• Korean Journal of Fisheries and Aquatic Sciences
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• v.20 no.2
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• pp.136-145
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• 1987

From the sea-urchin, Hemicentrotus pulcherrismus, we have purified by four column chromatographic steps for DNA polymerase $\alpha$ activity. The molecular weight of DNA polymerase u was determined to be around 137,000-138,000 by Sephadex G-200 gel filtration and SDS-polyacrylamide gel electrophoresis. The purified enzyme had the optimal activity at pH 7.4. This enzyme showed to be a function of the metal ion $K^+,\;Na^+$\;and\;Mg^{2+}$ employed as activators, the optimum $K^+$\;or\;Na^+ concentration were 20 mM or 25mM and the optimum $Mg^{2+}$ concentration was 10 mM. The enzyme activity was inhibited by N-ethyl-maleimide, aphidicolin, cytosine $\beta-D-arabinofuranoside$ 5'-triphoshate (ara CTP) and phosphonoacetic acid.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.62-69
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• 1999

In order to investigate the critical amino acid residues involved in the catalytic activities of $\beta$-cyclodextrin glucanotransferase ($\beta$-CGTase) excreted by Bacillus firmus var. alkalophilus, the amino acid residues in $\beta$-CGTase were modified by various site-specific amino acid modifying reagents. The cyclizing and amylolytic activities of $\beta$-CGTase were all seriously reduced after treatment with Woodward's reagent K (WRK) modifying aspartic/glutamic acid, N-bromosuccinimde (NBS) modifying tryptophan, and diethylpyrocarbonate (DEPC) modifying histidine residues. The roles of tryptophan and histidine residues in $\beta$-CGTase were further investigated by measuring the protection effect of various substrates during chemical modification, comparing protein mobility in native and affinity polyacrylamide gel electrophoresis containing soluble starch, and comparing the $K_m$ and $V_{max}$ values of native and modified enzymes. Tryptophan residues were identified as affecting substrate-binding ability rather than influencing catalytic activities. On the other hand, histidine residues influenced catalytic ability rather than substrate-binding ability, plus histidine modification had an effect on shifting the optimum pH and pH stability.

• Microbiology and Biotechnology Letters
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• v.22 no.2
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• pp.134-142
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• 1994

The neucleotide sequences of the xylA gene encoding $\beta $-xylosidase of Bacillus stearothermophilus and is its flanking regions were datermined. Three open reading frame(ORFs) were found, one of which(ORF1) appeared to code for the $\beta $-xylosidase. The 1830 base pair ORF1 encoded 609 amino acids starting from a TTG initiation codon. The molecular weight deduced from the nucleotide sequence(68 KD) was in agreement with that estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme(66 KD). The Shine-Dalgarno sequence(5'-AGGAGG-3') was found 11 bp upstream of the initiation codon. Further 15 bp upstream, there observed a potential transcription initiation signals. The putative -10 sequence(CATAAT) and -35 sequence(TTGTTA) coresponded closely to the consensus sequences for Bacillus subtilis RNA polymerase with major sigma factor. The guanine-plus-cytosine content of the coding region of the xylA gene was 56mol% while that of the third position of the codons was 63 mol%. Based on the comparison with the amino acid sequences of several other carbohydrate degrading enzymes, two conserved regions, possibly participating in the catalytic mechamism of $\beta $-xylosidase xylA, were identified in 278-298 and 329-350 regions of the translated xylA gene. The nucleotide sequence of the xylA was found to exhibit no homology to any other genes so far reproted.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.887-892
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• 2015

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuDof P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

• The Korean Journal of Physiology
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• v.25 no.2
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• pp.115-123
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• 1991

Molecular association of glucose transporters with the other proteins in the plasma membrane was assessed by gel electrophoresis and immunoblot techniques. Approximately $31.5{\pm}5.1%$ of GLUT-4, $64.8{\pm}2.7%$ of clathrin, 48.7% of total protein in the plasma membrane (PM) were found insoluble upon extraction with 1% Tx-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the Tx-100 insoluble PM fraction contained about 4 major polypeptides with apparent molecular weight of above 200, 100-120, 80 and 30-35 KDa that were readily removed upon wash with a high pH buffer which is known to remove clathrin and 0.5 M Tris-buffer which is known to remove assembly proteins (AP). Immunoblotting of GLUT4 and clathrin against specific antibodies showed that GLUT-4 and clathrin were co-solubilized up to 84.6% and 82.7% respectively by wash with a high pH buffer and 1% Tx-100. When the membrane was pre-washed with a high pH buffer and 0.5 M Tris solution, GLUT4 and clathrin were not solubilized further suggesting that GLUT4 molecules are in molecular association with clathrin, AP and/or other extrinsic membrane proteins in plasma membrane and the formation of clathrin-coated structures might be involved in insulin stimulated glucose transporter translocation mechanism.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.14 no.8
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• pp.4106-4110
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• 2013

