• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1001-1010
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• 2005

Bacillus megaterium KL39, an antibiotic-producing plant growth promoting rhizobacterium (PGPR), was selected from soil. The antifungal antibiotic, denoted KL39, was purified from culture filtrate by column chromatography using Dion HP-20, Silica gel, Sephadex LH-20, and prep-HPLC. Thin layer chromatography, employing the solvent system of ethanol:ammonia:water=8:1:1, showed the $R_{f}$. value of 0.32. The antibiotic KL39 showed a negative reaction with ninhydrin solution, positive with iodine vapor, and also positive with Ehrlich reagent. It was soluble in methanol, ethanol, butanol, and acetonitrile, but insoluble in chloroform, toluene, hexane, ethyl ether, or acetone. Its UV spectrum had the maximum absorption at 208 nm. Amino acid composition, FAB-mass, $^{1}H-NMR,\;^{13}C-NMR$, and atomic analyses showed that the antibiotic KL39 (MW=1,071) has a structure very similar to iturin E. The antibiotic KL39 has a broad antifungal spectrum against a variety of plant pathogenic fungi including Rhizoctonia solani, Pyricularia oryzae, Monilinia froeticola, Botrytis cinenea, Altenaria kikuchiana, Fusarium oxysporum, and F. solani. An MIC value of $10\;{\mu}g/ml$ was determined for Phytophthora capsici. Macromolecular incorporation studies with P. capsici using radioactive [$^{3}H-adenine$] as the precursor, indicated that the antibiotic KL39 strongly inhibits the DNA biosynthesis of the fungal cell. Microscopic observation of the antifungal action showed abnormal hyphal swelling of P. capsici. The purified antibiotic KL39 was very effective for the biocontrol of in vivo Phytophthora-blight disease of pepper.

• Mycobiology
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• 제41권1호
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• pp.47-55
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• 2013

Rhizoctonia solani, Fusarium solani, F. oxysporum, and Macrophomina phaseolina were found to be associated with root rott and wilt symptoms of faba bean plants collected from different fieldes in New Valley governorate, Egypt. All the obtained isolates were able to attack faba bean plants (cv. Giza 40) causing damping-off and root rot/wilt diseases. R. solani isolates 2 and 5, F. solani isolate 8, F. oxysporum isolate 12 and M. phaseolina isolate 14 were the more virulent ones in the pathogenicity tests. Biocontrol agents (Trichoderma viride and Bacillus megaterium) and chemical inducers (salicylic acid [SA] and hydrogen peroxide) individually or in combination were examined for biological control of damping-off and root rot/wilt and growth promoting of faba bean plants in vitro and in vivo. Both antagonistic biocontrol agents and chemical inducers either individually or in combination inhibited growth of the tested pathogenic fungi. Biocontrol agents combined with chemical inducers recorded the highest inhibited growth especially in case SA + T. viride and SA + B. megaterium. Under green house and field conditions, all treatments significantly reduced damping-off and root rot/wilt severity and increased of survival plants. Also, these treatments increased fresh and weights of the survival plants in pots compared with control. The combination between biocontrol agents and chemical inducers were more effective than used of them individually and SA + T. viride was the best treatment in this respect. Also, under field conditions, all these treatments significantly increased growth parameters (plant height and number of branches per plant) and yield components (number of pods per plant and number of seeds per plant, weight of 100 seeds and total yield per feddan) and protein content in both seasons (2010~2011 and 2011~2012). Faba bean seeds soaked in SA + T. viride and SA + B. megaterium were recorded the highest growth parameters and yield components. Generally, the combination between biocontrol agents and chemical inducers recorded the best results for controlling damping-off and root rot/wilt diseases in greenhouse and field with addition improved plant growth and increased yield components in field.

• Journal of Microbiology and Biotechnology
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• 제30권5호
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• pp.777-784
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• 2020

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named 'CYP102A15 and CYP102A170,' from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724,respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C₁₂-C₂₀, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with K_m 15.3 μM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 μM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.

• Preventive Nutrition and Food Science
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• 제4권2호
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• pp.97-102
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• 1999

Antimicrobial activity and chemical composition of volatile flavor extracts from Agastache rugosa were investigated. The volatile flavor extracts were obtained from leaves and stems of Agastache rugosa by simultaneous distillation extraction (SDE) method. Antimicrobial activity was investigated by disc diffusion and broth dilution methods against several microorganisms of Bacillus cereus, bacillus megaterium, Bacillus subtilis, Corynebacterium xerosis, Staphylo coccus aureus, Staphylococcus epidermidis, Agrobacterium rhizogenes , Agrobacterium tumefaciences, Enterobacter cloacae, Escherichia coli, Salmonella typhi, Vibrio parahaemolyticus, Candida utilis and Saccharomyces cerevisiae. Volatile flavor extractsfrom leaves have strong antimicrobial activity against C.utilis and S.cerevisiae. When 0.12% volatile flavor extracts from fresh leaves were included in the medium, lag phase of C. utilis was extended 6 hr and that of S.utilis and S.cerevisiae was extended 2hr. Further analyses were performed to elucidatethe effective component of the extracts. The major component of volatile flavor was estragole, a phenolic compound. Minor components were determined to be terpenes , alcohols, acids , esters, ketones and aldethydes.

