scholarly journals A Potent Peroxidase from Solid Cell Culture of Ocimum Basilicum with High Sensitivity for Blood Glucose Determination

2021 ◽  
Author(s):  
Parvin Mohammadnejad ◽  
Saeed Soleimani Asl ◽  
Zahra Rasoulian ◽  
Saeed Aminzadeh ◽  
Jaleh Ghashghaie ◽  
...  
Keyword(s):  
Cell Culture ◽  
Large Scale ◽  
Specific Activity ◽  
High Sensitivity ◽  
High Specificity ◽  
Global Market ◽  
Ocimum Basilicum ◽  
Scale Production ◽  
Market Demand ◽  
Glucose Determination

Abstract Objectives Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020 - 2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. In pursuit of this trend; Results, A novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of Ocimum basilicum L. in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg-1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (Km = 21.5 mM), but obtained an exceptionally high specificity constant (kcat/Km = 66743.7 s-1M-1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Conclusion Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L-1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.

scholarly journals A Potent Peroxidase from Solid Cell Culture of Ocimum Basilicum with High Sensitivity for Blood Glucose Determination

2021 ◽  
Author(s):  
Parvin Mohammadnejad ◽  
Saeed Soleimani Asl ◽  
Zahra Rasoulian ◽  
Saeed Aminzadeh ◽  
Jaleh Ghashghaie ◽  
...  
Keyword(s):  
Cell Culture ◽  
Large Scale ◽  
Specific Activity ◽  
High Sensitivity ◽  
High Specificity ◽  
Global Market ◽  
Ocimum Basilicum ◽  
Scale Production ◽  
Market Demand ◽  
Glucose Determination

Abstract Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020 - 2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. In pursuit of this trend, a novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of Ocimum basilicum L. in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg-1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (Km = 21.5 mM), but obtained an exceptionally high specificity constant (kcat/Km = 66743.7 s-1M-1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L-1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.

Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

scholarly journals Infection with intracellular parasite Amoeboaphelidium protococcarum induces shifts in associated bacterial communities in microalgae cultures

2021 ◽  
Vol 33 (5) ◽  
pp. 2863-2873
Author(s):  
Anna-Lena Höger ◽  
Carola Griehl ◽  
Matthias Noll
Keyword(s):  
Bacterial Communities ◽  
Large Scale ◽  
Algal Growth ◽  
Dry Weight ◽  
Illumina Miseq ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Scale Production ◽  
Market Demand ◽  
Freshwater Microalgae

AbstractIn recent years microalgae products have developed increasing market demand, but sustainable industrial production is still challenged by biological stability of large-scale production plants. Yet the relationships between algal hosts, associated microbiomes, and contaminants in photobioreactors remains widely understudied. The aim of this study was to investigate the temporal development of microbiomes of four freshwater microalgae species Scenedesmus vacuolatus, Desmodesmus quadricauda, Chlorella sorokiniana, and Botryococcus braunii, in presence and absence of the zoosporic parasite Amoeboaphelidium protococcarum. To compare the effects of sterile and nonsterile culture conditions, infection experiments were performed in sterile laboratory (sterile) and simulated industrial conditions (open). Algal growth (dry weight, optical density, and nutrient consumption) was observed for 21 days, and samples of the associated microbiome were collected for bacterial 16S rRNA gene Illumina MiSeq sequencing. Infection patterns of A. protococcarum were algae species-specific, irrespectively of culture conditions. Bacterial community analysis demonstrated distinct and stable bacterial communities for each algae species, which were mostly dominated by α- and γ-Proteobacteria. Upon aphelid parasitosis, bacterial diversity increased, and community compositions diverged algae-specific over time. Moreover, bacterial functional traits shifted to detoxification, degradation, and cellulolysis once algae were infected. This study provides a first insight into the close connection between algae, associated bacterial microbiomes and appearing contaminants in photobioreactor systems.

scholarly journals Vibrio Proteases for Biomedical Applications: Modulating the Proteolytic Secretome of V. alginolyticus and V. parahaemolyticus for Improved Enzymes Production

2019 ◽  
Vol 7 (10) ◽  
pp. 387 ◽  
Author(s):  
Monica Salamone ◽  
Aldo Nicosia ◽  
Giulio Ghersi ◽  
Marcello Tagliavia
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Biomedical Applications ◽  
Large Scale ◽  
Proteolytic Enzymes ◽  
High Specificity ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Scale Production ◽  
Biological Responses ◽  
Large Scale Production

