Novozym 234에 의한 Kluyveromyces fragilis N100과 Candida pseudotropicalis CBS607의 원형질체 형성과 재생

배석,전순배,Bai, Suk,Chun, Soon-Bai 한국미생물학회 1984 미생물학회지 Vol.22 No.1

Novozym 234에 의한 K. fragilis와 C. pseudotropicalis의 원형질체 형성과 재생에 관한 연구를 하였다. intact cell을 원형질체로 전환시키는데 정지기의 세포보다 대수기에 있는세포가 효과적이었으며 osmotic stabilizer로는 $(NH_4)_{2}SO_4$가 두 종 똑같이 원형질체 형성이나 재생에 있어서 적합했고 최적 효소농도는 K. Fragilis에서는 3mg/ml, C. pseudotropicalis에서는 1~3mg/ml이었다. 반응 후 관찰된 세포 중의 원형질체 형성 수율은 K. fragilis에서 약 95%, C. pseudotropicalis에서 약 100%였으나 원형질체의 재생은 Glusulase와 비교해 볼 때 매우 낮았다. 그러나 세포 이물질이 없는 원형질체를 얻는데는 Novozym 234가 적합한 것 같다. Formation and regeneration of protoplasts by Novozym 234 from Kluyveromyces fragilis N100 and Candida pseudotropicalis CBS607 were studied. This enzyme was more effective on cells grown at exponential phase than those at stationary one to convert intact cells into protoplasts. As osmotic stabilizer, ammonium sulphate was suitable for not only protoplast formation but also regeneration in K. fragilis as well as in C. pseudotropicalis. Optimal enzyme concentration was 3mg per ml in K. fragilis and 1~3mg per ml in C. pseudotropicalis, respectively. After the exposure of K. fragilis cells to 3mg per ml of enzyme for 3hr at 30$^{\circ}C$ , approximately 95% of protoplast formation of all observed cells was obgained, while about 100% from C. pseudotropicalis under the same condition was produced. The regeneration frequency of protoplasts by this enzyme was much lower than that by snail enzyme(Glusulase) although Novozym 234 converted cells from above two species into protoplasts free of cell debris effectively, compared with Glusulase. Novozym 234 appears to be suitable for subcellular fractionation to obtain nuclei or other organelles rather than protoplast regeneration.

Schwanniomyces castellii Glucoamylase의 정제 및 성질

배석,박종천,김동호,김강화,전순배,Bai, Suk,Park, Jong-Chun,Kim, Dong-Ho,Kim, Kang-Hwa,Chun, Soon-Bai 한국미생물학회 1991 미생물학회지 Vol.29 No.2

The glucoamylase of Schwanniomyces castellii was purified to homogeneity from the culture filtrate. the purified enzyme was a glycoprotein with a molecular mass of about 145 KDa, which was monomeric protein with an isoelectric point of 4.3. The pH and temperature optima were 5.5 and 40.deg.C, respectively. The enzyme was fairly stable up to 50.deg.C and at acid pH range (pH 4.5-6.0). The apparent Km of the enzyme toward soluble starch, isomaltose and pullulan were 3.84, 0.51 and 13.7 mg/ml, respectively. The analysis of amino acid composition on this enzyme was found to be acidic protein like other fungal glucoamylase. The amino acid sequence of N-terminal peptide consisted of Ala-Pro-Ala-Asp-Gly-Ile-Gly-Asp-X-Ala-X-Ala.

Phaffia rhodozyma의 원형질체 융합

배석(Suk Bai),김문휘(Moon-Whee Kim),박종천(Jong-Chun Park),김재형(Jae-Hyung Kim),전순배(Soon-Bai Chun) 한국생물공학회 1990 KSBB Journal Vol.5 No.3

원형질체 융합 방법을 이용한 전분 발효성 효모의 개발

전순배,배석,이기영,김수현,이진종,김상문,Chun, Soon-Bai,Bai, Suk,Lee, Kee-Young,Kim, Soo-Hyun,Lee, Jin-Jong,Kim, Sang-Moon 한국미생물학회 1990 미생물학회지 Vol.28 No.2