• Natural Product Sciences
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• 제13권4호
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• pp.279-282
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• 2007

The aim of the present study was to investigate the various chemical components present in the cone volatile oil of Cupressus macrocarpa and also determine its antimicrobial activity. Totally 13 compounds were identified with 99.99% by GC-MS analysis. The major compounds identified were terpinene-4-ol (19.42%), dinopol (15.63%), ${\alpha}$-pinene (13.58%), and ${\beta}$-pinene (12.16%). The antimicrobial activity was carried out for the oil and a 2% cream formulation using cup plate method by measuring the zone of inhibition. The gram positive organisms used were Bacillus subtilis, Staphylococcus aureus, Bacillus megaterium, and Bacillus cogulans. The gram negative organisms used were Escherichia coli, Kleibseilla pneumonia, Pseudomonas aeruginosa and Salmonella typhi. In vitro antifungal studies were also carried out by using organisms, Candida albicans, Aspergillus flavus, Trichoderma lignorum and Cryptococcus neoformans. The standard drugs used were penicillin ($100{\mu}g/mL$), gentamycin ($100{\mu}g/mL$) and griseofulvin ($100{\mu}g/mL$) for gram positive bacteria, gram negative bacteria and fungi respectively. Both oil and cream formulation showed good activity against fungi than bacteria. This study is being reported for the first time on cone volatile oil of this plant.

• 한국식품저장유통학회지
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• 제10권1호
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• pp.80-88
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• 2003

대두단백 가수분해 산물의 맛과 향을 개선하기 위해 효소에 의한 가수분해 system을 확립하기 위하여 단백질 가수분해 패턴이 서로 다른 효소로 생산된 단백 분해산물의 가수분해도와 표면 소수도 등을 측정하였다. 이들 분해물의 pattern을 SDS 전기영동으로 조사하였고, 효소반응에 의한 단백질분해물의 관능검사를 실시하였다. 각 균주가 생산한 단백질 분해효소의 pH 변화에 따른 효소의 활성은 No. 16 효소(Bacillu megarerium Bl6)와 No. 4효소(Aspergillus oryzae M4)는 pH 7.0에서 No. 95효소(Bacillu subtilis YG 95)와 No. 5효소(Mucor circinelloides M5)는 8.0에서 가장 높은 효소반응 활성을 보였다. 또한 반응온도에 따른 효소활성의 크기는 4가지 효소 모두 45$^{\circ}C$에서 가장 높은 활성을 나타내었다. 대두단백질 분해물의 SDS 전기영동 pattern변화에서, Bacillus megaterium Bl6과 Mucor circinelloides M5의 효소 (No. 16, No. 5)는 반응 후 분자량이 비교적 큰 peptide가 많이 생성되었으며, 효소반응 3시간 경과 후에도 분자량 66KD의 peptide를 확인할 수 있었다. 반면에 Bacillus subtilis YG 95와 Aspergillus oryzae M4의 효소(No. 95, No. 4)는 분자량 15KD∼45KD 미만의 작은 분자량의 peptide 물질이 주로 생성되었으며, 반응 2시간 경과후에는 30KD 미만의 저분자 Peptide가 주로 생성되었다. 이와 같은 결과는 HPLC 분석 결과와 일치하였다. 가수분해가 진행됨에 따라서 SDS 표면소수도가 크게 저하되었으며, Aspergillus oryzae M4 효소의 분해물의 가수분해도가 가장 높았다. 관능검사 결과, No. 4(Aspergillus oryzae M4)와 No. 95(Bacillus subtilis YG 95) 가 강한 쓴맛을 나타내었다. 각각의 효소들을 조합해서 분해한 단백분해물을 관능검사한 결과, Aspergillus oryzae M4 효소와 조합된 것의 대두단백질 분해물이 비교적 강한 쓴맛을 나타내었다. 분해물의 단맛은 시료별로 큰 차이가 나타나지 않았으나 Bacillu megarerium Bl6과 Aspergillus oryzae M4를 조합하였을 때 상대적으로 단맛의 정도가 높게 나타났다. 따라서 이와 같은 효소특성을 이용하여 대두단백질을 가수분해를 하였을 때 다양한 단백질 분해물의 제조에 이용이 가능할 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제35권4호
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• pp.261-271
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• 2007

