• Journal of Microbiology and Biotechnology
• /
• 제8권2호
• /
• pp.141-145
• /
• 1998

Overproduction of intracellular ${\beta}$-glucosidase was attempted by modifying the promoter region of a ${\beta}$-glucosidase gene cloned from Cellulomonas fimi and expressing it in Bacillus subtilis DB 104. A strong engineered promoter, BJ27UΔ88, was fused to the ${\beta}$-glucosidase gene after removing its native promoter. An effective Shine-Dalgamo sequence (genel0 of phage T7) was inserted between the promoter and the ${\beta}$-glucosidase structural gene. The modified gene was overexpressed in B. subtilis and produced 1121.5 units of ${\beta}$-glucosidase per mg protein which is about $12\%$ of total intracellular protein. Secretion of overproduced intracellular ${\beta}$-glucosidase was attempted by using the signal sequence of the Bacillus endoglucanase gene as well as an in-frame hybrid protein of endoglucanase. The hybrid protein was normally secreted into the culture medium and still retained ${\beta}$-glucosidase activity.

• Journal of Microbiology and Biotechnology
• /
• 제9권3호
• /
• pp.255-259
• /
• 1999

A 6.9-kb DNA fragment including the minimal Bacillus pasteurii urease gene cluster was subcloned into a high-copy-number plasmid vector, pUC19, and the recombinant B. pasteurii urease was overexpressed in Escherichia coli. The recombinant urease was purified 25.9-fold by using combinations of anion-exchange and gel-filtration chromatography followed by Mono-Q chromatography on a FPLC. N-terminal peptide sequencing analyses revealed that two distinct smaller peptide bands resolved on a 10-18％ gradient SDS-PAGE corresponded to UreA and UreB peptides, respectively. It was also shown that the ureB gene was translated from a GUG codon and the first methionine residue was post-translationally cleaved off. The native molecular weight of the recombinant urease was 176,000 and 2 nickel atoms were present per catalytic unit. pH stability studies of the purified enzyme showed that the recombinant Bacillus pasteurii urease is stable in alkaline pH range, which is similar to the enzyme of the evolutionarily related bacterium, Sporosarcina ureae.

• Journal of Microbiology and Biotechnology
• /
• 제15권3호
• /
• pp.537-543
• /
• 2005

Subtilisin (EC 3.4.21.14) is the major extracellular alkaline serine protease of Bacillus species. Previously, we found that subtilisins did not migrate in the electrophoretic field in the Laemmili buffer system due to their high pI values (over 8.8); however, it formed a 'binding mode' at the top of the separating gel [5]. Utilizing this characteristic, four subtilisins from Bacillus sp. strains (e.g., B. subtilis 168, B. subtilis KCTC 1021, B. amyloliquefaciens KCTC 3002, and Bacillus sp. DJ-1 and DJ-4) were easily and quickly identified by an over-running electrophoretic technique with a miniscale culture supernatant (less than 20 ml) without any column chromatographic steps. Two subtilisins (DJ-l and a recombinant version) from Bacillus sp. DJ-l were characterized, and the enzymatic properties were determined by SDS-fibrin zymography and densitometric analysis. Based on this observation, the recombinant pro-subtilisin DJ-l showed the same 'binding mode,' similar to native subtilisin DJ-l. On the other hand, mature subtilisin DJ -1 without pro-peptide showed no enzymatic activity.

• 한국환경농학회지
• /
• 제20권1호
• /
• pp.63-66
• /
• 2001

토양으로 부터 분리한 Bacillus sp. WRD-l의 토마토 생육에 미치는 영향을 조사하기 위하여 미생물 배양액을 100배, 300배, 500배의 희석 비율에 따라 토마토재배지에 관주를 실시하여 토마토 생육 및 토양미생물상 변화를 관찰 하였다. 대조구로는 실제 미생물의 영향을 확인하기 위하여 Bacillus sp. WRD-1 배양액을 멸균하여 사용하였다. 토마토 생육은 전체적으로 100배> 300배> 500배> 대조구 순으로 희석 비율과 비례하였다. 시간별 토양미생물 수는 세균과 방선균의 수는 증가하는 경향을 보였으며, 곰팡이수는 감소하는 경향을 보였다. 이러한 결과로 토양활성개량미생물제로의 개발 가능성을 확인하였다.

