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Site-directed mutagenesis was employed to investigate the structure-function relationship of nattokinase (NK) and its effect on the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK. Specific mutations (T270S, V271I, E262D, and A259T) were introduced within the nk gene, targeting regions predicted to be involved in substrate binding. The NK(E262D) mutant exhibited a significant increase in enzymatic activity (2-fold) and catalytic efficiency (2.2-fold) as assessed by N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis, compared to the wild type. In silico analysis supported these findings, demonstrating lower binding energy for the NK(E262D) mutant, suggesting stronger fibrin affinity. Thermostability assays revealed that NK(E262D) and NK(A259T) displayed exceptional stability, retaining enzyme activity at 60 °C. All mutants exhibited a broader pH tolerance range (pH 5.0–10.0) compared to the wild-type NK. The fibrinolytic activity assay revealed that the E262D mutant possessed the highest fibrinolytic activity (2414 U/mg), surpassing the wild-type. This study reported an NK variant with improved enzymatic activity, thermostability, and fibrinolytic properties. [Display omitted] • Nattokinase mutant generated. • Biochemical characterization of mutant and wild type nattokinase. • Enzyme kinetics carried out. • Clot lysis time determined. • E262D demonstrated enhanced proteolytic activity, fibrinolytic activity and thermostability. [ABSTRACT FROM AUTHOR]

Himachal Pradesh, a state of India, is the treasure of unique reservoirs in the structure of hot water springs. These hot water springs are the substantial unexplored waterbodies of microbial biodiversity on the Earth and have opportunities for industrial applications. The microorganisms living in these hot water springs have unique characteristics that help them survive under extreme environments like high-temperature conditions and are known as extremophiles. Extremophiles can grow and survive at high temperatures, low pH, highly saline conditions, low temperatures, etc., and produce industrially important enzymes and various thermostable proteins that can control metabolic and other biological activities and can perform under extremely adverse environments. The enzymes produced from extremophiles are highly thermostable and are acceptable for industrial applications. In this review, we mentioned the significance of thermostable microbes and explored the industrially important microbial diversity of thermal hot springs in Himachal Pradesh, India. In conclusion, hot water springs offer a rich, yet underutilized reservoir of extremophiles with considerable industrial potential. Advancing our understanding of these organisms and their habitats will not only enhance the efficiency and sustainability of industrial processes but also expand the scope of biotechnological applications. Future research and exploration are essential for unlocking the full potential of extremophiles from hot water springs. [ABSTRACT FROM AUTHOR]

Background: Protein instability has been reported to be an important cause of haze in white wines; to investigate the feasibility of protease as a substitute for bentonite in the protein stability of white grape liqueurs, the effect of protease combined with heat treatment on the composition and protein stability of liqueur wines was investigated by chemical analysis, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and amino acid analysis. Results: The results showed that the contents of total acid, volatile acid, pH, and glycerol were not significantly different from the blank control; the effect on total protein content and thermal stability were similar to the bentonite treatment; the L∗, b∗, and Cab∗ values of liqueur wines increased by 5.59%, 4.33%, and 4.33% after protease combined with heat treatment (p < 0.05); SDS‐PAGE results confirmed that protease combined with heat treatment had good hydrolysis for thaumatin‐like proteins (TLPs) and chitinases (CHIs) in liqueur wines. Amino acid analysis revealed that the levels of threonine, serine, tyrosine, arginine, and proline had increased, respectively, and isoleucine (1.26 mg/L) and leucine (1.16 mg/L) were newly detected after protease combined with heat treatment. Particle size analysis showed that the protease combined with heat treatment had less effect on the particle size distribution of the wines. Conclusion: Protease combined with heat treatment had a certain protein stabilizing effect and had no significant negative impact on other nutrients of liqueur wines. Thus, it can be used as an alternative to bentonite for the protein stabilization of liqueur wines. [ABSTRACT FROM AUTHOR]

Thermophilic microorganisms possess several adaptations to thrive in high temperature, which is reflected as biosynthesis of proteins and thermostable molecules, isolation and culture represent a great methodological challenge, therefore High throughput sequencing enables screening of the whole bacterial genome for functional potential, providing rapid and cost-effective information to guide targeted cultures for the identification and characterization of novel natural products. In this study, we isolated two thermophilic bacterial strains corresponding to Bacillus LB7 and Streptomyces LB8, from the microbial mats in the Atacama Desert. By combining genome mining, targeted cultures and biochemical characterization, we aimed to identify their capacity to synthesize bioactive compounds with antimicrobial properties. Additionally, we determined the capability to produce bioactive compounds under controlled in vitro assays and detected by determining their masses by Thin-Layer Chromatography/Mass Spectrometry (TLC/MS). Overall, both isolates can produce antimicrobial (e.g., Myxalamide C by-product) and antioxidants (e.g. Dihydroxymandelic Acid, Amide biotine and Flavone by-products) compounds. Bacillus LB7 strain possesses a more diverse repertoire with 51.95% of total metabolites unmatched, while Streptomyces LB8 favors mainly antioxidants, but has over 70% of unclassified compounds, highlighting the necessity to study and elucidate the structure of novel compounds. Based on these results, we postulate that the uncultured or rare cultured thermophiles inhabiting high-altitude hydrothermal ecosystems in the Atacama Desert offer a promising opportunity to the study of novel microbial bioactive compounds. [ABSTRACT FROM AUTHOR]

Introduction: Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. Methods: We expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Results: Of the tested proteins, thermal green protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. Discussion: These initial results in live protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms. [ABSTRACT FROM AUTHOR]

