• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.109-115
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• 2000

FtsH is a membrane-bound, ATP-dependent metalloprotease that is involved in a variety of cellular functions including the regulation of responses to heat and stress shock. Previously, we had cloned and sequenced pneumococcal ftsH gene whose deduced amino acid sequence was very similar to those of several gram-positive bacteria and Escherichia coli, except for the N-terminal domain that was responsible for membrane anchoring. In order to better understand the role of Streptococcus pneumoniae FtsH, we expressed pneumococcal ftsH gene in Escherichia coli. When it was expressed from a strong promoter, $P_{tac}$, a considerable amount of the recombinant FtsH was produced, although the prolonged induction resulted in not only accumulation of breakdown products but also ceasing of the further growth of E. coli host. This indicated that the expression of the exogenous ftsH gene was tightly regulated since the excessive FtsH appeared detrimental to bacterial cells. In Western blotting, the pneumococcal FtsH protein, whether native or recombinant, was reactive to anti-E. coli FtsH serum. The observation that FtsH proteins were well conserved throughout the bacterial kingdom and its expression level was fine-tuned suggests an important role for this protein in the stress adaptation which may be related to infecting process by pneumococci.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.446-453
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• 1995

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.482-487
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• 1999

Periodate-oxidized soluble starch and maltohexaose reacted with ${\alpha}-NH_2$ group of free amino acids and ${\varepsilon}-NH_2$ group of peptidyl lysine. The result shows that periodate-oxidized soluble starch and maltooligosaccharides reacted with protein and formed Schiff base between CHO group of oxidized sugar and ${\varepsilon}-NH_2$ group of surface lysine of protein molecule. Carbon and hydrogen composition of sweet potato ${\beta}-amylase$ modified with oxidized soluble starch increased and it's nitrogen composition decreased. Carbohydrate contents of sweet potato ${\beta}-amylase$ modified with oxidized soluble starch were 13.2% (pentamer), 13.4% (monomer), and with oxidized maltohexaose were 9.7% (pentamer), 9.3% (monomer) by $phenol-H_2SO_4$ method. Alpha-amino group of N-terminal, and ${\varepsilon}-NH_2$ group of lysine, of sweet potato ${\beta}-amylase$ were reacted with oxidized soluble starch by dinitrophenylation were 70% (pentamer), 73% (monomer) and 33% (pentamer), 26% (monomer), respectively, in comparison with native enzyme.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.636-643
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• 2001

Bacillus sp. AIR-5, a strain from soil, produced an extracellular maltopentaose-forming amylase from amylose and soluble starch. This bacterium produced 8.9 g/l of maltopentaose from 40 g/l of soluble starch in a batch fermentation and the maltopentaose made up 90 % of the maltooligosaccharides produced (from maltose to maltoheptaose). The culture supernatant was concentrated using a 30 K molecular weight cut-off membrane and purified by DEAE-Cellulose and Sephadex G-150 column chromatographies. The purified protein showed one band on a native-PAGE and its molecular mass was estimated as 250 kDa. The 250-kDa protein was composed of tetramers of a 63-kDa protein. the isoelectric point of the purified protein was pH 6.9, and the optimum temperature for the enzyme activity was $45^{\circ}C$. The enzyme was quickly inactivated above $55^{\circ}C$, and showed a maximum activity at pH 8.5 and over 90% stability between a pH of 6 to 10. The putative N-terminal amino acid sequence of AIR-5 amylase, ATINNGTLMQYFEWYVPNDG, showed a 96% sequence similarity with that of BLA, a general liquefying amylase.

• Molecules and Cells
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• v.23 no.3
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• pp.370-378
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• 2007

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.1
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.