Ammonium Sulfate Precipitation Research Articles

Generation of Chicken-Based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms

The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 μg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

Synergistic and Cytotoxic Effects of the Combination of Inerolysin and Vaginolysin Toxins on VK2 and HeLa Cells Isolated from an Iraqi Woman Who had an Abortion

Objective: The goal of this study is to purify inerolysin produced by Lactobacillus iners and vaginolysin produced by Gardnerella vaginalis, and to investigate the cytotoxic effects of the combination of these purified toxins on VK2 and HeLa cells. Methods: Out of 592 specimens from Baghdad teaching hospitals, 101 isolates were identified as L. iners and 165 isolates as G. vaginalis. The toxins were purified using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The cytotoxic effects of the combined inerolysin and vaginolysin on VK2 and HeLa cells were studied. Results: For inerolysin, the optimal precipitation saturation ratio was 70%, with ion exchange chromatography yielding a specific activity of 14 mg and gel filtration increasing the specific activity to 40 U/mg. For vaginolysin, the optimal precipitation saturation ratio was 50%, with ion exchange chromatography yielding a specific activity of 4 mg and gel filtration increasing the specific activity to 10 U/mg. The combination of toxins showed a viability ratio of 38% for VK2 cells and 31% for HeLa cells at higher toxin concentrations. Conclusion: Inerolysin and vaginolysin cause lysis of vaginal and cervical cells, which may lead to abortion. The killing ratio increases with higher concentrations of the toxins.

Optimization and purification of L-methioninase enzyme purified from clinical samples of Pseudomonas spp.

Background: This study focused on Pseudomonas aeruginosa, which secrete important enzymes in medical and pharmaceutical applications and are isolated from clinical samples. One of these enzymes is L-methioninase, the most desirable enzyme in the medical and industrial fields today since it converted L-methionine into methanethiol, ammonia, and alpha-ketobutyrate. Objectives: Evaluated the optimum conditions of L-methioninase production and then purified it. Methods: Pseudomonas samples were identified by microscopic and biochemical tests and confirmed with the VITEK2 system. After the L-methioninase production was examined, the optimal conditions for the production of L-methioninase (MGL) were established using clinical samples of P. aeruginosa. Additionally, MGL was purified from the supernatant of P.aeruginosa using ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G150 accordingly. Results: There were 33 isolates of P.aeruginosa according to microscopic characteristics and biochemical tests, followed by the VITEK2 system to ensure identification. The optimization conditions were achieved when supplement media with sucrose 1% w\v, casein 1% w\v, pH 7, temperature 37oC, and incubation for 48hrs within the specific activity of 1.6, 3.3, 3.4, 3.4, and 3.5U\mg protein, respectively, and 4.6 fold the amount of the purified enzyme as compared to the crude enzyme. A single 55 kDa band on the SDS-PAGE indicates the enzyme is purified. Conclusions: P.aeruginosa can be an efficient source of L-methioninase, and this enzyme had better and higher specific activity after applying optimum conditions for it and purifying it.

Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae)

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed Km and Vmax to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl2 significantly inhibited the enzyme, while the Na+, Ca++, and Mg++ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.

Purification and characterization of a thermostable Galium aparine β-galactosidase: A competent agent with enhanced cytotoxic activity against MCF-7 cell line