Background: The important challenge to the biotechnology is to find new effective fusion partners that enable to improve solubility, expression, and optimize the subsequent fine purification of the target protein. Results: The most invariant part of the most immunogenic region of the surface virulence factor A of Streptococcus pneumoniae was selected as a model target protein, while the thermostable chemotaxis polypeptide of W-type from Thermotoga petrophila was used as a fusion partner. The genes encoding fusion variants of these proteins were constructed and cloned into a plasmid vector under the control of the strong bacteriophage T7 transcription regulatory system. Effective Escherichia coli producer strains were obtained, and optimal conditions were chosen for the production of resulting constructs. The optimal pH and temperature ranges of recombinant proteins were determined, and three-dimensional shapes of their molecules were also predicted. Methods of low-stage protein purification were improved. Some of the isolated proteins demonstrated a high level of expression, solubility and purity. Conclusions: Novel chimeric proteins were obtained which had not previously been observed in nature in such domain combinations. It was shown that the biotechnologically valuable characteristics of the hybrid proteins were more marked when the thermal-resistant partner was combined with the Nterminus of pneumococcal protein. The principles of their low-stage purification were performed which does not require any special equipment. That is a basis for significant reduction of prices for diagnostic test-systems components and subunit vaccine production. How to cite: Grishin DV, Sidorov NG, Parfenova OK, et al. Construction, heterological expression and a simple purification of the BP region of the pneumococcal surface protein a fused in different orientations to the chemotaxis adaptor protein CheW from Thermotoga petrophila. Electron J Biotechnol 2024;71. [ABSTRACT FROM AUTHOR]

Thermal green protein Q66E (TGP-E) has previously shown increased thermal stability compared to thermal green protein (TGP), a thermal stable fluorescent protein produced through consensus and surface protein engineering. In this paper, we describe the protein crystal structure of TGP-E to 2.0 Å. This structure reveals alterations in the hydrogen bond network near the chromophore that may result in the observed increase in thermal stability. We compare the very stable TGP-E protein to the structure of a yellow mutant version of this protein YTP-E E148D. The structure of this mutant protein reveals the rationale for the observed low quantum yield and directions for future protein engineering efforts. [ABSTRACT FROM AUTHOR]

Green fluorescent protein has long been a favorite protein for demonstrating protein purification in the biochemistry lab course. The protein's vivid green color helps demonstrate to students the concept(s) behind affinity or ion exchange chromatography. We designed a series of introduction to biochemistry labs utilizing a thermostable green protein (TGP‐E) engineered to have unusually high thermostability. This protein allows students to proceed through purification and characterization without the need to keep protein samples on ice. The 5‐week lab series begins with an introduction to molecular biology techniques during weeks 1 and 2, where site‐directed mutagenesis is used introduce, a single nucleotide change that shifts the fluorescent spectra of TGP‐E to either cyan (CTP‐E) or yellow (YTP‐E). Students identify successful mutagenesis reaction by the color of a small expression sample after induction with IPTG. Next, students purify either the TGP‐E (control—typically one group volunteers), YTP‐E, or CTP‐E protein as a 1‐week lab. During the following week's lab, students run SDS‐PAGE to verify protein purity, bicinchoninic acid assay to quantify protein yield, and absorbance and fluorescence spectra to characterize their protein's fluorescent character. The final lab in the series investigates the thermostability of YTP‐E and CTP‐E compared with TGP‐E using a fluorescence plate reader. This 5‐week series of experiments provide students with experience in several key biochemistry techniques and allows the students to compare properties of mutations. At the end of the course, the students will write a research report and give a short presentation over their results. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) contamination is a food safety issue threatening human health globally. Biodegradation is an effective method for overcoming this problem, and many microorganisms have been identified as AFB1-degrading strains. However, the response mechanisms of these microbes to AFB1 remain unclear. More degrading enzymes, especially of new types, need to be discovered. In this study, a novel AFB1-degrading strain, DDC-4, was isolated using coumarin as the sole carbon source. This strain was identified as Bacillus halotolerans through physiological, biochemical, and molecular methods. The strain's degradation activity was predominantly attributable to thermostable extracellular proteins (degradation rate remained approximately 80% at 90 °C) and was augmented by Cu2+ (95.45% AFB1 was degraded at 48 h). Alpha/beta hydrolase (arylesterase) was selected as candidate AFB1-degrading enzymes for the first time as a gene encoding this enzyme was highly expressed in the presence of AFB1. Moreover, AFB1 inhibited many genes involved in the nucleotide synthesis of strain DDC-4, which is possibly the partial molecular mechanism of AFB1's toxicity to microorganisms. To survive under this stress, sporulation-related genes were induced in the strain. Altogether, our study identified a novel AFB1-degrading strain and explained its response mechanisms to AFB1, thereby providing new insights for AFB1 biodegradation. [ABSTRACT FROM AUTHOR]

Thermophilic cyanobacteria are prokaryotic photoautotrophic microorganisms capable of growth between 45 and 73 °C. They are typically found in hot springs where they serve as essential primary producers. Several key features make these robust photosynthetic microbes biotechnologically relevant. These are highly stable proteins and their complexes, the ability to actively transport and concentrate inorganic carbon and other nutrients, to serve as gene donors, microbial cell factories, and sources of bioactive metabolites. A thorough investigation of the recent progress in thermophilic cyanobacteria reveals a significant increase in the number of newly isolated and delineated organisms and wide application of thermophilic light-harvesting components in biohybrid devices. Yet despite these achievements, there are still deficiencies at the high-end of the biotechnological learning curve, notably in genetic engineering and gene editing. Thermostable proteins could be more widely employed, and an extensive pool of newly available genetic data could be better utilised. In this manuscript, we attempt to showcase the most important recent advances in thermophilic cyanobacterial biotechnology and provide an overview of the future direction of the field and challenges that need to be overcome before thermophilic cyanobacterial biotechnology can bridge the gap with highly advanced biotechnology of their mesophilic counterparts. Key points: • Increased interest in all aspects of thermophilic cyanobacteria in recent years • Light harvesting components remain the most biotechnologically relevant • Lack of reliable molecular biology tools hinders further development of the chassis [ABSTRACT FROM AUTHOR]

