• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.40-45
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• 2010

The gene encoding $\beta$-agarase (agaB) which hydrolyzes $\beta$-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal ($MF{\alpha}1$), in which the transcription of $MF{\alpha}1$-AgaB was under the control of AOX1 (alcohol oxidase 1, methanol inducible) promoter. The constructed plasmid pPIC-AgaB (9 kb) was integrated into HIS4 gene locus of Pichia pastoris genome. Successful integration was confirmed by performing colony PCR. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the active expression of agaB in P.pastoris. By SDS-PAGE and zymographic analysis, the molecular weight of $\beta$-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular $\beta$-agarase reached 1.34, 1.42 and 1.53 units/mL by inducing 0.1, 0.5, and 1% methanol, respectively, at baffled flask culture of P.pastoris GS115/pPIC-AgaB for 48 hr. Most of the enzyme activity was found in the extacellular fraction and the secretion efficiency showed 98%. Thermostability of recombinant $\beta$-agarase was also increased by glycosylation.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.271-279
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• 2015

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in physiological secretions of mammals. LF shows antibacterial, antiviral and antifungal activities. In the present study, a gene encoding the N-terminal lobe of human lactoferrin (hLF) was isolated, cloned and expressed in methylotrophic yeast, Pichia pastoris. The recombinant hLF-N (rhLF-N) protein was secreted into the culture medium at the level of $458{\mu}g/ml$ in 3 L fermentor. The size of purified hLF-N was estimated as 35 kDa when analyzed by SDS-PAGE and western blotting. The rhLF-N was further confirmed by immunodiffusion using the anti-hLF polyclonal antibody. The expression profile analysis by qRT-PCR showed that the relative mRNA expression of rhLF-N was maximal after 2-3 days of methanol induction and reduced gradually at 4 days. The purified rhLF-N showed broad antibacterial activities against the pathogens such as Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, and Salmonella typhimurium. However, rhLF-N showed relatively lower activity when compared to peptides derived from LF. In spite of this weak activity, the rhLF-N expressed in P. pastoris might be more advantageous for the industrial application, because rhLF-N is secreted into the culture medium and the production can also be increased by optimization of culture conditions.

• Journal of Life Science
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• v.22 no.4
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• pp.508-515
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• 2012

In this study, DEAE-cotton [derivatized by 2-(diethylamino)ethyl chloride hydrochloride (DEAE HCl)] was prepared as a carrier for immobilized $Pichia$ $stipitis$ for ethanol production. When cotton was derivatized with 0.5 M DEAE HCl, the yeast cell suspension was adsorbed at 100% of the initial cell $OD_{600}$. The adsorbed yeast cells were estimated to be 101.8 mg-dry cells/g-DEAE-cotton. In particular, when a flask culture using the immobilized yeast cells was conducted in a glucose and xylose-containing medium, the yeast cells on the DEAE-cotton gradually produced ethanol, according to glucose and xylose consumption; the ethanol yield was approximately 0.33 g-ethanol/g-monosaccharide. Because DEAE-cotton was successfully used as a carrier for ethanol production from a glucose and xylose-containing medium, we expect that this bioethanol production process may be used for the bioethanol production process from the hydrolysate of lignocellulosic biomass. All the results of DEAE-cotton were compared with those of DEAE-cellulose as a carrier for immobilization.

• Food Science and Preservation
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• v.14 no.1
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• pp.94-99
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• 2007

To investigate the effects of Pichia anomala SKM-T and Galactomyces geotrichum SJM-59 on Baechu kimchi fermentation, lyophilized yeasts were added to Baechu kimchi and co-cultured at room temperature ($20{\pm}2^{\circ}C$) for 7 days. Desirable pH and acidity levels appeared by 3 days of fermentation in both the control culture and that with added G. geotrichum SJM-59. Furthermore, the culture with G. geotrichum SJM-59 sustained a desirable pH and acidity level until 5 days of co-culture. The pH of the culture with P. anomala SKM-T decreased slowly and was significantly higher than that of control throughout the experimental period. As fermentation time increased, the acidity of the culture with P. anomala SKM-T increased gradually. However, this culture maintained a desirable acidity level throughout the experiment. The number of lactic acid bacteria in the culture with P. anomala SKM-T was higher than in the culture with G. geotrichum SJM-59, or the control culture, throughout the experiment. The highest LA/TM ratio appeared after 3 nays of fermentation in the control culture, and on the 5 day of the yeasts added co-cultures. On sensory evaluation, no differences were detected between control and the culture with G. geotrichum SJM-59 arter 3 days of fermentation. The co-cultures with yeasts received high marks in umami taste. The co-culture with P. anomala SKM-T showed better texture properties than did the control culture. It was considered that fermentation times were delayed by addition of G. geotrichum SJM-59 or P. anomala SKM-T to Baechu kimchi fermentation.

• Journal of the Korean Wood Science and Technology
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• v.33 no.4 s.132
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• pp.45-52
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• 2005

The manganese peroxidase (mnp5) from white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. The majority of the rMnP5 (recombinant MnP5) produced by P. pastoris exhibited an approximate molecular mass 45 kDa considerably larger than that of the predicting mnp5 due to two glycosylation sites of mnp5. After site direct mutation treatment, the effect of N-linked hyperglycosylation was examined by enzyme activity. Analysis by sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with a molecular mass of 37 kDa. Enzyme activity of M-rMnP5 (mutant recombinant MnP5) was similar to that of rMnP5, indicating that hyperglycosylation did not affect the active site. In this work, active mnp5 was successfully expressed in P. pastoris, suggesting that P. pastoris has potential capability of producing active heme-containing proteins.

