• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.327-336
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• 2010

Cysteine proteases have been known as a critical factor in plant defense mechanisms in pineapple, papaya, or wild fig. Papain or ficin is one kind of cysteine proteases that shows toxic effects to herbivorous insects and pathogenic bacteria. However, resistance to bacterial soft rot of plants genetically engineered with cysteine protease has been little examined thus far. We cloned a cysteine protease cDNA from Ananas comosus and introduced the gene into Chinese cabbage (Brassica rapa) under the control of the cauliflower mosaic virus 35S promoter. The transgene was stably integrated and actively transcribed in transgenic plants. In comparisons with wild-type plants, the $T_2$ and $T_3$ transgenic plants exhibited a significant increase in endo-protease activity in leaves and enhanced resistance to bacterial soft rot. A cDNA microarray analysis revealed that several genes were more abundantly transcribed in the transgenic than in the wild type. These genes encode a glyoxal oxidase, PR-1 protein, PDF1, protein kinase, LTP protein, UBA protein and protease inhibitor. These results suggest an important role for cysteine protease as a signaling regulator in biotic stress signaling pathways, leading to the build-up of defense mechanism to pathogenic bacteria in plants.

• Microbiology and Biotechnology Letters
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• v.8 no.2
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• pp.77-85
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• 1980

This experiment was conducted to investigate the conditions for production and the characteristics of pretenses. Aspergillus oryzae KC-15, which is selected as a superior strain for the production of the protease, was used in this study. The results obtained were as follows: 1. The optimum culture time for the production of acid, neutral and alkaline protease on wheat bran medium were about 48, 48 and 72hr, respectively. The protease-produced by the strain were mainly alkaline and neutral one, but the production of acid protease was feeble extremely. 2. The addition of NaH$_2$PO$_4$, Na$_2$HPO$_4$, glucose, rice powder and Na-glutamate respectively to wheat bran media were effective for the production of alkaline and neutral protease, and the addition of (NH$_4$)$_2$HPO$_4$, glucose and rice powder respectively were effective for the production of acid protease. 3. Characteristics of professes(equation omitted) 4. As a heat resistance agent, NaH$_2$PO$_4$was the most effective one. The optimum amount of NaH$_2$PO$_4$was 10mg for alkaline and neutral protease, and 5mg for acid protease. 5. The heat resistance of the Protease by NaH$_2$PO$_4$was not recognized mostly above 6$0^{\circ}C$. 6. After the treatment of enzyme solution with 10mg of NaH$_2$PO$_4$for 30 minutes at 55$^{\circ}C$, the residual activities measured for alkaline, neutral and acid protease were 58, 57 and 55％ respectively.

• Food Science and Preservation
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• v.7 no.4
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• pp.414-417
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• 2000

Competent cell transformation of B. subtilis AC819 was carried out using phenotypic protease-defective(Npr-) DNA of B. subtilis MT-2. An obtained transformant, designated B. subtilis HL-1, was obtained by homologous DNA recombination. Phenotypes of B. subtilis HL-1 were characterized histidine requirement streptomycin-resistance, tetracyclin resistance and non-producing protease. Protoplast transformation frequency of B. subtilis HL-1 by plasmid pUB110 was higher than that of B. subtilis MT-2. From this result, B. subtilis HL-1 is useful for protease gene transformation and thermostable protease gene cloning as a host.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.458-463
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• 2022

We evaluated the antibiotic susceptibilities, hemolytic activities, and technological properties of 36 Staphylococcus xylosus strains and 49 S. pseudoxylosus strains predominantly isolated from fermented soybean foods from Korea. Most of the strains were sensitive to chloramphenicol, erythromycin, gentamycin, kanamycin, lincomycin, oxacillin, tetracycline, and trimethoprim. However, 23 strains exhibited potential phenotypic acquired resistance to erythromycin, lincomycin, and tetracycline. Based on breakpoint values for staphylococci from the Clinical and Laboratory Standards Institute, >30% of the isolates were resistant to ampicillin and penicillin G, but the population distributions in minimum inhibitory concentration tests were clearly different from those expected for acquired resistance. None of the strains exhibited clear α- or β-hemolytic activity. S. xylosus and S. pseudoxylosus exhibited salt tolerance on agar medium containing 20% and 22% (w/v) NaCl, respectively. S. xylosus and S. pseudoxylosus strains possessed protease and lipase activities, which were affected by the NaCl concentration. Protease activity of S. pseudoxylosus was strain-specific, but lipase activity might be a characteristic of both species. This study confirms the potential of both species for use in high-salt soybean fermentation, but the safety and technological properties of strains must be determined to select suitable starter candidates.

• Proceedings of the Korea Crystallographic Association Conference
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• 2000.05a
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• pp.10-10
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• 2000

No Abstract, See Full-text

• Microbiology and Biotechnology Letters
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• v.5 no.3
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• pp.153-158
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• 1977

These experiments were conducted to investigate the condition of the production and the heat resistance of the acid protease by Rhizopus japonicus S-62. The results obtained were as follows: 1) The optimum concentrations of sucrose, yeast, ammonium chloride and sodium phosphate monobasic added to the wheat bran medium in the acid protease production were 0.5％, 2.0％, 0.4％, and 0.4％, respectively. 2) KH$_2$PO$_4$ and NaH$_2$PO$_4$ were the most effective as the heat resistant agents. 3) When the enzyme solutions added with KH$_2$PO$_4$ and NaH$_2$PO$_4$ to the concentration of 2％ were heated for 10 min, at 50$^{\circ}C$, their residual activities were 100％, respectively. 4) The heat resistant effects of KH$_2$PO$_4$ and NaH$_2$PO$_4$ were not observed almost above 55$^{\circ}C$.

• Proceedings of the Microbiological Society of Korea Conference
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• 1998.04a
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• pp.41.1-41.1
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• 1998

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.410-416
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• 2008

The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-${\beta}$-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and $60^{\circ}C$, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by $Fe^{2+}$ and strongly inhibited by $Hg^{2+}$ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.

• KSBB Journal
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• v.9 no.3
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• pp.332-338
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• 1994

Solubilization phenomena of a protein in a reversed micellar solution were investigated and a hollow fiber membrane extractor was tested for reversed micellar protein extraction equipment. Alkaline protease was used as a model protein compound and the reversed micellar solution was consisted of AOT and isooctane. It was found that protein solubilization was strongly influenced by ionic strength and pH. The distribution coefficient of the protease between the aqueous solution and the AOT/isooctane solution was also observed to be as high as 4.0 within the scope of this experiment. A hollow fiber membrane extractor was constructed and tested for the protein extraction. The overall mass transfer coefficient at a typical experimental condition of this study was observed to be $6.7{\times}10^{-5}cm/s$. It was also found that the mass transfer resistance on reversed micellar solution was the dominant resistance for the protein transfer.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1532-1539
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• 2012

The ${\alpha}$-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ${\leq}97.2%$ with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 ${\alpha}$-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas ${\alpha}$-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas ${\alpha}$-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-${\alpha}$-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and $60^{\circ}C$ and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant ${\alpha}$-galactosidases showing thermolability at $50^{\circ}C$ or $60^{\circ}C$ and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at $60^{\circ}C$) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas ${\alpha}$-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.