Protein structure has a direct impact on thermostability. Deviations in the primary sequence can affect structural changes, leading to alterations in thermostability properties. However, the molecular basis of protein thermostability is unspecified; thus, elucidation of key factors that role particular protein thermostability is required when engineering proteins to be thermostable. To address this challenge, the amino acid composition, hydrophobicity/hydrophilicity ratio, cysteine bridges, and intrinsic features of two structurally homologous but different thermostability, poly(ethylene terephthalate) hydrolase (PETase) were compared. According to the findings, thermostable and thermolabile PETases have similar folds, compactness, and disulfide bridges. Interestingly, an abundance gap of aromaticity, hydrophobic cluster area, polar amino acid and hydrogen bond network compositions demonstrated dominant trends of variations for both PET hydrolases, indicating a pivotal role of these features in the thermostability of PET hydrolase. Furthermore, increased hydrophobic amino acid frequency in the inner surface of thermostable proteins contributed significantly to thermostability by forming more internal hydrophobic interactions and a less hydrophobic patch. There are no consistent trends in insertions and deletions between both PETases. Taken together, these observations demonstrate that hydrophobicity and hydrogen bond networks are essential factors in thermostability of thermostable PETase. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA), a naturally occurring polysaccharide with recognized health benefits, has gained approval for use in the food industry as a food additive, ingredient, and health supplement in numerous countries. HA can increase viscosity in solutions and is available commercially in various molecular weights (MW) depending on end applications. Nevertheless, no research has explored the impact of different MW HAs on functionality, rheological properties, and texture-building benefits in the dairy product matrix wherein they are incorporated. Therefore, the objective of this study was to evaluate how varying MWs of HA—specifically 8 kDa, 320 kDa, 980 kDa, and 2550 kDa at 0.25% (w/w) concentration—impact rheological characteristics, functional attributes, heat stability, protein stability, protein structure, and protein fractions within skim milk. The addition of HA led to an increase in the apparent viscosity of all samples. A higher G″ value over G′ values for all HA samples was observed in frequency sweep, indicating the absence of interparticle interactions between HA particles. Protein stability and heat stability were significantly lower for 980 kDa and 2550 kDa HA as compared to the control and 8 kDa HA samples. As the MW increased, WHC, emulsion properties, and foaming stability notably increased. However, reversed results were found in the case of foaming activity. Moreover, no significant changes were observed in the percent area of individual protein fractions and the hydrodynamic diameter of protein particles. This study would help to understand the effect of HA when incorporated in dairy products for water binding or enhancement in viscosity-based applications. [ABSTRACT FROM AUTHOR]

Milk protein concentrates (MPCs) possess significant potential for diverse applications in the food industry. However, their heat stability may be a limitation to achieving optimal functional performance. Shearing, an inherent process in food manufacturing, can also influence the functionality of proteins. The aim of this research was to examine the heat stability of reconstituted MPCs prepared at two protein concentrations (4% and 8% w/w protein) when subjected to varying levels of shearing (100, 1000, or 1500 s−1) during heating at 90 °C for 5 min or 121 °C for 2.6 min. While the impact of shear was relatively minor at 4% protein, it was more pronounced in 8% protein MPC suspensions, leading to a considerable decline in heat stability. An increase in protein concentration to 8% amplified protein interactions, intensified by shearing. This, in turn, resulted in comparatively higher aggregation at elevated temperatures and subsequently reduced the heat stability of the reconstituted MPCs. [ABSTRACT FROM AUTHOR]

Proteins from hyperthermophiles often contain a large number of ionic interactions. Close examination of the previously determined crystal structure of the ATPase domain of MutL from a hyperthermophile, Aquifex aeolicus , revealed that the domain contains a continuous ion-pair/hydrogen-bond network consisting of 11 charged amino acid residues on a β-sheet. Mutations were introduced to disrupt the network, showing that the more extensively the network was disrupted, the greater the thermostability of the protein was decreased. Based on urea denaturation analysis, a thermodynamic parameter, energy for the conformational stability, was evaluated, which indicated that amino acid residues in the network contributed additively to the protein stability. A continuous network rather than a cluster of isolated interactions would pay less entropic penalty upon fixing the side chains to make the same number of ion pairs/hydrogen bonds, which might contribute more favorably to the structural formation of thermostable proteins. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we present the characterization of Aedes aegypti mosquito salivary protein, LTRIN (lymphotoxin β receptor inhibitor), a key facilitator of ZIKV transmission. Injected into the human host during blood-feeding, LTRIN interacts with the human receptor LTβR. We found that LTRIN exists as a thermostable protein with homodimers consisting of an alpha helix-dominant secondary structure with two EF-hand motifs, crucial for withstanding temperature fluctuations during blood-feeding. Despite being an EF-hand protein, its secondary structure remains unaffected by Ca2+ binding. ELISA assays and HDX-MS experiments identified the binding region of LTRIN with LTβR. Disrupting the calcium-binding ability in the second EF-hand motif significantly impacted the interaction between LTRIN, particularly the 15 kDa C-terminal protein, ΔLTRIN, and LTβR. Moreover, the decrease in binding affinity of full-length LTRIN suggests the presence of only the truncated form, ΔLTRIN, in the mosquito's saliva, as the N-terminal region likely covers the interaction site. This insight provides a basis for developing inhibitors against LTRIN as a potential treatment or vaccine target for ZIKV. Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV. [ABSTRACT FROM AUTHOR]

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively. [ABSTRACT FROM AUTHOR]

Thermostable proteins find their use in numerous biomedical and biotechnological applications. However, the computational design of stable proteins often results in single-point mutations with a limited effect on protein stability. However, the construction of stable multiple-point mutants can prove difficult due to the possibility of antagonistic effects between individual mutations. FireProt protocol enables the automated computational design of highly stable multiple-point mutants. FireProt 2.0 builds on top of the previously published FireProt web, retaining the original functionality and expanding it with several new stabilization strategies. FireProt 2.0 integrates the AlphaFold database and the homology modeling for structure prediction, enabling calculations starting from a sequence. Multiple-point designs are constructed using the Bron–Kerbosch algorithm minimizing the antagonistic effect between the individual mutations. Users can newly limit the FireProt calculation to a set of user-defined mutations, run a saturation mutagenesis of the whole protein or select rigidifying mutations based on B-factors. Evolution-based back-to-consensus strategy is complemented by ancestral sequence reconstruction. FireProt 2.0 is significantly faster and a reworked graphical user interface broadens the tool's availability even to users with older hardware. FireProt 2.0 is freely available at http://loschmidt.chemi.muni.cz/fireprotweb. [ABSTRACT FROM AUTHOR]