전분 분해능이 있는 담자균성 효모, Filohasidium capsuligenum의 종내 및 Saccharomyces cerevisiae와의 속간 원형질체융합을 시도하였다. F capsuligenum CBS 6122-2의 종내 융합율은 각각 $2.9\times10^{-3}$ (ade+mel trp), $8.5\times10^{-3}$(ade+met)이고, F. capsuligemum CBS 4381 (cys his)과 S.cerevisiae 262-12-1 (hom3 thr1), X2180-1A (ade thr)와의 속간 융합율은 각각 $8.8\times10^{-6}$, $9.5\times10^{-4}$이었다. DNA 정량, 핵 염색, 자외선 조사에 따른 생존력 비교 그리고 형질 분리 분석 결과 등을 통해 종내와 속간에서 핵 융합이 일어난 것을 알 수 있었다. 또한, 종내 융합체의 경우 세포당 $\alpha$-amylase와 glucoamylase 생성능이 양천형에 비해 1.6-2.1배의 증진 효과가 있었다. Intraspecific fusion frequency between Filobasidium capsuligenum CBS6122-2 ade and met trp or met strains was $2.9\times 10^{-3}$ and $8.5\times 10^{-3}$, respectively. And intergeneric fusion frequency between Folobadidium capsuligenum CBS 4318 (cys his) and Saccharomyces cerevisiae 262-12-1 (hom3 thr1) or X2180-1A (ade thr) was $8.8\times 10^{-6}$ and $9.5\times 10^{-4}$, respectively. Nuclear fusion appears to occru in fusants between intraspecies and intergenera as strongly suggested by DNA content, nuclear staining, comparison of survival rate to UV light and mitotic segregation analysis. It was also found that $\alpha$-amylase and glucoamylase activity from intraspecific hybrids was 1.6-2.1 fold increased when compared with that from thier parents.

Bovine serum albumin, Myoinositol과 Ergosterol에 의한 Candida pseudotropicalis의 원형질체 재생 및 융합증진

전순배,배석,Chun, Soon-Bai,Bai, Suk 한국미생물학회 1987 미생물학회지 Vol.25 No.4

Candida pseudotropicalis의 영양요구성 돌연변이 균주들에 대한 원형질체 형성, 재생 그리고 융합에 있어서 bovine serum albumin (BSA), myoinositol 그리고 ergosterol의 증진효과를 연구하였다. 원형질체 형성율은 영양요구성 균주의 type에 따라 48%-98%였다. O. 5mg/ml의 myoinositol과 0.1mg/ml의 ergosterol을 배양액에 첨가하고 4mg/ ml의 BSA을 원형 질체 형성 완충용액에 첨가했을 때는 50~100%의 원형절체 형성윷을 얻었다. 영양요구성 균주의 생장배지에 myoinositol과 ergosterol을 동시에 첨가하고 원형질체 형성 완충용액에 BSA를 침가하면 2.2-3.0애의 원형질체 재생율 증진을 보여주었다. 원형질체 융합의 최적조건에서 상보적 영양요구성 균주간의 융합율은 $7.0\times 10^{-4}$ ~ $1.5\times 10^{-3}$ 이였으며 상기화합물을 처리하지 않고 얻은 융힐l율과 비교해서 1. 9-2. 3배의 증가를 보였다. 이러힌 결과로 본 연구에서 사용된 균주들에서는 상기화합물 처리 에 의해 원형질체 재생은 물론 세포융합율도 증진됨을 알 수 있었다. The effects of bovine serum albumin, myoinositol and ergosterol on protoplast formation, regeneration and fusion from auxotrophic mutants of Candida pseudotropicalis were examined. Frequency of protoplast formation ranged from 48 to 98% depending on auxotrophic types. When myoinositol (0.5mg/ml) and ergosterol (0.1mg/ml) were supplemented in the medium of cell growth, and bovine serum albumin (4mg/ml)was added to protoplasting buffer, 50-100% of cells were converted to protoplasts. Such a treatment of three additives improved 2.2-3.0 fold of regeneration rate of protoplasts. The fusion frequencies between complementary auxotrophs ranged from $7.0\times 10^{-4}$ to $1.5\times 10^{-3}$ in the optimal conditions. These values showed 1.9-2.3 fold increase when compared with fusion frequencies obtained without the treatment of additives. These results suggested that these comsion frequencies obtained without the treatment of additives. These results suggested that these xompounds may improve protoplast regeneration and fusion between complementary auxotrophs used in this study.

