Molecular Weight Proteins Research Articles

Photo-Reactivity and Photo-Transformation of Algal Dissolved Organic Matter Unraveled by Optical Spectroscopy and High-Resolution Mass Spectrometry Analysis

The rapid proliferation of planktonic algae induced by eutrophication and climate warming make algae dissolved organic matter (AOM) an important source of dissolved organic matter (DOM) in surface waters, but the understanding of the link between AOM composition and photo-reactivity/photo-transformation of DOM in aquatic systems is limited. Here, intracellular organic matter (IOM) from Microcystis aeruginosa was extracted and subjected to molecular weight (MW) fractionation. Results indicated that IOM had lower aromaticity and higher photosensitive activity compared to Suwannee River fulvic acid (SRFA). The photosensitive activity of IOM relied on both its molecular weight distribution and fluorescence components. The IOM fraction with the highest MW proteins had the lowest quantum yields of reactive intermediates (ΦRIs), which increased with the decrease of MW, while the fractions with more low-excitation tyrosine-like components had relatively higher ΦRIs. Parallel factor analysis and high-resolution mass spectrometry revealed that light radiation of IOM resulted in the composition transformation from tryptophan-like and tyrosine-like components to humic-like components, forming less aromatic and more saturated recalcitrant dissolved organic carbon. Our findings provide new insights into the photo-reactivity and photo-transformation of algae-derived organic matters and help to predict DOM formation involved in carbon cycling in water environment.

Production and Characterization of a SARS-CoV-2 Nucleocapsid Protein Reference Material.

Rapid antigen tests have become a widely used COVID-19 diagnostic tool with demand accelerating in response to the highly contagious SARS-CoV-2 Omicron variant. Hundreds of such test kits are approved for use worldwide, predominantly reporting on the presence of the viral nucleocapsid (N) protein, yet the comparability among manufacturers remains unclear and the need for reference standards is recognized. To address this lack of standardization, the National Research Council Canada has developed a SARS-CoV-2 nucleocapsid protein reference material solution, NCAP-1. Reference value determination for N protein content was realized by amino acid analysis (AAA) via double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in conjunction with UV spectrophotometry based on tryptophan and tyrosine absorbance at 280 nm. The homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. The molar concentration of the N protein in NCAP-1 was 10.0 ± 1.9 μmol L-1 (k = 2, 95% confidence interval). Reference mass concentration and mass fraction values were subsequently calculated using the protein molecular weight and density of the NCAP-1 solution. Changes to protein higher-order structure, probed by size-exclusion liquid chromatography (LC-SEC) with UV detection, were used to evaluate transportation and storage stabilities. LC-SEC revealed nearly 90% of the N protein in the material is present as a mixture of hexamers and tetramers. The remaining low molecular weight species (<30 kDa) were interrogated by top-down mass spectrometry and determined to be autolysis products homologous to those previously documented for N protein of the original SARS-CoV [Biochem. Biophys. Res. Commun.2008t, 377, 429-433].

Genome-Wide Identification and Expression Analysis of the NAC Gene Family in Alfalfa Revealed Its Potential Roles in Response to Multiple Abiotic Stresses.

NAC (NAM, ATAF1/2, and CUC2) transcription factors compose one of the largest families of plant-specific transcription factors; they are widely involved in plant growth and development and have especially important roles in improving stress resistance in plants. However, NAC gene family members in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed genome-wide due to the complexity of the alfalfa reference genome. In this study, a total of 421 M. sativa NAC genes (MsNACs) were identified from the alfalfa “Xinjiangdaye” reference genome. Basic bioinformatics analysis, including characterization of sequence length, protein molecular weight and genome position and conserved motif analysis, was conducted. Expression analysis showed that 47 MsNACs had tissue-specific expression, and 64 MsNACs were expressed in all tissues. The transcriptomic profiles of the genes were very different, indicating that these MsNACs have various functions in alfalfa growth and development. We identified 25, 42 and 47 MsNACs that respond to cold, drought and salt stress based on transcriptome data analysis and real-time quantitative PCR (RT–qPCR). Furthermore, 22 MsNACs were found to respond to both salt and drought stress, and 15 MsNACs were found to respond to cold, salt and drought stress. The results of this study could provide valuable information for further functional analysis of MsNACs and for the improvement of stress resistance in alfalfa.