Milk protein dispersions containing added cocoa powder (1.5% (w/w)) and sucrose (7% (w/w)) and varying levels of κ-carrageenan (0.01, 0.03, or 0.05% w/w) were subjected to combined heat treatment (90 °C/5 min or 121 °C/2.6 min) and shear (100 or 1000 s−1) to investigate the heat stability of milk proteins. The application of shear led to a notable reduction in non-sedimentable proteins, resulting in an increase in the average particle size and apparent viscosity of the dispersions, particularly at high concentrations of k-carrageenan and elevated temperatures. This indicates that shear forces induced prominent protein aggregation, especially at higher κ-carrageenan concentrations. This aggregation was primarily attributed to the destabilisation of micelles and presence of loosely bound caseins within the κ-carrageenan network, which exhibited increased susceptibility to aggregation as collision frequencies increased due to shear. [ABSTRACT FROM AUTHOR]

Sweet and, to a lesser extent, acid whey protein ingredients can be used for the formulation of infant nutritional products. Unlike acid whey, sweet whey contains caseinomacropeptide (CMP), a heat-stable peptide liberated from κ-casein during cheese and rennet casein manufacture. Four protein systems—sweet whey (SW) and acid whey (AW), with or without standardization for CMP protein content—were added to skim milk (50/50, wt/wt) and unheated or heated to 85 or 110°C. These 12 samples were assessed for physicochemical stability in the presence of added calcium at pH 6.8. The effect of CMP content on the physicochemical properties of the protein systems was also assessed. Without preheat treatment, mixtures of AW and skim milk (SM) were more heat stable than SW and SM, demonstrating the effect of whey protein type on heat stability. Preheat treatment of the SW in the presence of SM significantly improved the heat stability of the resultant protein systems on subsequent heating. All of the protein systems had significantly lower heat stability with the addition of Ca, although the reduction was significantly smaller for the heated protein systems than the unheated controls. The findings can help identify heating parameters and ingredients for optimizing processing stability and physicochemical characteristics of nutritional beverages such as infant formulations. [ABSTRACT FROM AUTHOR]

The structural comparative analysis of β-Galactosidase in Sulfolobus acidocaldarius and Escherichia coli highlights its thermostable nature and potential industrial applications under extreme conditions. This study investigates the 3D structure of β-Galactosidase in the thermophilic archaeon Sulfolobus acidocaldarius and compares it with the mesophilic bacterium Escherichia coli. The aim is to highlight the distinct structural features of the thermostable β-Galactosidase in S. acidocaldarius, emphasizing its potential for industrial applications under extreme conditions. The absence of a known 3D structure for this enzyme in S. acidocaldarius prompted modeling efforts. The findings reveal significant structural differences, particularly in thermal stability, making S. acidocaldarius β-Galactosidase promising for applications in the dairy industry, pharmaceuticals, and biotechnology. This study underscores the importance of understanding extremophile enzymes' adaptability to extreme environments and their potential for biotechnological advancements. The comparative analysis lays the foundation for future research aimed at harnessing thermostable βGalactosidase enzymes' unique properties, offering innovative possibilities across various industries. [ABSTRACT FROM AUTHOR]

Coagulation is an important process in the context of water purification; and the seed protein of the moringa tree (Moringa oleifera) is a remarkably effective coagulant. The laboratory course described here is designed to provide high‐school students with a stepwise, hands‐on experience in investigating the protein‐rich coagulant found in Moringa seeds. First, the seed powder was applied to model polluted water containing fine clay, food dyes, copper sulfate, and bacteria. This treatment changed the polluted water into clear water via coagulation; all students were convinced that the coagulation‐inducing agent was a thermostable cationic protein. Finally, basic biochemical techniques (e.g., chromatographic separation and electrophoresis) were used to show that the target coagulant is a dimeric protein composed of 6.5 and 4.5 kDa subunits. Overall, this made it possible for the students to gain a deeper understanding (more comprehensive than the information taught in formal classes) of protein structure and its real‐world implications. This stepwise exercise can be applied to research‐based learning programs in high school, as it is an effective learning tool. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recently, there is a growing interest in developing protein‐fortified liquid systems, which are formulated to provide special nutrient combinations to those with special dietary needs. The fabrication of heat‐stable protein for protein‐fortified liquid systems relies heavily on precise control of the edible protein‐building process. RESULTS: Results suggested that heat‐stable 7S protein particles (7SPPs) could be obtained by preheating at 100 °C for an extended time, whereas 7S proteins with better gelling properties were discovered after preheating at lower temperatures. According to the findings of the protein conformational and morphological characterization, the 7SPPs showed rather stable tertiary and secondary structures as well as size distributions, which might be responsible for their heat stability. Additionally, during the reheating test, suspensions of 7SPPs showed no signs of gelation and had a low viscosity even though the protein content was as high as 120 mg mL−1. However, 7S proteins with improved gelling properties were found to show rising aggregate size, higher susceptibility and larger conformational structure changing rates upon reheating treatment. CONCLUSION: Soy β‐conglycinin (7S) proteins with tunable heat stability were successfully prepared by preheating 10 mg mL−1 protein dispersions at various temperatures (80–120 °C) and durations (15–120 min). These findings provide fundamental insights for developing 7S‐based protein‐fortified systems. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The genome of Rhizobium etli, a nitrogen‐fixing bacterial symbiont of legume plants, encodes two l‐asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High‐resolution crystal structures of ReAIV now confirm that it is a class 3 l‐asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc‐binding site. ReAIV has a high pH optimum (9–11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser–Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme‐engineering experiments to improve the kinetic parameters for medicinal applications. [ABSTRACT FROM AUTHOR]

The DNA-binding protein from starved cells, known as DPS, has been characterized as a crucial factor in protecting E. coli from external stresses. The DPS functions in various cellular processes, including protein-DNA binding, ferroxidase activity, compaction of chromosome and regulation of stress resistance gene expression. DPS proteins exist as oligomeric complexes; however, the specific biochemical activity of oligomeric DPS in conferring heat shock tolerance has not been fully understood. Therefore, we investigated the novel functional role of DPS under heat shock. To elucidate the functional role of DPS under heat shock conditions, we purified recombinant GST-DPS protein and demonstrated its thermostability and existence in its highly oligomeric form. Furthermore, we discovered that the hydrophobic region of GST-DPS influenced the formation of oligomers, which exhibited molecular chaperone activity, thereby preventing the aggregation of substrate proteins. Collectively, our findings highlight the novel functional role of DPS, as a molecular chaperone and may confer thermotolerance to E. coli. • DPS is a thermostable oligomer protein. • DPS formed higher-order oligomer in response to heat shock. • DPS acts as a molecular chaperone through its hydrophobic region under heat shock. • This novel molecular chaperone activity of DPS may confer thermotolerance to E. coli. [ABSTRACT FROM AUTHOR]

