• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.579-586
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• 2010

A genomic DNA fragment encoding a putative maltooligosyltrehalose synthase (NfMTS) for trehalose biosynthesis was cloned by the degenerate primer-PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTS was 2,799 bp in length and encoded 933 amino acid residues constituting a 106.6 kDa protein. The deduced amino acid sequence of NfMTS contained 4 regions highly conserved for MTSs. By expression of NfMTS in E. coli, it was demonstrated that the recombinant protein catalyzed the conversion of maltohexaose to maltooligosyl trehalose. The $K_m$ of the recombinant enzyme for maltohexaose was 1.87 mM and the optimal temperature and pH of the recombinant enzyme was at $50^{\circ}C$ and 7.0, respectively. The expression of MTS of N. flagelliforme was upregulated, and both trehalose and sucrose contents increased significantly in N. flagelliforme during drought stress. However, trehalose accumulated in small quantities (about 0.36 mg/g DW), whereas sucrose accumulated in high quantities (about 0.90 mg/g DW), indicating both trehalose and sucrose were involved in dehydration stress response in N. flagelliforme and sucrose might act as a chemical chaperone rather than trehalose did during dehydration stress.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1022-1030
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• 2004

The gene encoding Aquifex pyrophilus (Apy) DNA polymerase was cloned and sequenced. The Apy DNA polymerase gene consists of 1,725 bp coding for a protein with 574 amino acid residues. The deduced amino acid sequence of Apy DNA. polymerase showed a high sequence homology to Escherichia coli DNA polymerase I-like DNA polymerases. It was deduced by amino acid sequence alignment that Apy DNA polymerase, like the Klenow fragment, has only the two domains, the $3'{\rightarrow}5'$ exonuclease domain and the $5'{\rightarrow}3'$ polymerase domain, containing the characteristic motifs. The Apy DNA polymerase gene was expressed under the control of T7lac promoter on the expression vector pET-22b(+) in E. coli. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $UNO^{TM}$ Q column chromatographies. The optimum pH of the purified enzyme was 7.5, and the optimal concentrations of KCl and $Mg^{2+}$ were 20 mM and 3 mM, respectively. Apy DNA polymerase contained a double strand-dependent $3'{\rightarrow}5'$ proofreading exonuclease activity, but lacked any detectable $5'{\rightarrow}3'$ exonuclease activity, which is consistent with its amino acid sequence. The somewhat lower thermostability of Apy DNA polymerase than the growth temperature of A. pyrophilus was analyzed by the comparison of amino acid composition and pressure effect.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.546-554
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• 2019

This study, for the first time, reports the functional expression of lipase B derived from the yeast Candida antarctica (CALB) in Corynebacterium strain using the Escherichia coli plasmid PK18. The CALB gene fragment encoding a 317-amino-acid protein was successfully obtained from the total RNA of C. antarctica. CALB was readily produced in the Corynebacterium strain without the use of induction methods described in previous studies. This demonstrated the extracellular production of CALB in the Corynebacterium strain. CALB produced in the Corynebacterium MB001 strain transformed with pEC-CALB recombinant plasmid exhibited maximum extracellular enzymatic activity and high substrate affinity. The optimal pH and temperature for the hydrolysis of 4-nitrophenyl laurate by CALB were 9.0 and 40℃, respectively. The enzyme was stable at pH 10.7 in the glycine-KOH buffer and functioned as an alkaline lipase. The CALB activity was inhibited in the presence of high concentration of Mg²⁺, which indicated that CALB is not a metalloenzyme. These properties are key for the industrial application of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.277-285
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• 2009

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-$\beta$-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, $\alpha$-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.214-221
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• 1998

A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme II ($EII^{Glc}$) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing an Escherichia coli mutation affecting a ptsG gene, and the complete DNA nucleotide sequence was determined. The cloned gene was identified to be a ptsG, which enables the E. coli transportment to use glucose more efficiently than mannose as the sole carbon source in an M9 minimal medium. The ptsG gene of B. ammoniagenes consists of an open reading frame of 1,983 nucleotides putatively encoding a polypeptide of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes $EII^{Glc}$ shows, at $46\%$, the highest degree of sequence similarity with the Corynebacterium glutamicum EII specific for both glucose and mannose. In addition, the $EII^{Glc}$ shares approximately $30\%$ sequence similarities with sucrose-specific and ${\beta}$-glucoside-specific EIIs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of $EII^{Glc}$ is also similar to that of E. coli enzyme $IIA^{Glc}$, specific for glucose ($EIIA^{Glc}$). The B. ammoniagenes $EII^{Glc}$ consists of three domains; a hydrophobic region (EIIC) and two hydrophilic regions (EIIA, EIIB). The arrangement of structural domains, IIBCA, of the $EII^{Glc}$ is identical to those of EIIs specific for sucrose or ${\beta}$-glucoside. While the domain IIA was removed from the B. ammoniagenes $EII^{Glc}$ the remaining domains IIBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking $EII^{Glc}$ activity with $EIIA^{Glc}$ of the E. coli mutant. $EII^{Glc}$ contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.251-259
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• 2013

