• 한국미생물·생명공학회지
• /
• 제30권4호
• /
• pp.298-304
• /
• 2002

PCR방법을 기본으로 한 in vitro recombination과 대장균 cdd 돌연변이주에서의 발현을 통하여 family shuffling이 수행되었다. 고온성 Bacillus caldolyticu와 B. stearothermophilus 유래의 시티딘 디아미나제을 코드하는 cdd 유전자를 shuffling하였다. 이것을 대장균 cdd 돌연변이주에 형질전환 시킨 후 uraci이 없는 AB배지에서의 생존을 통하여 150개의 돌연변이 균주를 얻을 수 있었으며, 그 중 연구를 위하여 4주(SH1067, SH1077, SH1086, 및 SH1118)를 선택하였다. 선택한 4주의 염기서열을 분석한 결과, 수 회의 point mutation과 recombination이 각각 일어났음을 확인 할 수 있었다. 특히 SH1067의 경우,$ 80^{\circ}C$에서 B. stearothemophilus 에서 유래한 7101의 시티딘 디아미나제 활성과 비교하여 770배 이상의 증가를 보여주었다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권12호
• /
• pp.1769-1774
• /
• 2001

Bacillus licheniformis strains L-25 and PWD-1 are two thermophilic feather-degrading bacteria. Despite isolated from different environmental conditions, they were both capable of breaking down chicken feathers and growing in a medium in which feather was the only source of carbon and nitrogen. A 1.46-kb keratinase gene (ker B) was isolated from strain L-25 by a polymerase chain reaction (PCR) using L-25 genomic DNA as templates. Sequencing results reveal that ker B shares great sequence identity with a previously published keratinase gene of B. licheniformis PWD-1 (ker A). Only two amino acids differences were found in the deduced amino acid sequence between the keratinases from L-25 and PWD-1. However several nucleotide changes were found upstream of the putative promoter region. Protease inhibition studies indicated that neutral protease activity accounted for approximate 25 to 30% of total extracellular proteolytic activity produced by strain L-25 in the feather medium. In contrast, no measurable neutral protease activity was produced by strain PWD-1 in the feather medium. When glucose (1%), a common catabolic repressor, was added into the feather medium, L-25 was still able to grow and produce keratinase. Strain PWD-1 produced no neutral protease activity and its growth was severely inhibited in the feather medium containing glucose. L-25 produced an enhanced level of keratinase in the feather medium in comparison with PWD-1.

• Journal of Microbiology and Biotechnology
• /
• 제24권11호
• /
• pp.1551-1558
• /
• 2014

The esterase gene Est8 from the thermophilic bacterium Bacillus sp. K91 was cloned and expressed in Escherichia coli. The monomeric enzyme exhibited a theoretical molecular mass of 24.5 kDa and an optimal activity around $50^{\circ}C$ at pH 9.0. A model of Est8 was constructed using a hypothetical YxiM precursor structure (2O14_A) from Bacillus subtilis as template. The structure showed an ${\alpha}/{\beta}$-hydrolase fold and indicated the presence of a typical catalytic triad consisting of Ser-11, Asp-182, and His-185, which were investigated by site-directed replacements coupled with kinetic characterization. Asp-182 and His-185 residues were more critical than the Ser-11 residue in the catalytic activity of Est8. A comparison of the amino acid sequence showed that Est8 could be grouped into the GDSL family and further classified as an SGNH hydrolase. Est8 is a new member of the SGNH hydrolase subfamily and may employ a different catalytic mechanism.

• Journal of Microbiology and Biotechnology
• /
• 제31권4호
• /
• pp.570-583
• /
• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Journal of Animal Science and Technology
• /
• 제50권5호
• /
• pp.713-720
• /
• 2008

본 연구는 톱밥주원료 버섯부산물의 효과적 사료화를 위한 균주개발을 목적으로 버섯부산물로부터 섬유소분해력이 높은 고온성 균주를 분리 동정하고 균주생산을 위한 배지의 최적화 조건을 도출하고자 실시하였다. Xylanase와 CMCase 활력이 상대적으로 우수한 것으로 선발된 고온성 균은 3, 201-7번으로서 동정한 결과 Bacillus spp.에 속하는 균주로 B. subtilis KU3, B. subtilis KU201-7로 각각 명명하였다. 균주생산을 위한 최적 액상배양조건에 있어서 B. subtilis KU3은 질소원으로 yeast extract 3%(w/v)에서, 탄소원으로 maltose 1%(w/v)에서 성장이 가장 좋았다. B. subtilis KU201-7은 질소원으로 yeast extract 0.5%(w/v)에서, 탄소원으로 CMC 0.5%(w/v)에서 성장이 가장 좋았다.

• BMB Reports
• /
• 제32권5호
• /
• pp.480-485
• /
• 1999

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, $K'_D$, for PLP was determined with the apoenzyme to be about $1.2\;{\mu}M$. The isoelectric point was 4.9. The optimal temperature and pH for the $\alpha,\beta$-elimination of L-tyrosine were found to be $80^{\circ}C$ and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, $\beta$-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, $\beta$-elimination, was the only D-amino acid racemized by the enzyme. The $K_m$ values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, $\beta$-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.