Simple Summary: Global climate change has a significant impact on soil decomposition processes through the alteration of temperature and precipitation and, in connection with them, through changes in the quantity and quality of biomass production in ecosystems. The role of soil organic matter (SOM) is particularly important, as the consequences might rapidly affect soil carbon stocks. These processes have a global impact on the CO2 content of the atmosphere and a local impact on the fertility of soils. In our research, which is based on a transcontinental litter manipulation, detritus input, and removal treatment (DIRT) project, we investigated how quantitative and qualitative changes in litter inputs can affect decomposition processes and carbon storage capacity of soils in relation to SOM content. The main question of the study is how various litter treatment sites respond to additional and/or removed organic matters through assessed soil-biological parameters. The changes were monitored with some potential soil-biological indicators, such as through examining enzyme activity, CO2 emissions, and labile carbon stocks (POXC) used by organisms in performing decomposition processes. The model experiment provided a great background highlighting organic matter's importance in soil-biological processes and soil ecosystem functioning. Soil organic matter is a biological system that functions as an integrated whole. These assemblies have different properties, functions, and decomposition times. SOM is one of the main determinants of soil productivity. Our studies were carried out in a temperate deciduous oak forest on Luvisols soil. In the DIRT Project (Detritus Input and Removal Treatments), the following treatments were applied: Double Litter, Double Wood, Control, No Litter, No Root and No Input. Our objective was to compare the effect of withdrawal or doubling of organic matter on the protein pattern of the soil and the biological activity and changes in labile C (permanganate-oxidizable carbon) content in a long-term organic matter manipulation experiment. Patterns of thermostable proteins, soil dehydrogenase enzyme activity, CO2 emission, and POXC content were measured at the most biologically active soil depth of 0–5 cm after 23 years of treatment. Our results show that the enzyme activities of the litter removal treatments were significantly reduced compared to the doubling treatments, as were the values of soil respiration. The same significant difference was also detected in the C content of the soils of the treatments. Based on cluster analysis of the protein profile of the soil samples, the No Litter and No Input treatments were significantly different from the other treatments. This shows that specific organic matter is needed to enhance soil biological activity and the associated POXC content. [ABSTRACT FROM AUTHOR]

The article discusses the use of artificial metalloenzymes for sustainable chemical production. Enzymes are often seen as ideal catalysts due to their ability to perform difficult reactions under mild conditions. The Jarvis group aims to bring metal-based catalytic processes into the biocatalytic toolbox by combining synthetic transition metal catalysts with protein scaffolds. They have successfully designed a rhodium-based metalloenzyme for biphasic hydroformylation of long chain alkenes, demonstrating good linear selectivity. The group is also working on expanding the substrate scope and conducting in vivo hydroformylation. [Extracted from the article]

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the μM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site. [ABSTRACT FROM AUTHOR]

This work evaluated the impact of the calcium reduction level (0 %, 10 % and 40 %) of micellar casein concentrates (MCC) and the presence of sugar and fat, on the fouling of the UHT plant and the heat stability of neutral high protein (14 %) beverages. The assessment of the heat stability was done using UHT pilot scale tubular heat exchanger (142 °C, few seconds). The aim of this study is to identify a casein modified structure suitable for the development of high protein beverages with acceptable functional properties and less fouling of the UHT plant. Our results showed that increasing the calcium reduction degree of MCC from 0 % to 40 % has drastically decreased the fouling rate by 4-fold for casein-based beverages without sugar and fat. In addition, the results of SDS-PAGE, HPLC and ICP-MS revealed that increasing the calcium reduction degree from 0 % to 40 % induced an increase of the mineral fraction from 13 % to 70 % and a decrease of the protein fraction from 77 % to 7 % in the fouling deposits. The HPLC results also showed that the increase of the calcium reduction appears to limit the deposition to dissociated protein from the micelles, in particular β-CN. Our results showed that the addition of sugar and fat induced an increase of the fouling deposition for all drinks and this increase was mostly linked to the increase of the protein deposition. Our results also showed that the calcium reduction degree doesn't have an impact on the particle size distribution. However, aggregates started to appear in the simple and complex beverages with the increase of the storage period. Overall, our results showed that the calcium reduction of MCC powders seems to be a good option to manufacture casein-based RTD beverages at high protein concentrations and to reduce the fouling of the UHT plant. [ABSTRACT FROM AUTHOR]

The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen. The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods. The allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αβα sandwich structure containing 4 α-helices and 6 antiparallel β-sheets, encompassing 4 conserved CAP domain. The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis. • The natural Hum j PR-1 was firstly purified and characterized as a major allergenic allergen in H. japonicus pollen. • Hum j PR-1 showed high sensitization rate of 76.6 % (23/30) in Humulus japonicus pollen-allergic patients. • Hum j PR-1 had a unique αβα sandwich structure with 4 conserved CAP domain. [ABSTRACT FROM AUTHOR]

The need to increase the COVID-19 vaccine manufacturing capacity at low to middleincome countries (LMIC) led to a growing focus on Novavax (NVX-CoV2373), a thermostable protein subunit vaccine manufactured using a baculovirus and insect cell system (BICS) platform. This study aimed to conduct a techno-economic analysis to assess the BICS platform of vaccine manufacturing and compare it to the mRNA and the saRNA platform. The data from the Novavax patent for the COVID-19 vaccine formulation and the manufacturing steps were used to simulate the BICS vaccine production in SuperPro Designer. From the techno-economic analysis, the productivity of all platforms was compared in terms of doses/day per L production scale. The saRNA platform’s productivity is about 1,000-fold of the BICS platform and 20-fold of the mRNA platform. BICS is a feasible option for LMIC to produce vaccines because the cost per dose is like the saRNA platform, while the mRNA platform’s cost per dose is 7 times higher than the BICS and saRNA platforms. However, further optimization is necessary to improve the productivity of the BICS platform to match saRNA’s platform. [ABSTRACT FROM AUTHOR]

