Modeling and Optimization of Methanol as a Cosolvent in Amoxicillin Synthesis and Its Advantage over Ethylene Glycol

Chow, Yvonne,Li, Ruijiang,Wu, Jinchuan,Puah, Sze Min,New, Shu Wei,Chia, Wei Qiang,Lie, Felicia,Rahman, Talukder Md Mahabubur,Choi, Won-Jae Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

The production of semi-synthetic beta-Iactam antibiotics such as Amoxicillin may be performed enzymatically using penicillin acylase under mild conditions. However, the thermodynamically favored hydrolysis of the antibiotic product and the acyl donor substrate needs to be minimized to use the kinetically controlled route. The addition of cosolvents such as ethylene glycol and methanol (the two best solvents identified so far for semi-synthetic beta-lactam antibiotics) can achieve this to some degree, but these additives also produce enzyme inhibition and deactivation. In this study, we compared ethylene glycol and methanol under various substrate conditions. Methanol gave a better synthesis to hydrolysis ratio, although its deactivating effects adversely affected production at lower cosolvent concentrations than ethylene glycol. This effect and its dependence on substrate concentration was further modeled and optimized. A few targets of optimization such as Amoxicillin level, the synthesis to hydrolysis ratio, or a combination, were employed. While maximum levels of Amoxicillin synthesis were achievable only at high substrate concentrations, improvements derived from cosolvents were most significant at low substrate concentrations.

Modeling and Optimization of Methanolas a Cosolvent in Amoxicillin Synthesisand Its Advantage over Ethylene Glycol

Yvonne Chow,Ruijiang Li,Jinchuan Wu,Sze Min Puah,Shu Wei New,Wei Qiang Chia,Felicia Lie,Talukder Md Mahabubur Rahman,Won Jae Choi 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

The production of semi-synthetic beta-lactam antibiotics such as Amoxicillin may be performed enzymatically using penicillin acylase under mild conditions. However, the thermodynamically favored hydrolysis of the antibiotic product and the acyl donor substrate needs to be minimized to use the kinetically controlled route. The addition of cosolvents such as ethylene glycol and methanol (the two best solvents identified so far for semi-synthetic beta-lactam antibiotics) can achieve this to some degree, but these additives also produce enzyme inhibition and deactivation. In this study, we compared ethylene glycol and methanol under various substrate conditions. Methanol gave a better synthesis to hydrolysis ratio, although its deactivating effects adversely affected production at lower cosolvent concentrations than ethylene glycol. This effect and its dependence on substrate concentration was further modeled and optimized. A few targets of optimization such as Amoxicillin level, the synthesis to hydrolysis ratio, or a combination, were employed. While maximum levels of Amoxicillin synthesis were achievable only at high substrate concentrations, improvements derived from cosolvents were most significant at low substrate concentrations.

Ketoprofen Resolution by Enzymatic Esterification and Hydrolysis of the Ester Product

Wu, Jin Chuan,Low, Hou Ran,Leng, Yujun,Chow, Yvonne,Li, Ruijiang,Talukder, MMR,Choi, Won-Jae The Korean Society for Biotechnology and Bioengine 2006 Biotechnology and Bioprocess Engineering Vol.11 No.3

Immobilized Candida antarctica lipase was used to catalyze the separation of ketoprofen into its components by means of esterification followed by the enzymatic hydrolysis of the ester product. In this study, ketoprofen underwent esterification to ethanol in the presence of isooctane. When the reaction was complete, 58.3% of the ketoprofen had been transformed into an ester. The ketoprofen remaining in solution after the reaction was complete consisted primarily of its S-enantiomer (83.0%), while the 59.4% of the ketoprofen component of the ester consisted of its R-enantiomer. We then subjected the ester product to enzymatic hydrolysis in the presence of the same enzyme and produced a ketoprofen product rich in the R-enantiomer; 77% of this product consisted of the R-enantiomer when 50% of the ester had been hydrolyzed, and 90% of it consisted of the R-enantiomer when 30% of the ester had been hydrolyzed. By contrast, the R-enantiomer levels only reached approximately 42 and 65%, respectively, when 50 and 30% of the racemic ester was hydrolyzed under the same conditions.

Insertional mutations exhibiting high cell-culture density HCD phenotypes are enriched through continuous subcultures in Chlamydomonas reinhardtii

Leena Thung,Jing He,Qingling Zhu,Zhenyu Xu,Jianhua Liu,Yvonne Chow 한국조류학회I 2018 ALGAE Vol.33 No.1

