• Microbiology and Biotechnology Letters
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• v.43 no.4
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• pp.330-335
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• 2015

The optimum conditions for the production of xylanase from Bacillus agaradhaerens DK-2386 have been previously investigated. In this study xylanase was purified by ammonium sulfate precipitation and CM-sepharose ion exchange chromatography. The molecular mass of the xylanase as determined by SDS-PAGE was 23 kDa in a form of monomeric enzyme. The optimum pH and temperature for xylanase activity was 6.0 and $60^{\circ}C$, respectively. Xylanase activity was increased by the addition of EDTA and then stabilized at $40^{\circ}C$ for 24 h. The maximum xylanase activity was obtained when Birchwood xylan was used as a substrate and the $V_{max}$ and $K_m$ were $49,724{\mu}mol/min$ and 6.08 mg/ml, respectively.

• Applied Biological Chemistry
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• v.47 no.1
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• pp.41-48
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• 2004

Cyclodextrin glucanotransferase (CGTase) of Bacillus sp. isolated from soil was purified and its enzymological characteristics were investigated. It was found that the production of CGTase reached to the maximum when the strain was cultured in the broth containing 0.1 % albumin, 2% $NH_4Cl$, 2% soluble starch and 0.2% $NH_2PO_4$ for 72 hrs at $37^{\circ}C$. The purity of CGTase was increased by 9.7 folds through purification procedures by the following column chromatography DEAE-cellulose ion exchange chromatography and Sephadex G-100, G-150 gel filtration and its specific activity was 528.0 unit/mg. The optimum pH and temperature for the CGTase activity were 8.0 and $80^{\circ}C$, respectively. The enzyme was stable in pH $8.0{\sim}11.0$ at $60{\sim}80^{\circ}C$. The activity of purified enzyme was inhibited by $Pb^{2+},\;Hg^{2+}$ and $Zn^{2+}$. When CGTase was treated with each 20.5 unit, 41 unit, 205 unit and 410 unit to investigate the transglucosylation to stevioside by purified cyclodextrin glucanotransferase, transglucosylation rate to stevioside was 74.9%, 75.7%, 68.7% and 57.9%. Brown effect was observed above the concentration amounting to 205 unit of our CGTase.

• Korean Journal of Food Science and Technology
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• v.9 no.2
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• pp.97-105
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• 1977

1) In order to obtain a microbial strain having a strong yeast cell wall lytic activity, about 156 isolates capable of forming clear zones on baker's yeast-peptone-bouillon agar plate were obtained from soil, mud and water samples and a strain K-42 with the highest lytic activity was identified as Bacillus circulans. 2) Effect of carbon sources on the lytic enzyme production by the K-42 strain was in the decreasing order of maltose>glucan>xylose>control in 2-day culture and of lactose>galactose>glucan>control in 3-day culture. Effect of inorganic nitrogen sources was in the decreasing order of ammonium acetate>sodium nitrate>control in 2-day culture and of ammonium chloride>ammonium oxalate>control in 3-day culture, whereas organic nitrogen sources except milk casein showed an increase in 2-day culture and a decrease in 3-day culture. Synergistic effect of carbon sources and nitrogen sources was not observed. 3) The enzyme production by the K-42 strain was greatly affected by pH change of the culture medium, thus a high lytic activity could be maintained by keeping the pH range of $7{\sim}8$ and adding carbon or nitrogen sources. 4) Optimum conditions for the lytic activity of the K-42 strain were obtained at $pH\;7{\sim}8$ and $60^{\circ}C$ and the extent of hydrolysis toward heated yeast cell wall was 65%.

• Journal of Environmental Science International
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• v.23 no.6
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• pp.1095-1100
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• 2014

Keratin wastes are generated in excess of million tons per year worldwide and biodegradation of keratin by microorganisms possessing keratinase activity can be used as an alternative tool to prevent environmental pollution. For practical use of keratinase, its physicochemical properties should be investigated in detail. In this study, we investigated characteristics of keratinase produced by Xanthomonas sp. P5 which is isolated from rhizospheric soil of soybean. The level of keratinase produced by the strain P5 increased with time and reached its maximum (10.6 U/ml) at 3 days. The production of soluble protein had the same tendency as the production of keratinase. Optimal temperature and pH of keratinase were $40^{\circ}C-45^{\circ}C$ and pH 9, respectively. The enzyme showed broad temperature and pH stabilities. Thermostability profile showed that the enzyme retained 94.6%-100% of the original activity after 1 h treatment at $10^{\circ}C-40^{\circ}C$. After treatment for 1 h at pH 6-10, 89.2%-100% of the activity was remained. At pH 11, 71.6% of the original activity was retained after 1 h treatment. Although the strain P5 did not degrade human hair, it degraded duck feather and chicken feather. These results indicate that keratinase from Xanthomonas sp. P5 could be not only used to upgrade the nutritional value of feather hydrolysate but also useful in situ biodegradation of feather.

