• Journal of Microbiology and Biotechnology
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• 제29권6호
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• pp.944-951
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• 2019

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of $80^{\circ}C$ at pH 11.0. The optimum substrate for enzyme activity was $C_{10}$, which is highly stable in methanol solvent. The enzyme was strongly activated by $Ca^{2+}$, $Mg^{2+}$, and strongly inhibited by $Fe^{2+}$ and $Zn^{2+}$. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

• Molecules and Cells
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• 제22권1호
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• pp.89-96
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• 2006

"Determinate" and "indeterminate" inflorescences in plants are controlled by a single recessive gene, for example, SELF-PRUNING (SP) in Solanum lycopersicum, TERMINAL FLOWER1 in Arabidopsis, CENTRORADIALIS in Antirrhinum, and CENTRORADIALIS-like gene in tobacco. Pepper (Capsicum annuum L.) is an indeterminate species in which shoots grow indefinitely. In this study, we cloned and characterized the pepper SP-like gene (CaSP). RT-PCR revealed that the CaSP transcript accumulates to higher levels in floral buds than in other organs. Comparison of genomic DNA and cDNA sequences from indeterminate and determinate pepper plants revealed the insertion of a single base in the first exon of CaSP in the determinate pepper plants. CaSP is annotated in linkage group 8 (chromosome 6) of the SNU2 pepper genetic map and showed similar synteny to SP in tomato. Transgenic tobacco plants overexpressing CaSP displayed late-flowering phenotypes similar to the phenotypes caused by overexpression of CaSP orthologs in other plants. Collectively, these results suggest that pepper CaSP is an ortholog of SP in tomato.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.930-935
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• 2015

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 µmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37℃. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

• 한국지하수토양환경학회지:지하수토양환경
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• 제19권3호
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• pp.47-55
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• 2014

Arthrobacter chlorophenolicus A6 possesses several monooxygenases (CphC-I, CphC-II, and CphB) that can catalyze the transformation of 4-chlorophenol (4-CP) to hydroxylated intermediates in the initial steps of substrate metabolism. The corresponding genes of the monooxygenases were cloned, and the competent cells were transformed with these recombinant plasmids. Although CphC-II and CphB were expressed as insoluble forms, CphC-I was successfully expressed as a soluble form and isolated by purification. The specific activity of the purified CphC-I was analyzed by using 4-CP, 4-chlorocatechol (4-CC), and catechol (CAT) as substrates. The specific activities for 4-CP, 4-CC, and CAT were determined to be 0.312 U/mg, 0.462 U/mg, 0.246 U/mg, respectively. The results of this study indicated that CphC-I is able to catalyze the degradation of 4-CC and CAT in addition to 4-CP, which is a primary substrate. This research is expected to provide the fundamental information for the development of an eco-friendly biochemical degradation of aromatic hydrocarbons.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.48-58
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• 2023

Interferon regulatory factor 3 (IRF3) integrates both immunological and non-immunological inputs to control cell survival and death. Small GTPases are versatile functional switches that lie on the very upstream in signal transduction pathways, of which duration of activation is very transient. The large number of homologous proteins and the requirement for site-directed mutagenesis have hindered attempts to investigate the link between small GTPases and IRF3. Here, we constructed a constitutively active mutant expression library for small GTPase expression using Gibson assembly cloning. Small-scale screening identified multiple GTPases capable of promoting IRF3 phosphorylation. Intriguingly, 27 of 152 GTPases, including ARF1, RHEB, RHEBL1, and RAN, were found to increase IRF3 phosphorylation. Unbiased screening enabled us to investigate the sequence-activity relationship between the GTPases and IRF3. We found that the regulation of IRF3 by small GTPases was dependent on TBK1. Our work reveals the significant contribution of GTPases in IRF3 signaling and the potential role of IRF3 in GTPase function, providing a novel therapeutic approach against diseases with GTPase overexpression or active mutations, such as cancer.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.973-978
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• 2001

The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation, mostly for driving rotation of the helical flagellar filament. In this study, we cloned a gene (motX) encoding a component of the sodium-driven flagellar motor from Vibrio fluvialis. The nucleotide sequence of the motX gene, composed of 633 bp and 211 amino acid residues, was determined. Overexpression of the motX gene in Escherichia coli using a strong promoter induced growth inhibition and cell lysis. The lethal effect of E. coli was suppressed by adding amiloride, as a potent inhibitor for the sodium channel. Electron microscopic observation of the expressed protein indicated that MotX protein induced by isopropyl ${\beta}$-D-thiogalactopyranoside caused the lysis of host cell.

