• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.298-304
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• 2002

A family shuffling associating PCR-based and in vitro recombination and expression in Escherichia coli cdd mutant was carried out. Two cdd genes encoding cytidine deaminases (CDase) from thermophilic Bacillus caldolyticus and B. stearothermophilus were shuffled. Around 150 viable mutant colonies screened on AB minimal medium without uracil by E. coli cdd complementation were selected for cytidine deaminase assay and 4 candidates (SH1067, SH1077, SH1086, and SH1118) were chosen for the detailed study. The nucleotide sequence analyses of 4 selected mutants revealed that they have several point mutations and recombinations. Surprisingly, the SH 1067 showed 770 fold more specific CDase activity at $80^{\circ}C$ than that of T101 from parental B. stearothermophilus.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.12
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• pp.1769-1774
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• 2001

Bacillus licheniformis strains L-25 and PWD-1 are two thermophilic feather-degrading bacteria. Despite isolated from different environmental conditions, they were both capable of breaking down chicken feathers and growing in a medium in which feather was the only source of carbon and nitrogen. A 1.46-kb keratinase gene (ker B) was isolated from strain L-25 by a polymerase chain reaction (PCR) using L-25 genomic DNA as templates. Sequencing results reveal that ker B shares great sequence identity with a previously published keratinase gene of B. licheniformis PWD-1 (ker A). Only two amino acids differences were found in the deduced amino acid sequence between the keratinases from L-25 and PWD-1. However several nucleotide changes were found upstream of the putative promoter region. Protease inhibition studies indicated that neutral protease activity accounted for approximate 25 to 30% of total extracellular proteolytic activity produced by strain L-25 in the feather medium. In contrast, no measurable neutral protease activity was produced by strain PWD-1 in the feather medium. When glucose (1%), a common catabolic repressor, was added into the feather medium, L-25 was still able to grow and produce keratinase. Strain PWD-1 produced no neutral protease activity and its growth was severely inhibited in the feather medium containing glucose. L-25 produced an enhanced level of keratinase in the feather medium in comparison with PWD-1.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1551-1558
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• 2014

The esterase gene Est8 from the thermophilic bacterium Bacillus sp. K91 was cloned and expressed in Escherichia coli. The monomeric enzyme exhibited a theoretical molecular mass of 24.5 kDa and an optimal activity around $50^{\circ}C$ at pH 9.0. A model of Est8 was constructed using a hypothetical YxiM precursor structure (2O14_A) from Bacillus subtilis as template. The structure showed an ${\alpha}/{\beta}$-hydrolase fold and indicated the presence of a typical catalytic triad consisting of Ser-11, Asp-182, and His-185, which were investigated by site-directed replacements coupled with kinetic characterization. Asp-182 and His-185 residues were more critical than the Ser-11 residue in the catalytic activity of Est8. A comparison of the amino acid sequence showed that Est8 could be grouped into the GDSL family and further classified as an SGNH hydrolase. Est8 is a new member of the SGNH hydrolase subfamily and may employ a different catalytic mechanism.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Journal of Animal Science and Technology
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• v.50 no.5
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• pp.713-720
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• 2008

This study was conducted to isolate and identify xylanase- and cellulase-producing thermophilic bacteria from stacked spent mushroom substrates and to determine the optimal medium conditions for their growth. Bacteria with the highest xylanase and CMCase activities were strain 3 and 201-7. Both of them were identified as Bacillus spp. and named Bacillus subtilis KU3 and Bacillus subtilis KU201-7. The optimal medium condition of Bacillus subtilis KU3 was obtained when 3%(w/v) of yeast extract and 1%(w/v) of maltose were used as nitrogen and carbon sources, respectively. That of Bacillus subtilis KU201-7 was obtained when 0.5%(w/v) of yeast extract and 0.5%(w/v) of CMC were used as nitrogen and carbon sources, respectively.

• BMB Reports
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• v.32 no.5
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• pp.480-485
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• 1999

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, $K'_D$, for PLP was determined with the apoenzyme to be about $1.2\;{\mu}M$. The isoelectric point was 4.9. The optimal temperature and pH for the $\alpha,\beta$-elimination of L-tyrosine were found to be $80^{\circ}C$ and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, $\beta$-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, $\beta$-elimination, was the only D-amino acid racemized by the enzyme. The $K_m$ values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, $\beta$-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.

