• Journal of Microbiology and Biotechnology
• /
• v.8 no.4
• /
• pp.378-387
• /
• 1998

The DNA sequence immediately following the xynA gene of Bacillus stearothermophilus 236 ［about l-kb region downstream from the translational termination codon (TAA) of the xynA gene］was found to have an ability to enhance the xylanase activity of the upstream xynA gene. An 849-bp ORF was identified in the downstream region, and the ORF was confirmed to encode a novel protein of 283 amino acids designated as XaiF (xylanase-activity-increasing factor). From the nucleotide sequence of the xaiF gene, the molecular mass and pI of XaiF were deduced to be 32,006 Da and 4.46, respectively. XaiF was overproduced in the E. coli cells from the cloned xaiF gene by using the T7 expression system. The transcriptional initiation site was determined by primer extension analysis and the putative promoter and ribosome binding regions were also identified. Blast search showed that the xaiF and its protein product had no homology with any gene nor any protein reported so far. Also, in B. subtilis, the xaiF trans-activated the xylanase activity at the same rate as in E. coli. In contrast, xaiF had no activating effect on the co-expressed ${\beta}-xylosidase$ of the xylA gene derived from the same strain of B. stearothermophilus. In addition, the intracellular and extracellular fractions from the E. coli cells carrying the plasmid-borne xaiF gene did not increase the isolated xylanase activity, indicating that the protein-protein interaction between XynA and XaiF was not a causative event for the xylanase activating effect of the xaiF gene.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2001.06a
• /
• pp.163-164
• /
• 2001

A number of bacterial species produce extracellular lipases. Among them, many lipase genes have been cloned and sequenced. A comparison of primary sequences revealed only very limited sequence homology among them. Based on the sequence homologies and molecular sizes (Mr), bacterial lipases were classified into four discrete groups. From soil samples taken around Taejon, five different lipase-producing bacteria were isolated; Proteus vulgaris K80, Bacillus stearothermophilus Ll, B. pumilus B26, Staphylococcus haemolyticus L62, S. aureus B56. Nucleotide sequence analysis showed that Staphylococcus lipase genes (L62 and B56) composed of pre-pro-mature parts, Bacillus lipase genes (Ll and B26) pre-mature parts, and Proteus lipase gene (K80) mature part only. In addition, the molecular sizes of their mature parts were quite different from 19,000 to 45,000. Finally, they had very little homology (less than 20％) in their amino acid sequences. Judging from the above results, lipase K80 belonged to bacterial lipase Group I, lipase L1 and lipase B26 Group III, and lipase L62 and lipase B56 Group IV. This diversity in their primary structures was also reflected in their enzymatic properties; temperature effects, pH effects, substrate specificity, detergent effects, and so on.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 1986.12a
• /
• pp.509.1-509
• /
• 1986

Recent advances in recombinant DNA techniques have provided a tool for breeding of microorganisms of hyper production. Enzyme production by cloned microorganism has some advantages. They are ⅰ) Enzymes can be produced by a microorganism easily cultured ⅱ) Hyper production. ⅲ) In some cases, such as thermophilic enzyme gene is cloned in a mesophilic bacteria, the enzyme purification procedure can be simplified. One example, production of thermophilic ${\beta}$-galactosidase in B. subtilis will be presented. Bacillus stearothermophilus IAM 11001 produced three ${\beta}$-galactosidases, ${\beta}$-galactosidase I, II and III (${\beta}$-gal-I, II and III). By connecting restriction fragments of the chromosomal DNA to plasmid vector, followed by transformation of Escherichia coli, two ${\beta}$-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were cloned.

• Food Science and Biotechnology
• /
• v.18 no.4
• /
• pp.1035-1037
• /
• 2009

In this study we analyzed the dynamic changes in microbiota composition during kochujang fermentation at $30^{\circ}C$. During fermentation, the viable cell counts slowly increased and reached $3.2{\times}10^7$ for aerobic bacteria, $8.3{\times}10^3$ for yeast, and $1.4{\times}10^3$ CFU/mL for fungi after 60 days. Bacilli were found to be the most dominant microorganisms throughout the fermentation process. Using the culture dependent method Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens were found to be the main species during the early stages of fermentation; however, Bacillus pumilus and Bacillus stearothermophilus became the most dominant species during the late stage of fermentation. In contrast, when the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was used Bacillus ehimensis was found to be the dominant species during the early stage of fermentation and Bacillus megaterium, B. pumilus, B. subtilis, and B. licheniformis were dominant in the ate stages. These results indicate various other Bacillus species rather than just B. subtilis and B. licheniformis might be involved in the fermentation of kochujang.

