• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1314-1321
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• 2010

Sulfur metabolism in S. cerevisiae is well established, but the mechanisms underlying the formation of sulfide remain obscure. Here, we investigated by real-time RT-PCR the dependence of expression levels of MET3, MET5/ECM17, MET10, MET16, and MET17 along with SSU1 on nitrogen availability in two wine yeast strains that produce divergent sulfide profiles. MET3 was the most highly expressed of the genes studied in strain PYCC4072, and SSU1 in strain UCD522. The strains behaved differently according to the sampling times, with UCD522 and PYCC4072 showing the highest expression levels at 120 h and 72 h, respectively. In the presence of 267 mg assimilable N/l, the genes were more highly expressed in strain UCD522 than in PYCC4072. MET5/ECM17 and MET17 were only weakly expressed in both strains under any condition tested. MET10 and SSU1 in both strains, but MET16 only in PYCC4072, were consistently upregulated when sulfide production was inhibited. This study illustrates that strain genotype could be important in determining enzyme activities and therefore the rate of sulfide liberation. This linkage, for some yeast strains, of sulfide production to expression levels of genes associated with sulfate assimilation and sulfur amino acid biosynthesis could be relevant for defining new strategies for the genetic improvement of wine yeasts.

• Journal of Applied Biological Chemistry
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• v.56 no.3
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• pp.171-177
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• 2013

Efficient extraction of total proteins from soil microorganisms is tedious because of small quantity. In this regard, an improved method for extraction of whole cell proteins is developed from soil microorganisms, Saccharomyces cerevisiae and Pichia pastoris. of which the cell wall are very strong. Pretreatment with NH4OH prior to the final extraction using NaOH/SDS was tried under the basis that ammonium ion was possible to enhance the permeability and/or to weaken the yeast cell walls. The pre-treatment of yeast cells with NH4OH drastically enhanced the protein extraction when it was compared with control (without NH4OH pre-treatment). At the pre-treatment of 0.04 N NH4OH at pH 9.0, about 3 fold of proteins was obtained from p. pastoris. Ammonium hydroxide appears to penetrate into the yeast cell walls more readily at basic pH. The effect of NH4OH pretreatment was pH dependent. The methods developed in this experiment might be applicable for an effective extraction of yeast proteins for the purpose of biochemical studies, especially proteomic analysis.

• Applied Chemistry for Engineering
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• v.23 no.3
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• pp.326-332
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• 2012

Among the samples prepared by various pre-treatment methods, the one pretreated by dilute sulfuric acid showed the highest glucose yield in the enzymatic hydrolysis. Statistical analysis of enzymatic hydrolysis revealed that the glucose yield was in proportion to the enzyme dosage, the ratio of the pre-treated sample to the buffer solution, and the reaction time and that the effect of enzyme dosage was predominant in the experiment range. In addition, the glucose yield was estimated to be 76.1% at an optimal enzymatic hydrolysis condition. In a separate hydrolysis and fermentation (SHF), Saccharomyces cerevisiae converted over 80% of glucose from the enzymatic hydrolysis of pre-treated wasted corn stalk by dilute sulfuric acid to bioethanol with 37% of ethanol yield and 0.42 $g/L{\cdot}hr$ of productivity. In the simultaneous saccharification and fermentation (SSF), 59.5% of conversion from glucan to ethanol and 0.20 $g/L{\cdot}hr$ of productivity were achieved. In both SHF and SSF, approximately 88 g of bioethanol could be obtained from 1 kg of wasted corn stalk. The possible amount of bioethanol in Gangwon province were estimated to be 1.9 kiloton with the assumption of the 50% of collection ratio.

• Korean Journal of Food Science and Technology
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• v.26 no.3
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• pp.204-212
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• 1994

