• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.305-311
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• 2002

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ$\alpha$C-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOXI promoter and connected downstream of mating factor u-1 signal sequence. The plasmid linearized by Sacl was integrated into the 5'AOXI region of the chromosomal DNA of P. pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/1 and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/1 of AXEase protein was produced in the extracellular medium.

• Journal of the East Asian Society of Dietary Life
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• v.11 no.5
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• pp.393-399
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• 2001

Effects of various types of salts (commercial low salt, sea salt, refined salt, and bamboo salt) and concentrations (0, 3.0 and 5.0% ) on the growth of yeasts isolated from Kimchi were investigated. The isolated yeasts used in the study are as follows : Saccharomyces cerevisia, Sporobolomyces albo-rubescens, Issatchenkia orientalis Cryptococcus luteolous, Ustilago maydis, Candida humilis, Pichia onychis, Cadida nitratophila, and Pichia jadinii. The growths of the yeasts were inhibited against each salt concentration. The growths of Candida sp. isolated from the later stage of fermentation was strongly inhibited against 5% concentration of salt. On the other hand the induction phase of Issatchenkia orientalis isolated at the early stage of fermentation was the quickest among all the tested yeasts. Among the salts, bamoo salt was found to be strongest inhibitor of the growth of yeasts.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.9
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• pp.1363-1370
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• 2016

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector $pPICZ{\alpha}A$, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1938-1944
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• 2008

The procpb gene encoding human procarboxypeptidase B (proCPB, GeneBank access code AJ224866) was cloned and its Pichia expression plasmid, $pPIC9{\alpha}$/hproCPB (9.2 kb), was constructed, in which procpb was under the control of the AOXl promoter and connected to the downstream of the mating factor ${\alpha}$-1 ($MF{\alpha}1$) signal sequence. The plasmid was linearized by digestion with Sacl, and integrated into the genome of P. pastoris strain GS115. By culturing of Pichia transformant on methanol medium, the human proCPB was successfully expressed and secreted into the culture supernatant. Moreover, Western blot analysis of the extracellular proteins showed proCPB bands clearly at a molecular mass of 45 kDa, confirming the expression of proCPB with its right size. The CPB activity reached about 3.5 U/ml and 12.7 U/ml in the flask and fermentor batch cultures of Pichia transformant, respectively. No CPB enzyme activity was found in the intracellular fraction. When the fed-batch cultivation was performed with methanol and glycerol mixture as a feeding medium, the extracellular CPB activity was increased to 42.0 U/ml, which corresponds to a 3.3-fold higher level of CPB activity than that of batch culture. The $K_m$ and $k_{cat}$ values of recombinant human CPB enzyme for hippuryl-$_L$-Arg as a substrate were estimated to be 0.16 mM and $11.93\;sec^{-1}$, respectively.

• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.254-257
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• 2011

A 2-year-old bearded dragon was referred to the Veterinary Medical Center at the College of Veterinary Medicine, Chungbuk National University with reduced activity and anorexia. On fecal examination, over growth of a bacteria and the proliferation of a yeast-like organism were found. The patient diagnosed with enteritis. By using fungal cultures and molecular typing, the yeast was identified as Pichia (P.) burtonii. The bearded dragon was treated with oral ketoconazole and trimethoprim/sulfamethoxazole. After 3 days, the dragon was recovered and fecal examination showed that the yeast had disappeared from the feces. The strain P. burtonii is supposed opportunistic pathogen in bearded dragon with enteritis according to its reports in a human. This report is the first paper about overgrowth of P. burtonii in a bearded dragon.

• The Korean Journal of Mycology
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• v.40 no.4
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• pp.254-257
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• 2012

The different microbial species were isolated from soy bean paste samples. A yeast strain NASS-2 was effective to decrease odor activity on pig excreata was identified as Pichia farinosa based on nucleotide sequences of 18S ribosomal DNA and Internal transcribed space (ITS). The extracellular fraction of P. farinosa NASS-2 was effective to decrease odor activity of pig excrements. Optimal medium component for decreasing order activity on odor material composed of soluble starch 2.0% (w/v) and yeast extract 0.8% (v/v). The decrease of odor material was maximum at $28^{\circ}C$ for 72 hours with pH 5.5. When the P. farinosa NASS-2 culture broth was treated to pig excrements, the removal efficiency was an average concentration with 1.38 ppm of ammonia gas.

• Microbiology and Biotechnology Letters
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• v.50 no.4
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• pp.508-511
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• 2022

In this study, Phaseolus vulgaris (kidney bean) leghemoglobin a (PhLba) gene was cloned into pPICZαA and expressed in Pichia pastoris to sustainably produce a heme-carrying protein for organoleptic use in plant-based meat. The recombinant PhLba protein was secreted into the culture medium in a solubilized form, and the molecular weight of the purified PhLba was estimated to be 16.5 kDa using SDS-PAGE. In addtion, the yield of recombinant PhLba holoprotein was enhanced by supplementation of the cultivation medium with hemin. This result indicates that the apo-forms of PhLba can be effectively saturated with cofactor.

• Food Science and Biotechnology
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• v.14 no.4
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• pp.487-492
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• 2005

Simple competition plate bioassays of Pichia anomala SKM-T and Galactomyces geotrichum SJM-59 were conducted to evaluate their potential as biological control agents that inhibit growth of Botrytis cinerea during post-harvest storage of strawberries (Fragaria ${\times}$ ananassa Duche, Red-Pearl). Occurrence rates of fungi on the surface of yeast-treated strawberries were evaluated during storage at $4^{\circ}C$. P. anomala SKM-T and G. geotrichum SJM-59 showed antifungal activities on agar plate, and P. anomala SKM-T maintained its desirable antifungal activity on surface of strawberries and its physicochemical properties during storage. Sensory evaluation was based on kinesthetics and consumer acceptability. Due to its potential antifungal activity, P. anomala SKM-T could function as biological control agent against spoilage fungi during post-harvest storage of strawberries.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.574-579
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• 2014

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.370-373
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• 2001

Experiments were carried out to develop of mutant strain from Pichia ciferrii ATCC 14091 and investigate the effect of sodium acetate on the production of tetraacetylphytosphingosine (TAPS). A high-TAPS producing mutant was simply selected by staining with sudan black B and colony shape after the treatment of UV and NTG. Sodium acetate enhanced the TAPS production.