Expression of enzymes for the usage in food and feed industry with Pichia pastoris

Spohner, Sebastian C.; Müller, Hagen; Quitmann, Hendrich; Czermak, Peter

doi:10.1016/j.jbiotec.2015.01.027

Cited by 123 publications

(68 citation statements)

References 210 publications

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“…15 P. pastoris secretes only very low levels of endogenous protein into the growth medium; thus, the secreted TmPLB1 protein constituted the majority of the total protein in the medium (Figure 3A); this advantage allows for the purification of the expressed protein. 29 Ni-affinity chromatography was used to capture TmPLB1 and allow contaminants to be washed away (Figure 4). After gradient dialysis, the final concentration of highly purified TmPLB1 reached 240.4 mg/L of culture supernatant, and the PLA1 and PLA2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively, an amount sufficient for subsequent functional studies.…”

Section: Discussionmentioning

confidence: 99%

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

Yan

et al. 2016

Emerging Microbes & Infections

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Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS–polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.

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Section: Discussionmentioning

confidence: 99%

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

Yan

et al. 2016

Emerging Microbes & Infections

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“…The yeast P. pastoris is widely used as a host for recombinant protein expression . Pichia belongs to the same family of yeast ( Saccharomycetaceae ) as several yeast genera widely used in food: Saccharomyces , Torula , Yarrowia , Dekkera , and Brettanomyces .…”

Section: Introductionmentioning

confidence: 99%

Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris

Yuan

Andoh-Kumi

et al. 2017

Molecular Nutrition Food Res

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ScopeThe Soybean (Glycine max) leghemoglobin c2 (LegHb) gene was introduced into Pichia pastoris yeast for sustainable production of a heme‐carrying protein, for organoleptic use in plant‐based meat. The potential allergenicity and toxicity of LegHb and 17 Pichia host‐proteins each representing ≥1% of total protein in production batches are evaluated by literature review, bioinformatics sequence comparisons to known allergens or toxins, and in vitro pepsin digestion.Methods and resultsLiterature searches found no evidence of allergenicity or toxicity for these proteins. There are no significant sequence matches of LegHb to known allergens or toxins. Eleven Pichia proteins have modest identity matches to minor environmental allergens and 13 Pichia proteins have significant matches to proteins from toxic sources. Yet the matched allergens and toxins have similar matches to proteins from the commonly consumed yeast Saccharomyces cerevisiae, without evidence of food allergy or toxicity. The demonstrated history of safe use indicates additional tests for allergenicity and toxicity are not needed. The LegHb and Pichia sp. proteins were rapidly digested by pepsin at pH 2.ConclusionThese results demonstrate that foods containing recombinant soy LegHb produced in Pichia sp. are unlikely to present an unacceptable risk of allergenicity or toxicity to consumers.

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“…The P. pastoris systems are a good option for demonstrating MBSP expression as they are suitable for recombinant protein expression, especially for proteins requiring correct folding . In the present study, SDS‐PAGE and Western blot were used to confirm that recombinant MBSP was successfully expressed (Fig.…”

Section: Discussionmentioning

confidence: 99%

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

Mei

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Myofibril‐bound serine proteinase (MBSP) comprises a class of trypsin‐type serine proteinases that can specifically cleave the carboxyl side of peptide bonds of arginine or lysine residues. To obtain higher specificity MBSPs, mutants were designed based on the substrate‐binding pocket and constructed through site‐directed mutagenesis. RESULTS The wild‐type (WT) and mutants were expressed in Pichia pastoris, and its enzymatic properties indicated that the mutants D170S, D170T, D170K and D170T/G203A were not biologically active. However, S171A specifically cleaved arginine residues and G203A preferably hydrolyzed lysine residues. The secondary structures of mutants were approximately similar to that of the WT according to the results of circular dichroism spectroscopy. Additionally, molecular docking showed that substrates were able to combine with S171A within the critical areas through hydrophobic interaction, hydrogen bonds and van der Waals electrostatic force. CONCLUSION The structural stability of MBSP was consistent using amino acid substitution. Due to the interaction pattern of substrates and mutant enzyme changes, only S171A was selective in cutting Arg residues, which illustrates how the catalytic triad and substrate‐binding pocket of MBSP plays an important role during enzymatic hydrolysis of substrates. © 2018 Society of Chemical Industry

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Expression of enzymes for the usage in food and feed industry with Pichia pastoris

Cited by 123 publications

References 210 publications

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

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