• 생명과학회지
• /
• 제16권3호
• /
• pp.415-419
• /
• 2006

방향족 에폭사이드 기질에 대한 입체선택적 가수분해능이 우수한 Rhodotorula glutinis의 epoxide hydrolase (EH)를 codon usage를 고려한 Escherichia coli 균주에서 고효율로 발현할 수 있었다. 효모인 R. glutinis와 박테리아인 E. coli에서의 codon usage 선호도를 분석하고 그 차이를 고려하여 E. coli 에서 잘 사용되지 않는 rare codon에 대한 tRNA유전자정보가 들어 있는 pRARE plasmid를 함유한 E. coli 균주인 Rosetta(DE3)pLysS를 숙주세포로 사용하였다. R. glutinis EH를 발현시킨 재조합 E. coli를 생촉매로 사용하여 라세믹 styrene oxide 혼합물과 반응시켰을 때, (R)-styrene oxide에 대한 입체선택적 가수분해활성이 wild type R. glutinis 대비 매우 향상됨을 관찰할 수 있었다. 또한 라세믹 기질로부터 입체적으로 고순도인 99% ee 값을 갖는 광학적으로 순수한 (S)-styrene oxide를 얻을 수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권4호
• /
• pp.299-305
• /
• 2006

Aromatic L-amino acid transaminase is an enzyme that is able to transfer the amino group from L-glutamate to unnatural aromatic ${\alpha}-keto$ acids to generate ${\alpha}-ketoglutarate$ and unnatural aromatic L-amino acids, respectively. Enrichment culture was used to isolate thermophilic Bacillus sp. T30 expressing this enzyme for use in the synthesis of unnatural L-amino acids. The asymmetric syntheses of L-homophenylalanine and L-phenylglycine resulted in conversion yields of >95% and >93% from 150 mM 2-oxo-4-phenylbutyrate and phenylglyoxylate, respectively, using L-glutamate as an amino donor at $60^{\circ}C$. Synthesized L-homophenylalanine and L-phenylglycine were optically pure (>99% enantiomeric excess) and continuously pre-cipitated in the reaction solution due to their low solubility at the given reaction pH. While the solubility of the ${\alpha}-keto$ acid substrates is dependent on temperature, the solubility of the unnatural L-amino acid products is dependent on the reaction pH. As the solubility difference between substrate and product at the given reaction pH is therefore larger at higher temperature, the thermophilic transaminase was successfully used to shift the reaction equilibrium toward rapid product formation.

• Journal of Microbiology and Biotechnology
• /
• 제20권4호
• /
• pp.659-665
• /
• 2010

A cis-epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52 was purified 51.4-fold with a yield of 27.1% using ammonium sulfate precipitation, ionic exchange, hydrophobic interaction, molecular sieve chromatography and an additional anion-exchange chromatography. The CESH was stable in a broad range of temperature (up to $50^{\circ}C$) and pH (4.0-10.0) with optima of $40^{\circ}C$ and pH 6.5, respectively. It could be partially inhibited by EDTA-$Na_2$, $Ag^+$, SDS, and DTT, and slightly enhanced by $Ba^{2+}$ and $Ca^{2+}$. The enzyme exhibited high stereospecificity in D(-)-tartaric acid (enantiomeric excess value higher than 99%) with $K_m$ and $V_max$ values of 18.67 mM and $94.34\;{\mu}M$/min/mg for disodium cis-epoxysuccinate, respectively. The Bordetella sp. BK-52 CESH gene, which contained 885 nucleotides (open reading frame) encoding 294 amino acids with a molecular mass of about 32 kDa, was successfully overexpressed in Escherichia coli using a T7/lac promoter vector and the enzyme activity was increased 42-times compared with the original strain. It may be an industrial biocatalyst for the preparation of D(-)-tartaric acid.

• Bulletin of the Korean Chemical Society
• /
• 제26권9호
• /
• pp.1359-1365
• /
• 2005

A series of new chiral [iminophosphoranyl]ferrocenes, {${\eta}^5-C_5H_4-(PPh_2=N-2,6-R_2-C_6H_3)$}Fe{${\eta}^5-C_5H_3-1-PPh^2-2-CH(Me)NMe_2$} (1: R = Me, $^iPr$), {${\eta}^5{-C_5H_4-(PPh_2=N-2,6-R_2}^1-C_6H_3)$}Fe{${\eta}^5-C_5H_3-1-(PPh_2=N-2,6-R_2-C_6H_3)-2-CH(Me)R_2$} (2: $R^1\;=\;Me,\;^iPr;\;R^2\;=\;NMe_2$, OMe), and $({\eta}^5-C_5H_5)Fe${${\eta}^5-C_5H_4-1-PR_2-2-CH(Me)N=PPh_3$} (3:R = Ph, $C_6H_{11}$) have been prepared from the reaction of [1,1'-diphenylphosphino-2-(N,N-dimethylamino) ethyl]ferrocene with arylazides (1 & 2) and the reaction of phosphine dichlorides ($R_3PCl_{2}$) with [1,1'-diphenylphosphino-2-aminoethyl]ferrocene (3), respectively. They form palladium complexes of the type $[Pd(C_3H_5)(L)]BF_4$ (4-6: L = 1-3), where the ligand (L) adopts an ${\eta}^2-N,N\;(2)\;or\;{\eta}^2$-P,N (3) as expected. In the case of 1, a potential terdentate, an ${\eta}^2$-P,N mode is realized with the exclusion of the –=NAr group from the coordination sphere. Complexes 4-6 were employed as catalysts for allylic alkylation of 1,3-diphenylallyl acetate leading to an almost stoichiometric product yield with modest enantiomeric excess (up to 74% ee). Rh(I)-complexes incorporating 1-3 were also prepared in situ for allylic alkylation of cinnamyl acetate as a probe for both regio- and enantioselectivities of the reaction. The reaction exhibited high regiocontrol in favor of a linear achiral isomer regardless of the ligand employed.