• The Korean Journal of Mycology
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• v.47 no.3
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• pp.259-269
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• 2019

Brown spot and sheath rot of rice are caused by fungal pathogens such as Curvularia lunata, Cochliobolus miyabeanus, and Sarocladium oryzae, and cause losses such as reduced rice yield and quality, which is an enormous problem with serious long-term effects. To search biological control agents of phytopathogenic fungi, five kinds of useful Bacillus-like isolates which are excellent in extracellular enzyme activity and produce siderophore were selected from paddy soil of Sunchang in Korea. The selected isolates were tested for excellent antifungal activity against three of the phytopathogenic fungi that frequently occur in rice, and JSRB 177 strain had the most excellent antifungal activity. Based on the experimental results, JSRB 177 is finally selected as a candidate for biological control and identified to Bacillus subtilis through 16S rRNA sequence analysis. In addition, physiological characteristics of JSRB 177 confirmed by analysis of carbohydrate fermentation patterns and enzyme production ability. Based on the above results, JSRB 177 is expected to be used as a biological control agent for the rice pathogenic fungi. In the future, further studies related to industrialization such as port test and establishment of mass production process are needed.

• Korean Journal of Agricultural Science
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• v.15 no.2
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• pp.153-163
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• 1988

This experiment was carried out to obtain the thermophilic fungus producing amylase and to investigate properties of the amylase. The selected strain, L-11 was obtained from soil in the vicinity of a hot spring and identified as Mocor sp.. And then the conditions for enzyme production in wheat bran cultures and properties of the crude enzyme were investigated. Furthermore, the enzyme was purified and the characteristics of purified enzyme were studied. The results obtained were as follows: 1. On the wheat bran medium added 80-100% water, amylase was effectively produced by the selected strain, L-11 for 48 hrs incubation at $50^{\circ}C$. 2. When the crude enzyme solution of the strain L-11 was passed through DEAE-Sephadex A-50 column chromatography, two peaks having amylase activity were obtained, and one peak was that of the main enzyme (enzyme of B peak). 3. The purified enzyme (enzyme of B peak) was recognized as single protein band on polyacrylamide disc gel electrophoresis. 4. In the hydrolysis reaction of soluble starch by the enzyme of main amylase, oligosaccharides produced at early stage were maltose and maltotriose mainly and procedure of the reaction maltose amount of maltose and glucose was increased. 5. The strain L-11 was recognized as a special strain producing ${\alpha}-amylase$ mainly and scarcely glucoamylase. 6. The optimum pH, optimum temperature, pH stability, and temperature stability of ${\alpha}-amylase$ were pH 4.0, $60-65^{\circ}C$, pH 4.0-9.0, and below$70^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Applied Biological Chemistry
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• v.41 no.2
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• pp.125-129
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• 1998

Xanthomonas sp. isolated from soil exhibited cell wall lytic activity of Candida albicans and secreted chitinase in chitin media. Especially, the chitinase activity was induced by chitin and reached a maximum level at 3 days culture in chitin media. We constructed genomic library of Xanthomonas sp. using cosmid vector in E. coli. Oligonucleotide probe was synthesized from the consensus sequence corresponding to chitinase active site, which was derived from the comparison of amino acid sequences of bacterial chitinase genes. Using this oligonucleotide probe, we screened the genomic library. By restriction enzyme mapping of the positive clones, we identified 4 independent clones which may contain the chitinase gene. One of the clones, named pXCH1 (1.2 kb insert), was further analyzed. Northern blot analysis indicated that is transcripts, 1 kb and 0.8 kb, were induced by chitin. When the cloned gene was induced by IPTG in E.coli cell, chitinase activity which was secreted onto culture media was not observed. However, when the cell was disrupted by using sonicator and then centrifuged, the supernatant exhibited chitinase activity. SDS-PAGE of the supernatant indicated that about 35 kDa protein was induced by IPTG. From these results, it was concluded that the cloned DNA was one of the chitinase genes of Xanthomonas sp.

• Journal of Microbiology
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• v.42 no.4
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• pp.319-327
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• 2004

An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a malto­genic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ struc­tural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The pro­moter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regu­latory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.238-243
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• 2000

An exoinulinase (${\beta}-D-fructofuranosidase$) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. the exoinulinase gene consisted of an ORF of 0,506 bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli $DH5{\alpha}$ strain was also purified to homogeneity as determined by SDS-PAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90 min at $55^{\circ}C$. Taken together, these results indicate that the purified ${\beta}-D-fructofuranosidase$ was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and $55^{\circ}C$, respectively. the enzymehad no apparent requirement for a cofactor, and its activity was completely inactivated by $Ag^{+},{\;}Hg^{2+},{\;}and{\;}Zn^{2+}$. Kinetic experiments gave $K_m,{\;}V_{max},{\;}and{\;}K_{cat}$ values for inulin of 11.5 mM, 18 nM/s, and $72{\;}s^{-1}$, respectively. the exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4 h incubation at $55^{\circ}C$.