Functional Properties of Glycosylated Lysozyme Secreted in<i>Pichia pastoris</i>

Saito, Akira; Sako, Yukikazu; Usui, Masakatsu; Azakami, Hiroyuki; Kato, Akio

doi:10.1271/bbb.67.2334

Cited by 11 publications

(9 citation statements)

References 18 publications

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“…Provided that the proteins secreted carry the appropriate sites for N-linked glycosylation, secretion in the yeast is accompanied by the addition of core oligosaccharides on an asparagines residue of nascent polypeptide in the recognition sequence of Asn-X-Ser/Thr. 33) In the case of PmHNL2, the enzyme has 16 sites of potential glycosylation patterns (Fig. 4B).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

Fukuta

Nanda

Kato

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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Section: Discussionmentioning

confidence: 99%

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

Fukuta

Nanda

Kato

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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“…Glycosylation appears to have a potential to modulate the physical properties of proteins without compromising enzymatic activity [25,38,[45][46][47][48]. This makes controlled glycosylation an interesting avenue for the engineering and formulation of pharmaceutical peptides and industrial enzymes.…”

Section: Discussionmentioning

confidence: 99%

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Bagger

Hoffmann

Fuglsang

et al. 2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractThe interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24°C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships.The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants.The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants.The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.

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“…Construction of a recombinant expression vector (pPICZαA) containing the functional yam chitinase gene and electroporation of the expression cassette into competent P. pastoris X-33 were done by the method of Saito et al 19) Large-scale expression of recombinant Pichia strain. A 3 mL preculture of recombinant P. pastoris was developed in YPDM + Zeocine TM medium (1% yeast extract, 2% peptone, 2% dextrose, 0.5% methanol, and 100 μg/mL of Zeocin TM ) in a 50 mL test tube at 30°C for 48 h at 140 strokes/min in a shaker water bath.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33

Akond

Matsuda

Ishimaru

et al. 2014

Bioscience, Biotechnology, and Biochemistry

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A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of culture medium. The chitinase was purified by column chromatography after Endoglycosidase H treatment and then characterized. It showed properties similar to the original chitinase E purified from the yam tuber reported by Arakane et al. (2000). This Pichia-produced chitinase also showed strong lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability, optimum activity at higher temperature such as 70 °C, and high substrate affinity, indicating that one can use this Pichia-produced yam chitinase as a bio-control agent.

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Functional Properties of Glycosylated Lysozyme Secreted inPichia pastoris

Cited by 11 publications

References 18 publications

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33

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