Molecular mechanisms of steric pressure generation and membrane remodeling by disordered proteins

Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Förster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape.

Proteinaceous Matter and Liquid Water in Fine Aerosols in Nanchang, Eastern China: Seasonal Variations, Sources, and Potential Connections

AbstractThe transformation of aerosol proteinaceous matter (PrM) has potentially adverse environmental and health effects. The origins and atmospheric processes of PrM have obtained an increasing interest, but there are still large uncertainties. Although aerosol liquid water (ALW) has been found to be vital in numerous atmospheric processes, its impact on atmospheric degradation of PrM remains poorly understood. ALW, proteins, and low molecular weight PrM (LMW PrM, &lt;10 kDa) were investigated in fine aerosols (PM2.5) collected in urban and suburban Nanchang (Eastern China) over a 1‐year period. The average concentrations of proteins and LMW PrM in the urban center were higher than those in the suburban area. Urban PrM tended to increase from cool season to warm season, which was opposite to the case of suburban PrM. These differences could be attributed to varied sources and atmospheric processes of PrM. Increase in ALW was prominent from the suburban site to the urban site, which was attributable to increased anthropogenic nitrate and sulfate. LMW PrM can be degraded from higher molecular weight PrM by ozone. Furthermore, correlation analysis between LMW PrM and ozone and ALW suggested that the enhanced ALW in the urban center facilitated the ozone‐induced LMW PrM release. The ALW‐related protein degradation was further supported by ozone exposure experiments with bovine serum albumin and PM2.5 samples under dry condition. Our findings suggest that ALW is a crucial promoter during PrM degradation by ozone, providing new insights into atmospheric transformation of proteins.

Bioinspired stretchable molecular composites of 2D-layered materials and tandem repeat proteins

Protein based composites, such as nacre and bone, show astounding evolutionary capabilities, including tunable physical properties. Inspired by natural composites, we studied assembly of atomistically thin inorganic sheets with genetically engineered polymeric proteins to achieve mechanically compliant and ultra-tough materials. Although bare inorganic nanosheets are brittle, we designed flexible composites with proteins, which are insensitive to flaws due to critical structural length scale (∼2 nm). These proteins, inspired by squid ring teeth, adhere to inorganic sheets via secondary structures (i.e., β-sheets and α-helices), which is essential for producing high stretchability (59 ± 1% fracture strain) and toughness (54.8 ± 2 MJ/m3). We find that the mechanical properties can be optimized by adjusting the protein molecular weight and tandem repetition. These exceptional mechanical responses greatly exceed the current state-of-the-art stretchability for layered composites by over a factor of three, demonstrating the promise of engineering materials with reconfigurable physical properties.

A cold-water polysaccharide-protein complex from Grifola frondosa exhibited antiproliferative activity via mitochondrial apoptotic and Fas/FasL pathways in HepG2 cells

Grifola frondosa (G. frondosa) is widely known for its anti-tumor potential, which has been demonstrated by numerous scientific researches. In this study, two water soluble polysaccharide-protein complexes were extracted from G. frondosa at 4 °C (GFG-4) and 100 °C (GFG-100) and purified. Compared with GFG-100, GFG-4 had a higher protein content and molecular weight. The main monosaccharides of GFG-4 and GFG-100 were rhamnose, glucose, and galactose, with an approximate ratio of 3.00: 1.00: 0.86 and 2.85: 1.00: 0.94, respectively. The Fourier transform infrared spectra indicated that the two polysaccharide-protein complexes displayed characteristic functional groups of polysaccharides and proteins, and mainly contain pyranose ring with α-glycosidic linkage. Atomic force microscope images showed that both GFG-4 and GFG-100 exhibited straight chains, and GFG-4 possessed a relatively abundant fraction of branched chains. Intriguingly, GFG-4 showed a stronger antiproliferative activity against HepG2 cells than GFG-100. The mechanisms were further investigated by quantitative real-time PCR and western blot, it found that GFG-4 inhibited the proliferation of HepG2 cells mainly through the intrinsic activation of mitochondrial pathway and the Fas/FasL-mediated Caspase-8/-3 pathway. Conclusively, G. frondosa cold-water extracted polysaccharide-protein complexes could be used as a functional food for preventing or treating hepatocellular carcinoma.

