Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Heterologous Expression of Polygalacturonase Genes Isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris: Il Jae Cho , In-Cheol Yeo , Nam Keun Lee , Suk Hee Jung , Young Tae Hahm; J. Microbiol. 2012;50(2):332-340. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1290-7

Abstract: The objective of this work was to isolate the polygalacturonase genes of Galactomyces citri-aurantii IJ-1 harvested from rotten citrus peels and to heterologously express these genes in Pichia pastoris. Two polygalacturonase (PG) genes from G. citri-aurantii IJ-1 were obtained and tentatively named PG1 and PG2. The genes were cloned into pPICZαC, and expressed in Pichia pastoris strain GS115 with a native signal peptide or the α-factor secretion signal peptide of Saccharomyces cerevisiae. All of the recombinant proteins were successfully secreted into the culture media and confirmed as a single band with a molecular weight of 35 to 38 kDa by SDS-PAGE. The specific enzyme activities of recombinant PG1 and PG2 purified by His-tag affinity resin were 4,749 and 6,719 U/mg, respectively, with an optimal pH and temperature of pH 4.0 and 50°C. The Michaelis- Menten kinetic constants for PG1 and PG2, Km, were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn2+, the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu2+ and Fe3+ acted as strong inhibitors to the PGs.

A Thermostable Phytase from Neosartorya spinosa BCC 41923 and Its Expression in Pichia pastoris: Patcharaporn Pandee , Pijug Summpunn , Suthep Wiyakrutta , Duangnate Isarangkul , Vithaya Meevootisom; J. Microbiol. 2011;49(2):257-264. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0369-x

Abstract: A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its Km and Vmax for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe2+, Fe3+, and Al3+. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).

Journal Article

Cloning, Expression, and Characterization of Serine Protease from Thermophilic Fungus Thermoascus aurantiacus var. levisporus: An-Na Li , Chen Xie , Jie Zhang , Jia Zhang , Duo-Chuan Li; J. Microbiol. 2011;49(1):121-129. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-9355-6

Abstract: The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Asp183, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70°C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.

Research Support, Non-U.S. Gov't

Note] Antibacterial Activity of Recombinant hCAP18/LL37 Protein Secreted from Pichia pastoris: Soon-ja Kim , Renshu Quan , Sung-Jin Lee , Hak-Kyo Lee , Joong-Kook Choi; J. Microbiol. 2009;47(3):358-362. Published online June 26, 2009; DOI: https://doi.org/10.1007/s12275-009-0131-9

Abstract: Human antimicrobial peptide CAP18/LL37 (hCAP18/LL37) was expressed in Pichia pastoris and its antibacterial activity was tested against pathogenic bacteria. The full length ORF of hCAP18/LL37 was cloned into the pPICZαA vector followed by integration into the genomic AOX1 gene of P. pastoris. Agar diffusion assay demonstrated that the different hCAP18/LL37 transformants showed various antibacterial activities against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis. The secreted form of hCAP18/LL37 exhibited its maximum activity after 72 h incubation with 2% methanol in MM media, not in BMM. This result suggests that the yeast secreted expression system can be used as a production tool of antimicrobial peptides for industrial or pharmaceutical application.

Expression and Characterization of the Human rpS3 in a Methylotrophic Yeast Pichia pastoris: Jae Yung Lee , Sang Oun Jung , BuHyun Youn , Oh Sik Kwon , Joon Kim; J. Microbiol. 2000;38(2):88-92.

Abstract: A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme (UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was previously characterized, and this activity of mammalian rpS3 was found to be non-specific upon purification and storage. Under the Pichia expression system, the subcloned cDNA of the human rpS3 gene revealed a peptide of 42 kDa by SDS-PAGE and Western blot. The secreted form of human rpS3 rendered no endonuclease activity while the intracellular form showed UV specific endonuclease activity by the nick circle assay.

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