Proteolytic enzymes are of great interest for biotechnological purposes, and their large-scale production, as well as the discovery of strains producing new molecules, is a relevant issue. Collagenases are employed for biomedical and pharmaceutical purposes. The high specificity of collagenase-based preparations toward the substrate strongly relies on the enzyme purity. However, the overall activity may depend on the cooperation with other proteases, the presence of which may be essential for the overall enzymatic activity, but potentially harmful for cells and tissues. Vibrios produce some of the most promising bacterial proteases (including collagenases), and their exo-proteome includes several enzymes with different substrate specificities, the production and relative abundances of which strongly depend on growth conditions. We evaluated the effects of different media compositions on the proteolytic exo-proteome of Vibrio alginolyticus and its closely relative Vibrio parahaemolyticus, in order to improve the overall proteases production, as well as the yield of the desired enzymes subset. Substantial biological responses were achieved with all media, which allowed defining culture conditions for targeted improvement of selected enzyme classes, besides giving insights in possible regulatory mechanisms. In particular, we focused our efforts on collagenases production, because of the growing biotechnological interest due to their pharmaceutical/biomedical applications.

Development of a Large Scale Production Process for BAX 855, a PEGylated rFVIII Product with Prolonged Half-Life

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4362-4362
Author(s):  
Jürgen Siekmann ◽  
Purtscher Martin ◽  
Oliver Zöchling ◽  
Robert Kellerer ◽  
Hanspeter Rottensteiner ◽  
...  
Keyword(s):  
Manufacturing Process ◽  
Large Scale ◽  
Specific Activity ◽  
Drug Product ◽  
Scale Production ◽  
Drug Products ◽  
Large Scale Production ◽  
Bulk Drug ◽  
Recombinant Fviii ◽  
Conjugation Process

Abstract Abstract 4362 Baxter and Nektar have developed BAX 855, a PEGylated form of Baxter’s recombinant FVIII (rFVIII) product based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses proprietary stable PEGylation from Nektar Therapeutics. Similar Nektar technology has been successfully employed for marked and licensed PEGylated drug products and drugs in clinical use. The manufacturing process for BAX 855 comprises several steps, including chromatographic purification on a MacroCap SP resin, a strong cation exchanger, which allows the fractionation of species with different PEGylation degrees and concentration of the conjugate collected by an ultra- / diafiltration step leading to the pre-formulated bulk drug substance (BDS). Final formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process described is suited to manufacture BAX 855 in gram scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch. BAX 855 manufactured by this process has a specific activity similar to that of rFVIII in ADVATE™ and PEGylation degrees in the narrow range of 2 to 3 mols PEG / mol rFVIII. SDS-PAGE and Western blot analysis of BAX 855 confirm the stable PEGylation and demonstrate an increase in the molecular weight of the various FVIII domains. PK studies in different species display longer survival of BAX 855 compared to ADVATE™. Disclosures: Siekmann: Baxter Innovations GmbH: Employment. Martin:Baxter Innovations GmbH: Employment. Zöchling:Baxter Innovations GmbH: Employment. Kellerer:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Bossard:Nektar Therapeutics: Employment. Phillips:Nektar Therapeutics: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

scholarly journals Large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience

2015 ◽  
Vol 13 (8) ◽  
pp. 1199-1208 ◽  
Author(s):  
Yoram Tekoah ◽  
Avidor Shulman ◽  
Tali Kizhner ◽  
Ilya Ruderfer ◽  
Liat Fux ◽  
...  
Keyword(s):  
Cell Culture ◽  
Plant Cell ◽  
Plant Cell Culture ◽  
Large Scale ◽  
Scale Production ◽  
Large Scale Production ◽  
Pharmaceutical Proteins

scholarly journals Upstream process optimization and micro- and macrocarrier screening for large-scale production of the oncolytic H-1 protoparvovirus

2021 ◽  
Author(s):  
Daniel Wohlfarth ◽  
Veronika Frehtman ◽  
Marcus Müller ◽  
Martin Vogel ◽  
Linh Minh Phuc Phan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Production Process ◽  
Large Scale ◽  
Culture Medium ◽  
Feeding Strategy ◽  
Virus Yield ◽  
Seeding Density ◽  
Scale Production ◽  
Cell Culture Medium ◽  
Transfer Capability

Abstract The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. Key points • Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™. • We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield. • High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.

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