scholarly journals Integration of GC-MS in identification of possible final metabolites from phytase production in Pichia Pastoris based on sorbitol induction optimization

2021 ◽  
Vol 5 (1) ◽  
pp. 020-025
Author(s):  
Jean Bernard Ndayambaje ◽  
Gratien Habarurema ◽  
Janvier Habinshuti ◽  
Angelique Ingabire ◽  
Sabine Ingabire Ange ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Large Scale ◽  
Culture Media ◽  
Induction Phase ◽  
Scale Production ◽  
Significant Activity ◽  
Fed Batch ◽  
Phytase Production ◽  
Methanol Induction ◽  
Induction Technique

The isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction technique was investigated. The biological activity of extracellular phytase after optimization with co-substrates induction in 4 liters fermentor (NBS) increased to 13250 U/ml. This led to a 509 fold increases in comparison to the other type of phytase. This effect was studied via induction with sorbitol/methanol in fermentation by Pichia Pastoris GS115 (Mut+) at 20 °C. The interference of by products; methylal, hexamine and (S)-(+)-1,2-propanediol with release of phytase in Pichia Pastoris under methanol induction were detected and cannot be repressed by methanol induction alone. The TLC was used for glycerin analysis under methanol/sorbitol induction and the results were lesser compare to that obtained during phytase production under methanol induction alone. This work showed the higher expression of heterologous proteins and by fed batch fermentation; the expression identified an advantage of producing a significant activity of phytase. Practical applications Plant derived products including sorbitol have been used as alternative medicines for the therapeutic treatment of various diseases, food supplements and could be used in many manufacturing processes. It serves as a culture media for bacteria, and helps to distinguish the pathogenic E. coli O157:H7 from its most other strains. Cells growing on methanol require high oxygen consumption. Sorbitol was used as an alternative cheap co-feeding for the production of proteins and is a non-repressing carbon source for AOX1 promoter with no effect on the level of r-protein at its induction phase. This report describes the isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction techniques, and sorbitol showed to be a promising co-substrate, as it could enhance both cell growth and targeted protein productivity. This co-feeding and fed-batch induction technique was used for recombinant phytase production in a small and large scale production and the metabolites were analyzed.

Large-Scale Production of Glaciozyma antarctica Antifreeze Protein 1 (Afp1) by Fed-Batch Fermentation of Pichia pastoris

2017 ◽  
Vol 43 (1) ◽  
pp. 133-141 ◽  
Author(s):  
Mahzan Md Tab ◽  
Noor Haza Fazlin Hashim ◽  
Nazalan Najimudin ◽  
Nor Muhammad Mahadi ◽  
Farah Diba Abu Bakar ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Batch Fermentation ◽  
Large Scale ◽  
Antifreeze Protein ◽  
Scale Production ◽  
Fed Batch ◽  
Large Scale Production ◽  
Fed Batch Fermentation

scholarly journals Evaluation of the Antioxidant Activities and Tyrosinase Inhibitory Property from Mycelium Culture Extracts

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Ki Moon Park ◽  
Kyung Min Kwon ◽  
Seung Ho Lee
Keyword(s):  
Inhibitory Activity ◽  
Large Scale ◽  
Antioxidant Activities ◽  
Culture Media ◽  
Antioxidant Properties ◽  
Radical Scavenging ◽  
Reducing Power ◽  
Ethanol Extract ◽  
Scale Production ◽  
Morchella Esculenta

Since mushrooms have many bioactive components, they have been used as components in folk medicine. Because mycelium has an advantage when it comes to large-scale production, this study aimed to evaluate the antioxidant properties and anti-tyrosinase activity from 55 mycelia in culture media. Relatively high 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity was detected from the ethanol extract of culture media including mycelium (EECiM) ofMorchella esculentavar.esculenta(MEVE),Auricularia polytricha(APO),Tremella aurantia(TAU),Volvariella bombycina(VBO), andOudemansiellasp. (Osp), which also showed strong reducing power and inhibitory activity in relation to the thiobarbituric acid (TBA) value. On the other hand, relatively high tyrosinase inhibitory activity was detected inInonotus mikadoi(IMI),Coriolus versicolor(CVE),Volvariella volvacea(VVO),Panellus serotinus(PSE),Auricularia auricula(AAU), andFomitopsissp. (Fsp). Interestingly, the APO EECiM exhibited the highest DPPH radical scavenging rate (77.5 ± 4.3%) and reducing power (1.18 ± 0.041), while the highest inhibitory power of the TBA value and antityrosinase activity were detected in that of TAU (64.5 ± 4.1%) and IMI (46.0 ± 7.5%), respectively. Overall, our study suggested potential candidates for EECiMs that exhibited powerful antioxidant and tyrosinase inhibitory properties and might be used as natural antioxidant tyrosinase inhibitor.

scholarly journals Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris — functional and kinetic asymmetry revealed in proteoliposomes

