Abstract
Two microorganisms showing high ω-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-α-methylbenzylamine (α-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of ω-transaminases were investigated. ω-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of α-MBA and showed remarkable stability. However, ω-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of α-MBA. The reaction rate of ω-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (V(org): V(aq) = 1:4) in the biphasic system with cyclohexanone, the reaction rate of ω-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM α-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-α-MBA with ca. 51% conversion.
| Original language | English |
|---|---|
| Pages (from-to) | 348-358 |
| Number of pages | 11 |
| Journal | Biotechnology and Bioengineering |
| Volume | 55 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 1997 Jul 20 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology
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Kinetic resolution of α-methylbenzylamine with ω-transaminase screened from soil microorganisms : Application of a biphasic system to overcome product inhibition. / Shin, Jong Shik; Kim, Byung Gee.
In: Biotechnology and Bioengineering, Vol. 55, No. 2, 20.07.1997, p. 348-358.Research output: Contribution to journal › Article
TY - JOUR
T1 - Kinetic resolution of α-methylbenzylamine with ω-transaminase screened from soil microorganisms
T2 - Application of a biphasic system to overcome product inhibition
AU - Shin, Jong Shik
AU - Kim, Byung Gee
PY - 1997/7/20
Y1 - 1997/7/20
N2 - Two microorganisms showing high ω-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-α-methylbenzylamine (α-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of ω-transaminases were investigated. ω-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of α-MBA and showed remarkable stability. However, ω-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of α-MBA. The reaction rate of ω-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (V(org): V(aq) = 1:4) in the biphasic system with cyclohexanone, the reaction rate of ω-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM α-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-α-MBA with ca. 51% conversion.
AB - Two microorganisms showing high ω-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-α-methylbenzylamine (α-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of ω-transaminases were investigated. ω-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of α-MBA and showed remarkable stability. However, ω-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of α-MBA. The reaction rate of ω-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (V(org): V(aq) = 1:4) in the biphasic system with cyclohexanone, the reaction rate of ω-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM α-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-α-MBA with ca. 51% conversion.
UR - http://www.scopus.com/inward/record.url?scp=0343907783&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0343907783&partnerID=8YFLogxK
U2 - 10.1002/(SICI)1097-0290(19970720)55:2<348::AID-BIT12>3.0.CO;2-D
DO - 10.1002/(SICI)1097-0290(19970720)55:2<348::AID-BIT12>3.0.CO;2-D
M3 - Article
C2 - 18636493
AN - SCOPUS:0343907783
VL - 55
SP - 348
EP - 358
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 2
ER -