• Journal of Microbiology
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• v.41 no.1
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Microbiology and Biotechnology Letters
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• v.50 no.1
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• pp.71-80
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• 2022

Lipases (triacylglycerol acylhydrolases [EC 3.1.1.3]) are water-soluble enzymes. They catalyze the hydrolysis of fats and oils. A cold-active lipase from marine Bacillus cereus HSS, isolated from the Mediterranean Sea, Alexandria, Egypt, was purified and characterized. The total purification depending on lipase activity was 438.9 fold purification recording 632 U/mg protein. The molecular weight of the purified lipase was estimated to be 65 kDa using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The optimum substrate concentration, enzyme concentration, pH, and temperature were 1.5 mM, 100 µl, pH 6 and 10℃, respectively. The lipase was tolerant to NaCl concentrations ranging from 1.5 to 4.5%. The lipase was affected by the tested metal ions, and its activity was inhibited by 16% in the presence of 0.05 M SDS. The application of the cold-active lipase for the removal of an oil stain from a white cotton cloth showed that it is a promising biological agent for the treatment of oily wastes and other related applications. To the best of our knowledge, this is the first report of the purification and characterization of a lipase from marine B. cereus HSS isolated from the Mediterranean Sea.

• Bulletin of the Korean Chemical Society
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• v.31 no.4
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• pp.887-890
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• 2010

The family of ClpB protein is a molecular chaperone which protects cellular proteins from being aggregated upon exposure to severe environmental stresses in association with DnaK/DanJ/GrpE in the ATP-dependent manner. In a psychrophilic bacterium which survives at a subzero temperature, any functional role of cold-active ClpB protein can be rather crucial. In order to identify a ClpB encoding gene from a cold-adapted bacterium whose genome sequence has not been fully discovered, we have employed a series of PCR technologies, including a gradient PCR with homologous primers, an inverse PCR and a cassette PCR. The full sequence of PaclpB gene was successfully identified and compared with those of other psychrophilic species. We have further cloned the gene in E.coli expression systems and were able to induce PaClpB protein expression by IPTG, which help us understand a molecular mechanism for survival against extremely cold environments.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.5
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• pp.721-733
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• 2019

Objective: The objectives of this study were to investigate the thermal gelation properties and molecular forces of actomyosin extracted from two classes of chicken breast meat qualities (normal and pale, soft and exudative [PSE]-like) during heating process to further improve the understanding of the variations of functional properties between normal and PSE-like chicken breast meat. Methods: Actomyosin was extracted from normal and PSE-like chicken breast meat and the gel strength, water-holding capacity (WHC), protein loss, particle size and distribution, dynamic rheology and protein thermal stability were determined, then turbidity, active sulfhydryl group contents, hydrophobicity and molecular forces during thermal-induced gelling formation were comparatively studied. Results: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that protein profiles of actomyosin extracted from normal and PSE-like meat were not significantly different (p>0.05). Compared with normal actomyosin, PSE-like actomyosin had lower gel strength, WHC, particle size, less protein content involved in thermal gelation forming (p<0.05), and reduced onset temperature ($T_o$), thermal transition temperature ($T_d$), storage modulus (G') and loss modulus (G"). The turbidity, reactive sulfhydryl group of PSE-like actomyosin were higher when heated from $40^{\circ}C$ to $60^{\circ}C$. Further heating to $80^{\circ}C$ had lower transition from reactive sulfhydryl group into a disulfide bond and surface hydrophobicity. Molecular forces showed that hydrophobic interaction was the main force for heat-induced gel formation while both ionic and hydrogen bonds were different significantly between normal and PSE-like actomyosin (p<0.05). Conclusion: These changes in chemical groups and inter-molecular bonds affected protein-protein interaction and protein-water interaction and contributed to the inferior thermal gelation properties of PSE-like meat.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1353-1360
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• 2007

Three csp-like genes were identified in the Corynebacterium glutamicum genome and designated cspA, cspB, and cspA2. The genes cspA and cspA2 encode proteins, comprising of 67 amino acid residues, respectively. They share 83% identity with each other. Identity of those proteins with Escherichia coli Csp proteins was near 50%. The cspB gene encodes a protein composed of 127 amino acids, which has 40% and 35% sequence identity with CspA and CspA2, respectively, especially at its N-terminal region. Analysis of the gene expression profiles was done using transcriptional cat fusion, which identified not only active expression of the three genes at the physiological growth temperature of $30^{\circ}C$ but also growth phase-dependent expression with the highest activity at late log phase. The promoters of cspA and cspA2 were more active than that of cspB. The expression of the two genes increased by 30% after a temperature downshift to $15^{\circ}C$, and such stimulation was more evident in the late growth phase. In addition, the cspA gene appeared to show DNA-binding activity in vivo, and the activity increased at lower temperatures. Interestingly, the presence of cspA in multicopy hindered the growth of the host C. glutamicum cells at $20^{\circ}C$, but not at $30^{\circ}C$. Altogether, these data suggest that cspA, cspB, and cspA2 perform functions related to cold shock as well as normal cellular physiology. Moreover, CspA and its ortholog CspA2 may perform additional functions as a transcriptional regulator.

• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1260-1268
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• 2014

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative ${\beta}$-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Ser-x-x-Lys motif that is conserved among all ${\beta}$-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme is a serine protein and was active against p-nitrophenyl esters of $C_2$, $C_4$, $C_8$, and $C_{10}$. The optimum pH and temperature for enzyme activity were pH 9.0 and $30^{\circ}C$, respectively. Relative activity of 55% remained at up to $5^{\circ}C$ with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, and $Hg^{2+}$ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

• Fisheries and Aquatic Sciences
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• v.16 no.4
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• pp.311-318
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• 2013

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.236-242
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• 2011

A psychrotrophic bacterium, Arthrobacter sp. ON14, isolated from Antarctica, was shown to exhibit a high ${\beta}$-galactosidase activity at a low temperature. A genomic library of ON14 was constructed and screened for ${\beta}$-galactosidase genes on functional plates containing 5-bromo-4-chloro-3-indolyl-${\beta}$-D-galactopyranoside (X-gal) as the substrate. Two different ${\beta}$-galactosidase genes, named as galA, galB, were found in ON14. Computational analyses of the genes revealed that the encoded protein GalA belongs to family 2 of glycosyl hydrolysases and is a cold-active protein, whereas GalB belongs to family 42 of glycosyl hydrolysases and is a mesophilic protein. Reverse transcription analyses revealed that the expression of galA is highly induced at a low temperature ($4^{\circ}C$ ) and repressed at a high temperature ($28^{\circ}C$ ) when lactose is used as the sole carbon source. Conversely, the expression of galB is inhibited at a low temperature and induced at a high temperature. The purified GalA showed its peak activity at $15^{\circ}C$ and pH 8. The mineral ions $Na^+$, $K^+$, $Mg^{2+}$, and $Mn^{2+}$ were identified as enzyme activators, whereas $Ca^{2+}$ had no influence on the enzyme activity. An enzyme stability assay revealed that the activity of GalA is significantly decreased when it is incubated at $45^{\circ}C$ for 2 h, and all its activity is lost when it is incubated at $50^{\circ}C$.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.20-20
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• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.