An endoxylanase gene (PoxynA) that belongs to the glycoside hydrolase (GH) family 11 was cloned from a xylanolytic strain, Penicillium oxalicum B3-11(2). PoxynA was overexpressed in Trichoderma reesei QM9414 by using a constitutive strong promoter of the encoding pyruvate decarboxylase (pdc). The high extracellular xylanase activities in the fermentation liquid of the transformants were maintained 29~35-fold higher compared with the wild strain. The recombinant POXYNA was purified to homogeneity, and its characters were analyzed. Its optimal temperature and pH value were $50^{\circ}C$ and 5.0, respectively. The enzyme was stable at a pH range of 2.0 to 7.0. Using beechwood as the substrate, POXYNA had a high specific activity of $1,856{\pm}53.5$ IU/mg. In the presence of metal ions, such as $Cu^{2+}$, and $Mg^{2+}$, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of $Mn^{2+}$ and $Fe^{2+}$. The recombinant POXYNA hydrolyzed birchwood xylan, beechwood xylan, and oat spelt xylan to produce short-chain xylooligosaccharides, xylopentaose, xylotriose, and xylobiose as the main products. This is the first report on the expression properties of a recombinant endoxylanase gene from Penicillium oxalicum. The properties of this endoxylanase make it promising for applications in the food and feed industries.

• Proceedings of the Korean Society of Life Science Conference
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• 2002.12a
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• pp.13-19
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• 2002

To elucidate the regulatory mechanism for expression of sialyl-glycoconjugates and their biological functions, ninetheen sialyltransferase cDNAs including eleven by our group or co-works have been cloned and characterized so far. The cloned sialyltransferases are classified into four families according to the carbohydrate linkages they synthesize: ${\alpha}2,3-sialyltransferase$ (ST3Gal I-VI), ${\alpha}$ 2,6-sialyltransferase (ST6Gal I), GalNAc ${\alpha}$ 2,6-sialyltransferase (ST6GalNAc I-VI), and ${\alpha}2,8-sialyltransferase$ (ST8Sia I-VI). Each of the sialyltransferase genes is differentially expressed in a tissue-, cell type-, and stage-specific manner. These enzymes differ in their substrate specificity and various biochemical parameters. However, enzymatic analysis conducted in vitro with recombinant enzyme revealed that one linkage can be synthesized by multiple enzymes. We present here an overview of structure and function of sialyltransferases performed by our group and co-works. Genomic structures and transcriptional regulation of two kinds of human sialyltransferase gene are also presented.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.2
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• pp.189-195
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• 2008

The sequence of hydroxypyruvate isomerase gene was obtained in NCBI. In this study, the hydroxypyruvate isomerase gene of Bombyx.mori was identified and annotated with bioinformatics tools. The result was confirmed by RT-PCR, prokaryotic expression, mass spectrographic analysis and sub-cellular localization. The hydroxypyruvate isomerase cDNA comtains a 783bp ORF, and has 4 exons. The deduced protein has 260 amino acid residues with the predicted molecular weight of 29169.30 Da, isoelectric point of 6.10, and contains conserved PRK09997 and Hfi domains. The hydroxypyruvate isomerases of Nasonia vitripennis and Bombyx mori have a high homology. Through RTPCR analysis, we found that this transcript was present in testis, ovary, blood-lymph, fat body, midgut, silk gland and tuba Malpighii. This protein was located in cytoplasm through immunohistochemistry. We submitted the cloned gene under the accession number EU344910. The enzyme has been classified under accession number EC 5.3.1.22.

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Journal of Photoscience
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• v.4 no.3
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• pp.141-145
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• 1997

Polymerase chain reaction(PCR) was conducted with a pea cDNA library using two primers synthesized from homology analysis of amino acid sequences for animal and plant cytosolic FBPases. A PCR product with 650 bp long was cloned into pGEM-T vector and sequenced. The deduced amino acid sequence of the cDNA fragment was 98, 91, and 85% homologous with those of cytosolic FBPases from spinach, sugarbeet, and sugarcane, respectively. It was 51% homologous with amino acid sequence of FBPase from pea chloroplasts. Northern blot analysis was proceeded with the cDNA clone resulting that 1.2 kb transcript was highly expressed in light-grown pea leaves but almost not expressed in dark-grown etiolated pea seedlings. When peas grown in the light for 10 days were transferred to darkness, the transcript was gradually decreased with dark treatment, indicating that the expression of the enzyme was induced by continuous white light but suppressed by dark treatment. Pea cytosolic FBPase was highly expressed in leaves with trace amounts in stems. but almost not expressed in roots.