• BMB Reports
• /
• 제40권1호
• /
• pp.22-28
• /
• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• Mycobiology
• /
• 제35권2호
• /
• pp.54-61
• /
• 2007

Two isolates of Tricholoma matsutake T-008 and T-034, preserved in Entomopathogenic Fungal Culture Collection (EFCC) of Korea, were used in the present study. The isolates had 100% Bootstrap homology with Tricholoma matsutake U62964 and T. matsutake AB188557 and AF309538 preserved in Gene Bank of NCBI. Mycelial growth of T. matsutake was highest in TMM and MYA at $25^{\circ}C$. The highest dry wt. of mycelium was obtained after 65 days of culture, when 6 mycelial discs were inoculated in 100 ml of broth in 250 ml shaking flask. Mycelial mats were observed in clumped condition at the inoculation sites of pine forest after two weeks of inoculation. After 5 months of inoculation, mycelia mats were observed growing inside soil and walls of a few inoculation sites, while mycelial mats growth up to $5{\sim}8$ cm were observed in the roots of pine tree after 6 months. The survival rate of the inoculum was about 40% of the total inoculation sites. The survival rate was found below 20% when the mycelium was inoculated in the summer. The reasons for low survival rates of the mycelium were mainly due to dry season and the soil-borne small animals such as earthworm and mole. After one year of inoculation, no external difference was observed between the artificially inoculated mycelia and the naturally existing mycelia of T. matsutake. The present study showed that fruiting bodies of T. matsutake could be produced by artificial inoculation under the appropriate environmental conditions.

• 한국미생물·생명공학회지
• /
• 제13권4호
• /
• pp.321-327
• /
• 1985

색소를 형성하지 못하는 Serratia sp. LW-1 균주의 균체의 protease생성조건을 검토하고 효소를 간단한 방법으로 정제하였다. 효소 생산을 위한 최적온도는 brain heart infusion배지에서 $25^{\circ}C$이었으며, 배양후 76-80시간에 최고의 균체의 효소활성을 나타냈다. Aeration효과는 5$\ell$용 flask에 배지량을 1$\ell$주입하여 180 cycles/min으로 진탕배양 하였을 때가 aeration하지 않았을 경우보다 약 8배의 균체의 효소생산을 보였다. 효소의 정 제는 ammonium sulfate침 전, ammonium sulfate분별염석 및 두번의 DEAE-cellulose column 크로마토그라피에 의하여 수행하였으며, 정제된 효소는 정제도가 약 100배, 회수율이 16%이었다. 정제된 효소는 analytical ultracentrifuge pattern에서 단일 단백질로 나타났으며 최고 활성을 나타내는 pH는 vitamin free casein을 substrate로 사용하였을 때 pH 8.5-9.5이었고 최적온도는 4$0^{\circ}C$ 근처이었다

• 자원리싸이클링
• /
• 제14권5호
• /
• pp.24-31
• /
• 2005

폐전자스크랩에서 유가금속을 회수하기 위하여 곰팡이균으로 Aspergillus niger를 이용한 Cu, Zn, Al, Co, Ni, Fe, Sn, Pb의 침출 거동을 조사하였다. Aspergillus niger는 전자스크랩의 존재 하에 배양이 가능하였고 신진대사 작용에 의해 유기산(구연산 및 옥살산)을 생성함으로써 폐전자스크랩에서 각 금속들을 침출시키는 것을 알 수 있었다. 예비실험으로 먼저 구연산 및 옥살산을 이용한 화학침출 실험으로부터 Cu, Zn, Al, Co, Fe, Sn, Pb의 침출거동을 조사하였다. Aspergillus niger를 이용한 미생물 침출 실험의 결과 전자스크랩의 농도가 50g/L인 경우 Cu 및 Co의 침출율은 95%이상이었고, Al, Zn, Pb 및 Sn의 경우는 15-35%의 침출율을 나타냈으며 Ni 및 Fe의 경우는 10%이하의 침출율을 보였다.