• 한국해양바이오학회지
• /
• 제16권1호
• /
• pp.1-18
• /
• 2024

Coastal dunes must be conserved. Their native halophytes support coastal geography while their symbiotic microorganisms help vegetation thrive. The Goraebul coast has the largest, well-conserved dune system on the East Sea of the Korean Peninsula due to a climax mixed halophyte (C. soldanella, C. kobomugi, and E. mollis) vegetation support. This study identified rhizobacteria and their diversity in mixed halophyte communities unique to Goraebul. Five phyla, 12 genera, and 21 species were identified based on 16S rDNA sequences from 65 isolates. The phylum Bacillota, class Bacillota, order Bacillales, and family Bacillaceae were identified, with Bacillus as the dominant genus (46.15%). The richness and Shannon's diversity were higher at the species than at the genus level due to the dominance of Bacillus; however, various Bacillus species (7) were identified. Therefore, the climax mixed vegetation adapted to the Goraebul coast may exert natural selection pressure in favor of the common characteristics of Bacillus. However, despite this advantage, the Shannon equitability (0.86), Simpson (0.08), and Shannon diversity (2.79) indexes indicate a stable rhizosphere cluster and the climax mixed vegetation is affected by symbiotic relationships between healthy rhizosphere microbiota.

• BMB Reports
• /
• 제37권6호
• /
• pp.642-647
• /
• 2004

The lysine residues of Bacillus licheniformis $\alpha$-amylase (BLA) were chemically modified using citraconic anhydride or succinic anhydride. Modification caused fundamental changes in the enzymes specificity, as indicated by a dramatic increase in maltosidase and a reduction in amylase activity. These changes in substrate specificity were found to coincide with a change in the cleavage pattern of the substrates and with a conversion of the native endo- form of the enzyme to a modified exo- form. Progressive increases in the productions of $\rho$-nitrophenol or glucose, when para nitrophenyl-maltoheptaoside or soluble starch, respectively, was used as substrate, were observed upon modification. The described changes were affected by the size of incorporated modified reagent: citraconic anhydride was more effective than succinic anhydride. Reasons for the observed changes are discussed and reasons for the effectivenesses of chemical modifications for tailoring enzyme specificities are suggested.

• Journal of Microbiology
• /
• 제37권4호
• /
• pp.213-217
• /
• 1999

An extracellular endopeptidase from Bacillus cereus SH-7 was purified to homogeneity. The protease was most active at pH 8 and $40{\circ}C$, respectively. The molecular mass of the protease was 40 kDa on SDS-PAGE, and 120 kDa by gel filtration, suggesting that the native enzyme is composed of three homogeneous subunits. The $K_m\;and\;V_{max}$ values of the protease for N-succinyl-$(Ala)_2$-Pro-Phe-p-nitroanilide were 11.11 mM and 170 nmol/mg of protein/min, respectively. The protease was also identified as a metalloprotease. The bioactivity of the SH-7 protease will need further study in the future.