Structural Regulation at Poly(ethylene glycol) Termini Facilitates the Formation of Injectable Hydrogels with Modulated Degradation and Release of Biomacromolecules

Hydrogels possessing modulated degradation and release behaviors are beneficial for the biological field. However, modulating the degradability and release behavior of a particular class of hydrogels requires a major structural alteration of a cross-linker as well as an adjustment in concentration, both of which affect the initial modulus and gelation kinetics. Herein, we address this shortcoming by a single step installation of a leaving nucleophile at the termini of PEG via the ether (O–CH2–Ph–, Cl′-PEG-Cl′) and ester (−Ph–COO–, Cl-PEG-Cl) spacers separately. These two PEG derivatives separately or in combination lead to the rapid formation of hydrogels when combined with a variety of macromolecules with tertiary amine functional group. The hydrogels exhibit a wide variation in degradation properties and time-dependent water swelling depending on the type and proportion of the PEG derivatives. The gelation kinetics is faster when the termini of the PEG derivative contain benzyl chloride groups connected through the ether spacer and is explained by density functional theory. Importantly, the ultimate moduli of the as prepared hydrogels are almost similar irrespective of the termini structure of the PEG derivatives and their proportion. The hydrogels exhibit modulated release profiles of model dextran and bovine serum albumin at pH 7.4. Prolonged delivery of biomacromolecules is achieved with the hydrogel prepared with Cl′-PEG-Cl′ or the combination Cl′-PEG-Cl′+Cl-PEG-Cl. More importantly, hydrogels prepared with Cl′-PEG-Cl′ or the Cl′-PEG-Cl′+Cl-PEG-Cl combination exhibit triggered release (60–100%) of a model protein (isoelectric point ≤ 7.4) and high molecular weight dextran (neutral) at a constant pH of 5 when compared to that at pH 7.4. The key advantages of our design are the access to a library of biological relevant hydrogels from easily available/synthesized amine-functional macromolecules and PEG derivatives with slight variations in the termini structures.

Blood glucose level, blood profile and ectoparasite infestation of cantang grouper fish commercially fed with the addition of marine leeches Zeylanicobdella

The main problem that often occurs in grouper cultivation is the appearance of disease attacks caused by pathogens due to water quality that has not been optimal during maintenance. There are common problems in grouper cultivation, that is unstable environmental conditions that cause accumulation of organic matter which can cause fish to be stress, which is characterized by increased blood glucose levels and susceptible to disease. The types of ectoparasites that commonly attack groupers are Trichodina, Cryptocaryon, Zeylanicobdella and Neobenedenia. An alternative to control pathogen attacks on fish is by increasing the body&#39;s defense cells of fish using immunostimulants. Zeylanicobdella known to have molecular weight of proteins that are immunogenic, and is thought to increase the activity of cells defense of the fish body as an immunostimulant. This study aims to find out the blood glucose level, blood profile and ectoparasite infestation of cantang grouper fish given a marine leeches Zeylanicobdella on feed. The method in this research used an experimental method with a factorial pattern consisting of two factors. Factor P is the dose given and factor Q is the maintenance time. Statistical test results showed there was an influence (p < 0.01) between the doses of marine leeches Zeylanicobdella on the number of erythrocytes and leukocytes, there was also interaction (p > 0.01) between the doses of marine leeches Zeylanicobdella with different maintenance times to the number of erythrocytes, leukocytes, differential leukocytes including lymparhocytes, neutrophils and monocytes. The results showed that there was a very significant difference between blood glucose levels and ectoparasite infestation of cantang grouper maintenance at different doses and times and there was an interaction effect (p < 0.01). The highest blood glucose level of 67.00 ± 5.148 mg/dL occurred in cantang grouper with a moderate degree of ectoparasite infestation of 36.20 ± 2.387 parasites on day 0 of maintenance with a dose of 0 ppm. The lowest blood glucose level of 28.80 ± 0.837 mg/dL occurred in cantang grouper with a mild degree of ectoparasite infestation, that is 12.60 ± 1.517 parasites on the 28th day of rearing with a dose of 9 ppm. The results showed that the highest of total erythrocytes was at a dose of 7 ppm with a maintenance time of the 21th day are 2,534 × 106 cell/ml. Total Leukocyte was at a dose of 9 ppm with a maintenance time of the 28th day are 1,4902 × 106 cell/ml. The percentage of lymphocytes is the highest with a range of 58-93%, neutrophils range from 5-38% and monocytes range from 1-9%.

