• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.164-169
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• 1995

An endogenous cryptic plasmid, pBL1, which has been used to construct plasmid vectors for coryneform bacteria producing amino acids, was eliminated from Brevibacterium lactofermentum. The pBL1 was partially digested with Sau3AI and the resulting DNA fragments were subcloned into a suicide vector pEM1 which contains a kanamycin-resistant (km$^{r}$) gene. KM$^{r}$ B. lactofermentum transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. A km$^{r}$ transconjugant was analyzed to contain a plasmid pEB14, which occurred in vivo by homologous recombination between pBL1 and the conjugal-transferred plasmid. The pEB14 including the pEM1-derived km$^{r}$ gene was found to be lost concomitantly with km$^{r}$ phenotype, resulting in the construction of a pBL1-free strain of B lactofermentum. Based on transformation efficiencies and plasmid stability, the resultant pBL1- free strain is more useful than wild strain as a host cell for genetic manipulation. It could be concluded that foreign plasmid DNAs are efficiently isolated and analyzed from the pBL1-free strain because of the absence of endogenous pBL1 plasmid.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.328-332
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• 2018

Soybean [Glycine max (L.) Merr.] seed is an important dietary source of protein, oil, carbohydrate, isoflavone and other various nutrients for humans and animals. However, there are anti-nutritional factors in the raw mature soybeans. Kunitz trypsin inhibitor (KTI) protein and stachyose are the main anti-nutritional factors in soybean seed. The ${\alpha}^{\prime}$-subunit of ${\beta}$-conglycinin protein exhibit poor nutritional and food processing properties. The genetic removal of the KTI and ${\alpha}^{\prime}$-subunit proteins will improve the nutritional value of the soybean seed. The objective of this research was to develop a new soybean strain with KTI and ${\alpha}^{\prime}$-subunit protein free ($titicgy_1cgy_1$ genotype) and proper agronomic traits. A breeding population was developed from the cross of the Bl-1 and 15G1 parents. A total of 168 $F_2$ seeds from the cross of the BL-1 and 15G1 parents were obtained. The segregation ratios of 9: 3: 3: 1 ($104Ti\_Cgy_{1\_}:\;30Ti\_cgy_1cgy_1:\;21cgy_1cgy_1Ti\_:\;13titicgy_1cgy_1$) between the Ti and $Cgy_1$ genes in the $F_2$ seeds were observed (${\chi}^2=5.12$, P=0.5-0.10). Two $F_4$ plant strains with proper agronomical traits and $titicgy_1cgy_1$ genotype (free of both KTI and ${\alpha}^{\prime}$-subunit protein) were selected and harvested. 2 strains (S1 and S2) had yellow seed coats and hilum. The plant height of the S1 strain was 65 centimeters. The 100-seed weight was 29.2 g. The plant height of the S2 strain was 66 centimeters and 100-seed weight was 26.2 g. The two strains selected in this research will be used to improve the new cultivar that will be free of the KTI and ${\alpha}^{\prime}$-subunit proteins.

• Microbiology and Biotechnology Letters
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• v.43 no.3
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• pp.300-305
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• 2015

The glutamate decarboxylase gene (gadB) from Lactobacillus plantarum WCFS1 was cloned and expressed as an N-terminal hexa-histidine-tagged fusion protein in Escherichia coli BL21 (DE3) as the host strain. Purified glutamate decarboxylase (GAD) was immobilized onto porous silica beads by covalent coupling. The pH dependence of activity and stability of the immobilized GAD was significantly altered, when compared to those of the free enzyme. Immobilized GAD was stable in the range of pH 3.5 to 6.0. The resulting packed-bed reactor produced 41.7 g of γ-aminobutyric acid/l·h at 45℃.

• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.83-89
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• 2001

Newcastle disease virus, CBP-1 strain isolated from Korean pheasants was passaged for 173 times by 9-day-old specific pathogenic free (SPF) embryonated eggs at $37^{\circ}C$ (parent strain) and subsequently passaged for 15 (cold attenuation (CA) -15) and 30(cold attenuation (CA) -30) times by 10-day-old of commercial broiler chicks embryonated eggs at $29^{\circ}C$, respectively, The Physical and chemical properties (sensitivity to lipid solvents, low pH and thermostability), pathogenicity (mean death time, intracerebral pathogenic index and intravenous patho-genic index), safety, booster or protective effect and characterization of temperature sensitivity were measured in cold attenuated CA-15 or 30 strain and compared to those of parent CBP-1 strain. NDV, CBP-1 CA-30 strain acquired cold attenuation and decreased infectivity at $41^{\circ}C$ compared to those of parent strain grown at $37^{\circ}C$. It lost hemagglutination activity (HA) and cell infectivity at $56^{\circ}C$ for 30, 60, and 120 Min. CA-30 strain treated with ethyl ether also lost its HA and cell infectivity. Both CA-30 and parent strains exhibited a little resistant to HA at pH 3.0 glycine HCI buffer. Intracerebral pathogenic index (ICPI) and intravenous pathogenic index (IVPI) of parent strain were 1.12 and 1.45, but decreased to 0.75 and 0.00 in CA-30 strain, respectively. The safety was evaluated by mortality in chicks inoculated with 10$^{4.0}$ EID$_{50}$ /0.1 ml. The mortalities of parent, CA-30 and commercial Bl strains were 17.5, 12.0 and 0.0%, respectively. The safety of CA-30 strain was higher than that of parent strain. The booster effects of CA-30 strain and parent strain performed in 4-week-old chicks after being vaccinated with primary commercial Bl strain.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.930-938
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• 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA-335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Ni^{2+}$, $Fe^{2+}$, $Fe^{3+}$, $Zn^{2+}$, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of $60^{\circ}C$ and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of $70^{\circ}C{\sim}80^{\circ}C$), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.