• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1559-1565
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• 2017

Naturally occurring ginsenoside F1 (20-O-${\beta}$-$\text\tiny{D}$-glucopyranosyl-20(S)-protopanaxatriol) is rare. Here, we produced gram-scale quantities of ginsenoside F1 from a crude protopanaxatriol saponin mixture comprised mainly of Re and Rg1 through enzyme-mediated biotransformation using recombinant ${\beta}$-glucosidase (BgpA) cloned from a soil bacterium, Terrabacter ginsenosidimutans Gsoil $3082^T$. In a systematic step-by-step process, the concentrations of substrate, enzyme, and NaCl were determined for maximal production of F1. At an optimized NaCl concentration of 200 mM, the protopanaxatriol saponin mixture (25 mg/ml) was incubated with recombinant BgpA (20 mg/ml) for 3 days in a 2.4 L reaction. Following octadecylsilyl silica gel column chromatography, 9.6 g of F1 was obtained from 60 g of substrate mixture at 95% purity, as assessed by chromatography. These results represent the first report of gram-scale F1 production via recombinant enzyme-mediated biotransformation.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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• pp.1310-1315
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• 2019

Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The present study aimed to develop a novel, highly active hyaluronidase and assess its function. Therefore, in order to find the most efficient active hyaluronidase, we produced several shortened hyaluronidases with partial removal of the N- or C-terminal regions. Moreover, we created an enzyme that connected six histidines onto the end of the hyaluronidase C-terminus. This simplified subsequent purification using $Ni^{2+}$ affinity chromatography, making it feasible to industrialize this highly active recombinant hyaluronidase which exhibited catalytic activity equal to that of the commercial enzyme. Therefore, this simple and effective isolation method could increase the availability of recombinant hyaluronidase for research and clinical purposes.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.689-698
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• 2013

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.

• BMB Reports
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• 제31권3호
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• pp.240-244
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• 1998

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and in a potent immunogen. In order to express the recombinant urease at a higher level, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a high copy-number vector. The recombinant H. pylori urease expressed in an E. coli strain that was grown in a rich medium supplemented with added nickel was purified to near homogeneity by using DEAE-Sepharose, Superdex HR200, and Mono-Q (FPLC) columns and the purified enzyme possessed the specific activity of 1255 U/mg. Polyclonal antibodies raised against the purified recombinant H. pylori urease were shown to be very specific when subjected to Western blot analysis, in which crude extracts from the H. pylori ATCC strain and the recombinant E. coli strains expressing various bacterial ureases were exnmined for cross-reactivity.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.604-605
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• 2005

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.258-263
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• 1998

${\beta}$-Xylosidase was purified from the recombinant Escherichia coli carrying the Bacillus sp. KK-1 ${\beta}$-xylosidase gene (xylB). The molecular mass of the purified enzyme was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the apparent molecular mass of the ${\beta}$-xylosidase was 140 kDa, indicating that the native ${\beta}$-xylosidase has an oligomeric structure composed of two identical subunits. The isoelectric point was determined to be pH 5.5. The enzyme was highly active on p-nitrophenyl-$\beta$-D-xylopyranoside but it barely hydrolyzed xylan substrates, and did not exhibit activity towards carboxymethylcellulose and p-nitrophenyl-${\beta}$-D- glucopyranoside. The enzyme had a pH optimum for its activity at pH 6.5 and a temperature optimum at $40^{\circ}C$. The enzyme activity was completely inhibited by the presence of $Hg^{++}$, and also markedly inhibited by D-xylose and D-glucose.

• Journal of Interdisciplinary Genomics
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• 제1권1호
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• pp.6-9
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• 2019

In this report, the phenotypes of three patients from two families with mucopolysaccharidosis type II (MPS II) are compared: a novel variant and recombinant variant of IDS gene. The results of urine in patients showed a pronounced increase in glycosaminoglycan excretion with decreased iduronate-2-sulfatase enzyme activity in leukocyte, leading to a diagnosis of MPS II. A patient has a novel variant with 1 bp small insertion, c.1224_1225insC in exon 9, which caused frameshifts with a premature stop codon, and two patients have a recombination variant, c.418+495_1006+1304del, leading to the loss of exons 4, 5, 6, and 7 in genomic DNA, which is relatively common in Korean patients. They had different phenotypes even in the same mutation. The patients have now been enzyme replacement therapy with a significant decrease in glycosaminoglycan excretion. Further study on residual enzyme activity, as well as experience with more cases, may shed light on the relationship between phenotypes in MPS II and gene mutations.

• 자연과학논문집
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• 제16권1호
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• pp.1-13
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• 2005

안지오텐신(ACE) 저해제는 항고혈 효과를 갖고 있으므로 오랫동안 고혈압의 예방이나 치료에 이용되어 왔다. 본 연구는 재조합 대장균으로부터 새로운 ACE 저해제를 생산하고 정제하며 나아가 이들이 구조-기능 관P를 규명하기 위해 수행되었다. Saccharomyces cerevisiae의 ACE 저해 펩타이드 유전자를 함유하고 있는 재조합 pGEX-4T-3을 대장균 BL21(DE3)로 형질전환 시켰다. 재조합 pGEX-4T-3을 갖고 있는 대장균 BL21(DE3)로부터 생산된 Glutathione-s 전이효소(GST) 융합 단백질을 얻어서 그중 ACE저해 펩타이드를 Sephadex G-25 컬럼 크로마토그래피로 정제하였다. 정제된 ACE 저해 펩타이드는 타이로신-아스파틱엑시드-그리신-글리신-발린-페닐알라린-아르기닌-발린-타이로신-트레오닌의 서열을 가진 새로운 decapeptide이었고 ACE에 대하여 경쟁적으로 저해하였다.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.792-799
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• 2007

A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae(abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at $85^{\circ}C$. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and $\alpha$-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotriose, and $6-O-\alpha-maltosyl-\beta-cyclodextrin(G2-\beta-CD)$ to maltose and $\beta$-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to $G2-\beta-CD$. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.

• 미생물학회지
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• 제26권1호
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• pp.6-12
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• 1988

Cellobiase ($\beta$-glucosidase) is an enzyme of the cellulase system in cellulolytic microor-ganisms. The chromosomal DNA fragment which include cellobiase gene of Cellulomonas biazotea was cloned in Eschericia coli via plasmid pBR 322 vector. Restriction enzyme Sal I was used to obtain adequate size of fragments from C. biazotea. chromosomal DNA. The transformant of E. coli HB101 with recombinant plasmid pBG101 showed cellobiase activity, which is not ordinary in E. coli HB101. The enzyme activity of the transformant was as of 20% lower than that of C. biazotea.