• Korean Journal of Microbiology
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• v.48 no.4
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• pp.293-297
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• 2012

Of the 169 strains of microorganisms stored in Polar and Alpine Microbial Collection of Korea Polar Research Institute, 27 strains were selected for their chitinase activity on ZoBell plates supplemented with 0.4% colloidal chitin. Among them, PAMC 21693 strain have shown the highest chitinolytic enzyme activity toward pNP-$(GlcNAc)_1$ at low temperature and the highest growth rate at $4^{\circ}C$. We cloned a full-length chitinase gene of 2,857 bp which contains an open reading frame of 2,169 bp encoding 872-amino acid polypeptide. Recombinant chitinase protein was expressed in E. coli and its molecular weight was confirmed 96 kDa. In this paper, we suggest the potential use of cold-active chitinase from polar microorganisms in the field of biotechnology.

• Journal of the East Asian Society of Dietary Life
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• v.19 no.2
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• pp.195-201
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• 2009

The antioxidative activities of two varieties of egg plant (Chunyang No 2: Dangaji, Jinju Janggaji: Janggaji) extracts were investigated. The total polyphenol contents of Dangaji peel hot water extract and the Janggaji flesh and fruit hot water extracts were higher than those of the other samples. However, the DPPH radical scavenging activities(electron donating activity) of Dangaji flesh-ethanol, peel-cold water, and fruit- ethanol extracts, as well as the Janggaji peel cold water extract, were higher than those of the other samples. Furthermore, the in vitro inhibitory effects of the Dangaji peel cold water and hot water extracts on rat hepatic xanthine oxidase were highest among the samples, and were exhibited in a dose dependant manners. Although there were marked changes in the xanthine oxidase Km values for xanthine as a substrate, the Vmax value changes by the addition of the Dangaji water extracts were minor compared with the control. This result suggests that Dangaji water extracts may regulate the activity of xanthine oxidase-via the inhibition of binding affinity between the enzyme and substrate. The present study provides experimental evidence that constituents of egg plant extracts may ameliorate reactive oxygen species(ROS)-induced oxidative stress via hepatic hepatic xanthine oxidase activity, but further studies to identify the active antioxidants and compounds and inhibitors of xanthine oxidase are required.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1484-1489
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• 2014

A novel esterase gene, est01, was successfully unearthed from a biogas digester microbiota metagenomic library. The 1,194 bp est01 gene encodes a protein of 44,804 Da (designated Est01). The amino acid sequence of Est01 shows only moderate (33%) identity to a lipase/esterase. Phylogenetic analysis and biochemical characterization confirmed that Est01 is a new member of family VIII esterases. The purified Est01 from recombinant Escherichia coli BL21 (DE3) showed high hydrolytic activity against short-chain fatty acid esters, suggesting that it is a typical carboxylesterase rather than a lipase. Furthermore, the Est01 was even active at $10^{\circ}C$ (43% activity remained), with the optimal temperature at $20^{\circ}C$, and had a broad pH range from 5.0 to 10.0, with the optimal pH of 8.0. These properties suggest that Est01 is a cold-adaptive esterase and could have good potential for low-temperature hydrolysis application.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.192-201
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• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.

• Plant Resources
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• v.7 no.3
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• pp.174-180
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• 2004

Calmodulin, a $Ca^{2+}$-binding protein, has no enzyme activity. It combines with $Ca^{2+}$ and makes variable proteins to an active form. Calmodulin 2 is a ubiquitous protein in plants. To investigate the defense mechanism against various stresses, a clone encoding a calmodulin 2 protein was isolated from a cDNA library prepared from taproot mRNAs of Codonopsis lanceolata. The cDNA, designated CICAM2, is 719 nucleotides long and has an open reading frame of 450 bp with a deduced amino acid sequence of 149 residues. The deduced amino acid sequence of CICAM2 showed a high similarity with calmodulins of P. x hybrida (P27163) 97%, N. tabacum (BAB61908) 97%, S. tuberosum (AAA74405) 96%, Z. mays (CAA74307) 92%, C. richardii (AF510075) 93%, M. truncatula (AAM81203) 91%, and G. max (P62163) 91%. The transcriptional expression of the CICAM2 gene, was gradually increased by the CaCl$_2$ treatment. Whereas its expression And it was gradually decreased in the cold stress treatment.ent.