• 한국약용작물학회지
• /
• 제15권1호
• /
• pp.46-50
• /
• 2007

하늘타리 (Trichosanthes kirilowii)로부터 조직배양 기법을 이용하여 약리작용을 나타내는 생리활성물질인 trichosantin을 생산하기 위한 연구로 Agrobacterium rhizogenes ATCC15834 의 감염을 통하여 기내 배양된 유묘의 잎절편으로부터 형질전환 모상근을 유도하였다. 유도된 모상근 clones으로부터 성장속도와 분지화 정도가 빠르며 단백질 합성능이 가장 우수한 TR-03 clones을 선발하였으며, 이를 사용하여 생체량과 배지내 가용성 단백질의 생산을 최대로 높일 수 있는 최적 배양조건을 조사하였다. TR-03 clone을 4% sucrose가 첨가된 MS 배지에 초기 접종농도 2 g (생중량)을 접종하여 100 rpm으로 배양시 생중량과 배지내 가용성 총단백질의 함량이 플라스크당 약 2.4 g (건중량)과 $28.3ug/\ell$으로 최대치를 나타내었다. 한편, 배지내 가용성 단백질에 대한 RIP활성 검정에서 배양 4주 후 21.3 unit로 최대치를 보였다.

• 한국자기공명학회논문지
• /
• 제17권2호
• /
• pp.67-75
• /
• 2013

ATP synthase produces ATP, a major energy source for metabolic processes in organisms, from ADP and inorganic phosphate in cellular membranes. ATP synthase is known as a rotary motor, in which the c-subunit ring functions as a rotor. In this work, we have tried to develop a more general preparation procedure of thermophilic $F_oc$-ring ($TF_oc$-ring) for NMR measurements. The expression of $TF_oF_1$ is easily affected by various experimental conditions such as temperature, shape and size of a flask, a volume of medium, and shaking rate of an incubator. Accordingly, we have tried to optimize the expression conditions of $TF_oF_1$. $TF_oc$-rings were purified from $TF_oF_1$ according to a reported method. We modified purification procedures to improve purity and yield of $TF_oc$. On top of them, we found a new combination of detergents for the purification at anion-exchange column chromatography. To examine the effect of lipid environments on the structure, the $TF_oc$-rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the $TF_oc$-ring structures in these membranes with solid-state NMR.

• Journal of Microbiology and Biotechnology
• /
• 제33권11호
• /
• pp.1531-1541
• /
• 2023

Lipase is a well-known and highly in-demand enzyme. During the last decade, several lipase optimization studies have been reported. However, production costs have always been a bottleneck for commercial-scale microbial enzyme production. This research aimed to optimize the conditions for lipase production by Limtongozyma siamensis DMKU-WBL1-3 via a One-Factor-At-a-Time (OFAT) approach combined with statistical methods while using a low-cost substrate. Results suggest that low-cost substrates can be substituted for all media components. An optimal medium was found, using response surface methodology (RSM) and central composite design (CCD), to consist of 0.50% (w/v) sweet whey, 0.40% (w/v) yeast extract (food grade), and 2.50% (v/v) palm oil with the medium pH adjusted to 4 under shaking flask cultivation. From an economic point of view, this work was successful in reducing production costs while increasing lipase productivity. The medium costs were reduced by 87.5% of the original cost while lipase activity was increased by nearly 6-fold. Moreover, lipase production was further studied in a 2-L stirred-tank fermentor. Its activity was 1,055.6 ± 0.0 U/ml when aeration and agitation rates were adjusted to 1 vvm and 170 rpm, respectively. Interestingly, under this optimal lipase production, the yeast showed accumulated lipids inside the cells. The primary fatty acid is a monounsaturated fatty acid (MUFA) that is typically linked to health benefits. This study hence reveals promising lipase production and lipid accumulation by L. siamensis DMKU-WBL1-3 that are worthy of further study.

• Journal of Microbiology and Biotechnology
• /
• 제13권6호
• /
• pp.960-968
• /
• 2003

