• Microbiology and Biotechnology Letters
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• v.44 no.2
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• pp.130-139
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• 2016

A bacterial strain, producing an excellent lipolytic enzyme, was isolated from the intestinal tracts of an earthworm (Eisenia fetida). The strain was identified as Aeromonas hydrophila by phenotypic, chemotaxonomic characteristics and 16S ribosomal DNA analysis, and was designated as Aeromona hydrophila PL43. The lipolytic enzyme from A. hydrophila PL43 was purified via 35−45% ammonium sulfate precipitation, DEAE-sepharose fast flow ion-exchange, and sephacryl S-300HR gel filtration chromatography. The yield of the purified enzyme was 3.7% and 2.5% of the total activity of crude extracts with p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as substrates, respectively. The molecular weight of the purified enzyme was approximately 74 kDa using gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and zymography. The optimal activity of purified enzyme was observed at 50℃ and pH 8.0 using pNPB, and 60℃ and pH 8.0 using pNPP. The purified enzyme was stable in the ranges 20− 60℃ and pH 7.0−10.0. The activity of purified enzyme was inhibited by PMSF, pepstatin A, Co²⁺, Cu²⁺, and Fe²⁺, but was recovered by metal chelating of EDTA. The K_m and V_max values of the purified enzyme were 1.07 mM and 7.27 mM/min using pNPB and 1.43 mM and 2.72 mM/min using pNPP, respectively.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.483-489
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• 1998

We carried out the expression and characterization of yeast thioredoxin system including thioredexin 1 (Trx1), Trx2, thioredoxin reductase (TR), and a novel thioredoxin (Trx3), which was reported in the data base of Saccharomyces genome. The Trx1, 2 and TR were expressed as soluble proteins in E. coli and the sizes of purified proteins were equal to the reported their molecular weights. The expressed Trx3 was found in both soluble fraction and precipitate. The size of Trx3 purified from soluble fraction of E. coli crude extracts was estimated as 14 kDa on SDS-PAGE instead of 18 kDa for Trx3 in precipitate. N-terminal amino acid sequence of the small size of purified Trx3 from soluble fraction was analyzed as FQSSYTS which is correspond to the sequence from 20 to 26 for Trx3. Trx3 together with thioredoxin reductase and NADPH was able to reduce the disulfide bridge of insulin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Trx3 stimulated the antioxidant effect of thioredoxin peroxidase 1 (TPx1) which inhibited inactivation of glutamine synthetase (GS) in dithiothreitol (DTT) containing metal catalyzed oxidation system. The stimulation effect of Trx3 was 10% of the effect of either Trx1 or Trx2. In addition, Trx3 could reduce the disulfide of TPx to thiol, so that the TPx had thioredoxin dependant peroxidase activity. In western blotting analysis, antibodies against purified Trx3 did not cross-react with crude extracts of yeast, purified Trx1, and Trx2 proteins. But, in PCR reaction using the cDNA library of yeast as a template, gene encoding of trx3 was amplified.

• Analytical Science and Technology
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• v.28 no.2
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• pp.117-124
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• 2015

Genetically modified (GM) papaya line 55-1, which is resistant to PRSV infection, has been marketed globally. Prompt and sensitive protocols for specific detections are essential for the traceability of this line. Here, an event- and construct-specific real-time polymerase chain reaction (RT-PCR) method was established to detect 55-1. Qualitative detection was possible for fresh papaya fruit up to dilutions of 0.005% and 0.01% for the homozygous SunUp and heterozygous Rainbow cultivars, respectively, in non-GM papaya. The method was applied in the qualitative detection of 55-1 in eight types of commercially processed papaya products. Additionally, papaya products were monitored to distinguish GM papaya using the P35S and T-nos RT-PCR detection methods. As expected, detection capacity was improved via modified sample preparation and the established RT-PCR detection method. Taking these results together, it can be suggested that a suitable method for the extraction and purification of DNA from processed papaya products was established for the detection of GM papaya.

• Korean Journal of Agricultural Science
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• v.23 no.1
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• pp.80-89
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• 1996

The expression and secretion of human lactoferrin (hLf) in Sacclnromyces diastaticus were performed. 1. For the secretion of hLf in yeast, recombinant plasmid pYEGLf was constructed using promoter, secretion signal sequence of glucoamylase I gene (STA1) and transcriptional terminator of GAL7 gene. 2. Each correct recombinant plasmid was selected by mini-preparation of plasmid DNA from E coli transformant and restriction enzyme digestion analysis. The selected plasmids, pYEGLf, were transformed into S. diastaticus YIY345 as a expression host, respectively. 3. Western blot analysis using rabbit anti-hLf was carried out to identify expressed hLf. Positive signals were shown in culture supernatant of pYEGLf transformant. 4. About $100{\mu}g-1mg$ of concentrated culture supernatant of positive clone were loaded on paper disc and tested for the antimicrobial activity against E coli. However, no activity was observed. We concluded that this fact results from low concentration of hLf secreted from yeast, compared with the fact that MIC of hLf is as high as $3mg/m{\ell}$. Therefore, the purification of secreted hLf may be require to investigate the antimicrobial activity. From this study, the feasibility of low-cost production of sufficient quantities of human lactofferin for nutritional and therapeutical applications were suggested.

