• Korean Journal of Veterinary Research
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• v.22 no.2
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• pp.139-147
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• 1982

A total of 133 isolates of yeast-like fungi was tested for sensitivity to seven different antifungal agents. The yeast-like fungi tested were isolated from the milk from normal or mastitic bovine quaters or from bovine feces. They were 5 Candida albicans (C. albicans) isolates, 63 C. krusei, 27 C. tropicalis, 5 C. parapsilosis, 10 Torulopsis glabrata, 6 Rhodotorula sp., 6 Hansenula sp. and 1 Pichia sp. isolate. The antifungal agents tested were nystatin, griseofulvin, cycloheximide, 5-fluorocytosine, miconazol, clotrimazole and tolnaftate. In general, clotrimazole, miconazol and 5-fluorocytosine were more effective in antifungal activity in vitro against the test organisms than the rest of the agents tested. However, some of the isolates showed higher resistance to certain antifungal agents compared to the other isolates of the some species. They were: 1 C. albicans isolate to 5-fluorocytosine; 1 C. albicans to 5-fluorocytosine, miconazol and clotrimazole; 1 C. krusei to 5-fluorocytosine and cycloheximide; and 11 C. tropicalis isolates to cycloheximide. The minimum inhibitory concentrations(MIC) of clotrimazole were $12.5{\mu}g/ml$ or lower for all isolates tested except one C. albicans isolate, for which MIC of the drug was $100{\mu}g/ml$. On the other hand, the MIC's of cycloheximide were $6.5{\mu}g/ml$ or lower for all isolates except the following; all isolates of C. albicans ($100{\mu}g/ml$), C. pseudotropicalis ($200{\mu}g/ml$) and Rhodotorula sp. ($25-50{\mu}g/ml$), 11 C. tropicalis isolates ($100{\mu}g/ml$) and 1 C. krusei isolate ($200{\mu}g/ml$).

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.347-355
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• 2016

An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50℃; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50℃ than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of V_max and k_cat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.545-555
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• 2014

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and $(NH_4)_2SO_4$ precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and $55^{\circ}C$ with 2,2'-azinobis-[ 3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and $60^{\circ}C$. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a $V_{max}$ value of 51.28 U/mg, and the Km and $V_{max}$ values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.483-488
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• 2014

In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times that of E. coli, and the $V_{max}$ and specific activity of XynB reached $2,547.7{\mu}mol/mg$ and 4,757 U/mg, respectively. XynB still had 74% residual enzyme activity after 30 min of heat treatment at $80^{\circ}C$. From the van der Waals force analysis of XYNB (ACN89393 and AAS67299), there is one more oxygen radical in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than in the 33rd Ala(ACN89393 ), and two H-bonds between Ser70 and Asp67.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1891-1907
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• 2016

Yeasts that are present in marine environments have evolved to survive hostile environments that are characterized by high exogenous salt content, high concentrations of inhibitory compounds, and low soluble carbon and nitrogen levels. Therefore, yeasts isolated from marine environments could have interesting characteristics for industrial applications. However, the application of marine yeast in research or industry is currently very limited owing to the lack of a suitable isolation method. Current methods for isolation suffer from fungal interference and/or low number of yeast isolates. In this paper, an efficient and non-laborious isolation method has been developed and successfully isolated large numbers of yeasts without bacterial or fungal growth. The new method includes a three-cycle enrichment step followed by an isolation step and a confirmation step. Using this method, 116 marine yeast strains were isolated from 14 marine samples collected in the UK, Egypt, and the USA. These strains were further evaluated for the utilization of fermentable sugars (glucose, xylose, mannitol, and galactose) using a phenotypic microarray assay. Seventeen strains with higher sugar utilization capacity than the reference terrestrial yeast Saccharomyces cerevisiae NCYC 2592 were selected for identification by sequencing of the ITS and D1/D2 domains. These strains belonged to six species: S. cerevisiae, Candida tropicalis, Candida viswanathii, Wickerhamomyces anomalus, Candida glabrata, and Pichia kudriavzevii. The ability of these strains for improved sugar utilization using seawater-based media was confirmed and, therefore, they could potentially be utilized in fermentations using marine biomass in seawater media, particularly for the production of bioethanol and other biochemical products.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.4
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• pp.789-799
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• 1998

Literatures on microorganisms of traditional Korean nuruk published since 1945 were reviewed in this paper. Traditional Korean nuruk consists of raw barley and various grains. Traditional Korean nuruk consists of unbolied raw barely and various grains. They are ground to paste and moistened, and then naturally inoculated by airborne microorganisms. Therefore, many kinds of microorganisms such as fungi, yeast, and bacteria grwo in nuruk. Since 1945, new 14 species of Aspergillus and 9 species of Penicillium have been identified from traditional Korean nuruk. Total number of fungal species identified so far is now up to 38 species among 12 different genus. Among newly isolated fungal species, Aspergillus penicilloides and Penicillium, expansum showed not only high production rate of acid and amylase but also extreme stability of the enzyme at room temperature for 3 months. As examples of newly isolated yeast species, there are 5 species of Candida, 4 species of Hansenula, 1 species of Pichia and 1 species of Schizosaccharomyces. Total number of yeast species isolated so far is up to 18 species from different 8 genus. Newly isolated bacteria, were Bacillus pumilus, Lactobacillus casei and Leuconostoc mesenteroides.