Protease from jute endophyte Micrococcus luteus MBL-Bac7 functions as a potential bating agent for the leather industry

Ever since the discovery of jute endophytes, testing their potential for commercial uses has been a matter of interest. Considering the same, jute endophyte Micrococcus luteus MBL-Bac7, capable of producing extracellular proteases, was selected for in vitro and in silico analysis to assess its role as a bating agent required in rawhide processing. The presence of extracellular protease was confirmed from the plate assay. As the enzyme is tested for commercial use, the effect of various metal ions and reaction conditions (pH, temperature) have been optimized. The protease activity appears to be retained even at 85°C. It also showed significant activity in a wide range of pH (pH 3.0-8.5). Metal ion Mn2+ increased the protease activity significantly, but Fe2+, Zn2+, and Co2+ ions showed the opposite effect. Molecular identification of the protease was done from the whole genome sequence data. Using PSORTb v.3.0.2, SecretomeP-1.0, TMHMM-2.0, and protein molecular weight software, the physicochemical properties of the protease were predicted. The isolated protease shared a strong evolutionary link with Micrococcus species' S8 family serine peptidase. Finally, in the bating of cowhide, effects similar to that of commercial agents were obtained during finger prick, lastometer, and tensile tests. The findings of this study corroborate the possibility of using this protease as a potential bating agent. However, further studies are necessary to reduce the production cost for higher yield and commercialization. J. Bangladesh Acad. Sci. 46(1); 31-43: June 2022

Levan-type fructooligosaccharides synthesis by novel levansucrase-inulosucrase fusion enzyme

A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.

Genome-Wide Identification and Analysis of the NF-Y Transcription Factor Family Reveal Its Potential Roles in Salt Stress in Alfalfa (Medicago sativa L.).

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that plays an important role in various biological processes in plants, such as flowering regulation, drought resistance, and salt stress. However, few in-depth studies investigated the alfalfa NF-Y gene family. In this study, in total, 60 MsNF-Y genes, including 9 MsNF-YAs, 26 MsNF-YBs, and 25 MsNF-YCs, were identified in the alfalfa genome. The genomic locations, gene structures, protein molecular weights, conserved domains, phylogenetic relationships, and gene expression patterns in different tissues and under different stresses (cold stress, drought stress, and salt stress) of these NF-Y genes were analyzed. The illustration of the conserved domains and specific domains of the different subfamilies of the MsNF-Y genes implicates the conservation and diversity of their functions in alfalfa growth, development, and stress resistance. The gene expression analysis showed that 48 MsNF-Y genes (7 MsNF-YAs, 22 MsNF-YBs, and 19 MsNF-YCs) were expressed in all tissues at different expression levels, indicating that these genes have tissue expression specificity and different biological functions. In total, seven, seven, six, and eight MsNF-Y genes responded to cold stress, the ABA treatment, drought stress, and salt stress in alfalfa, respectively. According to the WGCNA, molecular regulatory networks related to salt stress were constructed for MsNF-YB2, MsNF-YB5, MsNF-YB7, MsNF-YB15, MsNF-YC5, and MsNF-YC6. This study could provide valuable information for further elucidating the biological functions of MsNF-Ys and improving salt tolerance and other abiotic stress resistance in alfalfa.

