• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.666-670
• /
• 2001

Levofloxacin은 ofloxacin의 L형의 광학이성질체로 D형의 ofloxacin보다 뛰어난 향균활성을 갖는다. 본 연구에서는 광학선택성을 지닌 외래 효소를 재조합균주에서 발현시키고 고농도배양과 세포고정화를 통해 levofloxacin을 생산하였다. E. coli 고농도 배양을 수행하여 89 g/L의 세포농도를 얻었으며 IPTG 1 mM로 단백질 발현을 유도하여 levofloxacin 생산 반응에 사용할 때 최적 첨가시기를 결정하였다. 세포고정화를 위해서는 sodium alginate를 사용하였으며 1.5% 첨가시 가장 우수한 효소활성을 나타내었고 재사용시에도 효소활성과 광학선택성이 유지됨올 확인하였다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권4호
• /
• pp.367-371
• /
• 2005

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed high apparent enantioselectivity ($E_{app}>100$) for (R)-2PB-O-res and were identified as Exiguobacterium acetylicum. The JH13 strain showed high esterase activity on p-nitrophenyl acetate (pNPA), but showed low lipase activity on p-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.

• Journal of Microbiology and Biotechnology
• /
• 제16권11호
• /
• pp.1832-1836
• /
• 2006

Mesorhizobium sp. LUK, which utilizes 3-amino-3-phenylpropionic acid as the sole source of nitrogen with high enantioselectivity (E(S)>100), was isolated using enrichment culture. The enzyme involved in the utilization of (S)-3-amino-3-phenylpropionic acid was confirmed to be a transaminase and was purified by 235-folds with a specific activity of 0.72 U/mg. The molecular weight of the purified protein was ca. 47 kDa and the active enzyme was determined as a dimer on gel filtration chromatography. The N-terminal sequence was obtained from the purified protein. Spontaneous decarboxylation of produced ${\beta}-keto$ acids was observed during the chiral resolution of 3-amino-3-phenylpropionic acid.

• Journal of Microbiology and Biotechnology
• /
• 제25권12호
• /
• pp.2024-2033
• /
• 2015

A gene encoding lipolytic enzyme, lip7-3, was isolated from Bacillus sp. W130-35 isolated from a tidal mud flat. The gene encoded a protein of 215 amino acids with a signal peptide composed of 34 amino acid residues. Lip7-3 belonged to the family I.4 lipase and showed its maximal activity at pH 9.0 and 60℃. Its activity increased in the presence of 30% methanol and, remarkably, increased as well to 154.6% in the presence of Ca²⁺. Lip7-3 preferred p-nitrophenyl octanoate (C8) as a substrate and exhibited broad specificity for short- to long- chain fatty acid esters. Additionally, Lip7-3 showed a low degree of enantioselectivity for an S-enantiomer (e.g., (S)-methyl-3-hydroxy-2-methylpropionate). It efficiently hydrolyzed glyceryl tributyrate, but did not hydrolyze glyceryl trioleate, fish oil, or olive oil. Its substrate specificity and activation by the solvent might offer a merit to the biotechnological enzyme applications like transesterification in the production of biodiesel.

• 한국응용과학기술학회지
• /
• 제32권3호
• /
• pp.451-460
• /
• 2015

An enantioselective recognition of D- and L-tryptophan (Trp)-b-cyclodextrin (CD) inclusion complex was performed using electrochemical and FT-Raman spectroscopic analysis. From the electrochemical analysis, the selectivity coefficient ($K_{DL}$) of b-CD inclusion complexes was found higher than that of the D- and L-Trp in phosphate buffered saline (PBS, pH=7.0) solution. The percentage of enantioselectivity ($I_{%{ee}}$) for peak current of D-Trp-b-CD inclusion complexes was observed higher than that of L-Trp-b-CD inclusion complexes in PBS solution. From Raman spectroscopy, chemical shift difference (D, $cm^{-1}$) for the C=C stretch, ring vibration, and ring breathing of D-Try-b-CD inclusion complex were observed higher than that of L-Trp-b-CD inclusion complex. The electrochemical and Raman spectroscopic analyses were found very useful for chiral detection of racemic amino acid in the presence of b-CD.

