• Journal of Microbiology and Biotechnology
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• v.9 no.5
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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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• v.8 no.3
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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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• v.48 no.4
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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.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.141-147
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• 2003

The simultaneous expression levels of antifungal activity related genes was analyzed by DNA microarray. We constructed DNA chips contained 2,000 randomly digested genome spots of the antifungal bacterium of Bacillus lentimorbus WJ5 and compared its quantitative aspect with 7 antifungal activity deficient mutants induced by gamma radiation ($^{60}Co$). From the analysis of microarray hybridization by the Gene Cluster (Michael Eisen, Stanford Univ.), totally 408 genes were expressed and 20 genes among them were significantly suppressed in mutants. 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), K130l), and ftsY (signal recognition particle (docking protein), K868) were simultaneously down-regulated in all mutants. It suggested that they were supposed to be related to the antifungal activity of B. lentimorbus WJ5.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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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.

• Korean Journal of Food Science and Technology
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• v.11 no.4
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• pp.249-257
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• 1979

Using the whole cell immobilized glucose isomerase which was prepared in the previous work (Korean J. Food Sci. & Technol., 11(3), 192 (1979), the specific activity of the immobilized enzyme was 48.1 units in the batch reaction system and 114 units in the continuous reaction system per g of matrix, respectively. In the continuous reactor the voidity was 0.36, which was suitable for the packed bed reactor. This immobilized enzyme showed a good operational stability of 115 days of half life which was sufficient for the continuous operation. The experimental result showed that 55 % of the substrate was converted to the product in the packed bed reactor. The productivity was dependent on the flow rate, column geometry, enzyme loading, and substrate concentration. An intrapaticle diffusion was observed by the effectiveness factor of 0.75 and interparticle diffusion by the decrease of Km' with increasing the superficial velocity.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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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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• v.43 no.3
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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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• v.38 no.1
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• pp.42-48
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• 1995

The experiment was carried out to purify and to investigate the properties of the cyclodextrinase produced from Bacillus megaterium KFCC 11855. The enzyme was partially purified with $(NH_4)_2SO_4$ and chromatography on DEAE-trisacryl, Ultrogel AcA 34, DEAE-trisacryl and Ultrogel HA. The optimum temperature and pH of the purified enzyme were $60^{\circ}C$ and 6.0, respectively. The enzyme was stable at the temperature of $45^{\circ}C$ below and at the pH range of $6.0{\sim}9.0$, respectively. The Km value for ${\gamma}-cyclodextrin$ was 0.903 mM. The enzyme activity was increased by $Mg^{2+}$ and $Mn^{2+}$, but decreased by $Hg^{2+}$ and $Cu^{2+}$. The enzyme degraded ${\gamma}-cyclodextrin$ but not ${\alpha}-cyclodextrin$. The degree of ${\beta}-CD$ degradation by the enzyme was very low. The decomposed products of ${\gamma}-cyclodextrin$ by the enzyme were mainly glucose, maltose and a little amount of maltotriose.

• Journal of Life Science
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• v.20 no.5
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• pp.683-690
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• 2010

The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase I, II or III, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and $45^{\circ}C$, respectively, whereas those for the extracellular glucoamylase were 5.0 and $50^{\circ}C$. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.