Low efficiency in microalgal biomass production was largely attributed to the low density of algal cell cultures. Though mutations that reduced the level of chlorophyll or pigment content increased efficiency of photon usage and thus the cell-culture density under high-illumination growth conditions (e.g., >500 µmol photon m-2 s-1), it was unclear whether algae could increase cell-culture density under low-illumination conditions (e.g., ~50 µmol photon m-2 s-1). To address this question, we performed forward genetic screening in Chlamydomonas reinhardtii. A pool of >1,000 insertional mutants was constructed and subjected to continuous subcultures in shaking flasks under low-illumination conditions. Complexity of restriction fragment length polymorphism (RFLP) pattern in cultures indicated the degree of heterogeneity of mutant populations. We showed that the levels of RFLP complexity decreased when cycles of subculture increased, suggesting that cultures were gradually populated by high cell-culture density (HCD) strains. Analysis of the 3 isolated HCD mutants after 30 cycles of subcultures confirmed that their maximal biomass production was 50-100% higher than that of wild type under low-illumination. Furthermore, levels of chlorophyll content in HCD mutant strains were similar to that of wild type. Inverse polymerase chain reaction analysis identified the locus of insertion in two of three HCD strains. Molecular and transcriptomic analyses suggested that two HCD mutants were a result of the gain-of-function phenotype, both linking to the abnormality of mitochondrial functions. Taken together, our results demonstrate that HCD strains can be obtained through continuous subcultures under low illumination conditions.

Insertional mutations exhibiting high cell-culture density HCD phenotypes are enriched through continuous subcultures in Chlamydomonas reinhardtii

Thung, Leena,He, Jing,Zhu, Qingling,Xu, Zhenyu,Liu, Jianhua,Chow, Yvonne The Korean Society of Phycology 2018 ALGAE Vol.33 No.1

Low efficiency in microalgal biomass production was largely attributed to the low density of algal cell cultures. Though mutations that reduced the level of chlorophyll or pigment content increased efficiency of photon usage and thus the cell-culture density under high-illumination growth conditions (e.g., >$500{\mu}mol\;photon\;m^{-2}\;s^{-1}$), it was unclear whether algae could increase cell-culture density under low-illumination conditions (e.g., ${\sim}50{\mu}mol\;photon\;m^{-2}\;s^{-1}$). To address this question, we performed forward genetic screening in Chlamydomonas reinhardtii. A pool of >1,000 insertional mutants was constructed and subjected to continuous subcultures in shaking flasks under low-illumination conditions. Complexity of restriction fragment length polymorphism (RFLP) pattern in cultures indicated the degree of heterogeneity of mutant populations. We showed that the levels of RFLP complexity decreased when cycles of subculture increased, suggesting that cultures were gradually populated by high cell-culture density (HCD) strains. Analysis of the 3 isolated HCD mutants after 30 cycles of subcultures confirmed that their maximal biomass production was 50-100% higher than that of wild type under low-illumination. Furthermore, levels of chlorophyll content in HCD mutant strains were similar to that of wild type. Inverse polymerase chain reaction analysis identified the locus of insertion in two of three HCD strains. Molecular and transcriptomic analyses suggested that two HCD mutants were a result of the gain-of-function phenotype, both linking to the abnormality of mitochondrial functions. Taken together, our results demonstrate that HCD strains can be obtained through continuous subcultures under low illumination conditions.

Ketoprofen Resolution by Enzymatic Esterification and Hydrolysis of the Ester Product

Jin Chuan Wu,Hou Ran Low,Yujun Leng,Yvonne Chow,Ruijiang Li,MMR Talukder,Won Jae Choi 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.3

Immobilized Candida antarctica lipase was used to catalyze the separation of ketoprofen into its components by means of esterification followed by the enzymatic hydrolysis of the ester product. In this study, ketoprofen underwent esterification to ethanol in the presence of isooctane. When the reaction was complete, 58.3% of the ketoprofen had been transformed into an ester. The ketoprofen remaining in solution after the reaction was complete consisted primarily of its S-enantiomer (83.0%), while the 59.4% of the ketoprofen component of the ester consisted of its R-enantiomer. We then subjected the ester product to enzymatic hydrolysis in the presence of the same enzyme and produced a ketoprofen product rich in the R-enantiomer; 77% of this product consisted of the R-enantiomer when 50% of the ester had been hydrolyzed, and 90% of it consisted of the R-enantiomer when 30% of the ester had been hydrolyzed. By contrast, the R-enantiomer levels only reached approximately 42 and 65%, respectively, when 50 and 30% of the racemic ester was hydrolyzed under the same conditions.

Enhanced enantioselectivity of immobilized Candida antarctica lipase for hydrolysis of ketoprofen ethyl ester at pH 1

Jin Chuan Wu,Philip Ho,Tee Yuan Poh,Yvonne Chow,MMR Talukder,Won Jae Choi 한국화학공학회 2007 Korean Journal of Chemical Engineering Vol.24 No.4

immobilized Candida antarctica lipase, a commercially available and one of the most commonly usedenzymes, showed significantly improved enantioselectivity (twice) when used in a very acidic environment (pH 1.0)than in the normal pH 7.0 for the hydrolysis of ketoprofen ethyl ester at 45oC. The enzyme was still 60% active atpH 1.0 compared to the activity at pH 7.0 and its stabilities at the two pH values were almost the same. The improvedenantioselectivity was ascribed to the conformational change of the enzyme in the very acidic environment.