• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.582-588
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• 1999

A Brevibacterium lactofermentum gene coding for a glucose-specific permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned, by complementing an Escherichia coli mutation affecting a ptsG gene with the B. lactofermentum genomic library, and completely sequenced. The gene was identified as a ptsG, which enables an E. coli transformant to transport non-metabolizable glucose analogue 2-deoxyglucose (2DG). The ptsG gene of B. lactofermentum consists of an open reading frame of 2,025 nucleotides encoding a polypeptide of 674 amino acid residues and a TAA stop codon. The 3' flanking region contains two stem-loop structures which may be involved in transcriptional termination. The deduced amino acid sequence of the B. lactofermentum enzyme $II^{GIe}$ specific to glucose ($EII^{GIe}$) has a high homology with the Corynebacterium glutamicum enzyme $II^{Man}$ specific to glucose and mannose ($EII^{Man}$), and the Brevibacterium ammoniagenes enzyme $II^{GIc}$ specific to glucose ($EII^{GIc}$). The 171-amino-acid C-terminal sequence of the $EII^{Glc}$ is also similar to the Escherichia coli enzyme $IIA^{GIc}$ specific to glucose ($IIA^{GIc}$). It is interesting that the arrangement of the structural domains, IIBCA, of the B. lactofermentum $EII^{GIc}$ protein is identical to that of EIIs specific to sucrose or $\beta$-glucoside. Several in vivo complementation studies indicated that the B. lactofermentum $EII^{Glc}$ protein could replace both $EII^{ Glc}$ and $EIIA^{Glc}$ in an E. coli ptsG mutant or crr mutant, respectively.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.214-221
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• 1998

A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme II ($EII^{Glc}$) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing an Escherichia coli mutation affecting a ptsG gene, and the complete DNA nucleotide sequence was determined. The cloned gene was identified to be a ptsG, which enables the E. coli transportment to use glucose more efficiently than mannose as the sole carbon source in an M9 minimal medium. The ptsG gene of B. ammoniagenes consists of an open reading frame of 1,983 nucleotides putatively encoding a polypeptide of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes $EII^{Glc}$ shows, at $46\%$, the highest degree of sequence similarity with the Corynebacterium glutamicum EII specific for both glucose and mannose. In addition, the $EII^{Glc}$ shares approximately $30\%$ sequence similarities with sucrose-specific and ${\beta}$-glucoside-specific EIIs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of $EII^{Glc}$ is also similar to that of E. coli enzyme $IIA^{Glc}$, specific for glucose ($EIIA^{Glc}$). The B. ammoniagenes $EII^{Glc}$ consists of three domains; a hydrophobic region (EIIC) and two hydrophilic regions (EIIA, EIIB). The arrangement of structural domains, IIBCA, of the $EII^{Glc}$ is identical to those of EIIs specific for sucrose or ${\beta}$-glucoside. While the domain IIA was removed from the B. ammoniagenes $EII^{Glc}$ the remaining domains IIBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking $EII^{Glc}$ activity with $EIIA^{Glc}$ of the E. coli mutant. $EII^{Glc}$ contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.

• Journal of Applied Biological Chemistry
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• 제48권4호
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• pp.218-221
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• 2005

Nucleotide sequence of Brevibacterium flavum ptsG gene capable of complementing Escherichia coli ZSC113 mutations defective to glucose permease activity of phosphotransferase system was completely determined, and the gene product was compared with other glucose-specific enzyme II ($EII^{Glc}$). A ptsG gene of B. flavum consisted of open reading frame of 2,025 nucleotides putatively encoding polypeptide of 675 amino acid residues and TAA stop codon. Deduced amino acid sequence of B. flavum ($EII^{Glc}$) had high homology with ($EIIs^{Glc}$) of Corynebacterium glutamicum, C. efficiens, and B. lactofermentum. Arrangement of structural domains, IIBCA, of B. flanum ($EII^{Glc}$) protein was identical to that of EIIs belonging to glucose-phosphotransferase system.

