• 동아시아식생활학회지
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• 제14권2호
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• pp.131-134
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• 2004

This study was carried out to investigate the shelf life of Shepherd's purse(Capsella bursa-pastoris) Kimchi during fermentation at 1$0^{\circ}C$. Capsella bursa-pastoris was treated without or with blanching. The viable cells of lactic acid bacteria(LAB) of raw and blanched Kimchi after fermentation for 15 days at 1$0^{\circ}C$ were 7.91 log CFU/mL and 6.4 log CFU/mL, respectively. The viable cells of LAB of Capsella bursa-pastoris Kimchi at 1$0^{\circ}C$ were lower in the blanched one when compared to the raw one. The pH of raw Kimchi was lower than that of the blanched one during fermentation for 25 days at 1$0^{\circ}C$. The viable cells of total bacteria of the blanched Kimchi were lower than that of non-blanched one during fermentation at 1$0^{\circ}C$. The ascorbic acid and chlorophyll contents decreased more in the blanched Kimchi when compared to that treated without heat. The sensory quality of the blanched Kimchi was a little inferior to that treated without heat during fermentation.

• 한국식품과학회지
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• 제28권5호
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• pp.827-833
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• 1996

연속증류추출(SDE) 장치를 이용하여 냉이로부터 휘발성 향기성분을 추출할 때 분산매의 조성(pH, 당 및 염)에 따른 향기성분의 강도 및 회수율을 조사하였다. 분산매의 pH를 3, 5, 7및 9로 하여, 각 pH에 따른 휘발성 향기성분의 변화 패턴 및 냉이로부터의 휘발성 향기성분 추출에 가장 적합한 pH를 조사한 결과 분산매의 pH가 7일 때 총 51종의 가장 다양한 휘발성 성분이 확인되었으며 냉이로부터 휘발성 향기성분을 추출할 때 중성 pH 부근에서 수증기 증류하는 것이 향기성분의 구성 및 회수율 측면에서 가장 바람직함을 알 수 있었다. 냉이를 수증기 증류할 때 분산매의 sucrose 농도가 10%, 20% 및 40%로 증가함에 따라 획득된 정유량은 각각 18.1 mg, 29.0 mg 및 33.4 mg으로 40% sucrose용액에 의하여 증류할 때 정유 회수율이 가장 높게 나타났다. 또한 sucrose 농도가 증가할수록 확인된 휘발성 향기성분의 종류가 다양하였으며 10%, 20% 및 40% socrose 용액으로 증류할 때 각각 27종, 32종 및 38종이 확인되었다. 냉이의 수증기 증류시 분산매의 surcrose 농도가 증가함에 따라 향기를 구성하는 휘발성 성분의 수는 더욱 다양하였으나 양적인 측면에서는 각 성분마다 다른 양상을 보였다. 2%, 8% 및 15% NaCl용액으로 냉이를 수증기 증류하여 얻어진 정유의 함량은 각각 33.0 mg, 12.5 mg 및 22.4 mg으로 2% NaCl용액을 사용하였을 때 정유의 회수율이 가장 좋았으나 15% NaCl용액으로 증류할 때 가장 많은 수인 22종의 휘발성 성분이 검출 및 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1098-1102
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• 2009

Gamma-linolenic acid (GLA, C18:3 ${\Delta}^{6,9,12}$) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2 ${\Delta}^{9,12}$) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.

• Journal of Microbiology and Biotechnology
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• 제23권9호
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• pp.1197-1205
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• 2013

Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis. Targeting the excessive activation of this system as well as the proliferation of the tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt the tumor development. In this regard, the amino-terminal fragment (ATF) of urokinase has been confirmed as effective to inhibit the proliferation, migration, and invasiveness of cancer cells via interrupting the interaction of uPA and uPAR. Previous studies indicated that ATF expressed in Escherichia coli was mainly contained in inclusion bodies and also lacked posttranslational modifications. In this study, the biologically active and soluble ATF was cloned and expressed in Pichia pastoris. The recombinant protein was purified to be homogenous and confirmed to be biologically active. The yield of the active ATF was about 30 mg/l of the P. pastoris culture medium. The recombinant ATF (rATF) could efficiently inhibit angiogenesis, endothelial cell migration, and tumor cell invasion in vitro. Furthermore, it could inhibit in vivo xenograft tumor growth and prolong the survival of tumor-bearing mice significantly by competing with uPA for binding to cell surfaces. Therefore, P. pastoris is a highly efficient and cost-effective expression system for large-scale production of biologically active rATFs for potential therapeutic application.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1244-1251
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• 2020

Phospholipase A₂ (PLA₂) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA₂ in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA₂ was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA₂ (P-PLA₂), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA₂. Finally, we observed that the extracellular PLA₂ from the recombinant E. coli P-PLA₂ culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA₂ expression system led to extracellular production of PLA₂ to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA₂.

• Journal of Microbiology and Biotechnology
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• 제27권6호
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.644-648
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• 2005

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제22권3호
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• pp.331-338
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• 2012

A novel gene coding for an endo-${\beta}$-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The ${\beta}$-mannanase showed an identity of 90.2-92.9% ${\leq}95%$) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified ${\beta}$-mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDI-TOF mass spectrometry. The recombinant ${\beta}$-mannanase had an optimum temperature of $45^{\circ}C$ and optimum pH of 6.5. The enzyme was stable at temperatures up to $50^{\circ}C$ (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions ($Hg^{2+}$, $Pb^{2+}$, and $Co^{2+}$) substantially inhibited the recombinant ${\beta}$-mannanase. The chemical additives including detergents (Triton X-100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the ${\beta}$-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.269-273
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• 2005

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

• Korean Chemical Engineering Research
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• 제46권4호
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• pp.764-768
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• 2008

소 고환 유래의 hyaluronidase 효소 PH-20을 pPIC9 expression vector를 사용하여 Pichia pastoris에서 발현하였다. 생산된 재조합 단백질 rPH-20b는 75 kDa의 분자량을 보였고 7460 units/L의 효소활성을 보였다. 세포내보다 세포외의 효소활성이 두배 높았다. pH 의 경우 buffer를 사용 안 한 경우가, 또한 $30^{\circ}C$ 배양 조건에서 높은 활성을 보였다. 1 M sorbitol 삼투압조건과 0.3% 메탄올의 생산유도인자를 사용시 성장과 생산에 유리하였으며 0.4 M 아르기닌을 첨가시 재조합 단백질의 분해가 감소하였다.