• BMB Reports
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• v.35 no.3
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• pp.330-336
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• 2002

The lgtB genes that encode $\beta$-1,4-galactosyltransferases from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at $37^{\circ}C$ (33 kDa), most of the $\beta$-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to $25^{\circ}C$, however, the solubility of the $\beta$-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned $\beta$-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at $25^{\circ}C$. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the $Mn^{+2}$ ions for its action. The $Mg^{+2}$ and $Ca{+2}$ ions showed about half of the galactosyltransferase activities with the $Mn^{+2}$ ion. In the presence of the $Fe^{+2}$ ion, partial activation was observed with the $\beta$-1,4-galactosyltransferase from N. meningitidis(64% of the enzyme activity with the $Mn^{+2}$$Ni^{+2}$, $Zn^{+2}$, and $Cu^{+2}$ ions could not activate the $\beta$-1,4-galactosyltransferase activity. The inhibited enzyme activity with the $Ni^{+2}$ ion was partially recovered with the $Mn^{+2}$$Fe^{+2}$, $Zn^{+2}$, and $Cu^{+2}$ ions, the $Mn^{+2}$$\beta$-1,4-galactosyltransferase activity was 1.5-fold stimulated with the non-ionic detergent Triton X-100 (0.1-5%).

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1460-1468
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• 2007

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from $20^{\circ}C$ to $60^{\circ}C$C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's $K_m$ value and $V_{max}$ of the catalase for $H_2O_2$ were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of $A_{406}$ to $A_{280}$ for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1 in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.

• The Korean Journal of Food And Nutrition
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• v.17 no.1
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• pp.47-52
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• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• Journal of Life Science
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• v.23 no.2
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• pp.273-281
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• 2013

The purpose of this research was to investigate the production and characterization of alkaline protease from Micrococcus sp. PS-1 newly isolated from seawater. Micrococcus sp. PS-1 was grown in Luria-Bertani (LB) medium. Its optimal temperature and pH for growth were $30^{\circ}C$ and 7.0, respectively. The effect of nitrogen sources was investigated on optimal enzyme production. A high level of alkaline protease production occurred in LB broth containing 2% skimmed milk. The protease was purified in a 3-step procedure involving ultrafiltration, acetone precipitation, and dialysis. The procedure yielded a 16.43-purification fold, with a yield of 54.25%. SDS-PAGE showed that the enzyme had molecular weights of 35.0 and 37.5 kDa. Its maximum protease activity was exhibited at pH 9.0 and $37^{\circ}C$, and its activity was stable at pH 8.0-11.0 and $25-37^{\circ}C$. The protease activity was strongly inhibited by PMSF, EDTA, and EGTA. Taken together, the results demonstrate that the protease enzyme from Micrococcus sp. PS-1 probably belongs to a subclass of alkaline metallo-serine proteases.

• Journal of Life Science
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• v.29 no.10
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• pp.1126-1135
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• 2019

From a sample of bamboo byproduct, the protease-producing yeast strain CO-1 was newly isolated. Strain CO-1 is spherical to ovoid in shape and measures $3.1-4.0{\times}3.8-4.4{\mu}m$. For the growth of strain CO-1, the optimal temperature and initial pH were $30^{\circ}C$ and 4.0, respectively. The strain was able to grow in 0.0-15.0%(w/v) NaCl and 0.0-9.0%(v/v) ethanol. Based on a phylogenetic analysis of its 18S rDNA sequences, strain CO-1 was identified as Pichia anomala. The extracellular protease produced by P. anomala CO-1 was partially purified by ammonium sulfate precipitation, which resulted in a 14.6-fold purification and a yield of 7.2%. The molecular mass of the protease was recorded as approximately 30 kDa via zymogram. The protease activity reached its maximum when 1.0%(w/v) CMC was used as the carbon source, 1.0%(w/v) yeast extract was used as the nitrogen source, and 0.3%(w/v) $MnSO_4$ was used as the mineral source. The protease revealed the highest activity at pH 7.0 and $30^{\circ}C$. This enzyme maintained more than 75% of its stability at a pH range of 4.0-10.0. After heating at $65^{\circ}C$ for 1 hr, the neutral protease registered at 60% of its original activity. The protease production coincided with growth and attained a maximal level during the post-exponential phase.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.22 no.6
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• pp.542-549
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• 2021

The purpose of this study was to provide an economical and easy preparation method for recombinant human epidermal growth factor (rhEGF) without the need for an expensive enzyme to cleave the fusion part. However, the N-terminal fusion part is still useful for affinity chromatography. The hEGF is an important hormone in cell growth and proliferation in humans, and many studies on the expression and purification of this protein have been reported. In the present study, the hEGF gene was designed to be optimized with the E. coli codon usage preference and to contain Asn-Gly at the N-terminus of the protein. The gene was inserted into pRSET_A, an E. coli expression vector, and transformed into E. coli BL21 (DE3). The recombinant fusion protein was successfully co-expressed with pG-Tf2, a chaperone vector, in E. coli and purified by Ni-NTA column chromatography. The rhEGF was then released by hydroxylamine treatment and confirmed by SDS-PAGE. ELISA analysis showed that the activity of the free rhEGF was more than 92% similar to that of commercial EGF. The biological activity of the rhEGF was confirmed by a cell proliferation test with human skin fibroblasts.