β-galactosidases (EC 3.2.1.23) are capable of catalysing two distinct types of reactions: transgalactosylation and hydrolysis. It is very important for the senescence of cancer cells. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from various sources including plants, animals, bacteria, yeasts, and filamentous fungi. Purification and thermodynamic characterization of β-galactosidase from Galium aparine seeds, and in vitro efficacy assessment of a breast cancer cell line. The enzyme was purified by using ammonium sulphate precipitation, dialysis, DEAE cellulose, and Sephadex G-100 chromatography. The enzyme was purified to 6.4 fold with a yield of 14 % and specific activity of 11.6 U/mg of protein. SDS-PAGE and MALDI-TOF analysis revealed that β-galactosidase is a monomer with a molecular weight of 35 kDa. The optimum pH and temperature for the purified enzyme were 6.0 and 50 ºC. The enzyme had Km and Vmax values of 0.25 mM and 32 µmol/min, respectively. The thermodynamic parameters (ΔHº, ΔGº, ΔSº) for the irreversible denaturation of the enzyme were also investigated at different temperatures (50–75 ºC). Lactose in milk was successfully hydrolyzed using a purified enzyme at a rate of 0.0121 min−1 and 12.12 min. This is better than other studied β-galactosidases. These results suggest the use of β-galactosidase from G. aparine for producing delactosed milk for the lactose-intolerant population and prebiotic synthesis. pH and optimum temperature and its activation by Ca2+ shows that it is suitable for milk processing. As a result, the enzyme was stable even at higher temperatures and had the potential to undergo catalysis, making it economically viable, primarily in the food and pharmaceutical industries. The present study is aimed to purification and thermodynamic characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Tenderization of giant squid (Dosidicus gigas) meat pretreated with pulsed electric field and Asian seabass trypsin with the aid of vacuum impregnation

Giant squid (Dosidicus gigas) meat (GSM) generally has a tough texture, significantly lowering its acceptability by consumers. To enhance tenderness, GSM was pretreated with pulsed electric field (PEF) at 250 V for 5 min (PEF-GSM). Thereafter, PEF-GSM was soaked in partially purified trypsin (PPT) from pyloric caeca of Asian seabass obtained from ammonium sulfate precipitation (40%–60% saturation). The soaking was done at various concentrations of PPT (0, 5, 10 and 20 units/mL) with the aid of vacuum impregnation (VI) (2 cycles). Subsequently, PPT-soaked GSM samples were incubated at 60 °C for 5 and 10 min, followed by cooking at 100 °C for 2 min. For quality assessment, untreated cooked GSM (CON-GSM) was also prepared. Lightness of PEF-GSM samples increased (p < 0.05), compared to that of CON-GSM. After hydrolysis with PPT, myofibrillar proteins were degraded with coincidental upsurge in AAGC (α-amino group content). Protein degradation and AAGC became more pronounced with increasing PPT concentration and incubation time. PEF-GSM treated with PPT at 20 units/mL (PEF-GSM-20) and incubated for 10 min had the decreased firmness and toughness values, however its cooking loss slightly increased. Degradation of myofibrillar proteins was reconfirmed by FTIR spectra and microstructure alteration. PEF-GSM-20 with incubation at 60 °C for 10 min showed higher overall likeness score (p < 0.05), indicating a significant improvement in acceptability of consumers. Thus, combined application of PEF, PPT (20 units/mL) and VI before incubation at 60 °C for 10 min significantly enhanced the eating quality of GSM, making it more preferable for consumers.

Bioremoval of tannins and heavy metals using immobilized tannase and biomass of Aspergillus glaucus

BackgroundThe presence of inorganic pollutants and heavy metals in industrial effluents has become a serious threat and environmental issues. Fungi have a remarkable ability to exclude heavy metals from wastewater through biosorption in eco-friendly way. Tannase plays an important role in bioconversion of tannin, a major constituent of tannery effluent, to gallic acid which has great pharmaceutical applications. Therefore, the aim of the current study was to exploit the potential of tannase from Aspergillus glaucus and fungal biomass waste for the bioremediation of heavy metals and tannin.ResultsTannase from A. glaucus was partially purified 4.8-fold by ammonium sulfate precipitation (80%). The enzyme was optimally active at pH 5.0 and 40 °C and stable at this temperature for 1 h. Tannase showed high stability at different physiological conditions, displayed about 50% of its activity at 60 °C and pH range 5.0–6.0. Immobilization of tannase was carried out using methods such. as entrapment in Na-alginate and covalent binding to chitosan. The effects of Na-alginate concentrations on the beads formation and enzyme immobilization revealed that maximum immobilization efficiency (75%) was obtained with 3% Na-alginate. A potential reusability of the immobilized enzyme was showed through keeping 70% of its relative activity up to the fourth cycle. The best bioconversion efficiency of tannic acid to gallic acid by immobilized tannase was at 40 °C with tannic acid concentration up to 50 g/l. Moreover, bioremediation of heavy metal (Cr3+, Pb2+, Cu2+, Fe3+, and Mn2+) from aqueous solution using A. glaucus biomass waste was achieved with uptake percentage of (37.20, 60.30, 55.27, 79.03 and 21.13 respectively). The biomass was successfully used repeatedly for removing Cr3+ after using desorbing agent (0.1 N HCl) for three cycles.ConclusionThese results shed the light on the potential use of tannase from locally isolated A. glaucus in the bioremediation of industrial tanneries contained heavy metals and tannin.

