누룩으로부터 분리된 전분대사 효모 Saccharomycopsis fibuligera 균주의 생육특성

최다혜 ( Da Hye Choi ),박은희 ( Eun Hee Park ),김명동 ( Myoung Dong Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2014 한국미생물·생명공학회지 Vol.42 No.4

누룩으로부터 MBY1276, 1280, 1282, 1320, 1322, 1324 및 MBY 1327로 각각 명명된 전분을 분해하는 효모균주를 분리하였다. 이들 균주는 18S rRNA 영역의 ITS 단편 및 26S rDNA 영역의 D1/D2 단편의 염기서열 해석 및 탄소원 대사특성 분석을 통하여 S. fibuligera로 동정하였다. 상주지역에서 수집된 누룩으로부터 분리된 MBY1320 균주는 대조구 균주인 S. fibuligera KCTC7806 및 누룩으로부터 분리된 다른 S. fibuligera 균주에 비해 고온에서 상대적으로 높은 비성장속도를 나타내었다. 전분을 분해하는데 필요한 효소인 α-amylase 및 glucoamylase 효소활성을 측정한 결과 MBY1320 균주의 α-amylase 효소활성은 대조구 균주보다 낮지만 glucoamylase 효소활성은 고온에서 상대적으로 우수한 것으로 나타났다. 효소활성 분석결과는 MBY1320 균주가 표준균주 및 다른 S. fibuligera 균주와 비교하여 고온에서 상대적으로 높은 비성장속도를 나타내는 것을 뒷받침하는 결과로 판단되었다. 가용성 전분을 이용한 회분식 배양결과 MBY1320 균주는 표준균주 보다 42oC에서 우수한 성장속도 및 에탄올 생산속도를 나타내었다. 본 연구를 통하여 기존에 보고된 S. fibuligera 균주보다 고온에서 glucoamylase 효소활성 및 비성장속도가 우수한 S. fibuligera 균주를 보고하는 바이다. A number of Saccharomycopsis fibuligera strains that can hydrolyse and utilises starch as a carbon source were isolated from nuruk, a traditional Korean starter for rice wine fermentation, their specific growth rates on starch-containing medium were compared to choose a prominent strain. An S. fibuligera strain named MBY1320 showed higher growth rate at 42°C than that of a type strain S. fibuligera KCTC7806, indicating that S. fibuligera MBY1320 has a more thermo-tolerant machinery for starch hydrolysis and utilisation than KCTC7806. Although the activity of α-amylase at 30°C was significantly lower in S. fibuligera MBY1320 than in KCTC7806(3,812.5 U vs. 14,878.5 U), S. fibuligera MBY1320 showed a much higher glucoamylase activity at 42°C than S. fibuligera KCTC7806(5,048.9U vs. 13,152.3U). Thus, a new S. fibuligera strain with higher starch-hydrolysing activity at elevated temperature than that of a type strain was isolated in this study.

실험실 적응진화를 이용한 Saccharomycopsis fibuligera의 젖산에 대한 내성 증대

유병혁 ( Boung-hyuk Yoo ),박은희 ( Eun-hee Park ),김명동 ( Myoung-dong Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.4

S. fibuligera 균주는 생전분을 분해하는 활성이 있는 효모 균주이다. 본 연구에서는 첨가되는 젖산의 농도가 점차 증가하는 장기간 반복 회분식 배양 방법을 사용하는 적응진화를 통하여 S. fibuligera 균주의 젖산에 대한 내성을 증진 시키고자 하였다. 진화된 균주인 S. fibuligera MBY1940 균주는 비성장속도 측정과 평판배지를 사용하는 성장분석을 통하여 모균주가 성장할 수 없는 젖산 농도 2.5%에 대해서도 내성을 나타내는 것을 확인하였다. 전자현미경 관찰을 통하여 젖산이 첨가된 스트레스 조건에서 모균주는 신장된 세포 형태와 세포막에 손상을 입은 것으로 보아 진화된 균주에 비해 젖산에 대하여 상대적으로 취약한 것으로 판단되었다. Saccharomycopsis fibuligera is an amylolytic yeast that exhibits raw starch-degrading activity. In this study, adaptive laboratory evolution was performed to improve the tolerance of S. fibuligera to lactic acid by prolonged repeated batch fermentation in which the lactic acid concentration was gradually increased. The evolved S. fibuligera strain exhibited a significantly enhanced tolerance to lactic acid at concentrations up to 2.5% (w/v), as assessed by determining its specific growth rate using a plate assay. Scanning electron microscopy revealed an elongated and perforated morphology of the parent strain under lactic acid stress, indicating that its membrane might be more prone to damage caused by lactic acid than that of the evolved strain.