• 미생물학회지
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• 제39권3호
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• pp.141-147
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• 2003

여러 항진균 활성 관련 유전자들의 발현 수준을 동시에 연구하기 위하여 DNA microarray를 이용하여 유전자들의 발현 패턴을 비교 분석하였다. 본 연구에서는 항진균활성을 가지는Bacillus lentimorbus WJ5의 genomic DNA를 무작위 하게 제한효소로 절단하여 2,000개의 DNA단편을 microarray하였으며, 감마선($^{60}Co$)조사로 유도된 7종의 항진균 활성 결핍 돌연변이체와 발현양상을 정량적으로 비교하였다. Gene Cluster (Michael Risen, Stanford Uniy.)를 이용한 DNA microarray의 분석 결과, 총 408개의 DNA 단편이 발현되는 것을 확인할 수 있었으며, 이들 중 20개의 DNA단편이 항진균 활성 결핍 돌연변이체에서 발현이 억제되는 것으로 나타났다. 특히,pbuX (xanthine permease, K222), ywbA (phosphotransferase system enzyme II, K393), ptsG (PTS glucose specific enzyme II ABC component, K877), yufO (ABC transporter(ATP-binding protein), K1301), 그리고 ftsY (signal recognition particle (docking protein), K868)는 모든 돌연변이체에서 동시에 발현되는 down-regulation된 유전자들로서 물질 이동과 관련된 것으로 보고되어 있으며, 항진균 활성 관련 신호 및 물질의 이동에 관여할 것으로 사료되어진다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.665-672
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• 2004

D-Ribose is a five-carbon sugar used for the commercial synthesis of riboflavin, antiviral agents, and flavor enhancers. Batch fermentations with transketolase-deficient B. subtilis JY1 were carried out to optimize the production of D-ribose from xylose. The best results for the fermentation were obtained with a temperature of $37^{\circ}C$ and an initial pH of 7.0. Among various sugars and sugar alcohols tested, glucose and sucrose were found to be the most effective for both cell growth and D-ribose production. The addition of 15 g/l xylose and 15 g/l glucose improved the fermentation performance, presumably due to the adequate supply of ATP in the xylose metabolism from D-xylulose to D-xylulose-5-phosphate. A batch culture in a 3.7-1 jar fermentor with 14.9 g/l xylose and 13.1 g/l glucose resulted in 10.1 g/l D-ribose concentration with a yield of 0.62 g D-ribose/g sugar consumed, and 0.25 g/l-h of productivity. Furthermore, the sugar utilization profile, indicating the simultaneous consumption of xylose and glucose, and respiratory parameters for the glucose and sucrose media suggested that the transketolase-deficient B. subtilis JY1 lost the glucose-specific enzyme II of the phosphoenolpyruvate transferase system.

• 한국식품과학회지
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• 제11권4호
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• pp.249-257
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• 1979

전보에서 저자들을 물리적 견고성이 우수한 포도당 이성화 효소를 세포 고정화 시킨 제품을 얻었다.(한국 식품 과학 회지, 11(No. 3), 192(1979). 이 효소 제품을 사용하여 실험실 조건에서 회분식 반응조와 충진식 반응조를 운영하여 효소의 반응 특성과 생산성, 활성 감소 현상을 비교 하였다. 본 고정화 효소의 비활성 역가는 회분식 반응조와 충진식 반응조에서 1 g당 각각 48 및 114 units였으며 연속적인 공정에서 더 높은 생산성과 이성화율을 보였다. 효소의 생산성은 체장 시간, 기질의 농도, 효소 부하율(附荷率) 및 반응조의 외형에 영향을 받았으며 충진 밀도가 450 g/l일 때 실제 공간율은 0.36이었으며 비교적 좋은 충진 현상을 보였다. 연속 공정중 효소의 활성 감퇴 현상을 고찰하기 위하여 2.5 M 포도당 용액을 체장 시간이 5.3시간이 되도록 약 220시간 동안 반응시켜 본 결과, 효소 활성 감퇴 곡선은 일차 반응을 따르며 활성 반감기는 115일로 연속 공정에 이용 가능함을 알았다. 이 고정화 효소 제품은 물리적 안정성이 높은 반면 물질 전달 계수가 반응 속도에 큰 영향을 미치는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.489-498
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• 2013

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using $1.38{\times}10^9$ CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at $37^{\circ}C$, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and $70^{\circ}C$, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