• 한국식물생명공학회:학술대회논문집
• /
• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
• /
• pp.27-39
• /
• 2005

a-Ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, or 9-hydroxy-10 -oxo-12(Z), 15(Z)-octadecadienoic acid] was found as a stress-induced factor in Lemna paucicostata. KODA reacts with catecholamines to generate many products that strongly induce flowering in L. paucicostata, although KODA itself was inactive. KODA contains an asymmetric carbon at the 9-position in the molecule; the 9-hydroxyl group is predominantly 9R, with an enantiomeric excess of 40% (70% 9R and 30% 9S). We analyzed two major products of the reaction between KODA and norepinephrine, named FN1 and FN2. FN1 was identified as a tricyclic a-ketol fatty acid, 9(R)-11-{(2'R,8’R,10'S,11'S)-2',8'-dihydroxy-7'-oxo-11'-[(Z)-2-pentenyl]-9'-oxa-4'-azatricyclo[6.3.1.01.5]dodec-5'en-10'-yl}-9-hydroxy-10-oxoundecanoic acid. FN2 was the C-9 epimer of FN1. FN1 was derived from 9R-type KODA and FN2 from 9S-type. FN1 showed strong flower-inducing activity, but FN2 was inactive. Pharbitis nil (violet) is a typical short-day plant; flowering can be induced by exposing a seedling cultivated under continuous light to a single 16-h dark period. We analyzed endogenous KODA levels and showed that they were closely related to flower induction: KODA sharply increased in the later part of a 16-h dark period, on the other hand, it failed to increase in the night-break experiment. In addition to it, KODA increased transiently in immature flower buds in all the plants we examined, including P. nil. No such increase of KODA was seen in foliar buds of P. nil. When KODA was sprayed on seedlings of Pharbitis, flower induction was promoted only by the (R)-form of KODA. We also found that KODA enhances flowering in garden plants such as carnations and impatienses. These phenomena indicate that KODA may be involved in flowering formationg of plants and it is potentially useful for a regulating agent for commercial plant flowering.

• KSBB Journal
• /
• 제19권3호
• /
• pp.215-220
• /
• 2004

2-아릴프로피온산 계열의 키랄 의약품의 효소적 dynamic kinetic resolution (DKR) 공정에서 라세미화 염기촉매로 트리 옥틸아민이 지금까지 주로 사용되어 왔으나 반응매질에 녹은 상태로 작용해 회수 및 재사용이 어려웠다. 본 연구에서는 이를 개선하고자 라세미화 반응을 위한 고효율 고체 염기를 탐색해 보았다. 45$^{\circ}C$, 아이소옥탄 내에서 (S)-나프록센 2,2,2-트리플로로에틸 씨오에스터를 기질로 무기 염기류, 염기성음이온교환수지류, resin-bound 염기류 등을 시험한 결과, 약염기성 음이온교환수지인 DIAION WA30을 사용하였을 때 가장 효과적이었다. DIAION WA30의 2차 interconversion constant ( $k_{int}$$^＊/)는 8.6${\times}$$10^{-4}$ m $M^{-1}$ $h^{-1}$이며 동일한 실험조건하에서 수행한 트리옥틸아민 ( $k_{int}$$^＊/ = 2.5${\times}$$10^{-4}$ m $M^{-1}$ $h^{-1}$)에 비해 약 3배가 높았다. 효소 활성에 필수적인 물의 양에 따른 DIAION WA30의 라세미화 효율에 관하여 실험한 결과, 물의 양이 증가할수록 그 효율은 감소하였다. DIAION WA30을 라세미화 촉매로 사용하여 아이소옥탄 내에서 라세믹 나프록센 2,2,2-트리플로로에틸 씨오에스터의 효소적 DKR 반응을 수행해 보았다. 그 결과 DIAION WA30을 사용하지 않은 경우에 비해 반응 전환율과 생성물의 광학 순도는 급격히 향상되었다. 전통적 광학분할 반응의 최대 50%라는 전환율의 제한이 본 연구에서 찾은 DIAION WA30을 첨가함으로써 성공적으로 극복되었다. 또한 고체 염기촉매인 DIAION WA30의 사용은 라세미화 촉매의 회수 및 재사용이 가능하게 해준다.다.다.다.다.다.