Apolipophorin-III (apoLp-III) was isolated and purified from the last larval hemolymph of Galleria mellonella by the KBr gradient ultracentrifugation and gel chromatography (Sephadex G-100). In this paper, we examined that apoLp-III is taken up into the last larval fat bodies in Galleria mellonella. The last larval fat body tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)-labeled apoLp-III (FITC-apoLp-III). Fluorescein microscopy and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed that the last larval fat body tissues internalize FITC-apoLp-III. The results show that the apoLp-III is taken up by the last larval fat body.

• Fisheries and Aquatic Sciences
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• v.2 no.2
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• pp.218-225
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• 1999

A protease without tryptic and chymotryptic activities was purified from the hepatopancreas of shrimp, Penaeus orientalis, using Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, Mono-Q, and gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 27kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS­PAGE). The amino acid composition of the protease was different from that of protease from P. japonicus or trypsin from P. orientalis. The protease was completely inhibited by benzamidine, $N\alpha-p-tosyl-L-lysine$ chloromethyl ketone (TLCK), and phenylmethylsulfonyl fluoride (PMSF) and was not affected by leupeptin, pepstatin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetate, and ethylenediamine tetra acetate (EDTA). The enzyme did not have any activity against Na-benzoyl-DL-arginine p-nitroanilide (BAPNA) or N-benzoyl-L-tyrosine ethyl ester (BTEE) which are specific substrates of trypsin and chymotrypsin, respectively. However, the protease showed hydrolytic activity for a carboxyl terminal of Tyr, Trp, Phe, Glu, and Cys.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.244-250
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• 2000

An ${\alpha}-glucosidase$ gene (aglA) from thermophilic Bacillus sp. DG0303 was cloned, sequenced, and expressed in Escherichia coli. The aglA was localized to the 2.1-kb PvuI-XmnI region within the 5.9-kb DNA insert of the gybrid plasmid pAG1. The gene consisted of an open reading frame of 1,686 bp with an unusual GTG initiation codon and TGA termination codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 562 amino acid residues with a M, of 66,551 dalton. A comparative amino acid sequence analysis revealed that DG0303 ${\alpha}-glucosidase$ is related to bacillary oligo-1, 6-glucosidases. The Bacillus sp. DG0303 ${\alpha}-glucosidase$ showed a high sequence identity (36-59%) to the B. flavocaldarius, B. cereus, and B. thermoglucosidasius oligo-1, 6-glucosidases. The number of prolines in theses four ${\alpha}-glucosidases. was observed to increase with increasing thermostability of these enzymes. The cloned ${\alpha}-glucosidase was purified from E. coli $DH5{\alpha}$ bearing pAG1 and characterized. The recombinant enzyme was identical with the native enzyme in its optimum pH and in its molecular mass, estimated by sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The temperature optimum of the cloned ${\alpha}-glucosidase$ was lower than that of the native enzyme.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.73-79
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• 1994

The optimum cultural temperature and time for the pullulanase production by Bacillus cereus subsp. mycoides were 35$\circ $C and 48 hrs, respectively. The addition of egg albumin and casein to the basal medium increased the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. specific activity of the purified enzyme was 82.37 U/mg protein and yield of theenzyume activity was 62.1%. The purified enzuyme showed a single band on ployacrylamide disc gel electrophoresis and its molecular weight was estimated to be 66.,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelcular point for the purified enzyme was pH 5.0. The optimum temperature and pH were 50$\circ $C and pH 6.5, respectively. The purified enzyme was stable below 40$\circ $C and in the pH range of 6.5~10.0 The pullulanase activity was greatly inhited by Ag$^{+}$, Hg$^{2+}$ and EDTA, and its heat stability was increased by the addition of Ca$^{2+}$. The tydrolysis product with the enzyme on pullulan was maltotriose.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.221-226
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• 2000

Cephalosporin-C deacetylase (CAH) catalyzes the deacetylation of cephalosporin derivatives. A novel gene encoding the CAH from Bacillus sp. KCCM10143 was cloned and sepuenced. The uncleotide sequence contained an open reading frame encoding a polypeptide consisting of 217 amino acids and a molecular weight of 24 kDa which was in good agreement with the value obtained by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An expression plasmid was constructed by inserting the CAH gene into the region of the pTrc99A expression vector. An active from of the CAH protein was expressed in the soluble fraction obtained after cell disruption. in fermentation using a 5-1 jar fementer, the transformant E. coli JM109 (pDST654) produced 4.12 U of CAH per ml of culture during 16 h of incubation.