The FT-IR study results were correlated with the adhesive systems thermal behavior. Thermogram images showed loss of mass, polymerization efficiency and residual mass amount. The different degradation dynamics were analyzed according to curve designs and baseline changes. Both groups of adhesive systems revealed high polymerization degrees. Nevertheless, the effect produced by the conditions in which they were subjected depends on the type of adhesive. Conventional adhesives, in contrast to self-etch adhesives, degraded in a minor extension as a result of their higher thermal stability.