The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N‐terminally His‐tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer‐of‐dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N‐terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits where the nucleoside ring of the substrate would normally be located. Tag binding appears to be driven by a combination of enthalpic, entropic and proximity effects, which convey a particularly high affinity in the crystallized form. Attempts to cleave the tag in solution yielded only a small fraction of untagged protein, suggesting that the enzyme predominantly exists in the tag‐bound form in solution, preventing rTEV from accessing the cleavage site. However, the tagged protein retained some activity in solution, suggesting that the tag does not completely block the active site, but may act as a competitive inhibitor. This serves as a warning that it is prudent to establish how affinity tags may affect protein structure and function, especially for industrial biocatalytic applications that rely on the efficiency and convenience of one‐pot purifications and in cases where tag removal is difficult. [ABSTRACT FROM AUTHOR]

α-synuclein (α-syn) is a highly conserved and thermostable protein that is widely distributed in human brain. An intracellular aggregation of α-syn in dopaminergic neurons is the hallmark of a group of neurodegenerative diseases including Parkinson's disease. Interestingly, α-syn is also highly expressed in red blood cells and is considered as one of the most abundant proteins in red blood cells. Moreover, α-syn is thought to play a regulatory role during normal erythropoiesis. However, whether α-syn participates in the pathogenesis of erythroid diseases has not been reported. In this review, we discuss the protein structure of α-syn and the importance of α-syn in erythropoiesis. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases (NDs) commonly present misfolded and aggregated proteins. Considerable research has been performed to unearth the molecular processes underpinning this pathological aggregation and develop therapeutic strategies targeting NDs. Fibrillary deposits of α-synuclein (α-Syn), a highly conserved and thermostable protein, are a critical feature in the development of NDs such as Alzheimer's disease (AD), Lewy body disease (LBD), Parkinson's disease (PD), and multiple system atrophy (MSA). Inhibition of α-Syn aggregation can thus serve as a potential approach for therapeutic intervention. Recently, the degradation of target proteins by small molecules has emerged as a new therapeutic modality, gaining the hotspot in pharmaceutical research. Additionally, interest is growing in the use of food-derived bioactive compounds as intervention agents against NDs via functional foods and dietary supplements. According to reports, dietary bioactive phospholipids may have cognition-enhancing and neuroprotective effects, owing to their abilities to influence cognition and mental health in vivo and in vitro. However, the mechanisms by which lipids may prevent the pathological aggregation of α-Syn warrant further clarification. Here, we review evidence for the potential mechanisms underlying this effect, with a particular focus on how porcine liver decomposition product (PLDP)-derived lysophospholipids (LPLs) may inhibit α-Syn aggregation. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) move through an ensemble of conformations which allows multitudinous roles within a cell. Keratinocytes, the predominant cell type in mammalian epidermis, have had only a few individual proteins assessed for intrinsic disorder and its possible contribution to liquid–liquid phase separation (LLPS), especially in regard to what functions or structures these proteins provide. We took a holistic approach to keratinocyte IDPs starting with enrichment via the isolation of thermostable proteins. The keratinocyte protein involucrin, known for its resistance to heat denaturation, served as a marker. It and other thermostable proteins were identified by liquid chromatography tandem mass spectrometry and subjected to extensive bioinformatic analysis covering gene ontology, intrinsic disorder, and potential for LLPS. Numerous proteins unique to keratinocytes and other proteins with shared expression in multiple cell types were identified to have IDP traits (e.g., compositional bias, nucleic acid binding, and repeat motifs). Among keratinocyte-specific proteins, many that co-assemble with involucrin into the cell-specific structure known as the cornified envelope scored highly for intrinsic disorder and potential for LLPS. This suggests intrinsic disorder and LLPS are previously unrecognized traits for assembly of the cornified envelope, echoing the contribution of intrinsic disorder and LLPS to more widely encountered features such as stress granules and PML bodies. [ABSTRACT FROM AUTHOR]

Aflatoxins, which are produced mainly by Aspergillus flavus and A. parasiticus, are recognized as the most toxic mycotoxins, which are strongly carcinogenic and pose a serious threat to human and animal health. Therefore, strategies to degrade or eliminate aflatoxins in agro-products are urgently needed. We investigated 65 Trichoderma isolates belonging to 23 species for their aflatoxin B1 (AFB1)-degrading capabilities. Trichoderma reesei CGMCC3.5218 had the best performance, and degraded 100% of 50 ng/kg AFB1 within 3 days and 87.6% of 10 µg/kg AFB1 within 5 days in a liquid-medium system. CGMCC3.5218 degraded more than 85.0% of total aflatoxins (aflatoxin B1, B2, G1, and G2) at 108.2-2323.5 ng/kg in artificially and naturally contaminated peanut, maize, and feed within 7 days. Box-Behnken design and response surface methodology showed that the optimal degradation conditions for CGMCC3.5218 were pH 6.7 and 31.3°C for 5.1 days in liquid medium. Possible functional detoxification components were analyzed, indicating that the culture supernatant of CGMCC3.5218 could efficiently degrade AFB1 (500 ng/kg) with a ratio of 91.8%, compared with 19.5 and 8.9% by intracellular components and mycelial adsorption, respectively. The aflatoxin-degrading activity of the fermentation supernatant was sensitive to proteinase K and proteinase K plus sodium dodecyl sulfonate, but was stable at high temperatures, suggesting that thermostable enzymes or proteins in the fermentation supernatant played a major role in AFB1 degradation. Furthermore, toxicological experiments by a micronucleus assay in mouse bone marrow erythrocytes and by intraperitoneal injection and skin irritation tests in mice proved that the degradation products by CGMCC3.5218 were nontoxic. To the best of our knowledge, this is the first comprehensive study on Trichoderma aflatoxin detoxification, and the candidate strain T. reesei CGMCC3.5218 has high efficient and environment-friendly characteristics, and qualifies as a potential biological detoxifier for application in aflatoxin removal from contaminated feeds. [ABSTRACT FROM AUTHOR]