• KSBB Journal
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• v.15 no.2
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• pp.174-180
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• 2000

Pichia pastoris and Hansenula polymorpha, the methylotrophic yeasts have been widely used as a host for the production of e eudaryotic proteins due to the advantages related to their inherited characters. This paper describes the method to enhance t the productivity of recombinant proteins by P. pastoris and H. po$\psi$morpha. In the production of recombinant proteins using a f fed batch fermentation system, the effects of specific growth rate on the specific expression rate of re$\infty$mbinant proteins w were studied. In both species, the expression system of recombinant proteins using the fed batch fermentation was optimezed.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.348-354
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• 2004

The expression and antibacterial. activity of recombinant human lactoferrin (hLf) was studied from meth­ylotrophic yeast Pichia pastoris. The gene encoding hLf, isolated from human breast cDNA library, was subcloned into the expression vector, pPIC3.5K under the control of AOX1 promoter. The gene was integrated into the host chromosome and was identified by Southern blotting. The expression of the integrated gene was investigated by RT-PCR, Northern blotting, SDS-PAGE and Western blotting. Discrete band corresponding to hLf was detected from the SDS-PAGE, which was confirmed by Western blotting. The expression was also confirmed by RT-PCR and Northern blotting. The antibacterial activity of the recombinant hLf (rhLf) was investigated using Staphy­lococcus aureus ATCC 6538P and Micrococcus flavus ATCC 10240 as test organisms. The rhLf showed strong antibacterial activities against the bacteria. Furthermore, many Gram-negative animal pathogens such as E.coli ATCC8739, 25922, and Salmonella typhimurium 114 and 115, Pseudomonas fluorescens ID 963 I, P. aeruginosa KCCM 11802, and Gram-positive bacteria Bacillus mesentericus were also inhibited in their growth by the rhLf.

• Journal of Life Science
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• v.21 no.3
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• pp.335-340
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• 2011

Glucose and xylose are the most abundant materials in nature which can be used to produce ethanol by yeast fermentation. Three combinations of cultivation with glucose and xylose were carried out; separated, co-culture, and sequential fermentation with Saccharomyces cerevisiae and Pichia stipitis. In the separated fermentation, S. cerevisiae fermented glucose to produce 14.5 g/l ethanol from 29.4 g/l glucose but hardly used xylose. However, P. stipitis utilized not only glucose but also xylose to produce ethanol 11.9 g/l and 11.6 g/l from 29.4 g/l glucose and 29.0 g/l xylose, respectively. In the mixture of glucose and xylose, P. stipitis fermented both sugars, producing 21.1 g/l ethanol while S. cerevisiae fermented only glucose, producing 13.4 g/l ethanol. In the co-culture and sequential fermentation, the co-culture showed more efficient ethanol productivity with 18.6 g/l ethanol than the sequential fermentation with 12.4 g/l ethanol. To investigate the effect of nutrients in the growth of microorganisms and ethanol production, yeast nitrogen base (YNB) was used in the sequential fermentation with S. cerevisiae and P. stipitis. YNB supplemented some nutrients which S. cerevisiae used up in the broth and the culture showed increased growth rate, increased consumption of xylose, and increased ethanol productivity producing 22.5 g/l ethanol from 54.6 g/l sugar with a yield of 0.41 g/g.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.243-248
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• 2011

We studied the repeated-batch process for the bioethanol production from the hydrolysate of Ulva pertusa Kjellman using yeast Pichia stipitis, which is able to assimilate C6- and C5-monosaccharides. During 180-hour operations, the repeated-batch process was carried out stably, and the average bioethanol concentration reached 11.9 g/L from about 30 g/L of reducing sugar in the hydrolysate. Meanwhile, the bioethanol yields, based on the reducing sugar and the quantitative TLC analysis, were 0.40 and 0.37, respectively, which corresponded to 78.4% and 72.5% of theoretical value, respectively. Throughout the quantitative process analysis, it was also demonstrated that 39.67 g-bioethanol could be produced from 1 kg of dried Ulva pertusa Kjellman. In this study, we verified that the bioethanol production from the hydrolysate of Ulva pertusa Kjellman was feasible using a yeast Pichia stipitis, particularly during the repeated-batch operation.

• Food Science and Preservation
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• v.22 no.5
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• pp.768-777
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• 2015

Persimmon contains high levels of vitamins and phenolic compounds, as well as soluble solids, necessary for the fermentation of persimmon wine. Co-fermentation of persimmon wine was carried out using a mixed culture of Pichia anomala JK04, a Korean indigenous yeast that improves wine quality and flavor, and Saccharomyces cerevisiae Fermivin, an industrial wine yeast, in the following ratios: 9:1 (v/v), 5:5 (v/v), 1:9 (v/v) and 0:10 (v/v). During fermentation, the alcohol contents increased more slowly in samples of mixed culture than in samples of the single culture of S. cerevisiae Fermivin. The alcohol contents of all samples reached 12~13% (v/v) after 15 days. All samples of the mixed culture showed greater variety in flavor and taste than S. cerevisiae Fermivin only. In the sensory evaluation, mixed culture samples had higher scores in terms of flavor and overall preference than the single culture samples. Therefore, P. anomala JK04 is thought to improve the wine flavor of Korean domestic persimmon wine.