• Applied Biological Chemistry
• /
• 제19권2호
• /
• pp.82-92
• /
• 1976

재래식메주에 있어서 자연서식한 microflora의 상태에 따라 외부(外部) 내부(內部) 외부(外部)와 내부(內部)의 혼합으로 각각 3구의 재래식 고추장을 담가 숙성과정중의 제화학성분 및 미생물의 동태(動態)를 조사하고 숙성과정에 관여한 미생물을 순수분리하여 이들의 효소활성을 screening하여 우수균주를 선정 및 동정 하였으며 효소적 특성을 비교하여 다음과 같은 결과를 얻었다. 1. 제화학 성분변화 (1) pH의 변화는 숙성 10일에 급격히 강하하였고 숙성 60일까지 점차 완만한 감소를 보이다가 그 이후는 약간 증가하였다. 메주외부고추장구(區)의 pH가 가장 낮았으며 메주내부고추장구(區)의 메주혼합고추장구(區)의 pH는 서로 비슷하였다. (2) 총산의 면화는 숙성 60일까지 3구(區) 공히 서서히 증가하다가 그 이후 점차 감소하였다. (3) 총질소의 변화는 숙성 60일까지 3구(區) 공히 점진 증가하다가 그후 약간 감소하였다. (4) Trichloroacetic acid에 가용(可容)인 질소의 변화는 숙성 10일에 3구(區) 공히 급격히 증가하였고 그후는 완만한 증가를 보였다. (5) amino태(態) 질소의 변화는 숙성 30일까지 3구(區) 공히 크게 증가하다가 그 이후는 완만하게 증가하였다. (6) 환원당의 변화는 숙성 50일까지 3구(區) 공히 증가하다가 그 이후는 서서히 감소하였다. 2. Microflora의 변화 (1) 호기성세균군(群), 혐기성세균군(群), mold군(群)의 변화는 숙성 $30{\sim}40$일까지 3구 공히 증가하였으나 그 이후는 감소하는 경향이었다. (2) Yeast군(群)의 변화는 3구 공히 숙성 20일까지는 보이지 않다가 pH가 5.4이하로 강하된 때부터 나타나기 시작하며 메주내부 고추장구(區)에서 처음에는 많이 나타났으나 점차로 감소하였고 메주 외부고추장구(區), 메주혼합고추장구(區)는 처음에는 적게 나타났으나 점차로 증가하였다. 3. 효소활성이 우수한 균주 및 그 효소특성 (1) amylase와 protease를 분비하는 우수균주는 Bacillus subtilis-P, Bacillus subtilis-G, Bacillus licheniformis-K, Aspergillus oryzae-B로 동정되었다. (2) amylase activity가 가장 강한 것은 Aspergillus oryzae-B였고 그 다음이 Bacillus subtilis-P Bacillus licheniformis-K, Bacillus subtilis-G의 순서였다. protease activity에 있어서는 Aspergillus oryzae-B와 Bacillus subtilis-P가 거의 비슷하게 우세하였고 Bacillus licheniformis-K, Bacillus subtilis-G의 순서였다. (3) 효소활성에 대한 식염농도 영향은 amylase가 protease보다 식염에 의한 저해를 많이 받고 있어서 고추장 식염농도인 15% NaCl에서 amylase activty는 $45{\sim}65%$, protease activity는 $40{\sim}46%$의 저해를 받았다.

• 한국미생물·생명공학회지
• /
• 제28권4호
• /
• pp.195-201
• /
• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.

• 농약과학회지
• /
• 제2권3호
• /
• pp.28-35
• /
• 1998

전국에서 분리한 세균으로부터 항균력이 있는 길항세균들을 먼저 분리한 다음 그중 배양배지 속으로 강력한 항균력을 나타내는 peptide를 분비하는 A405균주를 선발, Biolog System 및 rDNA 염기서열을 이용하여 Bacillus subtilis로 동정하고 이 균주가 생성하는 항균 peptide의 항균력을 조사한 바 Botrytis cinerea, Cercospora sp., Fusarium oxysporum, Penicillium digitatum, Celletotrichum gloeosporioides, Rhizoctonia solani, Pythium ultimum, Pyricularia oryzae등의 식물병원균과 Escherichia coli, Pseudomonas spp. 등의 세균 및 효모인 Candida albicans의 성장도 억제하는 강력한 항균력을 보였다. 또한 이 균주가 생산하는 항균 peptide는 알콜, 아세톤과 같은 유기용매에 내성을 가지고 있었으며 proteinase K와 phenol 처리시에는 항균력이 감소되거나 소실되는 특성을 가지고 있었다. 이 항균 peptide를 SDS-PAGE, Native-PAGE 및 Tris-Tricine 전기영동으로 분자량이 3kDa인 작은 peptide임을 확인하였고 aspartic acid, glycine, serine, glutamine, valine, leucine, isoleucine, proline, tyrocine 등의 9가지 아미노산으로 구성되어 있음을 Amino Acid Analyzer로 확인하였으며 냉동건조한 항균 peptide 50 ug/ml을 체리토마토에 처리하여 과일의 선도유지를 조사한 결과 대조구에 비하여 월등한 선도유지 효과가 있음을 입증하였다.