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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]

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]

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]

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]

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]

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]

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]

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 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]

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]

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]

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]

Natural proteins are often only slightly more stable in the native state than the denatured state, and an increase in environmental temperature can easily shift the balance toward unfolding. Therefore, the engineering of proteins to improve protein stability is an area of intensive research. Thermostable proteins are required to withstand industrial process conditions, for increased shelf-life of protein therapeutics, for developing robust 'biobricks' for synthetic biology applications, and for research purposes (e.g., structure determination). In addition, thermostability buffers the often destabilizing effects of mutations introduced to improve other properties. Rational design approaches to engineering thermostability require structural information, but even with advanced computational methods, it is challenging to predict or parameterize all the relevant structural factors with sufficient precision to anticipate the results of a given mutation. Directed evolution is an alternative when structures are unavailable but requires extensive screening of mutant libraries. Recently, however, bioinspired approaches based on phylogenetic analyses have shown great promise. Leveraging the rapid expansion in sequence data and bioinformatic tools, ancestral sequence reconstruction can generate highly stable folds for novel applications in industrial chemistry, medicine, and synthetic biology. This review provides an overview of the factors important for successful inference of thermostable proteins by ancestral sequence reconstruction and what it can reveal about the determinants of stability in proteins. [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]

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]

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]

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]

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]

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50–52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. Results: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at − 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. Conclusions: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis. [ABSTRACT FROM AUTHOR]

The engineered three-helix bundle, UVF, is thermostabilized entropically due to heightened, native-state dynamics. However, it is unclear whether this thermostabilization strategy is observed in natural proteins from thermophiles. We performed all-atom, explicit solvent molecular dynamics simulations of two three-helix bundles from thermophilic H. butylicus (2lvsN and 2lvsC) and compared their dynamics to a mesophilic three-helix bundle, the Engrailed homeodomain (EnHD). Like UVF, 2lvsC had heightened native dynamics, which it maintained without unfolding at 100°C. Shortening and rigidification of loops in 2lvsN and 2lvsC and increased surface hydrogen bonds in 2lvsN were observed, as is common in thermophilic proteins. A buried disulfide and salt bridge in 2lvsN and 2lvsC, respectively, provided some stabilization, and addition of a homologous disulfide bond in EnHD slowed unfolding. The transferability and commonality of stabilization strategies among members of the three-helix bundle fold suggest that these strategies may be general and deployable in designing thermostable proteins. [ABSTRACT FROM AUTHOR]

In any case, as expected, TMP was the most abundant cyclic in these products.[[28]] The cyclic content for the Fisher HMP was quantified from both the titration and SP 31 sp P-NMR spectroscopy results, giving values of 1.65 and 3.61% respectively. The value from the SP 31 sp P-NMR spectroscopy is likely more accurate because the approximation for the fractioned HMP titration assumes that the linear polyphosphates in the residual fraction would have the same average chain length as the previous fraction (n SB 9 sb in Equation 2), while it is probable that the linear phosphates remaining in the residual fraction will in fact be shorter. Hexametaphosphate (HMP) is an inorganic condensed phosphate, which has been used in a wide variety of industries for nearly a century, originally patented to deflocculate clay and soften hard water.[[1]] Today, HMP is still used in the minerals processing industry as a dispersant to improve separation.[[3], [5]] It is also used in the food industry, as additive E452i, to improve the stability of whey protein drinks,[[6]] prevent efflorescence in fermented sausages,[[8]] and as an emulsifying salt in processed cheese.[[9]] HMP is used in some toothpastes, to prevent caries and reduce the amount of fluorine required.[[10]] HMP is particularly useful in these applications due to its ability to bind to surfaces to provide steric and electrostatic repulsion, and to form strong soluble complexes with multivalent cations. [Extracted from the article]

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins. [ABSTRACT FROM AUTHOR]