• Journal of Microbiology
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• 제43권3호
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• pp.244-250
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• 2005

The effect of cytochrome $c_{550}$ encoded by cccA in Bacillus subtilis during the event of sporulation was investigated. The sporulation of cccA-overexpressing mutant was significantly accelerated, while disruptant strain showed delayed sporulation in spite of the same growth rate. Activity of sporulation stage-0-specific enzyme, extracellular $\alpha-amylase$ of mutant strains was similar to that of the control strain, but cccA-overexpressing mutant exhibited higher activity of stage-II-specific alkaline phosphatase and stage-III-specific glucose dehydrogenase when compared to deletion mutant and control strain. Northern blot analysis also revealed that cccA-overexpressing mutant showed high level of spo0A transcripts, while the disruptant rarely expressed spo0A. These results suggested that although cytochrome $c_{550}$ is dispensable for growth and sporulation, expression of cccA may play an important role for initiation of sporulation through regulation of spo0A expression.

• Applied Biological Chemistry
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• 제38권1호
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• pp.42-48
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• 1995

Bacillus megaterium이 생산하는 ${\gamma}-cyclodextrinase({\gamma}-CDase)$ 염석, DEAE-trisacryl, Ultrogel AcA 24 및 Ultrogel HA column chromatography 등의 방법으로 부분정제한 결과 specific activity는 120.4 units/mg protein으로 조효소액에 비하여 125.4배 정제되었다. 부분정제한 ${\gamma}-CDase$는 SDS-ployacrylamide gel 전기영동에 의해 2개의 band로 나타났으며 band I과 band II의 분자량은 각각 64,000과 50,000이었다. ${\gamma}-CDase$의 최적 pH는 6.0, 최적 온도는 $60^{\circ}C$이었고, $45^{\circ}C$ 이하의 온도와 pH $6.0{\sim}9.0$에서 안정하였으며, ${\gamma}-CD$에 대한 Km값은 0.903 mM이었다. $Mg^{2+}$와 $Mn^{2+}$ 이온에 의해 활성이 증가한 반면, $Hg^{2+}$와 $Cu^{2+}$에 의해서는 활성이 현저하게 감소되었다. ${\gamma}-CDase$는 ${\alpha}-CD$와 ${\beta}-CD$에는 거의 활성이 없었고, ${\gamma}-CD$에는 매우 높은 활성이 나타내었으며, 이의 분해 생성물은 주로 glucose와 maltose이었다.

• 생명과학회지
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• 제20권5호
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• pp.683-690
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• 2010

효모 Saccharomyces diastaticus 는 세포 외로 분비되는 glucoamylase I, II, III 동위효소 중 하나를 생산하여 전분을 가수분해하여 포도당을 생성할 수 있다. Glucoamylase I, II, III는 STA1, STA2, STA3 유전자에 의해 각각 암호화된다. 효모 Saccharomyces 속이 포자가 형성되는 시기에 세포 내에서 특이적으로 발현된다고 알려진 glucoamylase (SGA)의 분자생물학적 및 생화학적 연구를 수행하기 위한 일환으로 S. diastaticus YIY 345 형질전환체의 배양 상등액으로부터 SGA 정제를 시도하였다. 황산암모늄 침전, DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-200 chromatography 등의 정제과정을 거쳐서 비특이 활성이 174배 증가된 0.22 mg의 순수한 SGA를 얻었다. HPLC와 SDS-PAGE 분석을 통해 이 효소는 63, 68 kDa의 단위체로 구성된 이합체임을 확인할 수 있었다. Con-A Sepharose 친화성 크로마토그피와 탈당쇄 효소를 처리한 결과로부터 SGA는 N-연결형 당쇄로 수식되었으며 단백질 부분은 59 kDa이었다. 정제한 SGA와 세포 외 분비성 glucoamylase의 효소학적 특성을 조사하고 비교한 결과 SGA의 최적 pH와 온도는 각각 5.5와 $45^{\circ}C$로 나타났으며 세포 외 분비성 glucoamylase는 5.0과 $50^{\circ}C$로 나타났다. SGA는 세포 외로 분비되는 glucoamylase에 비해 열처리 및 SDS에 대해 더 민감한 반응성을 나타내었다.