• KSBB Journal
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• v.21 no.4
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• pp.260-266
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• 2006

Methanol induction conditions with various additives for the enhanced production of recombinant hepatitis B surface antigen(HBsAg) were investigated in Pichia pastoris, which can utilize methanol as a carbon source and produce recombinant proteins under the control of strong, tightly-regulated alcohol oxidase(AOX) promoter. The presence of non-methanol carbon sources such as glycerol and glucose fully repressed the expression of AOX promoter. Various additives were tested to improve the production of recombinant protein and it was found that sorbitol could be a good carbon source during methanol induction period. An optimized concentration of amino acid mixture enhanced the production of HBsAg significantly. Pluronic F-68, a non-ionic surfactant, also improved the production of HBsAg without inhibiting cell growth. Addition of oleic acid at 0.01%(v/v) during the induction period showed positive effect on the production of HBsAg. Finally, 1.2%(v/v) of trace salts enhanced the production of HBsAg 1.9 times compared to that of control culture.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.651-654
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• 2005

Analysis of extra- and intra- cellular metabolites is very important to study cell metabolish. Intracellular amino acids in yeast are of great interest as precursors of desired products in biomass production of the feed industries and the production of glutamate. In this study, the fermentations of P. pastoris X-33 and KM71H were carried out in shake flasks. After centrifugation, the harvested cells were mechanically disrupted by using glass bead. The supernatants were used to analyze intracellular amino acid by HPLC system. For HPLC analysis, Resolve C18 column was used with Fluorescence detector. The OPA(o-phthalaldehyde) derivation reaction was employed for analysing amino acids at 30 $^{\circ}C$, 1 ml/min and gradient mode. The concentration of intracellular protein was measured by spectrophotometer.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.864-871
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• 2013

The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was $60^{\circ}C$, but it was not stable at high temperature. The enzyme could remain stable at $30^{\circ}C$ for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system ($k_{obs}$ = 0.151) than that in the buffer solution ($k_{obs}$ = 0.028).

• BMB Reports
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• v.40 no.1
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• pp.22-28
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• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• KSBB Journal
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• v.29 no.4
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• pp.303-306
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• 2014

Antifreeze proteins (AFP) inhibit ice growth to permit the survival of polar organisms in the cold environments. The recombinant AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris ice-binding protein), was produced using Pichia pastoris expression system. The optimum fermentation temperature ($30^{\circ}C$) and pH (5) for FfIBP production were determined using a fed-batch culture system. The maximal cell density and purified FfIBP were 112 g/L and 70 mg/L, respectively. The thermal hysteresis (TH) activity (0.85) of FfIBP obtained using a glycerol-methanol fed-batch culture system was 2-fold higher than that of the LeIBP (Leucosporidium ice-binding protein). This work allows for large-scale production of FfIBP, which could be extended to further application studies using recombinant AFPs.

• Biomolecules & Therapeutics
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• v.14 no.1
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• pp.30-35
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• 2006

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

• Journal of Animal Science and Technology
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• v.51 no.2
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• pp.177-182
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• 2009

The glycosylated human MINPP (multiple inositol polyphosphate phosphatase), which was recombinantly over-expressed by using industrial host, Pichia pastoris, showed the phytase activity against phytate ($InsP_6$) and the enzyme activity of the unglycosylated counterpart was decreased to 30%. The optimal phytase activity occurred at pH 7.4. The human MINPP showed high substrate specificity for $InsP_6$ with little activity on other organic phosphate conjugates such as para-nitrophenylphosphate (pNPP), ATP, and ribose-1-phosphate (R-1-P). The phosphatase activity against 2,3-bisphosphoglycerate (2,3-BPG) by human MINPP was increased to 1.2-fold in the presence of stimulator, 1 mM 2-phosphoglycolate (2-PG) but the phytase activity against $InsP_6$ was not affected by addition of 1 mM 2-PG. The phosphatase activity against 2,3-BPG by human MINPP was not increased in the presence of 2 mM $Mg^{2+}$ or 100 mM $Cl^-$.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.456-460
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• 2006

We investigated the activity and stability of alcohol oxidase from Hansenula sp., Pichia pastoris, and Candida boidinii under the electric stimulation. The activity and stability of alcohol oxidase depended on electric output voltage, electric stimulation time. This inactivation of the enzyme under electric stimulation could be recovered by stabilizing additives such as sugars, surfactants and hydrogels. These alcohol oxidase was more stable in trehalose, Triton X-100, Brji solution and alcohol oxidase from Hansenula is more stable than that from P. pastoris, and C. boidinii. The stabilizing of enzymes against electric stimulation showed a great potential use of enzymes in biotechnology and medical engineering fields.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Korean Journal of Food Science and Technology
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• v.48 no.4
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• pp.341-346
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• 2016

The AMY2B gene, encoding human pancreatic ${\alpha}$-amylase isozyme (HPA II), was expressed in Pichia pastoris, and the effects of chloride ions on HPA II activity toward starch substrates were investigated. As seen with chloride ion-dependent ${\alpha}$-amylases-including HPA I, the isozyme of HPA II-chloride ions increased enzyme activity and shifted the optimal pH to an alkaline pH. The activity enhancement by chloride was more significant at pH 8 than that at pH 6, suggesting that the protonation state of the general acid/base catalyst of HPA II was important for the hydrolysis of starches at an alkaline pH because of the increase in its $pK_a$ by chloride ions. The turnover values for cereal starches as the substrates markedly increased in the presence of chloride by up to 7.2-fold, whereas that for soluble starch increased by only 1.7-fold. Chloride inhibited substrate hydrolysis at high substrate concentrations, with $K_i$ values ranging from 6 to 15 mg/mL.