Candida pseudotropicalis 융합세포의 유전적 분석

전순배,배석,Chun, Soon-Bai,Bai, Suk 한국미생물학회 1988 미생물학회지 Vol.26 No.2

The genetic analysis and characterization of protoplast fusion hybrids between complementary auxotrophic mutants of Candida pseudotropicalis were carried out. Nuclear fusion appeared to occur in fusion hybrids (e.g., F15 and F33), as strongly suggested by isolation of recombinants after mitotic segregation of parental genetic markers. This was confirmed by KNA content, nuclear staining and comparison of survival rate to UV light. After keeping fusion hybrids for approximately one year, the frequency of spontaneous mitotic segregation was $3.0\times 10^{-4}$ - $8.1\times 10^{-4}$ while that of induced mitotic segregation was $1.4\times 10^{-3}$- $1.7\times 10^{-3}$. These results suggested that they maintained stable karyogamy state. It was also found that the production of $\beta$-D-galactosidase from F15, F33 and F158 was somewhat increased when compared with that from either auxotrophic parents or wild type.

B16 Melanoma Cell에서 티로시나아제 유전자 발현에 황금(Scutellaria baicalensis) 추출물이 미치는 효과

조남철,배석,진종언,Cho, Nam-Chul,Bai, Suk,Chin, Jong-Eon 한국식품영양학회 2010 韓國食品營養學會誌 Vol.23 No.1

유전자 발현 조절 수준에서 멜라닌 색소의 생합성을 조절하는 천연물질을 탐색하고자 황금으로부터 유용성 물질을 추출 및 분획하여 티로시나아제 프로모터 부위를 도입하여 형질전환된 B16 melanoma cell에 처리한 결과, 그 메탄올 추출물의 티로시나아제 유전자 발현율은 $10\;{\mu}g/m{\ell}$와 $100\;{\mu}g/m{\ell}$의 농도에서 대조군 세포에 비해 각각 약 231과 256%로 매우 크게 증진하는 효과를 보여 주었다. 극성도가 서로 다른 용매 분획물들을 형질전환된 세포에 처리하였을 때 methylene chloride와 ethyl acetate 용매 분획물들은 $10\;{\mu}g/m{\ell}$의 농도에서, butyl alcohol 용매 분획물은 $10\;{\mu}g/m{\ell}$와 $100\;{\mu}g/m{\ell}$의 농도에서, 그리고 물 용매 분획물은 $500\;{\mu}g/m{\ell}$의 고농도에서 티로시나아제 유전자의 발현을 증진시키는 효과를 보여 주었다. 그러나 그들의 티로시나아제 발현 증진 효과는 메탄올 추출물보다 낮았다. 황금 메탄올 추출물을 B16 melanoma cell에 처리한 다음 MTT assay를 실시한 결과, $10\;{\mu}g/m{\ell}$와 $100\;{\mu}g/m{\ell}$의 농도에서는 대조군 세포와 세포활성능이 유사할 정도로 세포독성이 매우 낮게 나타났다. 그러나 그 이상의 농도에서는 세포 활성능이 현저하게 낮았으며, 특히 $1,000\;{\mu}g/m{\ell}$의 농도에서는 세포 독성이 심하여 세포의 활성능을 측정하는 것이 불가능하였다. 본 연구결과, 황금 메탄올 추출물 내에는 티로시나아제 유전자의 발현을 증진시키는 데에 관여하는 성분들이 함유되어 있는 것으로 추측된다. 따라서 황금 추출물은 멜라닌 색소의 생합성에 관여하는 유전자의 발현하는 증진시키는 목적으로 이용함이 바람직하며, 이를 산업적으로 응용하기 위해서는 앞으로도 보다 많은 연구가 필요하다. To estimate the regulatory effects of Scutellaria baicalensis extracts on melanin biosynthesis, we evaluated the regulatory effects of the tyrosinase gene on B16 melanoma cells. The results revealed that methanolic extracts of Scutellaria baicalensis resulted in a high increase in the expression of the tyrosinase gene. Specifically, treatment with extracts at concentrations of $10\;{\mu}g/m{\ell}$ and $100\;{\mu}g/m{\ell}$ resulted in increases in tyrosinase gene expression rates of approximately 231% and 256%, respectively, when compared to the control. Moreover, the solvent fraction layers(methylene chloride, ethyl acetate, butyl alcohol, water) improved the expression of the tyrosinase gene, but to a lesser degree than the methanolic extracts. An MTT assay revealed, that the methanolic extract exhibited very low cytotoxicities at $10\;{\mu}g/m{\ell}$ and $100\;{\mu}g/m{\ell}$. Taken together, the results of this study indicated that the methanolic extracts of Scutellaria baicalensis was a very effective positive regulator of tyrosinase gene expression.