Cloning of the LEU2 Gene from the Amylolytic Yeast Saccharomycopsis fibuligera

박은희,여수환,김명동 한국식품과학회 2015 Food Science and Biotechnology Vol.24 No.6

The SfLEU2 structural gene of LEU2 encoding β-isopropylmalate dehydrogenase was cloned from the amylolytic yeast Saccharomycopsis fibuligera isolated from Nuruk, a Korean traditional starter for rice wine production. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 1,107 bp, including a stop codon, encoding a 368 amino acid residue. The deduced amino acid sequence of Leu2 in S. fibuligera was 76.4% homologous with Candida boidinii and Kluyveromyces marxianus sequences. The SfLEU2 gene complemented leu2Δ mutation in Saccharomyces cerevisiae, suggesting that the gene encodes a functional LEU2 in S. fibuligera. The GenBank Accession No. for SfLEU2 is KR820563.

Arabinoxylo- and Arabino-Oligosaccharides- Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera

( Tae Hyeon Park ),( Chang-yun Choi ),( Hyeon Jin Kim ),( Jeong-rok Song ),( Damee Park ),( Hyun Ah Kang ),( Tae-jip Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.2

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45oC and pH 7.0 in sodium phosphate buffer and at 50oC and pH 6.0 in sodium acetate buffer, respectively. These exoacting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylooligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)- and α-(1,3)-L-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)- linkages of linear and branched AOS. On the contrary, they have much lower activity against the α- (1,2)- and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

Comparison of volatile and non-volatile metabolites in rice wine fermented by <i>Koji</i> inoculated with <i>Saccharomycopsis fibuligera</i> and <i>Aspergillus oryzae</i>

Son, Eun Yeong,Lee, Sang Mi,Kim, Minjoo,Seo, Jeong-Ah,Kim, Young-Suk Elsevier 2018 Food Research International Vol.109 No.-

<P>This study investigated volatile and nonvolatile metabolite profiles of makgeolli (a traditional rice wine in Korea) fermented by koji inoculated with Saccharomycopsis fibuligera and/or Aspergillus oryzae. The enzyme activities in koji were also examined to determine their effects on the formation of metabolites. The contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09, and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids. On the other hand, major volatile metabolites were fusel alcohols, acetate esters, and ethyl esters. The contents of most fusel alcohols and acetate esters were the highest in makgeolli fermented by S. fibuligera CN2601-09, for which the protease activity was the highest, leading to the largest amounts of amino acods. The makgeolli samples fermented only by koji inoculated with S. fibuligera could be discriminated on PCA plots from the makgeolli samples fermented in combination with A. oryzae. In the case of nonvolatile metabolites, all amino acids and some metabolites such as xylose, 2-methyl benzoic acid, and oxalic acid contributed mainly to the characteristics of makgeolli fermented by koji inoculated with S. fibuligera and A. oryzae. These results showed that the formations of volatile and nonvolatile metabolites in makgeolli can be significantly affected by microbial strains with different enzyme activities in koji. To our knowledge, this study is the first report on the effects of S. fibuligera strains on the formation of volatile and nonvolatile metabolites in rice wine, facilitating their use in brewing rice wine.</P>

Isolation of the Phosphoribosyl Anthranilate Isomerase Gene (TRP1) from Starch-Utilizing Yeast Saccharomycopsis fibuligera

( Eun Hee Park ),( Myoung Dong Kim ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.8

The nucleotide sequence of the TRP1 gene encoding phosphoribosyl anthranilate isomerase in yeast Saccharomycopsis fibuligera was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 759 bp, including the stop codon, encoding a 252 amino acid residue. The deduced amino acid sequence of Trp1 in S. fibuligera was 43.5% homologous to that of Komagataella pastoris. The cloned TRP1 gene (SfTRP1) complemented the trp1 mutation in Saccharomyces cerevisiae, suggesting that it encodes a functional TRP1 in S. fibuligera. A new auxotrophic marker to engineer starch-degrading yeast S. fibuligera is now available. The GenBank Accession No. for SfTRP1 is KR078268.