• Journal of Plant Biotechnology
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• v.33 no.2
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• pp.123-131
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• 2006

Chinese cabbage (Brassica rapa ssp. pekinesis) is one of the most important vegetable crops in korea and other East Asian countries. Cytosolic fructose-1,6-bisphospha-tase (cytFBPase) is a key enzyme in sucrose biosyn-thesis, which controls the sucrose levels as well as the productivity at plants. The Chinese cabbage cytFBPase gene, BrFBPase, encodes the 340 amino acid polypep-tide, giving a theoretical molecular weight of 37.2 kD and a isolectric point of 5.4. BrFBPase showed high sequence identity with Brassica homologs and its functional domains, such as 12,6P$_2$ binding site or active site and F6P binding site, were highly conserved in diverse sources of organisms. Although the genome of Chinese cabbage seemed to be triplicated, BrFBPase appears to be a single copy gene. The expression of BrFBPase was examined at transcript and protein levels under various conditions. BrFBPase expression was observed only in photosynthetic source tissue, not in sink tissue. The expression was slightly higher during the day than at night, and it showed a diurnal cycle with circadian rhythmicity. Short-term exposure to low temperature inhibited the expression of the BrFBPase, while long-term exposure increased the expression, supporting that sugar levels are high in late autumn when temperature are low.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.162-167
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• 1991

In an attempt to explore the mechanistic aspects of chilling injury in plants and their defensive measures against the low temperature stress, the time sequential measurements of pyruvate, superoxide radicals$(O_{\overline{2}})$ and antioxygenic enzymes during whole period of injury-inducing treatment were performed using mostly rice seedlings. Pyruvate was substantialy accumulated in leaf tissues during the exposure period to $5^{\circ}C$ of the seedlings ; the relative extent of the accumulation was increased with increasing time of the cold treatment. When the cold-treated plants were translocated to ambient temperature$({\sim}25^{\circ}C)$, the accumulation started to dissipate, concomitantly accompaning a remarkable increase in the $O_{\overline{2}}$ level of tissues. Superoxide dismutase(SOD) and catalase were also activated during post-chilling period, although they showed a considerable lag time for activation. In contrast, glutathione peroxidase, another antioxygenic enzyme in cells, was not activated at all by preceding cold treatment of plants. The uptake of exogenous $O_{\overline{2}}$ by the roots of rice seedlings resulted in increase in the activities of SOD and catalase in root tissues. The supply of $H_2O_2$ to plan st brought about the activation of catalase in situ, while failing to exert any effect on the activation state of glutathione peroxidase. The results obtained in this work suggest that pyruvate accumulation in cells is the direct cause of the overproduction of $O_{\overline{2}}$ and thereby other toxic activated oxygen species, and that SOD and catalase may play a crucial role in the protection of plant cells against active oxygen-mediated chilling injury.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.168-173
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• 1991

The uptake of $Mn^{+2}$, a metal cofactor Mn-SOD, by rice seedings resulted in not only a substantial increase in SOD activity in leaf tissues of the plants, but also a significant enhancement of their cold tolerance : the relative extent of the cold tolerance appeared to accord with relative level of the SOD activity. In contrast, $Fe^{+3},\;Cu^{+2}$ and $Zn^{+2}$, which are the cofactors of Fe-SOD and Cu/Zn-SOD, were found to be ineffective for increasing the SOD activity as well as for improving the chilling-resistant capacity of the plants. The results suggest that Mn-SOD, which is most likely induced by its substrate(superoxide) and activated by the presence of $Mn^{+2}$a at high level, is the enzyme acting as an active component of the defense system against low temperature stress in rice plants. In addition, the application of abscisic acid which has been know to protect to some extent certain plants from chilling injury brought about an increase in SOD activity in rice tissues, providing another affirmative information for the crucial role of SOD under the circumstance of cold stress in plants.

• Applied Biological Chemistry
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• v.37 no.6
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• pp.433-440
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• 1994

The exposure of Clamydomonas reinhardtii to low temperatures resulted in an accumulation of cellular pyruvate that dissipated when the chilled cells returned to ambient temperature. The dissipation of pyruvate accumulation was accompanied by an increase in the production level of superoxide radicals $(O_2^-)$ in cells. The formation of $O_2^-$ at an excessive level during the post-chilling period was apparently countered by a substantial activation of superoxide dismutase (SOD). All these results are similar to those observed previously in rice seedlings subjected to the cold-treatment, implicating that a common mechanism is probably underlying for the primary processes of chilling injury both in higher plants and in algae. It was also observed that the activation of Mn-containing SOD contributes the major share in the increase of SOD activity of whole algal cells. Because Mn-SOD is present only in mitochondria, the observation corroborates the concept that the $O_2^-$ scavenging enzyme would be induced to cope with the cold treatment-caused adverse situation in mitochondria where the toxic active oxygen is produced at rates far exceeding the normal rate.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.110-115
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• 2016

A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas - Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli $DH5{\alpha}$ cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.