• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1218-1230
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• 2021

Cold-adapted plant growth-promoting bacteria (PGPB) with multiple functions are an important resource for microbial fertilizers with low-temperature application. In this study, culturable cold-adapted PGPB strains with nitrogen fixation and phosphorus solubilization abilities were isolated. They were screened from root and rhizosphere of four dominant grass species in nondegraded alpine grasslands of the Qilian Mountains, China. Their other growth-promoting characteristics, including secretion of indole-3-acetic acid (IAA), production of siderophores and ACC deaminase, and antifungal activity, were further studied by qualitative and quantitative methods. In addition, whether the PGPB strains could still exert plant growth-promoting activity at 4℃ was verified. The results showed that 67 isolates could maintain one or more growth-promoting traits at 4℃, and these isolates were defined as cold-adapted PGPB. They were divided into 8 genera by 16S rRNA gene sequencing and phylogenetic analysis, of which Pseudomonas (64.2%) and Serratia (13.4%) were the common dominant genera, and a few specific genera varied among the plant species. A test-tube culture showed that inoculation of Elymus nutans seedlings with cold-adapted PGPB possessing different functional characteristics had a significant growth-promoting effect under controlled low-temperature conditions, including the development of the roots and aboveground parts. Pearson correlation analysis revealed that different growth-promoting characteristics made different contributions to the development of the roots and aboveground parts. These cold-adapted PGPB can be used as excellent strain resources suitable for the near-natural restoration of degraded alpine grasslands or agriculture stock production in cold areas.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.681-686
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• 2015

The purpose of this study was to find a cold-adapted and surfactant-stable alginate lyase as a candidate for biotechnological and industrial applications. The gene for a new alginate lyase, AlyL1, from Agarivorans sp. L11 was cloned and expressed in Escherichia coli. The recombinant AlyL1 was most active at 40℃ (1,370 U/mg). It was a cold-adapted alginate lyase, which showed 54.5% and 72.1% of maximum activity at 15℃ and 20℃, respectively. AlyL1 was an alkaliphilic enzyme and most active at pH 8.6. In addition, it showed high stability in the presence of various surfactants at a high concentration (from 0.1% to 1% (w/v)). AlyL1 was an endo-type alginate lyase that degraded both polyM and polyG blocks, yielding disaccharides and trisaccharides as the main products. This is the first report of the cloning and functional expression of a cold-adapted and surfactant-stable alginate lyase. AlyL1 might be an interesting candidate for biotechnological and industrial applications.

• KSBB Journal
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• v.16 no.6
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• pp.626-631
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• 2001

Lactobacilli have been considered to play important roles in the health of human vagina. They secrete inhibitory substances to prevent vaginal infection by pathogenic organisms. In a previous study, we have isolated several lactobacilli from Korean woman and one of them (KLB46) was selected and indentified as Lactobacillu crispatus which showed high antimicrobial activity. In this study. cold adaptation prior to subsequent stresses exposure was examined whether L. crispatus KLB46 maintain the viability better than the non-adapted calls under stresses. For pharmaceutical formulation, the lyophilization process is required where stresses such as freezing/thawing and dehydration are routinely applied. Formulated L. crispatus KLB46 can be used for ecological treatment of bacterial vaginosis. The response of cold-adapted cells to other environmental stresses such as acid, heat, ethanol, NaCl, and H$_2$O$_2$ was also examined. The results showed that cold-adapted cells maintained higher survival rate compared with the non-adapted cells (freezing-thawing. 3-folds; dehydration: 3-folds; acid, 3-folds; heat, 10-folds). However, we did net observe any positive effect of cold adaptation on other stresses such as ethanol, NaCl and H$_2$O$_2$. When chloramphenicol was added during cold adaptation, adaptation effect was abolished. This confirms that de novo protein synthesis is necessary during the adaptation process. Moreover, we have identified cold shock protein homolog that codes for a major cold shock protein by PCR amplification using degenerate primers.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2087-2097
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• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.52.2-52.2
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• 2007

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• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.235-239
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• 2022

Psychrophiles have evolved to produce cold-adapted enzymes to enable survival in low-temperature environments. In this study, the cold adaptation of 5-enolpyruvylshikimate-3-phosphate synthase (CpsEPSPS) from Colwellia psychrerythraea, a model psychrophile, was analyzed. The optimum temperature for the activity of CpsEPSPS was found to be 25℃, with 35% activity remaining at 5℃. However, the unfolding temperature of CpsEPSPS was 54℃. This phenomenon is frequently observed in cold-active enzymes. As is the cases for most cold-active enzymes, the K_m values of CpsEPSPS for its substrates were higher than those of Escherichia coli EPSPS. These results indicate that CpsEPSPS is cold-adapted, but not perfectly.

• Journal of Microbiology and Biotechnology
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• v.28 no.3
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• pp.448-453
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• 2018

In this study, a 107 kDa protease from psychrophilic Janthinobacterium lividum PAMC 26541 was purified by anion-exchange chromatography. The specific activity of the purified protease was 264 U/mg, and the overall yield was 12.5%. The J. lividum PAMC 25641 protease showed optimal activity at pH 7.0-7.5 and $40^{\circ}C$. Protease activity was inhibited by PMSF, but not by DTT. On the basis of the N-terminal sequence of the purified protease, the gene encoding the cold-adapted protease from J. lividum PAMC 25641 was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3) as an intracellular soluble protein.

• Microbiology and Biotechnology Letters
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• v.46 no.2
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• pp.111-113
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• 2018

In this study, purification of cold-adapted protease from Janthinobacterium sp. was investigated. First, using gradient precipitation, protease was confirmed to be deposited in the 30-80% range of ammonium sulfate. Next, DEAE-Sepharose column was used for the binding of the protease under various conditions. The optimal binding condition was found to be pH 8.5 and flow rate of 30 ml/h. Under the optimal condition, the protease was purified with 29% recovery yield. This result can be useful for the purification of other cold-adapted protein.

• Genomics & Informatics
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• v.9 no.2
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• pp.79-84
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• 2011

Two initial models of cold-adapted lipase PsLip1 have been constructed, based on homology with the bacterial lipases Chromobacterium viscosum (CvLip) and Pseudomonas cepacia (PcLip), whose X-ray structures have been solved and refined to high resolution. The mature polypeptide chains of these lipases have 84% similarity. The models of Mod1 and Mod2 have been compared with the tertiary structures of CvLip and PcLip, respectively, and analyzed in terms of stabilizing interactions. Several structural aspects that are believed to contribute to protein stability have been compared: the number of conserved salt bridges, aromatic interactions, hydrogen bonds, helix capping, and disulfide bridges. The 3-dimensional structural model of PsLip1 has been constructed in order to elucidate the structural reasons for the decreased thermostability of the enzyme in comparison with its mesophilic counterparts.

• Journal of Veterinary Science
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• v.21 no.6
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• pp.85.1-85.6
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• 2020

A cold-adapted porcine reproductive and respiratory syndrome virus (CA-VR2332) was generated from the modified live virus strain VR2332. CA-VR2332 showed impaired growth when cultured at 37℃ with numerous mutations (^S731^F, ^E819^D, ^G975^E, and ^D1014^N) in the hypervariable region of the NSP2, in which the mutation ^S731^F might play a vital role in viral replication at 30℃. Conserved amino acid sequences of the GP5 protein suggests that CA-VR2332 is a promising candidate for producing an effective vaccine against PRRSV infection. Further studies on replication and immunogenicity in vivo are required to evaluate the properties of CA-VR2332.