누룩에서 분리한 Saccharomycopsis fibuligera 미강 발효물의 항염활성에 대한 연구

박용원,이상현 중소기업융합학회 2021 융합정보논문지 Vol.11 No.11

본 논문은 대체식품 및 화장품 원료로 사용되는 미강을 발효를 통해 기능성과 경쟁력을 증가시키는 것을 목표로 하였다. 미강 추출물과 누룩에서 분리된 Saccharomycopsis fibuligera A8로 발효된 미강 추출물을 이용 해 세포독성과 항염효과를 확인하였다. 세포 독성의 경우 미강 추출물의 경우 100 μg/mL에서 세포독성을 보였으 나 발효 미강 추출물의 경우에는 세포독성을 보이지 않았다. 한편 nitric oxide, IL-6, TNF-α, IL-1β와 같은 염증 지표를 통해 항염효과를 확인한 결과, 미강 추출물은 nitric oxide, TNF-α만 100 μg/mL이상의 농도에서 항염효과를 보였다. 발효 미강 추출물의 경우에는 각각 25 μg/mL, 25 μg/mL, 50 μg/mL, 50 μg/mL의 농도에 서 항염효과를 보여 더 낮은 농도로 항염효과를 나타내는 것을 확인하였다. 한편 S. fibuligera A8 사균체의 세포 독성 및 항염 효과를 실험한 결과 발효 산물에서 사균체 역시 유효하게 작용하는 것을 알 수 있었다. This paper aims to increase functionality and competitiveness through fermentation of rice bran, which is used as a raw material for alternative foods and cosmetics. Cytotoxicity and anti-inflammatory effects were confirmed using rice bran extract fermented with Saccharomycopsis fibuligera A8 isolated from Nuruk. In the case of cytotoxicity, cytotoxicity was shown at 100 μg/mL for rice bran extracts, but cytotoxicity was not shown for fermented rice bran extracts. Meanwhile, as a result of confirming anti-inflammatory effects through inflammatory indicators such as nitric oxide, TNF-α, IL-1β, and IL-6, rice bran extracts showed anti-inflammatory effects at concentrations of 100 μg/mL or higher only nitric oxide and TNF-α. And fermented rice bran extracts exhibited anti-inflammatory effects at concentrations of 25 μg/mL, 25 μg/mL, 50 μg/mL, and 50 μg/mL, respectively, exhibiting anti-inflammatory effects in lower concentration. Meanwhile, as a result of testing the cytotoxicity and anti-inflammatory effects of heat-killed S. fibuligera, it was found that the heat-killed S. fibuligera showed anti-inflammatory effect in fermented products.

