Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris

Cho, Il Jae; Yeo, In-Cheol; Lee, Nam Keun; Jung, Suk Hee; Hahm, Young Tae

doi:10.1007/s12275-012-1290-7

Cited by 3 publications

(3 citation statements)

References 34 publications

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“…For the secretory efficiency of recombinant xylanase xynB, the bovine β-casein signal peptide was less efficient than the α-factor prepro sequence (24). Polygalacturonase genes were expressed in P. pastoris with the native signal peptide or the α-factor secretion signal peptide, but only slight differences in expression were observed (25). As signal peptides secrete recombinant proteins with varying efficiencies, it is important to examine different signal peptides that secrete the same protein in P. pastoris.…”

Section: Discussionmentioning

confidence: 99%

Expression, purification and characterization of human interferon-γ in Pichia pastoris

Wang

Ren

et al. 2013

Molecular Medicine Reports

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Human interferon-γ (hIFN-γ) is a multifunctional protein known to possess immunoregulatory, antiviral and anticancer functions. In the present study, in order to explore the biological roles of hIFN-γ and its mechanisms of action, IFN-γ was cloned and expressed in Pichia pastoris (P. pastoris) under the control of alcohol oxidase promoter 1 (AOX1). The protein was secreted by two different signal peptides, the native secretion signal peptide of hIFN-γ and the Saccharomyces cerevisiae α signal peptide. Following 96 h of methanol induction, Tricine-SDS-PAGE Coomassie staining, western blot analysis and N-terminal protein sequencing revealed that the level of recombinant hIFN-γ (rhIFN-γ) secreted by the native secretion signal was barely detectable, while the α signal peptide secreted ~300 mg/l. rhIFN-γ was purified by Vivaflow 200, SP Sepharose Fast Flow and Vivaspin 2 ml, yielding >96% of a highly purified rhIFN-γ preparation, with a specific activity of 1 x 10(7)-1.4 x 10(7) IU/mg protein as determined by an antiviral assay. The results demonstrated that the experimental procedures developed are capable of producing a large quantity of active rhIFN-γ from P. pastoris.

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Section: Discussionmentioning

confidence: 99%

Expression, purification and characterization of human interferon-γ in Pichia pastoris

Wang

Ren

et al. 2013

Molecular Medicine Reports

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“…At the same concentration, CoCl2 enhanced the activity by 41% (Fig. 6) acted as a strong inhibitor (Cho et al 2012). The effect of metal ions appears with varying results depending on the source and the fine structure of the endo-PG enzyme.…”

Section: Bioresourcescommentioning

confidence: 91%

“…Endo-polygalacturonases (endo-PGases) from a number of fungi have been studied and some have been cloned and utilized (Cho et al 2012;Liu et al 2014). The enzymes of bacterial origin, however, have not received as much attention in cloning for large scale using GRAS microorganisms.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Expression of Pectobacterium carotovorum Endo-polygalacturonase Gene in Pichia pastoris for Production of Oligogalacturonates

Rafique

Tabassum

Awan³

et al. 2016

BioResources

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A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZαA vector and constitutively expressed in Pichia pastoris. The recombinant endoPGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included glycerol in replacement of glucose as the carbon source. The enzyme had optimum activity at pH 5.5 and 40 °C with stability between pH 5.0 and 8.0 and at temperatures up to 50 °C. The enzyme activity was enhanced by 41% with the addition of 1 mM Co ++ , and inhibited by Fe ++ with a 63% reduction. The mode of the enzyme action showed internal cleavage of α-1,4 glycoside bonds of polygalacturonic acid and citrus peel pectin. Trigalacturonate and hexagalacturonate were the main hydrolysis products, with a yield of 0.44±0.01 and 0.21±0.01 mg released per mg polygalacturonic acid substrate, respectively. This represents the first report of a microbial endo-PGase that produced trimer and hexamer uniquely as the end products of hydrolysis, in contrast to mixtures of mono-, di-, and trigalacturonates commonly observed for the action of fungal enzymes. Pectic oligosaccharides generated from native carbohydrate polymers offer the potential application as building blocks for value-added products.

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From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18

Karataş

Tülek

Çakar

et al. 2021

PPL

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Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo-PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

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Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris

Cited by 3 publications

References 34 publications

Expression, purification and characterization of human interferon-γ in Pichia pastoris

Expression, purification and characterization of human interferon-γ in Pichia pastoris

Cloning and Expression of Pectobacterium carotovorum Endo-polygalacturonase Gene in Pichia pastoris for Production of Oligogalacturonates

From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18

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