Extraction and Partial Purification of Glucoamylase from Rhizopus oryzae by Solid State Fermentation using Agro-Industrial Residues

Background: Filamentous fungi are used to produce many industrial enzymes because these fungi have a great ability to produce such enzymes, especially when using solid substrates that are usually low in cost. The widespread use of this enzyme has made it very important to work on improving its production to achieve the maximum possible benefit.Methods: Five fungal isolates were obtained from soil and decaying fruits and vegetables. All isolates were identified as Rhizopus spp. and cultured on Potato Dextrose Agar (PDA) plates escorted by starch 1%. The appearance of clearance zones around the fungal colonies represents the ability of fungi to produce Glucoamylase. Different solid substrates, nitrogen sources, and temperatures were used to improve enzyme production.Results: Wheat bran gave the highest enzyme production with specific activity 6.25 U/mg, and yeast extract was the potent inducer for enzyme production with specific activity 9.28 U/mg. The optimal temperature for enzyme production was 30°C with specific activity10.27U/mg. Maximum specific activity 18.40U/mg was recorded at 55% saturation with ammonium sulfate precipitation. The result showed increased specific activity 24.84U/mg with dialysis. Partial purification of the enzyme revealed that pH5 was optimum for enzyme-specific activity 9.2U/ml; higher enzyme-specific activity was found with MnSO4 (11.83U/ml) as the best enzyme inducer.Conclusion: This investigation attempted to examine R. oryzae as an effective maker of GA. Innovative work into the nearby creation of GA for industrial use utilizing local resources has demonstrated financial effectiveness. It requires improvement into consistency as needed by worldwide associations dealing with industrial enzymes. Keywords: Glucoamylase; Production; Partial purification; Rhizopus oryzae; Solid state fermentation

Novel cold-active amylase-producing bacterium from Chukchi Sea and its enzyme properties

A cold-active amylase was purified from Alteromonas sp. KS7913 isolated from the Chukchi Sea in the Arctic Ocean. After purification with use of ammonium sulfate precipitation, phenyl column chromatography, and size exclusion chromatography, 200.34 U mg−1 of purified amylase was obtained. The final yield was 3.4%, and the activity was 5.7-fold higher than that of the initial culture broth. KS7913 origin amylase showed a molecular weight of 70 kDa and optimal activity at 25℃, pH 7.0 in Tris-HCl buffer. The amylase was highly active, especially at 5℃, and maintained stability at basic conditions below 25℃. Copper and zinc ions inhibited enzyme activity, whereas manganese, barium, and calcium ions exhibited positive effects. This activity was maintained even in the presence of alcohol. The findings of this study supplement our understanding of cold-active amylases, and may have practical applications in low-temperature industries.