• The Korean Journal of Mycology
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• v.45 no.1
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• pp.43-53
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• 2017

In this study, we analyzed the densities and taxonomic characteristics of various microorganisms that play important roles in Agaricus bisporus culture medium composting, and examined changes in the levels of decomposition-related enzymes secreted by these microorganisms. Various microorganisms such as thermophilic bacteria, actinomycetes, fluorescent Pseudomonas spp., and filamentous bacteria are closely associated with culture medium composts of Agaricus bisporus. The population densities of microorganisms change, and harmful bacteria disappear during thermophilic composting. Psychrobacter sp., Pseudomonas sp., Bacillus sp., and Pseudoxanthomonas sp. accounted for the highest proportion of bacteria in the culture media during outdoor composting, whereas Bacillus sp. and Psychrobacillus sp. were dominant after pasteurization. Cellulose and hemicellulose enzymes of the microorganisms were important at an early stage of rice straw composting and after decomposition of carbon sources, respectively. Microorganisms that secreted these enzymes were present in the second and third turning stage of composting.

• Korean Journal of Food Science and Technology
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• v.20 no.2
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• pp.218-224
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• 1988

For the optimization of thermal processing conditions in soymilk process, 4 strains of thermoresistant flat-sour bacteria were isolated from soymilk. The isolates were aerobic spore-forming rods, and grew at $-65^{\circ}C$. Based on the morphological and physiological properties, all of the isolated strains were identified as Bacillus stearothermophilus. The heat resistance of spores of 3 isolates and Bacillus stearothermophillus ATCC 12980 as a reference was determined in soymilk(pH 7.0) and pH 7.0 buffer solution. For each of the spores studied, linear regression equations with standard error were presented for the thermal destruction at 110, 115, 121, and $125^{\circ}C$. It was not obvious that the components of soy milk increased the heat resistance of spores. Between the strains studied, variability was noted in the D values at the different temperature, and no one strain was consistently the most heat resistant at all the given temperatures. The average D value for the 4 strains was 77.27, 20.20, 2.76 and 1.39 min at 110, 115, 121 and $125^{\circ}C$, respectively, and the average z value was $8.36^{\circ}C$.

• Food Science and Biotechnology
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• v.14 no.4
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• pp.461-465
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• 2005

To determine the cause of wet noodle spoilage, microorganisms isolated from wet noodles were identified and characterized. In addition, the growth inhibitory effects of organic acid mixture (OA: lactic acid 27.8%, acetic acid 12.0%, succinic acid 1.0%) and sodium dehydroacetate (SD) on the isolated strain were estimated in nutrient broth medium. The isolated strain was Gram-positive, rod shaped, motile, and spore forming. Based on physiological characteristics and the API 50 CHB-kit test results for the assimilation of 49 carbohydrates, the isolated strain was identified as Bacillus amyloliquefaciens (92.6%), which is able to degrade starch. Decimal reduction times (D-values) at 100, 105, and $110^{\circ}C$ for spores of B. amyloliquefaciens were 8.5, 5.1, and 2.5 min, respectively, and the z-value was $12.8^{\circ}C$. We estimated that B. amylo-liquefaciens isolated from spoiled wet noodles was a thermophilic species having high heat-resistance. Viable cell numbers in wet noodles and broth medium inoculated with B. amyloliquefaciens were decreased by 2-4 log cycles by combined treatment with 0.03 or 0.05% OA and 0.3% SD. These results revealed that OA combined with SD could be used as a potential agent to inhibit B. amyloliquefaciens in wet noodles.

• Journal of Life Science
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• v.12 no.2
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• pp.129-135
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• 2002

Several kinds of thermophilic, aerobic microorganisms (Bacillus genus), metal leaching microorganisms (Thiobacillus, T. ferooxidans), and other nondegradable chemical-degrading microorganisms (Pseudomonas genus) were utilized to study the effect on composting livestock manure. Under the Carbon-Nitrogen ratio (C/N) of 35∼40 and water content of 50∼65% conditions, the composting in the cycling drum reactor showed slower composting and lower temperature increase than that of the manual reactor. Element analysis after composting indicated relatively high levels of mineral contents with the substitutional effect of chemical fertilizer. Metal analysis before and after composting showed lower As in all, Cr in pig, Pb in cow, Hg in chicken, and Cu in cow manure compost than the regulation values. Compost maturity was ascertained by the several maturity tests. Salmonella and E. cozi detection test by SS or EMB agar plate confirmed the safety from the pathogenic microorganisms. The results suggest that the inoculation of metal and some other chemical degrading microorganisms during composting might decrease metal contamination and increase composting rate.