• Mycobiology
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• v.48 no.2
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• pp.122-132
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• 2020

Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30-40 g/L) was greater than that of freely suspended cells (8-19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ~52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 ℃, with NH₄Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 ℃ with (NH₄)₂ SO₄ as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.501-508
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• 2003

Artificial coupling of one enzyme with another can provide an efficient means for the production of industrially important chemicals. Xylose reductase has been recently discovered to be useful in the reductive production of xylitol. However, a limitation of its in vitro or in vivo use is the regeneration of the cofactor NAD(P)H in the enzyme activity. In the present study, an efficient process for the production of xylitol from D-xylose was established by coupling two enzymes. A NADH-dependent xylose reductase (XR) from Pichia stipitis catalyzed the reduction of xylose with a stoichiometric consumption of NADH, and the resulting cofactor $NAD^+$ was continuously re-reduced by formate dehydrogenase (FDH) for regeneration. Using simple kinetic analyses as tools for process optimization, suitable conditions for the performance and yield of the coupled reaction were established. The optimal reaction temperature and pH were determined to be about $30^{\circ}C$ and 7.0, respectively. Formate, as a substrate of FDH, affected the yield and cofactor regeneration, and was, therefore, adjusted to a concentration of 20 mM. When the total activity of FDH was about 1.8-fold higher than that of XR, the performance was better than that by any other activity ratios. As expected, there were no distinct differences in the conversion yields of reactions, when supplied with the oxidized form $NAD^+$ instead of the reduced form NADH, as a starting cofactor for regeneration. Under these conditions, a complete conversion (>99%) could be readily obtained from a small-scale batch reaction.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.40-46
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• 2013

A variety of nuruk were collected from various provinces in Korea, and their microflora profiles were analyzed at the species level. A total of 42 nuruk samples were collected and when the viable cell numbers in these nuruk were enumerated, the average cell numbers of bacteria, fungi, yeast, and lactic acid bacteria from all nuruk were 7.21, 7.91, 3.49, and 4.88 log CFU/10 g, respectively. There were no significant differences in viable cell numbers of bacteria or fungi according to regions collected. Bacillus amyloliquefaciens and B. subtilis were the predominant bacterial strains in most samples. A significant portion, 13 out of 42 nuruk, contained foodborne pathogens such as B. cereus or Cronobacter sakazakii. There were various species of lactic acid bacteria such as Enterococcus faecium and Pediococcus pentosaceus in nuruk. It was unexpectedly found that only 13 among the 42 nuruk samples contained Aspergillus oryzae, the representative saccharifying fungi in makgeolli, whereas a fungi Lichtheimia corymbifera was widely distributed in nuruk. It was also found that Pichia jadinii was the predominant yeast strain in most nuruk, but the representative alcohol fermentation strain, Saccharomyces cerevisiae, was isolated from only 18 out of the 42 nuruk. These results suggested that a variety of species of fungi and yeast were distributed in nuruk and involved in the fermentation of makgeolli. In this study, a total of 64 bacterial species, 39 fugal species, and 15 yeast species were identified from nuruk. Among these strains, 37 bacterial species, 20 fungal species, and 8 yeast species were distributed less than 0.1%.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.464-472
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• 2022

An endo-polygalacturonase (endo-PGase) exhibiting excellent performance during acidic fruit juice production would be highly attractive to the fruit juice industry. However, candidate endo-PGases for this purpose have rarely been reported. In this study, we expressed a gene from Penicillium oxalicum in Pichia pastoris. The recombinant enzyme PoxaEnPG28C had an optimal enzyme activity at pH 4.5 and 45℃ and was stable at pH 3.0-6.5 and < 45℃. The enzyme had a specific activity of 4,377.65 ± 55.37 U/mg towards polygalacturonic acid, and the K_m and V_max values of PoxaEnPG28C were calculated as 1.64 g/l and 6127.45 U/mg, respectively. PoxaEnPG28C increased the light transmittance of orange, lemon, strawberry and hawthorn juice by 13.9 ± 0.3%, 29.4 ± 3.8%, 95.7 ± 10.2% and 79.8 ± 1.7%, respectively; it reduced the viscosity of the same juices by 25.7 ± 1.6%, 52.0 ± 4.5%, 48.2 ± 0.7% and 80.5 ± 2.3%, respectively, and it increased the yield of the juices by 24.5 ± 0.7%, 12.7 ± 2.2%, 48.5 ± 4.2% and 104.5 ± 6.4%, respectively. Thus, PoxaEnPG28C could be considered an excellent candidate enzyme for acidic fruit juice production. Remarkably, fruit juice production using hawthorn as an material was reported for the first time.