Characterization of raw starch hydrolysing and detergent-compatible amylase from newly isolated Bacillus subtilis from bakery chimney

Amylases are highly needful product in food, beverage, and detergent industry. With the objective of isolating α-amylases producing strains, samples were collected from local bakery chimneys and kitchens, Chennai, Tamil Nadu, India. The potential α-amylase producers were isolated and screened by Starch-Iodine plate assay method. The optimized parameters such as 1.5% of starch concentration in the media with 7.2-pH at 35 °C for 36 h enhanced the production of α-amylase. The strain showing high potential, identified by 16s rRNA sequencing was found to be Bacillus subtilis. The Bacillus subtilis strain produced extracellular α-amylase with activity of 1330.14 U/mL. Further partial purification by ammonium sulphate precipitation followed by dialysis and column chromatography enhanced the specific activity to 1695.306 U/mg. The molecular weight of enzymes was found to be around 36 kDa. The enzyme stability and activity characterization revealed that the enzyme was thermotolerant which was able to retain more than 70% activity at 70 °C, 80 °C and 90 °C. The purified enzyme was also found to be Ca2+ independent. The hydrolysis of raw starch from rice, wheat and corn flour using the purified α-amylase from Bacillus subtilis showed significant activity of 72.8%, 76.1% and 65.2% with 4 h incubation respectively. The potentiality of the enzyme as an additive with commercial detergent was also determined to be effective. This study favourably attracts the attention towards industrial applications.

Isolation and characterization of polygalacturonase producing thermophilic Aspergillus niger isolated from decayed tomato fruits

This study aimed to isolate a fungal strain capable of producing acidophilic and thermostable polygalacturonase. In this study, the fungal isolate was isolated from decaying tomatoes. Based on the colony characteristics, microscopic and morphological observations, the isolated fungal pathogen has been identified as Aspergillus niger. The isolated fungus was used in solid-state fermentation to produce an acidic polygalacturonase enzyme. The enzyme was then purified using ammonium sulphate precipitation and column chromatography, and its activity was assayed by measuring the releasing sugar group from citrus pectin using a 3, 5-dinitrosalicylic acid (DNSA) reagent assay. The crude extract obtained from solid-state fermentation had an activity of 94.6 U/mL. Ammonium sulphate precipitation increased the enzyme's specific activity from 6.89 U/mg to 12.42 U/mg. Sephadex G-200 was used to purify the enzyme 3.58 times, and its specific activity was determined to be 24.66 U/mg. The Sephacryl S-100 column achieved a final fold purification of 9.93 times and a specific activity of 68.41 U/mg. The purified enzyme performed best when polygalacturonic acid was used as a substrate. The enzyme's optimum temperature and pH were 55°C and 5, respectively. CaCl2 was found to be the best chelating ion for the enzyme. This enzyme is recommended for use in a variety of industrial applications as the enzyme was found to be stable at acidic pH and high temperature.

Purification and structural analysis of tannase from novel bacteria of Bacillus cereus strain KMS3-1 isolated in marine sediment

Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds of hydrolyzable tannins to release small compounds such as glucose. In this work, tannase production from Bacillus cereus strain KMS3-1 was purified and characterized. The standard methods, such as ammonium sulfate precipitation followed by gel filtration chromatography, were used to purify the enzyme tannase from KMS3-1. With 40% enzyme recovery, the purification process yielded a 1.93-fold increase in specific activity. Analysis using SDS-PAGE confirmed a molecular weight of about 40 kDa. Circular dichroism analysis revealed the secondary structure composition α-helix (12.03%), indicating a dominant role of β-turns (38.4%). A pH of 6.0 and a temperature between 50 and 60 °C were ideal for enzyme activity. Tannic acid has an enzyme Km value of 3.0 x 10−3 M and a Vmax of 4.45 U/mL. Surfactants, metal ions, organic solvents, and inhibitors significantly influence the enzyme activity. Cytotoxicity assays revealed the non-toxic nature of the purified enzyme on Vero cells and rat animal models. These results explore and contribute to a better understanding of tannase enzyme properties and their potential applications in the food and beverage industries.