Effects of Different Heat Processing on Molecular Weight and Allergenicity Profile of White Shrimp (Litopenaeus vannamei) and Mud Crab (Scylla serrata) from Indonesian Waters

Shrimp and crab are fishery products with high nutritional value, especially as protein sources. However, they belong to the crustacean group known to cause many allergies in Southeast Asian countries. Not only affecting the nutritional composition, processing also has the potential in reducing the allergenicity of a food ingredient. This study aims to analyze the effects of various heat processing on nutrient composition, soluble protein content, protein molecular weight profile, and allergenicity of white shrimp and mud crab. The processing was carried out by boiling, frying, and autoclaving. Changes in macronutrient content were determined using proximate analysis. Soluble protein content was analyzed using the Bradford method, while molecular weight and allergenicity profiles were analyzed using SDS-PAGE electrophoresis and ELISA techniques. Results showed that processing treatment could significantly reduce protein solubility. SDS-PAGE profile showed that heating shrimp and crab using autoclave at 121.1 oC and pressure of 0.2 MPa for 10 min was able to remove protein bands of 35-38 kDa, which were suspected as the allergen bands. However, boiling (100 oC for 10 min) and deep frying in palm oil (160 oC for 10 min) treatments of the samples gave stronger 35-38 kDa bands intensity than the unprocessed sample. Autoclaving was able to significantly reduce IgE reactivity to shrimp extract better than the boiling and frying process. In contrast, IgE reactivity of crab extract was increased by heat processing especially frying. Thus, autoclaving can be used in the white shrimp processing to produce hypoallergenic food.

Protein degradation and aggregation in silver carp (Hypophthalmichthys molitrix) muscle during hot air drying

This research was to investigate protein changes (protein molecular weight distribution, free amino acid (FAA), cathepsin activity) in silver carp (Hypophthalmichthys molitrix) muscle during the process of brine curing (10 g/L salt, 4 °C, 3 h) and air-drying (45 °C, 24 h). The results showed that after air-drying for 4 h, the proportion of protein aggregates with molecular weight >1000 kDa increased by 7.47% compared to fresh fish. Meantime, activities of cathepsin B and B + L increased from 1.52 ± 0.35 and 2.05 ± 0.45 U/g to 2.33 ± 0.22 and 3.27 ± 0.18 U/g, respectively. However, total FAA content had not significantly different (P > 0.05) in this drying period. Thus, although protein aggregation and protein degradation occurred simultaneously, protein aggregation was dominant in the early stage of drying processing. And the protein aggregation decreased, and total FAA content increased, activities of cathepsin decreased but remained relatively high with continuous drying. Thus, protein changes in silver carp muscle were mainly due to degradation. Protein analysis in this study favored to understand the silver carp muscle changes during drying processing and supported to improve freshwater fish drying processes.

Response surface methodology‐optimized co‐fermentation of pigeon pea okara by Rhizopus oligosporus and Yarrowia lipolytica and its application for vegetable paste

AbstractIntroductionPigeon pea (Cajanus cajan) is a perennial legume usually consumed directly or processed into pigeon pea milk. Pigeon pea okara (PPO) contains 15.5 – 28.8% protein, 56% carbohydrate, as well as vitamins, minerals, and dietary fiber, making it a valuable food material or other applications. This study examines co‐fermentation of PPO by Rhizopus oligosporus and Yarrowia lipolytica to maximize its nutritional value.MethodsThe response surface methodology (RSM) with Four variables and five levels central composite design was used to optimize the co‐fermentation conditions including fermentation temperature, R. oligosporus and Y. lipolytica inoculum concentrations, and fermentation time.ResultsSolid‐state co‐fermentation significantly increased the total phenolic, total flavonoid, and protein contents, with SDS‐PAGE revealing protein molecular weights generally &lt; 10 kDa. The optimal fermentation condition was using 5.05 × 105 spores/g R. oligosporus and 5.05 × 105 CFU/g Y. lipolytica at 39°C for 48 hrs. Sensory analysis and shelf‐life testing were then performed on vegetable pastes prepared using optimally fermented PPO. Vegetable paste prepared with 35% fermented PPO content had comparable overall acceptability to paste made with 35% unfermented PPO, and a 14‐day shelf‐life at 4°C.ConclusionIn summary, RSM‐optimized, solid‐state co‐fermentation is an effective method to improve the nutritional value of PPO.