2013 ◽  
Vol 1828 (9) ◽  
pp. 2238-2246 ◽  
Author(s):  
Piero Pingitore ◽  
Lorena Pochini ◽  
Mariafrancesca Scalise ◽  
Michele Galluccio ◽  
Kristina Hedfalk ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Large Scale ◽  
Scale Production ◽  
Large Scale Production

scholarly journals High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets

2019 ◽  
Vol 98 (1) ◽  
Author(s):  
Jian Wang ◽  
Yajing Liu ◽  
Yongzhi Yang ◽  
Chengling Bao ◽  
Yunhe Cao
Keyword(s):  
Pichia Pastoris ◽  
Growth Performance ◽  
Large Scale ◽  
Animal Feed ◽  
Nutrient Digestibility ◽  
Control Group ◽  
Scale Production ◽  
Thermostable Xylanase ◽  
Weaned Piglets ◽  
Degrading Bacteria

Abstract An acidic thermostable xylanase (AT-xynA) which was stable at low pH and high temperature was considered to have great potential in animal feed. For large-scale production, AT-xynA activity was enhanced about 1-fold in Pichia pastoris by constructing a double-copy expression strain in this study. Furthermore, impacts of different AT-xynA levels on growth performance, nutrient digestibility, short-chain fatty acids, and bacterial community in weaned piglets were determined. Compared with the control group, ADFI and ADG were higher for the pigs fed 4,000 or 6,000 U/kg AT-xynA (P < 0.05). AT-xynA supplementation also significantly increased the digestibility of OM, GE, and DM (P < 0.05). AT-xynA supplementation increased the concentrations of acetate in ileal (P < 0.01) and cecal digesta (P < 0.05). Isobutyrate (P < 0.05) and valerate (P < 0.05) concentrations in colonic digesta also significantly increased compared with the control group. AT-xynA supplementation increased the abundance of Lactobacillus in the ileal, cecal, and colonic digesta of weaned piglets (P < 0.05). AT-xynA alleviated anti-nutritional effects of nonstarch polysaccharides (NSP) by preventing the growth of Pateurella and Leptotrichia in the ileum (P < 0.05). AT-xynA increased the abundance of NSP-degrading bacteria, such as Ruminococcaceae, Prevotella in the cecum and colon (P < 0.05). In summary, AT-xynA addition could improve the growth performance of weaned piglets by altering gut microbiota.

scholarly journals High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia pastoris

2019 ◽  
Vol 21 (1) ◽  
pp. 279
Author(s):  
Qinghua Zhou ◽  
Zhixin Su ◽  
Liangcheng Jiao ◽  
Yao Wang ◽  
Kaixin Yang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Yarrowia Lipolytica ◽  
Large Scale ◽  
Gene Dosage ◽  
Life Time ◽  
Industrial Applications ◽  
Culture Conditions ◽  
Scale Production ◽  
Large Scale Production ◽  
High Level

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

scholarly journals NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections

Diagnostics ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 379
Author(s):  
Mariana Fonseca Xisto ◽  
John Willians Oliveira Prates ◽  
Ingrid Marques Dias ◽  
Roberto Sousa Dias ◽  
Cynthia Canedo da Silva ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Dengue Virus ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Limiting Factor ◽  
Scale Production ◽  
Global Public Health ◽  
Virus Infections ◽  
Correct Treatment ◽  
Diagnostic Kits

Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key point for the correct treatment and prevention of this disease. Currently, the limiting factor in the manufacture of dengue diagnostic kits is the large-scale production of the non-structural 1 (NS1) antigen used in the capture of the antibody present in the infected patients’ serum. In this work, we demonstrate the production of the non-structural 1 protein of dengue virus (DENV) serotypes 1–4 (NS1-DENV1, NS1-DENV2, NS1-DENV3, and NS1-DENV4) in the methylotrophic yeast Pichia pastoris KM71H. Secreted recombinant protein was purified by affinity chromatography and characterized by SDS-PAGE and ELISA. The objectives of this study were achieved, and the results showed that P. pastoris is a good heterologous host and worked well in the production of NS1DENV 1–4 recombinant proteins. Easy to grow and quick to obtain, this yeast secreted ready-to-use proteins, with a final yield estimated at 2.8–4.6 milligrams per liter of culture. We reached 85–91% sensitivity and 91–93% specificity using IgM as a target, and for anti-dengue IgG, 83–87% sensitivity and 81–93% specificity were achieved. In this work, we conclude that the NS1 recombinant proteins are efficiently produced in P. pastoris and have great potential for use in diagnostic kits for dengue virus infections. The transformed yeast obtained can be used for production in industrial-scale bioreactors.

scholarly journals Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Keyword(s):  
Pichia Pastoris ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Binding Domain ◽  
Size Exclusion ◽  
Scale Production ◽  
Receptor Binding Domain ◽  
Serological Tests

AbstractThe yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

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