토양오염을 복원하는 방법 중 식물상 복원은 식물을 이용하여 오염물을 제거하는 기술로, 환경 친화적이며, 경제적인 기술이기 때문에 많이 이용되고 있다. 이 연구에서는 식물상 복원에 있어 식물의 영향, 근권 세균과 물리화학적 제한인자에 대해 고찰하였다. 성공적인 식물상 복원을 위해서는 식물의 선택이 가장 중요하다. 유류(디젤) 분해를 위해 적용된 식물은 쥐보리(Lolium multiflorum lam), 베치(Vicia villosa), 버섯류(white mustard), 톨페스큐(Festuca arundinacea), 콩과식물(leguminosae), 포플러, 소나무(Pinus densiflora) 등이고, 유류 제거 효율은 68-99% 이었다. PAH(polycyclic aromatic hydrocarbons) 제거용으로는 옥수수(Zeo mays), 쥐보리, 베치, 버섯류, 토끼풀(Trifolium repens), 그리고 톨페스큐이 이용되었고, 50-98%의 제거 효율을 보였다. 식물의 성장을 향상시킬 뿐 만 아니라, 오염물질을 직접적으로 제거할 수 있는 근권 세균도 식물상 복원에 있어 중요한 역할을 한다. 식물상 복원에 이용된 근권 세균에는 Azospirillum lipoferum, Enterobactor cloacae, Azospirillum brasilense, Pseudomonas putida, Burkholderia xenovorans, Comamonas testosterone, Pseudomonas gladioli, Azotobacter chroococcu, Bacillus megaterium, Bacillus subtilis 등이 있다. pH, 온도, 영양물질, 최종전자수용체, 수분함량, 유기물 함량, 오염물질 종류와 물리화학적 인자도 식물상 복원에 있어 제한 요소로 작용한다.

• Food Science and Biotechnology
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• 제17권3호
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• pp.519-526
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• 2008

To improve the quality of traditional kochujang, strains with high protease and amylase activity were isolated and identified from Sunchang traditional kochujang. Twenty-three strains strongly producing protease and 16 strains strongly producing $\alpha$- and $\beta$-amylase were isolated by using 1% isolated soy protein agar medium and 2% starch agar medium, respectively. Protease activities of the IA7, I5, and IA2 strain were 22.5, 21.2, and 20.6 unit/mL, respectively, and were higher than those of the other strains. Stains with high $\alpha$-amylase activity included K9 (967.8 unit/mL), K14 (828.3 unit/mL), K13 (662.5 unit/mL), K8 (601.5 unit/mL), and K11 (405.9 unit/mL). The $\beta$-amylase activity of the K11 strain was the highest, 34.3 unit/mL, among the isolated strains. Based on morphological, physiological properties, and API 50CHB-kit test for assimilation of 49 carbohydrates, 8 strains selected according to protease, $\alpha$-amylase, and $\beta$-amylase activities were tentatively identified as Bacillus megaterium (IA2), Bacillus subtilis (IA7, 15), Bacillus amyloliquefaciens (K8, K9, K11, and K13), and Bacillus stearothermophillus (K14). The IA7, 15, and K11 strains were finally identified as B. subtilis (99% ID) based on 16S rDNA sequencing.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1472-1482
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• 2017

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ($K_d$ value) to 4-androstenedione was $387{\pm}37{\mu}M$. Moreover, the crystal structure of BaCYP106A2 was determined at $2.7{\AA}$ resolution. Structural analysis suggested that the ${\alpha}8-{\alpha}9$ loop region of BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were mono-hydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.437-447
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• 2012

Natural and beneficial associations between plants and bacteria have demonstrated potential commercial application for several agricultural crops. The sunflower has acquired increasing importance in Brazilian agribusiness owing to its agronomic characteristics such as the tolerance to edaphoclimatic variations, resistance to pests and diseases, and adaptation to the implements commonly used for maize and soybean, as well as the versatility of the products and by-products obtained from its cultivation. A study of the cultivable bacteria associated with two sunflower cultivars, using classical microbiological methods, successfully obtained isolates from different plant tissues (roots, stems, florets, and rhizosphere). Out of 57 plant-growth-promoting isolates obtained, 45 were identified at the genus level and phylogenetically positioned based on 16S rRNA gene sequencing: 42 Bacillus (B. subtilis, B. cereus, B. thuringiensis, B. pumilus, B. megaterium, and Bacillus sp.) and 3 Methylobacterium komagatae. Random amplified polymorphic DNA (RAPD) analysis showed a broad diversity among the Bacillus isolates, which clustered into 2 groups with 75% similarity and 13 subgroups with 85% similarity, suggesting that the genetic distance correlated with the source of isolation. The isolates were also analyzed for certain growth-promoting activities. Auxin synthesis was widely distributed among the isolates, with values ranging from 93.34 to 1653.37 ${\mu}M$ auxin per ${\mu}g$ of protein. The phosphate solubilization index ranged from 1.25 to 3.89, and siderophore index varied from 1.15 to 5.25. From a total of 57 isolates, 3 showed an ability to biologically fix atmospheric nitrogen, and 7 showed antagonism against the pathogen Sclerotinia sclerotiorum. The results of biochemical characterization allowed identification of potential candidates for the development of biofertilizers targeted to the sunflower crop.