To estimate the possibility of wine-making with Korean dried persimmon, its homogenized and filtered solution was fermented at $15^{\circ}C$ and $25^{\circ}C$ for 12 weeks with Saccharomyces cerevisiae (Japan Alcoholic Beverage Association N0.7). Sugars of dried persimmon were mainly composed of 27.02% of glucose, 19.81% of fructose and 5.12% of mannose. In the fermentation at $25^{\circ}C$, glucose was almost completely consumed in 8 days, but fructose and mannose were consumed up to 64% and 74%, respectively, in the same period and were not utilized any more afterwards. In the fermentation at $15^{\circ}C$, 75% of glucose, 20% of fructose and 49% of mannose were consumed in 8 days and these sugars were continuously utilized for 12 weeks. Organic acids in the homogenized and filtered solution were levulinic acid (148.6 mg%), 4-methylvaleric acid (73.5 mg%), oxalic acid (28.7 mg%), acetic acid (8.5 mg%), N-butyric acid (8.4 mg%) and succinic acid (6.7 mg%). Irrespective of fermentation temperature, levulinic acid rapidly reduced according to progression of fermentation. Oxalic acid, N-butyric acid and succinic acid decreased at 2nd day of fermentation, and then increased at 4th and 6th days and subsequently decreased again under the levels of the solution. Acetic acid and 4-methylvaleric acid increased with the proceeding of fermentation and at 12th week of fermentation these contents were more than those of the solution. The contents of total free amino acid significantly reduced at 2th day of fermentation and then increased to the level of the solution at 12th week irrespective of fermentation temperature. Ethanol content rapidly increased to the levels of 5.3(v/v) at $15^{\circ}C$ and 9.4%(v/v) at $25^{\circ}C$ to 8th day after fermentation, but at 12th week its content was 14.5%(v/v) at $15^{\circ}C$ and 9.4%(v/v) at $25^{\circ}C$. The higher alcohots identified were 2-methyl-l-propanol, 3-methyl-ibutanol, 2-methyl-l-butanol and 2-methyl-2-propanol and the range of those contents was from 0.001% (v/v) to 0.06%(v/v). The color of the wine fermented at $15^{\circ}C$ was slightly superior but flavor and taste were slightly superior in the wine fermented at $25^{\circ}C$.

• Korean Journal of Food Science and Technology
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• v.49 no.3
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• pp.279-285
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• 2017

This study reviews the manufacturing characteristics of distilled liquor prepared using Saccharomyces cerevisiae 88-4 separated from traditional Nuruk. From analysis of soju mash (sool-dut) during the fermentation process, 17.2% of alcohol was detected in the final fermentation with succinic acid present at the highest level ($7,164.3{\pm}85.2ppm$). From the analysis of alcohol content in different distillation conditions, distillation condition No. 6 showed the lowest amount of alcohol ($29.6{\pm}0.0%$), whereas distillation condition No. 4 showed the highest amount ($59.9{\pm}0.1%$). N-propanol has been detected at the highest level in distilled liquor under conditions No. 1 and 4, each being $163.4{\pm}18.3$ and $174.0{\pm} 0.1ppm$, respectively. Isobutanol showed a tendency similar to n-propanol. Distilled liquor in conditions No. 1 and 4 has shown the highest acetaldehyde level, each being $303.4{\pm}4.5$ and $325.4{\pm}13.1ppm$, respectively. After distillation, 14 volatile substances were found in common, with isoamyl alcohol present at the highest levels in all the distilled liquors. Distillation conditions No. 3, 5, 6, and 7 have shown high levels of isobutanol that emits a banana-like fragrance and ethyl octanoate that emits a pleasant fruity and floral fragrance.

• Korean Journal of Food Science and Technology
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• v.50 no.4
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• pp.407-413
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• 2018

The current study reviews the manufacturing characteristics, upon aging, using different distillation methods with Saccharomyces cerevisiae 88-4 isolated from traditional Nuruk. The alcohol content slightly decreased at 180 days of aging period, under the conditions: -60 cmHg, distilling temperature $50^{\circ}C$, head cut 7%, and body cut 30%. The alcohol content decreased by 2.2% at: -50 cmHg, distilling temperature $60^{\circ}C$, head cut 7%, and body cut 30%. From 2-thiobarbituric acid (TBA) analysis, the TBA values were not found to change significantly in most of the alcohols, but increased at conditions of: -60 cmHg, distilling temperature $60^{\circ}C$, head cut 7%, and body cut 50% and -50 cmHg, distilling temperature $50^{\circ}C$, head cut 7%, and body cut 50%, when initial alcohol levels were low. According to n-propanol, iso-butanol, and isoamyl alcohol analysis, with increasing aging period, the n-propanol and isoamyl alcohol content did not change, although that of iso-butanol decreased. The flavor components showed different results based on the aging period. Results of sensory evaluation showed no significant difference based on aging. The sensory evaluation test was continued for 180 days at a condition of: -60 cmHg, distilling temperature $60^{\circ}C$, and body cut 50%, which had the best overall evaluation ($5.8{\pm}0.9$).