• Bulletin of the Korean Chemical Society
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• 제30권11호
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• pp.2647-2650
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• 2009

Cold shock proteins (Csps) are a subgroup of the cold-induced proteins on reduction of the growth temperature below the physiological temperature. They preferentially bind to single-stranded nucleic acids to translational regulation via RNA chaperoning. Csp plays important role in cold adaptations for the psychrophilic microorganism. Recently, Cold shock protein from psychrophilic bacteria, Colwellia psychrerythraea (CpCsp) has been identified. Three dimensional structures of a number of Csps from various microorganisms have been solved by NMR spectroscopy or X-ray crystallography, but structures of psychrophilic Csps were not studied yet. Therefore, cloning and purification protocols for further structural study of psychrophilic Csp have been optimized in this study. CpCsp was expressed in E. coli with pET-11a vector system and purified by ion exchange, size exclusion, and reverse phase chromatography. Expression and purification of CpCsp in M9 minimal media was carried out and $^{15}N$-labeled proteins with high purity over 90% was obtained. Further study will be carried out to investigate the tertiary structure and dynamics of CpCsp.

• BMB Reports
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• 제39권3호
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• pp.311-318
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• 2006

One of the biggest group of proteins influenced by protein kinase CK2 is formed by factors engaged in gene expression. Here we have reported recently identified yeast transcription elongation factor Elf1 as a new substrate for both monomeric and tetrameric forms of CK2. Elf1 serves as a substrate for both the recombinant CK2$\alpha$' ($K_m$ 0.38 ${\mu}M$) and holoenzyme ($K_m$ $0.13\;{\mu}M$). By MALDI-MS we identified the two serine residues at positions 95 and 117 as the most probable in vitro phosphorylation sites. Co-immunoprecypitation experiments show that Elf1 interacts with catalytic ($\alpha$ and $\alpha$') as well as regulatory ($\beta$ and $\beta$') subunits of CK2. These data may help to elucidate the role of protein kinase CK2 and Elf1 in the regulation of transcription elongation.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.1995-1999
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• 2006

Pseudomonas sp. K82 cultured in p-hydroxybenzoate induces protocatechuate 4,5-dioxygenase (PCD 4,5) for p-hydroxybenzoate degradation. In this study, a 6.0-kbp EcoR1 fragment containing p-hydroxybenzoate degradation genes was cloned from the genome of Pseudomonas sp. K82. Sequence analysis identified four genes, namely, pcaD, pcaA, pcaB, and pcaC genes known to be involved in p-hydroxybenzoate degradation. Two putative 4-hydroxyphenylpyruvate dioxygenases and one putative oxidoreductase were closely located by the p-hydroxybenzoate degradation genes. The gene arrangement and sequences of these p-hydroxybenzoate degradation genes were similar to those of Comamonas testosteroni and Pseudomonas ochraceae. PcaAB (PCD4,5) was overexpressed in the expression vector pGEX-4T-3, purified using a GST column, and confirmed to have protocatechuate 4,5-dioxygenase activity. The N-terminal amino acid sequences of overexpressed PCD4,5 were identical with those of purified PCD4,5 from Pseudomonas sp. K82.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1660-1669
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• 2015

The present study describes the gene cloning, overexpression and characterization of a novel nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The nitrilase gene consisted of 804 base pairs, encoding a protein of 268 amino acid residues with a molecular mass of 30.07 kDa after SDS-PAGE analysis. The optimal temperature and pH of the purified enzyme were 45℃ and 7.5, respectively. The enzyme demonstrated good temperature tolerance, with 40% residual activity after 60 min of heat treatment at 75℃. The kinetic constants V_max and K_m of this nitrilase toward 3-cyanopyridine were 3.12 μmol/min/mg and 7.63 mM, respectively. Furthermore, this novel nitrilase exhibited a broad spectrum toward the hydrolysis of the aliphatic nitriles among the tested substrates, and particularly was specific to aliphatic dinitriles like succinonitrile, which was distinguished from most nitrilases ever reported. The catalytic efficiency k_cat/K_m was 0.44 /mM/s toward succinonitrile. This distinct characteristic might enable this nitrilase to be a potential candidate for industrial applications for biosynthesis of carboxylic acid.