A bacterial isolate showing a strong endochitosanase activity was isolated from soil and then characterized. The isolate was identified and designated as Bacillus cereus P16, based on morphological and biochemical properties, assimilation tests, cellular fatty acids pattern, along with 16S rRNA gene sequence. The optimized medium for producing extracellular chitosanase in a batch culture contained 1% tryptone, 0.5% chitosan, and 1% NaCl (pH 7.0). Powder chitosan and tryptone served the best as carbon and nitrogen sources, respectively, for the chitosanase production. Chitosanase activity was the highest when culture was completed at $37^{\circ}C$ among various temperatures ($20-42^{\circ}C$) tested in a shaking incubator (200 rpm). The levels of chitosanase activity in the culture fluid were 2.0 U/ml and 3.8 U/ml, respectively, when incubated in a flask for 60 h and in a jar fermenter for 24 h. The culture supernatant showed a strong liquefying activity on the soluble chitosan. The viscosity of 1% chitosan solution, that was incubated with the culture supernatant, was rapidly decreased, suggesting the secretion of endochitosanolytic enzymes by P16. The culture fluid revealed six endo-type chitosanase isozymes, two major (38 and 45 kD), and four minor (54, 65, 82, and 96 kD) forms by staining profile. The crude enzymes were very stable, and full activity was maintained for 4 weeks at $4^{\circ}C\;or\;-20^{\circ}C$ in the culture supernatant, suggesting a highly desirable stability rate for making an industrial application of the crude enzymes. The supernatant also cleaved the insoluble chitosan powder, but the hydrolysis rate was much lower. The enzymic degradation products of chitosan contained $(GlcN)_n$ (n=2-8). The concentration of chitosan in the reaction mixture of the crude enzyme affected the chitooligosaccharides composition of the hydrolysis products. When the higher concentration of chitosan was used, the higher degree of polymerized chitooligosaccharides were produced. By comparison with other commercial chitosanase preparations, P16 was indeed found to be a valuable enzyme source for industrial production of chitooligosaccharides from chitosan.

• KSBB Journal
• /
• 제25권6호
• /
• pp.520-526
• /
• 2010

토양 미생물로부터 스크리닝한 미생물 중 카탈라제 생산량이 높은 세 가지 미생물 (Bacillaceae bacterium BKBChE-1, Bacillus sp. BKBChE-2와 Bacillus flexus BKBChE-3)을 배양하여 얻은 카탈라제 효소가 과산화수소를 분해하는 것을 확인하고, 미생물 균체를 천연제올라이트에 정전기적 흡착법으로 고정하였다. 주사전자 현미경을 통해 각각의 미생물이 지지체에 잘 흡착된 것이 관찰되었고, 10.5 mM의 과산화수소를 1시간 이내에 완전히 분해되는 것을 확인하였다. 과산화수소 분해 속도가 가장 빠른 Bacillus flexus BKBChE-3은 온도와 pH 영향성 실험을 통해 고온과 pH 10 이상의 강알칼리 조건에서도 30일 이후까지 미생물이 생존하는 것을 관찰하였다. 또한 고정화된 미생물의 경우 온도 $25-40^{\circ}C$, pH 7-10의 조건에서 생균수가 계속하여 증가하는 것을 확인하였다. 본 실험을 통해 Bacillus flexus BKBChE-3이 생산한 카탈라제 효소 뿐만 아니라 고정화된 균체도 과산화수소의 제거에 효과적임을 확인할 수 있었다. 이들 결과로부터 미생물 발효를 통해 생산된 효소의 생산량 증가와 미생물 개체 수 증가를 이용하여 산업폐수에 적용하면, 과산화수소의 제거 효율을 높이고 균체 재활용을 통한 공정비용을 절감할 수 있을 것으로 사료된다.

• 생명과학회지
• /
• 제21권9호
• /
• pp.1244-1250
• /
• 2011

인삼의 효과적 이용을 위해 부산물로 취급되어 대부분 폐기되는 세근을 발효공학적 기법으로 항균성 물질을 증대하기 위한 연구이다. 이를 위해서 발효에 적합한 균주의 선별과 발효에 사용될 인삼 세근 분말의 농도를 선별하여 발효 시킨 후 항균성 물질을 탐색하였다. 그 결과 8종의 유용 미생물 중 L. plantarum이 가장 적절한 균주로 선정 되었으며, 영양원인 인삼 세근 분말의 농도는 5%로 밝혀졌다. 또한, 발효 추출물의 항균 및 항바이러스 활성분석 결과 항균성은 있으나 항바이러스 활성은 미약한 것으로 밝혀졌다. 항균 활성의 경우 비발효 추출물 보다 발효 추출물이 약 2배 정도 증가되었고, Gram Positive 균주에서 활성이 더 좋은 것으로 분석 되었다. 발효에 의한 항균 활성 물질의 성분 변화를 알아 보기 위해서 HPLC 분석법을 사용하였으며 그 결과 ginsenoside Rg1, Re, Rd 성분의 함량이 증가되었고, 이 중 가장 많은 함량 변화를 보인 성분은 Rd로 약 50 ${\mu}g/g$이 증가되었다. 이러한 결과로부터 인삼 세근 발효물 중 항균 활성을 보이는 주된 물질은 ginsenoside Rd 성분인 것으로 추정된다.