• Journal of Microbiology and Biotechnology
• /
• v.11 no.3
• /
• pp.508-514
• /
• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.10 no.6
• /
• pp.1287-1291
• /
• 2009

The genes of cyanide hydratase(CHT), a kind of nitrilases whichhydrolyze cyanide to formamide were extracted from N. crassa and A. nidulans, the two fungal strains. The recombinant forms of the CHT originated from N. crassa and A. nidulans were prepared with N-terminal hexahistidine purificationtags or no tags, and expressed in E. coli. The enzymes were purified using immobilized metal affinity chromatography. They were compared according to their pH activity profiles, and kinetic parameters. The N. crassa CHT has the wider pH range of activity above 50% and three-fold higher turnover rate (6.6 ${\times}$ $10^8$ $min^{-1}$) than the A. nidulans, meanwhile the CHT of A. nidulans has the higher $K_m$ value. Expression of CHT in both N. crassa and A. nidulans were induced by the presence of KCN, regardless of any presence of nitrogen sources. Max. 82% of KCN was degraded in 60 min for biological degradation tests.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.4
• /
• pp.483-488
• /
• 2014

In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times that of E. coli, and the $V_{max}$ and specific activity of XynB reached $2,547.7{\mu}mol/mg$ and 4,757 U/mg, respectively. XynB still had 74% residual enzyme activity after 30 min of heat treatment at $80^{\circ}C$. From the van der Waals force analysis of XYNB (ACN89393 and AAS67299), there is one more oxygen radical in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than in the 33rd Ala(ACN89393 ), and two H-bonds between Ser70 and Asp67.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.6
• /
• pp.1360-1364
• /
• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• International Journal of Oral Biology
• /
• v.36 no.2
• /
• pp.103-108
• /
• 2011

Measurement of estrogen concentration in bio-samples are very important for differential diagnosis of various disease or evaluation of health status. However, it is difficult to collect immediate data of estrogen concentration because they are measured by radioimmunoassay or chromatography which need time- and cost-consuming sample pre-treatment. This study was performed for development of new estrogen biosensor employing taste principles, and for evaluation of cross reactivity between various steroid hormones. Gene sequence of ligand binding domain of ${\alpha}$-human estrogen receptor (amino acid 302-553; hER-LBD) was cloned from human breast cancer cell line. The proteins of hER-LBD were produced by T7-E.coli expression system, and isolated by chromatography. hER-LBD were coated on the gold plated quartz crystal (AT-cut 9MHz), and resonance frequencies were measured by universal frequency counter. Estradiol, progesterone, testosterone, and aldosterone were used for cross reactivity of the hER-LBD. We also monitored influences of pH change in resonance frequency. The resonance frequencies of hER-LBD coated quartz crystal were decreased during increase of estrogen concentration from $15 \;{\mu}g/mL$ to $50\;{\mu}g/mL$. However, similar steroid hormones, progesterone and aldosterone, did not elicit the change in resonance frequency. Testosterone evoke weak change in resonance frequency. The new estrogen biosensor was more sensitive in pH 7.2 than in pH 7.6. These results suggest that hER-LBD coated quartz crystal biosensor is a probable estrogen biosensor.

• Molecules and Cells
• /
• v.23 no.3
• /
• pp.280-286
• /
• 2007

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and $NH_4{^+}$. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.

• Journal of Korean Neurosurgical Society
• /
• v.30 no.sup1
• /
• pp.13-19
• /
• 2001

Objective : We investigated the feasibility of a double suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase(TK) gene and the Escherichia coli cytosine deaminase(CD) gene, via a recombinant adenoviral vector and ganciclovir(GCV) and/or 5-fluorocytosine(5-FC) treatment, in C6 glioma cells. Methods : Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the ${\beta}$-galactosidase gene and then staining cells with 5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene(ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-${\Delta}E1$(as a control). After addition of a variety of concentrations of GCV and 5-FU, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. Result : C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity(>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30ug/ml or GCV at concentrations greater than 0.3ug/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after sinlge-prodrug treatments, indicating additive effects of the prodrug treatments. Conclusion : The administration of a double-suicide gene/prodrug therapy might have great potential in the treatment of brain tumors.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.4
• /
• pp.577-586
• /
• 2019

The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was $50^{\circ}C$. This enzyme also had a half-life of 33.3 min at $40^{\circ}C$. Kinetics assay showed that AoG6PDH was strictly dependent on $NADP^+$ ($K_m=6.3{\mu}M$, $k_{cat}=1000.0s^{-1}$, $k_{cat}/K_m=158.7s^{-1}{\cdot}{\mu}M^{-1}$) as cofactor. The $K_m$ and $k_{cat}/K_m$ values of glucose-6-phosphate were $109.7s^{-1}{\cdot}{\mu}M^{-1}$ and $9.1s^{-1}{\cdot}{\mu}M^{-1}$ respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where $NADP^+$ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.8
• /
• pp.1154-1162
• /
• 2021

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (P_k.rtufB, P_k.r1, P_k.r2, and P_k.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with P_k.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by P_k.r1 and P_k.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.

• Food Science of Animal Resources
• /
• v.42 no.6
• /
• pp.1031-1045
• /
• 2022

Postbiotics are defined as probiotics inactivated by heat, ultraviolet radiation, sonication, and other physical or chemical stresses. Postbiotics are more stable than probiotics, and these properties are advantageous for food additives and pharmacological agents. This study investigated the immunostimulatory effects of heat-treated Lactiplantibacillus plantarum LM1004 (HT-LM1004). Cellular fatty acid composition of L. plantarum LM1004 isolated form kimchi was analyzed by gas chromatography-mass spectrometry detection system. The nitric oxide (NO) content was estimated using Griess reagent. Immunostimulatory cytokines were evaluated using enzyme-linked immunosorbent assay. Relative protein expressions were evaluated by western blotting. Phagocytosis was measured using enzyme-labelled Escherichia coli particles. L. plantarum LM1004 showed 7 kinds of cellular fatty acids including palmitic acid (C16:0). The HT-LM1004 induced release of NO and upregulated the inducible NO synthase in RAW 264.7 macrophage cells. Tumor necrosis factor-α and interleukin-6 levels were also increased compared to control (non-treated macrophages). Furthermore, HT-LM1004 modulated mitogen-activated protein kinase (MAPK) subfamilies including p38 MAPK, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase. Therefore, these immunostimulatory effects were attributed to the production of transcriptional factors, such as nuclear factor kappa B (NF-κB) and the activator protein 1 family (AP-1). However, HT-LM1004 did not showed significant phagocytosis of RAW 264.7 macrophage cells. Overall, HT-LM1004 stimulated MAPK/AP-1 and NF-κB expression, resulting in the release of NO and cytokines. These results will contribute to the development of diverse types of food and pharmacological products for immunostimulatory agents with postbiotics.