Regular use of certain drugs and other antibiotics has led to the development of many side effects and drug resistance in the humans. But researchers have already started to find out various other alternatives to combat the negative effects. Among these, probiotics are being used up as an alternate and are consumed to great extent by humans in day to day life. Lactobacillus is the most common bacterial strain that is commercially used in various probiotic products. These LABs must bind to the human epithelial gut in order to perform their activity. For binding, mucin binding protein plays a major role by providing the attachment sites to the bacteria. In this paper we have analyzed the mucin-binding proteins in Lactobacillus plantarum using computational tools. L plantarum was chosen due to its ability to show strong binding towards epithelial surface. The FASTA sequences were extracted from UniProtkb database and were used for further comparison based on different parameters. Physicochemical analysis was done by ProtPARAM software, followed by secondary structure prediction (CFSSP software) and tertiary structure prediction (CASTp software). The MUBs motifs were also compared with the motifs of gut pathogens using TOMTOM tool. Moreover, the particular motifs responsible during adhesion were being searched in MUBs using Motif Finder. These motifs showed some similarity with the binding motifs of pathogens present in human gut. Physicochemical analysis showed that these mucin binding proteins are thermostable. GRAVY value indicates that these are soluble proteins which are hydrophilic in nature. These proteins do not have any effect of cellular proteases and hence are able to survive in the small intestine. These chosen strains have conserved sequences determined by their secondary structure analysis using the CFSSP server. Moreover, it was found that these mucin binding proteins has some of the motifs having the same sequences as present in that of pathogens. Hence, these are able to show the competitive adherence against the pathogens by blocking the binding sites for pathogens and resulting in their elimination. The study conducted showed the presence of MUBs in Lactobacillus plantarum and also same motifs were found in pathogen which proved the competitive adherence of L. plantarum against pathogens hence providing some light to the host-microbes interactions for other future studies too. [ABSTRACT FROM AUTHOR]

The research has proved the practical importance of using a biopolymer complex of thermostable animal proteins with functional and technological properties as a component of restructured ham-type products. It has been established that the water-holding and fat-holding capacity and the stabilising power of meat products significantly increase due to the introduction of the biopolymer complex developed by the authors, which contains sodium caseinate, buttermilk powder, and blood plasma Vepro 75 PSC. The experiments have confirmed that a protein-infat emulsion based on a biopolymer complex of thermostable animal proteins, a protein stabiliser obtained from turkey skin, and pork and turkey fats can be used as a component of restructured ham-type products from PSE pork and turkey meat. Thus, 5, 10, 15, and 20% of the proteinin-fat emulsion added to the formulations of restructured hams from lowfat pork and turkey meat increase the water-holding capacity of model meat systems by 7.49–9.05%, and their fat-holding capacity by 8.11–9.32%. Regular patterns have been established in how the stability of the functional and technological properties of the protein-infat emulsion changes throughout storage, depending on the nature and the content of a protein preparation (Vepro 75 PSC, sodium caseinate, buttermilk) and on the fat composition. It has been proved that the introduction of a protein-in-fat emulsion into the composition of hams allows obtaining products with improved and persistently high consumer properties. It has been confirmed that 0.35% of powdered sweet potato juice, in the presence of 0.025% of the starter culture of Staphylococcus carnosus, can be included in the composition of restructured ham-type products from PSE and turkey meat to colour them rosy-red, thus making it unnecessary to use sodium nitrite, a hazardous chemical. Sweet potato juice powder used in the presence of 0.025% of Staphylococcus carnosus has been found to reduce the residual amount of sodium nitrite to 3.10– 3.11 mg/100 g, because nitrate transforms into nitroso compounds more completely. The results of the study are of practical significance and will be helpful in expanding the range of restructured ham-type products of enhanced nutritional value. [ABSTRACT FROM AUTHOR]