There has been a growing demand for the development of high protein beverages in the food industry. However, during thermal processing, high-protein beverages undergo protein aggregation and gelation. In this study, thermostable soy protein was prepared by disulfide bond cleavage combined with preheating treatment. Thermostable soy protein had a higher denature extent than the control sample, which prevented the formation of larger aggregates upon reheating. Thermostable soy protein possessed lower viscosity (nearly 0 Pa·s) and an excellent fluidity index (around 0.8) after reheating at a concentration of 10 % (w / v), whereas the control sample had gelled. Moreover, rheology and dynamic light scattering collectively demonstrated that a minimum of 2.5 mM sodium metabisulfite (Na 2 S 2 O 5) was required to prepare thermostable protein solutions. The present study shows an innovative method to produce thermostable soy protein. Flowchart for Modified Protein Preparation. Diagram of Chemical Mechanism of Reductant Modification. [Display omitted] • Reducing and heat combined treatment was effective in preparing stabilized soy protein. • Combined treatment endowed proteins with larger particle size and denaturation degree than natural proteins. • Modified proteins (10 wt%) exhibited good fluidity and resisted gelling during heating. [ABSTRACT FROM AUTHOR]

[Display omitted] • Single-molecule microscopy revealed the sliding motion of Cren7 along DNA. • Cren7 does not impede the target search by a model transcription factor p53. • Cren7 form large clusters on DNA via bridging, which compacts DNA. A nucleoid protein Cren7 compacts DNA, contributing to the living of Crenarchaeum in high temperature environment. In this study, we investigated the dynamic behavior of Cren7 on DNA and its functional relation using single-molecule fluorescence microscopy. We found two mobility modes of Cren7, sliding along DNA and pausing on it, and the rapid dissociation kinetics from DNA. The salt dependence analysis suggests a sliding with continuous contact to DNA, rather than hopping/jumping. The mutational analysis demonstrates that Cren7 slides along DNA while Trp (W26) residue interacts with the DNA. Furthermore, Cren7 does not impede the target search by a model transcription factor p53, implying no significant interference to other DNA-binding proteins on DNA. At high concentration of Cren7, the molecules form large clusters on DNA via bridging, which compacts DNA. We discuss how the dynamic behavior of Cren7 on DNA enables DNA-compaction and protein-bypass functions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bacterial urea metabolism is a key component in the global nitrogen cycle. • Identification of urea-binding proteins in ABC transporters is challenging. • Genomic linkage of binding proteins with known functions reveals cognate ligands. • Structure-based sequence snippets extrapolate ligand assignments in homologs. • The discovered proteins reveal structure adaptions and enable biosensor engineering. ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here. [ABSTRACT FROM AUTHOR]

Nature has developed extremozymes that catalyze complex reaction processes in extreme environmental conditions. Accordingly, a combined approach consisting of extremozyme screening, ancestral sequence resurrection (ASR), and molecular dynamic simulation was utilized to construct a developed endoglucanase. The primary experimental and in-silico data led to the prediction of a hypothetical sequence of endoglucanase (EG5-G131) using Bacillus sp. G131 confirmed by amplification and sequencing. EG5-G131 exhibited noticeable stability in a broad-pH range, several detergents, organic solvents, and temperatures up to 80 °C. The molecular weight, V max , and K m of the purified endoglucanase were estimated to be 36 kDa, 4.32 μmol/min, and 23.62 mg/ml, respectively. The calculated thermodynamic parameters for EG5-G131 confirmed its intrinsic thermostability. Computational analysis revealed Glu142 and Glu230 as active-site residues of the enzyme. Furthermore, the enzyme remained bound to cellotetraose at 298 K, 333 K, 343 K, and 353 K for 300 ns, consistent with our experimental data. ASR of EG5-G131 led to the introduction of ancestral ANC204 and ANC205, which show similar thermodynamic characteristics with the last Firmicute common ancestor. Finally, truncating loops from the N-terminal of two sequences created two variants with desirable thermal stability, suggesting the evolutionary deciphering of the functional domain of the GH5 family in Bacillus sp. G131. [ABSTRACT FROM AUTHOR]

Background: Catalase (EC 1.11.1.6) is a heme-containing tetrameric enzyme that plays a critical role in signaling and hydrogen peroxide metabolism. It was the first enzyme to be crystallized and isolated. Catalase is a well-known industrial enzyme used in diagnostic and analytical methods in the form of biomarkers and biosensors, as well as in the textile, paper, food, and pharmaceutical industries. In silico analysis of CAT genes and proteins has gained increased interest, emphasizing the development of biomarkers and drug designs. The present work aims to understand the catalase evolutionary relationship of plant species and analyze its physicochemical characteristics, homology, phylogenetic tree construction, secondary structure prediction, and 3D modeling of protein sequences and its validation using a variety of conventional computational methods to assist researchers in better understanding the structure of proteins. Results: Around 65 plant catalase sequences were computationally evaluated and subjected to bioinformatics assessment for physicochemical characterization, multiple sequence alignment, phylogenetic construction, motif and domain identification, and secondary and tertiary structure prediction. The phylogenetic tree revealed six unique clusters where diversity of plant catalases was found to be the largest for Oryza sativa. The thermostability and hydrophilic nature of these proteins were primarily observed, as evidenced by a relatively high aliphatic index and negative GRAVY value. The distribution of 5 sequence motifs was uniformly distributed with a width length of 50 with the best possible amino residue sequences that resemble the plant catalase PLN02609 superfamily. Using SOPMA, the predicted secondary structure of the protein sequences revealed the predominance of the random coil. The predicted 3D CAT model from Arabidopsis thaliana was a homotetramer, thermostable protein with 59-KDa weight, and its structural validation was confirmed by PROCHECK, ERRAT, Verify3D, and Ramachandran plot. The functional relationships of our query sequence revealed the glutathione reductase as the closest interacting protein of query protein. Conclusions: This theoretical plant catalases in silico analysis provide insight into its physiochemical characteristics and functional and structural understanding and its evolutionary behavior and exploring protein structure-function relationships when crystal structures are unavailable. [ABSTRACT FROM AUTHOR]