Partial purification and pectinase activity of lactic acid bacteria and pectinolytic bacteria consortium

Abstract Pectinase is an enzyme that can degrade pectin and often applied to the food industry. Microbes predominantly produce pectinases. This study aims to obtain the partial pectinase enzyme activity of bacteria from Liberica coffee fermentation. Lactic acid bacteria (LAB) isolates and pectinolytic bacteria were tested qualitatively to determine their pectinolytic activity. The highest pectinolytic activity will be used as a consortium. Optimal growth of lactic acid bacteria consortium and pectinolytic bacteria consortium incubation results were centrifuged to obtain enzyme extracts. The pectinase enzyme extract was partially purified by ammonium sulfate precipitation and dialysis. Based on the analysis, the qualitative index of pectinolytic lactic acid bacteria with isolate code BAL 1, BAL 2, and BAL 3 was 3,62%, 5,17%, and 7,27%, respectively. In pectinolytic bacteria, the qualitative index of pectinolytic with isolate codes IF, 2C, and 3S was 14.56%, 6.26%, 6.74%. Precipitation 70% ammonium sulfate has the highest enzyme-specific activity.

Lipopeptides from Bacillus velezensis ZLP-101 and their mode of action against bean aphids Acyrthosiphon pisum Harris

BackgroundNatural products are important sources for the discovery of new biopesticides to control the worldwide destructive pests Acyrthosiphon pisum Harris. Here, insecticidal substances were discovered and characterized from the secondary metabolites of the bio-control microorganism Bacillus velezensis strain ZLP-101, as informed by whole-genome sequencing and analysis.ResultsThe genome was annotated, revealing the presence of four potentially novel gene clusters and eight known secondary metabolite synthetic gene clusters. Crude extracts, prepared through ammonium sulfate precipitation, were used to evaluate the effects of strain ZLP-101 on Acyrthosiphon pisum Harris aphid pests via exposure experiments. The half lethal concentration (LC50) of the crude extract from strain ZLP-101 against aphids was 411.535 mg/L. Preliminary exploration of the insecticidal mechanism revealed that the crude extract affected aphids to a greater extent through gastric poisoning than through contact. Further, the extracts affected enzymatic activities, causing holes to form in internal organs along with deformation, such that normal physiological activities could not be maintained, eventually leading to death. Isolation and purification of extracellular secondary metabolites were conducted in combination with mass spectrometry analysis to further identify the insecticidal components of the crude extracts. A total of 15 insecticidal active compounds were identified including iturins, fengycins, surfactins, and spergualins. Further insecticidal experimentation revealed that surfactin, iturin, and fengycin all exhibited certain aphidicidal activities, and the three exerted synergistic lethal effects.ConclusionsThis study improved the available genomic resources for B. velezensis and serves as a foundation for comprehensive studies of the insecticidal mechanism by Bacillus velezensis ZLP-101 in addition to the active components within biological control strains.

Chromatographic purification of the plasmin-like enzyme from clamworm (Perinereis aibuhitensis Grub) and its fibrinolytic activity by metal ions.

In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.

HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

Industrial Approach to Invertase Production from Fruit Waste for Enhanced Efficiency and Conservation.

This study investigates the commercial viability of repurposing fruit waste for enzyme production, specifically focusing on the invertase enzyme derived from Saccharomyces cerevisiae. By utilizing fruit pulp that incorporates mulberry, carob, Figure, and grape pulp as a nutrient source, it is observed that the culture medium containing carob pulp exhibits the highest invertase activity. Specifically, the invertase activity in this medium is approximately 2.5 times greater (12.90 U/mg protein) than that observed in the peptone medium (5.98 U/mg protein). The extract undergoes several purification steps, including ultrafiltration, ammonium sulfate precipitation, dialysis, and ion-exchange chromatography (purification ratio: 12.11 times, yield: 26.93%). The purified enzyme is immobilized using alginate beads, improving pH and thermal stability. The immobilized enzyme exhibits optimal activity between pH 3.50 and pH 7.00, thereby broadening the enzyme's high-activity pH range. The thermal stability of the immobilized invertase enzyme is significantly improved, especially at 65 °C. Activity studies in the presence of metal ions and certain chemicals have been conducted. The immobilized enzyme's activity increases by approximately 40% in the presence of Ca2+ and Mg2+, and the immobilized enzyme maintains its activity in the presence of detergents such as SDS, Tween-20, and organic solvents like ethanol and methanol. The potential for the reuse of immobilized invertase was investigated under standard assay conditions. After 20 cycles, the immobilized enzyme was found to retain 80% of its initial activity. Overall, the study establishes the commercial potential of fruit pulp, typically discarded in fruit juice production, as a valuable source for obtaining an invertase enzyme. Furthermore, this study also aims to develop a suitable purification process for invertase in the fruit juice industry. By harnessing fruit waste and implementing innovative enzyme production strategies, industries can enhance their efficiency, reduce their environmental footprint, and optimize resource utilization.