• Journal of Microbiology and Biotechnology
• /
• 제20권9호
• /
• pp.1300-1306
• /
• 2010

Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni-NTA and desalting column chromatography. It evidenced an optimum temperature of $45^{\circ}C$ and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVII)
• /
• pp.243-246
• /
• 2005

A novel esterase showing high enantioselectivity to (S)-ketoprofen ethyl ester was selected from fosmid environmental DNA library which is provided by Microbial Genomic & Applications Center. As a result of Blast search, the gene wasn't registerated in Gene Bank yet. And as we know, conserved domain region of esterase , G-X-S-X-G, wasn't discovered.$^{4)}$ And it is similar to Beta-lactamase. The DNA sequence of cloned esterase include an open reading frame consisting of 1170 bp, designated as EST-Y29, encoding a protein of 389 amino acids with a molecular mass of about 42.8 kDa. And amino acid sequence analysis revealed only a few identity (28%) to tile known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases. when being comparison to other esterase revealed , this enzyme seems to be classified as a new member of esterase family. EST-Y29 was functionally overexpressed in a soluble form in E. coli with maximum conversion yield of (S)-ketoprofen at $65^{\circ}C$. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzyme from a metagenome.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제6권3호
• /
• pp.179-182
• /
• 2001

Porcine liver esterase was immobilized in polyacrylamide gel for the enantioselective production of levofloxacin from ofloxacin butyl ester. The initial activity of immobilized esterase was found to be significantly affected by the polyacrylamide gel composition. The optimum concentrations of monomer and crosslinker were determined to be 20% and 8.3%, respectively. The activity of immobilized esterase was 55.4% compared to a free enzyme. Enantiomeric excess was maintained at 60%, almost the same level as that of free enzyme. In addition, the immobilized esterase could be used repeatedly up to 10 times without experiencing any severe loss of activity and enantioselectivity.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2002년도 생물공학의 동향 (X)
• /
• pp.445-448
• /
• 2002

The Pseudomonas fluorescens KCTC 1767, a selected and identified as potential candidate for stereo-specific resolution of rac-ketoprofen ethyl ester, was systematically investigated in order to induce the high level expression and detailed characterization of the expressing enzyme esterase. We cloned the esterase gene from chromosomal DNA of Pseudomonas fluorescens KCTC 1767 by PCR with two synthetic primers that desinged for simple purification. The recombinant esterase from Pseudomonas fluorescens KCTC 1767 exibited a high conversion rate and enantioselectivity to the (S)-ketoprofen ethyl ester as expected. The enzyme was easily purified to homogeniety by using a metal chelating affinity chromatography as a protein with poly histidine taq, and thus obtained 0.6 mg of protein from a 100 mL culture broth in a single step. The purified enzyme was steadily stable at the pH range from 7.0 to 10. The activity was also retained to be about 70% after the preincubation at $40^{\circ}C$ but over $50^{\circ}C$ lost the activity completely. The molecular mass of the esterase was estimated to be about 43 kDa on SDS-PAGE, and an identical result was also shown in gel filteration chromatography. The specific activity was calculated 27 mM/mg-protein/min by using the rac-ketoprofen ethly ester as a substrate.

• Korean Chemical Engineering Research
• /
• 제58권2호
• /
• pp.280-285
• /
• 2020

We have developed a constitutive display system of the Pseudomonas fluorescens SIK W1 TliA lipase on the cell surface of Escherichia coli using E. coli outer membrane protein C (OmpC) as an anchoring motif, which is an economical compared to induced system. For the constitutive expression of truncated OmpC-TliA fusion proteins, gntT104 promoter was employed. Cell growth was not affected by over expression of fusion protein during entire culture time, suggesting cell lysis was not a problem. The localization of truncated OmpC-TliA fusion protein on the cell surface was confirmed by immunofluorescence microscopy and measuring whole cell lipase activity. Constitutively displayed lipase was very stable, retaining activity enantioselectivity throughout the five repeated reactions. These results suggest that OmpC from E. coli be a useful anchoring motif for displaying enzymes on the cell surface without any inducers, and this stable surface display system can be employed for a broad range of biotechnological applications.