• 한국균학회지
• /
• 제45권1호
• /
• pp.43-53
• /
• 2017

본 연구는 양송이 배지의 관행적인 발효기술 개선에 대한 기초자료를 제공하기 위하여 배지 발효에 중요한 역할을 하는 다양한 미생물에 대한 밀도변화 및 분류학적인 특징을 밝혔고, 이들 미생물이 분비하는 세포벽 분해 관련 효소활성 변화를 조사하였다. 양송이 배지 발효와 밀접한 관련이 있는 미생물의 밀도 변화를 분석한 결과 고온성 세균, 방선균, 형광성 Pseudomonas spp., 사상균 등 다양한 미생물들이 분포하였으며, 고온성 발효가 진행됨에 유해균은 사멸되는 경향을 보였다. 야외발효 과정에서는 Psychrobacter속, Pseudomonas속, Bacillus속, Pseudoxanthomonas속이 가장 많이 분포하였고, 후발효 완료 배지에서는 Bacillus속, Psychrobacillus속이 우점하였다. 이러한 볏짚 발효 과정 중 분리한 미생물의 효소활성은 발효 초기에 cellulose 분해효소가 먼저 배지에 작용하여 탄소원을 분해한 후 hemicellulose 분해효소가 부차적으로 작용하는 양상을 보였고, 이들 효소를 분비하는 미생물들은 2차와 3차 뒤집기에서 많이 분포하였다.

• 한국식품과학회지
• /
• 제20권2호
• /
• pp.218-224
• /
• 1988

두유제조공정 중 열처리 공정을 최적화 하기 위하여, 두유에서 4균주의 열저항성이 큰 flat-sour 변질균을 분리하였다. 분리균주들은 호기성 포자형성간균으로서 $-65^{\circ}C$에서도 성장하였으며, 형태학적 및 생리학적인 특징들로부터 모두 Bacillus stearothermophilus로 동정할 수 있었다. 3분리균주 및 Bacillus stearothermophilus ATCC 12980의 두유(pH 7.0) 및 pH 7.0 완충용액 내 에서의 열저항성을 측정하기 위하여, 각각의 균주에 대한 110, 115, 121 및 $125^{\circ}C$에서의 열치사에 관한 직선회귀식을 구하였다. 두유의 구성성분이 포자의 열저항성을 증가시킨다는 점은 뚜렷하게 확인되지 않았으며, 균주간에 있어서도 D값의 대소관계가 온도에 따라 변화하여, 주어진 모든 온도에서 일관성있게 가장 큰 열 저항성을 보인 균주는 없었다. 4균주의 평균 D값은 110, 115, 121 및 $125^{\circ}C$에서 각각 77.27, 20.20, 2.76 및 1.39분이었으며, 평균 z값은 $8.36^{\circ}C$이었다.

• Food Science and Biotechnology
• /
• 제14권4호
• /
• pp.461-465
• /
• 2005

To determine the cause of wet noodle spoilage, microorganisms isolated from wet noodles were identified and characterized. In addition, the growth inhibitory effects of organic acid mixture (OA: lactic acid 27.8%, acetic acid 12.0%, succinic acid 1.0%) and sodium dehydroacetate (SD) on the isolated strain were estimated in nutrient broth medium. The isolated strain was Gram-positive, rod shaped, motile, and spore forming. Based on physiological characteristics and the API 50 CHB-kit test results for the assimilation of 49 carbohydrates, the isolated strain was identified as Bacillus amyloliquefaciens (92.6%), which is able to degrade starch. Decimal reduction times (D-values) at 100, 105, and $110^{\circ}C$ for spores of B. amyloliquefaciens were 8.5, 5.1, and 2.5 min, respectively, and the z-value was $12.8^{\circ}C$. We estimated that B. amylo-liquefaciens isolated from spoiled wet noodles was a thermophilic species having high heat-resistance. Viable cell numbers in wet noodles and broth medium inoculated with B. amyloliquefaciens were decreased by 2-4 log cycles by combined treatment with 0.03 or 0.05% OA and 0.3% SD. These results revealed that OA combined with SD could be used as a potential agent to inhibit B. amyloliquefaciens in wet noodles.

• 생명과학회지
• /
• 제12권2호
• /
• pp.129-135
• /
• 2002

여러 종류의 호열성, 호기성 간균(Bacillus genus)군, 중금속 leaching 미생물군(Thiobacillus, T. ferooxidans), 그리고 여러 가지 난분해성 물질을 분해하는 미생물군 (Pseudomonas genus)을 활용하여 퇴비화의 조건을 연구하고 이를 이용하여 축산폐기물의 퇴비화에 미치는 효과를 연구하였다. 35∼40의 C/N비, 50∼65%의 함수율 범위에서 실험실용 회전드럼형 반응조에서의 퇴비화는 온도상승이 수동식 반응조보다 낮으며 느리게 일어났다. 퇴비화 후 성분분석에서 높은 수준의 광물질을 함유하는 것으로 화학비료 대체효과를 보여주며 퇴비화 전후 중금속 분석에서 As는 모든 퇴비에서, Cr은 돈분, ph은 축분, Hg은 계분, 그리고 Cu는 축분퇴비에서 규제값 이하를 보여주었다. 여러 가지 부숙도 분석에서 퇴비의 숙성도를 나타내었다. SS 또는 EMB agar plate을 이용한 살모넬라균과 대장균의 검사에서 병원균에 대한 퇴비의 안전성이 확인되었다. 이러한 결과는 금속 및 다른 난분해성 물질을 생분해하는 미생물을 투입하여 퇴비의 중금속감소와 퇴비화 속도증가의 가능성을 시사한다.