Schwanniomyces castellii CBS 2863(ATCC 26077)으로부터 $\alpha$-Amylase 정제 및 특성

박종천,배석,임선영,이진종,이향,전순배,Park, Jong-Chun,Bai, Suk,Lim, Suhn-Young,Lee, Jin-Jong,Lee, Hyang,Chun, Soon-Bai 한국미생물·생명공학회 1993 한국미생물·생명공학회지 Vol.21 No.6

The extracellular alpha-amylase was purified to homogenity from the culture filtrate of starch grown Sch. castellii CBS 2863. The purified enzyme was glycoprotein with a molecular weight of about 56 kDa. The pH and temperature optimum were 5.5 and 40C, respectively. The enzyme was fairly stable up to 40C and at acid pH range (pH 4.0-7.0). The apparent Km and Vmax of the enzyme toward starch was 1.0mg/ml and 100U/mg protein, respectively. The analysis of amino acid composition was found to be acidic protein. The amino acid sequence of N-terminal peptide consisted of Asp-Val-Ser-Ser-Ala-X-X-Thr-Arg-Ser-Glu-Ser-Ile-Tyr.

Schwanniomyces castellii CBS 2863으로부터 ${\alpha}$-Amylase 유전자 Cloning

박종천,배석,전순배,Park, Jong-Chun,Bai, Suk,Chun, Bai-CHun 한국미생물학회 1994 미생물학회지 Vol.32 No.1

Schwanniomyces castellii의 제놈 DNA로 제조된 유전자 은행으로부터 cloning된 ${\alpha}$-amylase 유전자가 Sacchromyces cerevisiae에서 발현되었다. Cloning된 삽입 DNA 절편의 크기는 약 5.0 kb이었고, Southern 및 immunoblot 분석 결과 cloning된 ${\alpha}$-amylase 유전자가 Sch. Castellii로부터 유래되었음이 확인되었다. S. cerevisiae SHY3 형질전환체에서 Sch. Castellii ${\alpha}$-amylase 유전자발현은 모균주에 비해 낮았으나, 단백질의 분자량 및 효소의 성질은 Sch. Castellii에서 분리한 ${\alpha}$-amylase의 그것과 차이가 없었다. The gene encoding ${\alpha}$-amylase of Schwanniomyces castellii was cloned in Saccharomyces cerevisiae. The 5.0-kilobase insert was shown to direct the synthesis of ${\alpha}$-amylase. Southern blot analysis confirmed that this ${\alpha}$-amylase gene was derived from the genomic DNA of Sch. castellii. Immunoblot analysis showed that ${\alpha}$-amylase production from S. cerevisiae transformant was less than that of donor strain. The ${\alpha}$-amylase secreted from S. cerevisiae transformant was shown to be indistinguishable from that of Sch. castellii on the basis of molecular weight and enzyme properties.

Schwanniomyces occidentalis var. persoonii CBS 2169 $\alpha$-Amylase 유전자의 Nucleotide Sequence

박종천,배석,오상진,이진종,전순배,Park, Jong-Chun,Bai, Suk,Oh, Sang-Jin,Lee, Jin-Jong,Chun, Soon-Bai 한국미생물·생명공학회 1993 한국미생물·생명공학회지 Vol.21 No.6

The relationship between Schwanniomyces occidentalis CBS 2863 (formerly castellii) and CBS 1153 (formerly alluvius), and their variety persoonii was examined at alpha-amylase gene level. Using Sch. occidentalis alpha-amylase gene as probe, Sch. occidentalis alpha-amylase gene homologues were obtained from Sch. occidentalis CBS 1153 and Sch. occidentalis var. persoonii. The restriction analysis of these homologues showed that the restriction enzyme sites between Sch. occidentalis CBS 2863 and CBS 1153 was identical but different between these strains and Sch. occidentalis var. persoonii.