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.2
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• pp.298-305
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• 2016

Prior research has attempted to develop a method for fermentation of onion vinegar to satisfy customer quality standard. Onion wine (OW) and onion vinegar (OV) were produced by alcoholic and acetic fermentation of onion extracts (OE) using Saccharomyces cerevisiae and Acetobacter pasteurianus, and their taste compounds (non-volatile organic acids, non-protein N compounds, and free sugars) were determined. Main components of non-volatile organic acids were malic acid (50.1%) and citric acid (26.9%) in OE, whereas malic acid (28.1%), acetic acid (20.8%), lactic acid (20.1%), citric acid (13.3%), and succinic acid (12.0%) were detected in OW. Total concentrations of non-volatile organic acids in OV were 4,612.0 mg/100 g, which was 3.9 and 2.3 times higher than those of OE and OW, respectively. Non-volatile organic acids except malonic acid and acetic acid were reduced during acetic fermentation. Non-protein N compounds increased 4.23-fold ($41,526.8{\mu}g/100g$) during alcohol fermentation, and urea content was the highest of non-protein N compounds at $33,816{\mu}g/100g$. The reduced values in OV might be used as a nutritious element of Acetobacter pasteurianus. Free sugars (glucose, fructose, and sucrose) were detected in OE, whereas only fructose was absent in OW and OV.

• The Plant Pathology Journal
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• v.22 no.1
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• pp.75-89
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• 2006

The actinomycete strain JK-1 that showed strong inhibitory activity against some plant pathogenic fungi and oomycetes was isolated from Jung-bal Mountain in Ko-yang, Korea. The strain JK-1 produced spores singly borne on sporophores and the spores were spherical and 0.9-1.2 11m in diameter. The cell wall of the strain JK-1 contained meso-diaminopimelic acid. The actinomycete strain JK-1 was identified as the genus Micromonospora based on the morphological, physiological, biochemical and chemotaxonomic characteristics. From the 168 rDNA analysis, the strain JK-1 was assigned to M aurantiaca. The antibiotic MA-1 was purified from the culture broth of M aurantiaca JK-1 using various purification procedures, such as Diaion HP20 chromatography, C18 flash column chromatography, silica gel flash column chromatography and Sephadex LH-20 column chromatography. $^{1}H-$, $^{13}C-NMR$ and EI mass spectral analysis of the antibiotic MA-1 revealed that the antibiotic MA-1 is identical to phenylacetic acid. Phenylacetic acid showed in vitro inhibitory effects against fungal and oomycete pathogens Alternaria mali, Botrytis cinerea, Magnaporthe grisea, Phytophthora capsici and yeast Saccharomyces cerevisiae at < 100 $\mug$ $ml^{-1}$. In addition, phenylacetic, acid completely inhibited the growth of Sclerotinia sclerotiorum, Bacillus subtilis, Candida albicans, Xanthomonas campestris pv. vesicatoria at < $\mug$ $ml^{-1}$. Phenylacetic acid strongly inhibited conidial germination and hyphal growth of M grisea and C. orbiculare. Phenylacetic acid showed significantly high levels of inhibitory' effect against rice blast and cucumber anthracnose diseases at 250 $\mug$ $ml^{-1}$. The control efficacies of phenylacetic acid against the two diseases were similar to those of commercial compounds tricyclazole, iprobenfos and chlorothalonil .n the greenhouse.

• BMB Reports
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• v.28 no.5
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• pp.437-442
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• 1995

The potency of yeast-derived methionyl-free human growth hormone (rhGH), which was obtained by removal of the N-terminal Met from methionyl-hGH, was estimated by in vitro and in vivo assays. In radio-receptor assay where the binding affinity of growth hormone to the receptor was estimated, the recombinant hGH showed 2.9 international units (IU) per mg of specific activity. In contrast, pitUitary-derived human growth hormone had a slightly lower receptor binding activity (2.5 IU/mg) compared with recombinant growth hormone. For the in vivo assay, efficacy of rhGH was tested by use of hypophysectomized rats, in which pituitary organs were surgically removed, resulting in the termination of growth hormone secretion. The weight-increase in rats by the injection of rhGH was almost identical to the result obtained by the injection of the same amount of pituitary-derived (international standard) hGH. A comparision of the secondary structures of rhGH and rMet-hGH by circular dichroism spectrophotometer demonstrated that the removal of the methionyl residue from rMet-hGH did not exert any effect on the structure of the growth hormone. In conclusion, methionyl-free human growth hormone produced from yeast was highly potent in biological activity and maintained a legitimate three dimensional structure.