누룩에서 분리한 Saccharomycopsis fibuligera 미강발효물의 효소활성 및 항산화능에 대한 연구

박용원 한국융합학회 2021 한국융합학회논문지 Vol.12 No.11

본 논문은 대체식품 및 화장품 원료로 사용되는 미강을 발효를 통해 기능성과 경쟁력을 증가시키는 것을 목표로 하였다. 5종류의 누룩에서 분리한 Saccharomycopsis fibuligera 50종의 균주 중 효소 활성이 우수한 6 균주와 표준균주를 이용한 발효미강물의 α-amylase, CMCase, β-glucosidase, protease 등의 효소 활성을 통해 비교한 결과, A8균이 KCTC 7806 균주에 비하여 13.7%, 21.1%, 50.3%, 10.0%의 우수한 효소 활성을 보였다. 발효의 결과지표로서 ABTS, DPPH 검사를 시행한 결과, A8으로 발효한 미강이 KCTC 7806 균주로 발효한 미강 에 비하여 1.12배, 1.28배의 항산화능이 있음을 확인하였다. 본 논문으로, S. fibuligera 표준균주인 KCTC 7806 보다 우수한 S. fibuligera A8 균주를 확인할 수 있었다. 또한, 선행연구과제의 항염활성 및 항독성 결과와 더불어 우수한 효소 활성과 항산화능 결과를 통해 S. fibuligera A8 균주를 통한 미강 발효물이 대체식품 및 화장품 원료 로서의 기능성과 가격 경쟁력을 증가시킬 수 있음을 확인하였다. This paper aimed to increase the functionality and competitiveness of rice bran, which is used as an alternative food and cosmetic raw material, through fermentation. As a result of comparing the enzyme activity of α-amylase, CMCase, β-glucosidase, protease, etc. of fermented rice bran using standard strains with 6 strains with excellent enzyme activity among 50 strains of Saccharomycopsis fibuligera isolated from 5 types of Nuruk, A8 strain showed excellent enzyme activity of 13.7%, 21.1%, 50.3%, and 10.0% compared to the KCTC 7806 strain. As a result of the ABTS and DPPH tests as a result of fermentation, it was confirmed that rice bran fermented with A8 had 1.12 times and 1.28 times more antioxidant activity than rice bran fermented with KCTC 7806 strain. In this paper, it was possible to confirm the S. fibuligera A8 strain superior to the standard S. fibuligera strain KCTC 7806.

Overexpression of Acid Protease of Saccharomycopsis fibuligera in Yarrowia lipolytica and Characterization of the Recombinant Acid Protease for Skimmed Milk Clotting

Xin-Jun Yu,Jing Li,Zhen-Ming Chi,Hui-Juan Li 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.3

The gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 94.8 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5and 33oC, respectively, and the enzyme was very stable over a pH range of 1.0 ~ 3.0. The recombinant acid protease was activated by Zn2+, but was inhibited by Hg2+,Fe2+, Fe3+, and Mg2+, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity,suggesting that it may be used as a rennet substitute in the cheese industry.

Genetic diversity and population structure of the amylolytic yeast Saccharomycopsis fibuligera associated with Baijiu fermentation in China

Wang Ju-Wei,Han Pei-Jie,Han Da-Yong,Zhou Sen,Li Kuan,He Peng-Yu,Zhen Pan,Yu Hui-Xin,Liang Zhen-Rong,Wang Xue-Wei,Bai Feng-Yan 한국미생물학회 2021 The journal of microbiology Vol.59 No.8

The amylolytic yeast Saccharomycopsis fibuligera is a predominant species in starters and the early fermentation stage of Chinese liquor (Baijiu). However, the genetic diversity of the species remains largely unknown. Here we sequenced the genomes of 97 S. fibuligera strains from different Chinese Baijiu companies. The genetic diversity and population structure of the strains were analyzed based on 1,133 orthologous genes and the whole genome single nucleotide polymorphisms (SNPs). Four main lineages were recognized. One lineage contains 60 Chinese strains which are exclusively homozygous with relatively small genome sizes (18.55–18.72 Mb) and low sequence diversity. The strains clustered in the other three lineages are heterozygous with larger genomes (21.85–23.72 Mb) and higher sequence diversity. The genomes of the homozygous strains showed nearly 100% coverage with the genome of the reference strain KPH12 and the sub-genome A of the hybrid strain KJJ81 at the above 98% sequence identity level. The genomes of the heterozygous strains showed nearly 80% coverage with both the sub-genome A and the whole genome of KJJ81, suggesting that the Chinese heterozygous strains are also hybrids with nearly 20% genomes from an unidentified source. Eighty-three genes were found to show significant copy number variation between different lineages. However, remarkable lineage specific variations in glucoamylase and α-amylase activities and growth profiles in different carbon sources and under different environmental conditions were not observed, though strains exhibiting relatively high glucoamylase activity were mainly found from the homozygous lineage.