Expression analysis of squalene synthase gene in mevalonate pathway of Sanghuangporus baumii

Fungi produce a wide repertoire of secondary metabolites with pharmaceutical impacts on human health. However, the low expression of secondary metabolites limits their clinical application. Triterpenoids produced by Sanghuangporus baumii possess anti-cancer properties, however, their biosynthetic pathway and regulatory mechanism remain unclear. In this study, squalene synthase gene (SbSQS), a key gene involved in the mevalonate pathway, was cloned and analyzed. Sequence analysis identified SbSQS as a typical squalene synthase with three conserved catalytic structures related to binding and catalysis. The SbSQS gene encodes a 489 amino acid polypeptide with a predicted protein molecular weight of 55.05 kDa. The promoter of SbSQS contains cis-acting regulatory elements involved in the light, abscisic acid and MeJA responsiveness, which is consistent with the promoters of other genes in the mevalonate pathway. Furthermore, SbSQS was cloned into the pET-32a vector and overexpressed in Escherichia coli. During mycelium fermentation of S. baumii, the biomass increased as fermentation progressed, and peaked on day 18 (1.20 g/250 mL). The triterpenoid content did not accumulate during the fermentation process and was 20.8 mg · g −1 on day 18. The SbSQS expression and the triterpenoid content were peaked on day 6, suggesting a correlation between SbSQS expression level with triterpenoid content. This study proposes a key candidate gene for genetic manipulation of the triterpenoid synthesis to enhance the triterpenoid content in S. baumii.

THE USE OF BIO-MOLECULAR SUBSTANCE OF MARINE BIOTA AS AN ALTERNATIVE EARLY INDICATOR OF OIL POLLUTED ENVIRONMENT: A NEW APPROACH FOR MONITORING CONSIDERATION

THE USE OF BIO-MOLECULAR SUBSTANCE OF MARINE BIOTA AS AN ALTERNATIVE EARLY INDICATOR OF OIL POLLUTED ENVIRONMENT: A NEW APPROACH FOR MONITORING CONSIDERATION

Characterization of a SARS-CoV-2 spike protein reference material

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography–tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet–visible spectrophotometry (UV–Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L−1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL−1 (k = 2), respectively.

Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5.

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s−1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.

Functional Properties of Collagen Extracted from Catfish (Silurus triostegus) Waste.

Collagen is used for a variety of biomedical and pharmaceutical uses, such as osteoarthritis-related pain management, hypertension, tissue engineering, and human implants, and is generally derived from porcine or bovine. Collagen from these animals has limitations due to the risk of disease transmission and religious constraints. Therefore, this study investigated the extraction of collagen from catfish (Silurus triostegus) waste. Acid-solubilized collagen and pepsin-solubilized collagen were extracted from catfish skin, fin, head, bone, and muscle. SDS-PAGE patterns of the extracted collagen showed that the protein molecular weights ranged from 97 to 200 kDa and skin, bone, and fin collagen consisted of 2 distinct α chains, which is typical of type 1 collagen. The proximate composition (moisture, protein, fat, and ash) and yield of the obtained extracts were determined. Skin collagen extracts were selected for further investigation due to the high collagen yield. The effects of the pH and salt concentration on solubility, and the denaturation temperature, FTIR spectra, reverse-phase HPLC, and SEM analysis were investigated to characterize the collagen samples. Based on the characterization of catfish skin collagen, this waste material has potential for use in the pharmaceutical and food industries.