Syncollin is a 16‐kDa protein found predominantly in the zymogen granules of pancreatic acinar cells, with expression at lower levels in intestinal epithelial cells and neutrophils. Here, we used Strep‐tagged syncollin isolated from the supernatant of transiently transfected mammalian cells to test the hypothesis that syncollin has antibacterial properties, which might enable it to play a role in host defence in the gut and possibly elsewhere. We show that syncollin is an exceptionally thermostable protein with a circular dichroism spectrum consistent with a predominantly beta‐sheet structure. Syncollin binds to bacterial peptidoglycan and restricts the growth of representative Gram‐positive (Lactococcus lactis) and Gram‐negative (Escherichia coli) bacteria. Syncollin induces propidium iodide uptake into E. coli (but not L. lactis), indicating permeabilisation of the bacterial membrane. It also causes surface structural damage in both L. lactis and E. coli, as visualised by scanning electron microscopy. We propose that syncollin is a previously unidentified member of a large group of antimicrobial polypeptides that control the gut microbiome. Take Aways: Syncollin is a 16‐kDa protein found in pancreatic zymogen granules.Syncollin is highly thermostable and has a predominantly beta‐sheet structure.Syncollin binds peptidoglycan and restricts the growth of L. lactis and E. coli.Syncollin causes propidium iodide uptake into E. coli (but not L. lactis).Syncollin causes surface structural damage in both L. lactis and E. coli. [ABSTRACT FROM AUTHOR]

Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure–function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. Key points: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants [ABSTRACT FROM AUTHOR]

Filamentous fungi are known to biosynthesize an extraordinary range of azaphilones pigments with structural diversity and advantages over vegetal-derived colored natural products such agile and simple cultivation in the lab, acceptance of low-cost substrates, speed yield improvement, and ease of downstream processing. Modern genetic engineering allows industrial production, providing pigments with higher thermostability, water-solubility, and promising bioactivities combined with ecological functions. This review, covering the literature from 2020 onwards, focuses on the state-of-the-art of azaphilone dyes, the global market scenario, new compounds isolated in the period with respective biological activities, and biosynthetic pathways. Furthermore, we discussed the innovations of azaphilone cultivation and extraction techniques, as well as in yield improvement and scale-up. Potential applications in the food, cosmetic, pharmaceutical, and textile industries were also explored. [ABSTRACT FROM AUTHOR]

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis. [ABSTRACT FROM AUTHOR]

Evolution of proteins in bacteria and archaea living in different conditions leads to significant correlations between amino acid usage and environmental temperature. The origins of these correlations are poorly understood, and an important question of protein theory, physics-based prediction of types of amino acids overrepresented in highly thermostable proteins, remains largely unsolved. Here, we extend the random energy model of protein folding by weighting the interaction energies of amino acids by their frequencies in protein sequences and predict the energy gap of proteins designed to fold well at elevated temperatures. To test the model, we present a novel scalable algorithm for simultaneous energy calculation for many sequences in many structures, targeting massively parallel computing architectures such as graphics processing unit. The energy calculation is performed by multiplying two matrices, one representing the complete set of sequences, and the other describing the contact maps of all structural templates. An implementation of the algorithm for the CUDA platform is available at http://www.github.com/kzeldovich/galeprot and calculates protein folding energies over 250 times faster than a single central processing unit. Analysis of amino acid usage in 64-mer cubic lattice proteins designed to fold well at different temperatures demonstrates an excellent agreement between theoretical and simulated values of energy gap. The theoretical predictions of temperature trends of amino acid frequencies are significantly correlated with bioinformatics data on 191 bacteria and archaea, and highlight protein folding constraints as a fundamental selection pressure during thermal adaptation in biological evolution. [ABSTRACT FROM AUTHOR]

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins. Directed evolution commonly relies on point mutations but InDels frequently occur in evolution. Here the authors report a protein-engineering framework based on InDel mutagenesis and fragment transplantation resulting in greater catalysis and longer glow-type bioluminescence of the ancestral luciferase. [ABSTRACT FROM AUTHOR]