To tune the efficiency of oxidized cofactor recycling between alcohol dehydrogenase (ADH) and NADH oxidase (NOX) for the production of aromatic chiral alcohols, we designed and constructed four novel bifunctional fusion proteins composed of thermostable ADH and NOX from Thermococcus kodakarensis KOD1. ADH was linked to the N‐ or C‐terminus of NOX with a typical rigid linker (EAAAK)3 and a flexible linker (GGGGS)3, respectively. Compared with the parental enzymes, the NOX moieties in the four fusion proteins exhibited higher specific activities (141%–282%), while the ADH moieties exhibited varying levels of specific activity (69%–167%). All fusion proteins showed decreased affinities toward the cofactors, with increased Km values toward NADH (159%–406%) and NAD+ (202%–372%). In the enantioselective oxidation of (RS)‐1‐phenylethanol coupled with cofactor regeneration, the four fusion proteins displayed different positive and negative effects on the recycling efficiency of the oxidized cofactor. The two fusion proteins composed of NOX at the N‐terminus exhibited higher total turnover numbers than the corresponding mixtures of individual enzymes with equal activities, particularly at low cofactor concentrations. These findings suggest high cofactor recycling efficiencies of the fusion proteins with appropriate design and their potential application in the biosynthesis of chiral alcohols. [ABSTRACT FROM AUTHOR]

The TGF-β superfamily is ubiquitously distributed from invertebrates to vertebrates with diverse cellular functioning such as cell adhesion, motility, proliferation, apoptosis, and differentiation. The present study aimed to characterize the TGF-β gene superfamily in buffalo through evolutionary, structural, and single nucleotide polymorphism (SNPs) analyses to find the functional effect of SNPs in selected genes. We detected 32 TGF-β genes in buffalo genome and all TGF-β proteins exhibited basic nature except INHA, INHBC, MSTN, BMP10, and GDF2, which showed acidic properties. According to aliphatic index, TGF-β proteins were thermostable but unstable in nature. Except for GDF1 and AMH, TGF-β proteins depicted hydrophilic nature. Moreover, all the detected buffalo TGF-β genes showed evolutionary conserved nature. We also identified eight segmental and one tandem duplication event TGF-β gene family in buffalo, and the ratio of Ka/Ks demonstrated that all the duplicated gene pairs were under selective pressure. Comparative amino acid analysis demonstrated higher variation in buffalo TGF-β gene family, as a total of 160 amino acid variations in all the buffalo TGF-β proteins were detected. Mutation analysis revealed that 13 mutations had an overall damaging effect that might have functional consequences on buffalo growth, folliculogenesis, or embryogenesis. [ABSTRACT FROM AUTHOR]

Studies on the structure–function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a 'β-barrel' fold, formed by the double-stranded antiparallel β-sheet 1 tightly packing with the triple-stranded antiparallel β-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the β-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the β-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2 ° C), as compared with the wild-type Cren7 (Tm > 80 ° C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins. [ABSTRACT FROM AUTHOR]

BAG9 Interacts with Hsp20s and Maintains Hsps Stability under Heat Stress We next identified BAG9-interacting proteins by applying yeast two-hybrid screens. To further investigate whether heat-induced oxidative stress caused the oxidation of functional proteins, SDS-PAGE was used to analyze protein oxidation among proteins isolated from total proteins. Involvement of BAG9 in Tomato Thermotolerance Transcript analysis of 10 I SlBAGs i under heat stress was conducted to determine whether heat stress induced BAG gene expression. Thus, BAG9 played a crucial role in response to heat stress by regulating cellular redox homeostasis and the stability of heat shock proteins. [Extracted from the article]

The world today is battling with a coronavirus infection that is considered a global pandemic. Coronavirus infection is mainly attribute to the varying technique of the replication and release of different genomic components of the virus. The present study aims to establish the physical and chemical features, as well as the basic structural and functional properties of Coronavirus ORF1ab domain. A molecular approach was adopt in this study using the Swiss Model and Phyre2 server whereas the prediction of the active ligand binding sites was done using Phyre2. The analysis of the structure of the protein showed that it has good structural and heat stability, as well as better hydrophilic features and acidic in nature. Based on the Homology modeling, only two binding active sites were noted with catalytic function being mediated by Zn2+ as the metallic heterogeneous ligand for binding sites prediction. The proteins mostly exhibited helical secondary configurations. This study can help in predicting and understanding the role of this domain protein in active coronavirus infection. [ABSTRACT FROM AUTHOR]

Recently, the pursuit of having a healthy diet has heightened the superiority of protein‐enriched beverages; however, this is challenged by the heat‐induced aggregation and gelation of proteins at high concentration. In this study, a facile and efficient strategy of combined preheating and microfluidic homogenisation treatment (CPMHT) was tested to enhance the heat stability of soy proteins (SPs). Results revealed that the CPMHT promoted the denaturation and unfolding of SPs, evidenced by the reduction of β‐sheet content and a slight red shift of fluorescence intensity. Increasing the number of CPMHT cycle led to an apparent decrease in the surface hydrophobicity and total sulfhydryl groups of soy proteins. In addition, when the suspensions (10% (w/v)) of modified soy proteins at higher CPMHT cycle were reheated at 100 °C for 30 min, no gelation was found, along with lower viscosities and higher flow behaviour index. The present study provides an approaching to enhance the heat stability of soy proteins by CPMHT, which might expand the application of soy proteins in protein‐enriched beverages. [ABSTRACT FROM AUTHOR]

Protein based vaccines are the most safe and affordable strategy to combat pneumococcal disease circumventing the limitations of conventional polysaccharide-based vaccines like serotype dependence, high cost and inability to be administered to immunocompromised. SP0845 is a highly conserved vaccine candidate shown to provide protection against heterologous strains of Streptococcus pneumoniae , the primal cause of pneumonia. However, the associated poor immunogenicity warrants the need for adjuvants and multiple doses to mount desired responses. The present study relates to improve the immunogenicity of pneumococcal protein SP0845 by use of poly lactic acid biodegradable polymer microparticles. The immunization studies showed that microparticles elicited higher antibody response compared to alum adjuvanted protein and this immunopotentiation was achieved without the use of any additional adjuvant. They were also capable of eliciting secondary antibody response upon boosting after four months. Further, the particles upon storage at 25 and 37 °C for one month were still capable of mounting an immune response equivalent to those stored in cold chain. Thus, using microparticles entrapping SP0845 for immunization not only improve the immunogenicity but also offer better temperature stability. This can greatly reduce the cost and increase access of protein-based vaccine to resource limited settings. [Display omitted] • Protein entrapped polymer particles offer improved immunogenicity. • Dry powder formulation of protein increases thermal stability. • Thermostable pneumococcal protein vaccine for resource limited settings • Cost effective pneumococcal vaccine for broader coverage [ABSTRACT FROM AUTHOR]