Stability of R-phycoerythrin from Furcellaria lumbricalis – Dependence on purification strategies and purity

R-phycoerythrin (R-PE) is the most abundant, naturally occurring phycobiliproteins found in red algae. The spectroscopic and structural properties of phycobiliproteins exhibit unique absorption characteristics with two significant absorption maxima at 498 and 565 nm, indicating two different chromophores of R-PE, phycourobilin and phycoerythrobilin respectively. This study aimed to clarify how the stability of R-PE purified from F. lumbricalis was affected by different purification strategies. Crude extracts were compared to R-PE purified by i) microfiltration, ii) ultrafiltration, and iii) multi-step ammonium sulphate precipitation followed by dialysis. The stability of the different R-PE preparations was evaluated with respect to pH (2, 4, 6, 7, 8, 10 and 12) and temperature (20, 40, 60, 80 and 100 °C). The absorbance spectra indicated higher stability of phycourobilin as compared to phycoerythrobilin for heat and pH stability in the samples. All preparations of R-PE showed heat stability till 40 °C from the findings of color, concentration of R-PE and fluorescence emission. The crude extract showed stability from pH 6 to 8, whereas R-PE purified by ultrafiltration and multi-step ammonium sulphate precipitation were both stable from pH 4 to 8 and R-PE purified by microfiltration exhibited stability from pH 4 to 10 from the results of color, SDS-PAGE, and concentration of R-PE. At pH 2, the color changed to violet whereas a yellow color was observed at pH 12 in the samples along with the precipitation of the protein.

Biochemical characterization of purified phytase produced from Aspergillus awamori AFE1 associated with the gastrointestinal tract of longhorn beetle (Cerambycidae latreille)

ABSTRACT The need for industrially and biotechnologically significant enzymes, such as phytase, is expanding daily as a result of the increased use of these enzymes in a variety of operations, including the manufacture of food, animal feed, and poultry feed. This study sought to characterize purified phytase from A. awamori AFE1 isolated from longhorn beetle for its prospect in industrial applications. Ammonium sulfate precipitation, ion-exchange chromatography, and gel-filtration chromatography were used to purify the crude enzyme obtained from submerged fermentation using phytase-producing media, and its physicochemical characteristics were examined. The homogenous 46.8-kDa phytase showed an 8.1-fold purification and 40.7% recovery. At 70 C and pH 7, the optimum phytase activity was noted. At acidic pH 4–6 and alkaline pH 8–10, it likewise demonstrated relative activity of 88–95% and 67–88%, respectively. It showed 67–70% residual activity between 30 and 70 C after 40 min, and 68–94% residual activity between pH 2 and 12 after 2 h. The presence of Hg+, Mg2+, and Al3+ significantly decreased the enzymatic activity, whereas Ca2+ and Cu2+ enhanced it. Ascorbic acid increased the activity of the purified enzyme, whereas ethylenediaminetetraacetic acid (EDTA) and mercaptoethanol inhibited it. The calculated values for Km and Vmax were 55.4 mM and1.99 μmol/min/mL respectively. A. awamori phytase, which was isolated from a new source, showed unique and remarkable qualities that may find use in industrial operations such as feed pelleting and food processing.