• Microbiology and Biotechnology Letters
• /
• v.27 no.3
• /
• pp.184-190
• /
• 1999

To acquire an industrially useful biocatalyst for the enzymatic synthesis and production of various D-amino acid aminotransferase (D-AAT) activity. The enzyme activity was found from 110 strains of isolated thermophiles revealing its wide occurrence in thermophiles. Enzyme activity and thermal stability of the D-AAT producers were compared. Finally we have selected four thermophiles as producers of potent biocatalysts for the D-amino acid production; two thermophiles, Bacillus sp. Lk-1 and LK-2, having higher specific activity and two thermophiles, B. stearothermophilus KL-01 and Bacillus sp. KLS-01, having higher thermal stability than the D-AAT producers. Taxonomic and physiological characteristics of the four isolated thermophiles were described herein.

• Journal of the Korea Organic Resources Recycling Association
• /
• v.10 no.3
• /
• pp.126-131
• /
• 2002

For the probiotic feed production from residual food waste, aerobic liquid fermentation was conducted by thermophilic bacteria. 11 Strains of bacteria were isolated from several soil sources and residual food waste. Screening was carried by shaking incubator for the separation of thermophilic strain at $55^{\circ}C$. The isolated strains were tested for enzyme activities such as ${\alpha}$-amylase and protease. 6 Bacterial strains were chosen and were adapted by repeated fermentation processes in food waste substrate. The viable cell count of them at final fermentation stages were shown as $3-7{\times}10^9/ml$ in 2L-jar fermenter. Among them B3, B6 showed higher enzyme activity. By the mixed fermentation of B3, B6 and Bacillus stearothermophilus, the highest viable cell count reached to $1.4{\times}10^{10}/ml$ in 8 hours.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.9
• /
• pp.1579-1585
• /
• 2016

Sugar esters are valuable compounds composed of various sugars and fatty acids that can be used as antibacterial agents and emulsifiers in toothpaste and canned foods. For example, fructose fatty acid esters suppress growth of Streptococcus mutans, a typical pathogenic bacterium causing dental caries. In this study, fructose laurate ester was chosen as a target material and was synthesized by a transesterification reaction using Candida antarctica lipase B. We performed a solvent screening experiment and found that a t-butanol/dimethyl sulfoxide mixture was the best solvent to dissolve fructose and methyl laurate. Fructose laurate was synthesized by transesterification of fructose (100 mM) with methyl laurate (30 mM) in t-butanol containing 20% dimethyl sulfoxide. The conversion yield was about 90%, which was calculated based on the quantity of methyl laurate using high-performance liquid chromatography. Fructose monolaurate (M_r 361) was detected in the reaction mixture by high-resolution mass spectrometry. The inhibitory effect of fructose laurate on growth of oral or food spoilage microorganisms, including S. mutans, Bacillus coagulans, and Geobacillus stearothermophilus, was evaluated.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.1
• /
• pp.28-33
• /
• 1998

A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.3
• /
• pp.563-568
• /
• 2010

The Val289 residue in the $\alpha$-amylase of Bacillus amyloliquefaciens, which is equivalent to the Ala289 and Val286 residues in the $\alpha$-amylases of B. stearothermophilus and B. licheniformis, respectively, was studied by site-directed mutagenesis. This residue was substituted with 10 different amino acids by random substitution of the Val codon. In these mutant $\alpha$-amylases, Val289 was substituted with Ile, Tyr, Phe, Leu, Gly, Pro, Ser, Arg, Glu, and Asp. Compared with the wild-type $\alpha$-amylase, the mutant $\alpha$-amylase Val289Ile showed 20% more hydrolytic activity, whereas Val289Phe and Val289Leu showed 50% lesser activity. On the other hand, the mutant $\alpha$-amylases Val289Gly, Val289Tyr, Val289Ser, and Val289Pro showed less than 15% activity. The substitution of Val289 with Arg, Asp, or Glu resulted in complete loss of the $\alpha$-amylase activity. Interestingly, the mutant $\alpha$-amylase Val289Tyr had acquired a transglycosylation activity, which resulted in the change of product profile of the reaction, giving a longer oligosaccharide.

• Korean Journal of Food Science and Technology
• /
• v.32 no.6
• /
• pp.1266-1270
• /
• 2000

Quality properties of soy bean pastes made with Aspergillus oryzae and 5 Bacillus strains isolated from traditional soy bean pastes were examined. The pH decreased gradually and contents of amino-type nitrogen increased during fermentation. There were small differences in moisture and crude-protein contents, whereas big difference was observed in reducing sugar, isoflavones and organic acid contents. Isoflavones in the samples made with Bacillus licheniformis F2358 and Bacillus subtilis F2362 were high. Samples made with Bacillus licheniformis F2382 had high contents of organic acid and good score for taste and overall acceptability in sensory evaluation.