Candida Antartica Lipase B Mediated Kinetic Resolution Of Racemic Acebutolol

Rajin, Mariani (2015) Candida Antartica Lipase B Mediated Kinetic Resolution Of Racemic Acebutolol. PhD thesis, Universiti Sains Malaysia.

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Abstract

Asebutolol masih lagi dipasarkan dalam bentuk rasemik sehingga kini. Kecenderungan terhadap penghasilan enantiomer tunggal bagi ubat-ubatan berbentuk kiral adalah didorong oleh beberapa faktor antaranya kesan yang berbeza daripada enantiomer tersebut, permintaan dalam pasaran yang semakin meningkat, juga peraturan yang semakin ketat terhadap pengeluaran ubat-ubatan tersebut. Oleh itu, resolusi kinetik terhadap rasemik asebutolol dikaji menggunakan perantaraan lipase dalam reaktor kelompok dan reaktor membran berenzim. Kaedah sambutan permukaan dengan rekabentuk central composite design (CCD) digunakan untuk analisis data bagi reaktor kelompok. Faktor yang dikaji adalah merangkumi jumlah enzim, kepekatan substrat dan penderma asil dan suhu tindakbalas. Kajian mendapati bahawa tindabalas di dalam reaktor kelompok ini mencapai optimum dengan 320 mg enzim, 50 mM kepekatan Asebutolol, 140 mM kepekatan vinil asetat and suhu 40 oC, memberikan kadar pertukaran sebanyak 46% dengan nilai E dan ees masing-masing 15 and 73%. Tenaga pengaktifan dan penyahtabii bagi enzim di dalam reaktor kelompok dalam kajian ini dianggarkan masing-masing sebanyak 39.63 kJ/mol dan 54.90 kJ/mol. Pemalar pendeaktifan kd meningkat sebanyak 0.012-0.031 per jam dengan peningkatan suhu daripada 45 oC ke 60 oC. Nilai entalpi adalah 52.12 kJ/mol.K dan entropi adalah -0.18 kJ/mol.K. Berdasarkan dapatan daripada tindak balas di dalam reaktor kelompok, resolusi kinetik Acebutolol telah berjaya dilakukan di dalam reaktor membran berenzim. Kesan jumlah enzim, kepekatan substrat dan penderma asil, pH larutan penimbal, suhu tindakbalas, kadar aliran fasa organik dan tekanan transmembran. Tindakbalas di dalam reaktor membran berenzim mencapai nilai optimum pada pH 7, 40 oC dan TMP 6 psi, dengan kadar aliran fasa organik sebanyak 40 ml/min. Ia memberikan nilai pertukaran 40%, E sebanyak 23 dan ees sebanyak 84%. Tindakbalas enzim di dalam reaktor kelompok dan reaktor membran berenzim kedua-duanya mematuhi mekanisme Ping Pong Bi Bi. Parameter kinetik untuk enzim bebas di dalam reaktor kelompok adalah seperti berikut: KMace =8.53 mM, KMva =5.19 mM, dan Vmax =1.18 mM/h. Manakala parameter kinetik untuk enzim tersekatgerak di dalam reaktor membran berenzim adalah seperti berikut; Kmace app = 2.13 mM, KMvaapp = 1.23 mM dan Vmax app =2.33 mM/h. Nilai pemalar perencat pula adalah KIace=10.72 mM, KIva,= 3.71 mM, KIace app = 11.56 mM dan KIva app=3.89 mM. Prestasi CALB di dalam kedua-dua jenis reaktor telah dibandingkan. Enzim tersekatgerak di dalam reaktor membran berenzim memberikan kapasiti tindak balas yang lebih tinggi, kestabilan terma yang lebih baik, afiniti yang lebih tinggi kepada substrat dan juga menunjukkan rintangan tinggi terhadap kesan perencat berbanding enzim bebas di dalam reaktor kelompok. Kelebihan enzim tersekatgerak dilihat amat berpotensi untuk diaplikasikan dalam industri penghasilan enantiomer tunggal, terutamanya penyekat beta dalam masa terdekat. ________________________________________________________________________________________________________________________ Acebutotol is still available in racemic form. The increasing preference for single or pure enantiomer of chiral drugs is driven mainly by the different effects of the enantiomers, the high market demand and the guidelines issued by regulatory authorities. Therefore, the kinetic resolution of racemic acebutolol is studied in batch and enzymatic membrane reactor. The response surface methodology based on central composite design (CCD) was employed for optimization and analysis of kinetic resolution of racemic acebutolol in a batch reactor. The process variables which were taken into account include; enzyme loading, substrate concentration, acyl donor concentration and temperature. The optimum conditions were found to be 320 mg of enzyme loading, with acebutolol concentration of 50 mM, vinyl acetate concentration of 140 mM and temperature at 40 oC, giving the overall conversion of 46.6%. The value of enantioselectivity E and enantiomeric excess of the substrate ees were found to be 15 and 73%, respectively. Lipase activation and deactivation energy was estimated to be 39.63 kJ/mol and 54.90 kJ/mol, respectively. Denaturation constant, kd was increasing from 0.012-0.031 h-1 with the increasing temperature from 45 0C to 60 0C. The value of enthalpy and entropy for free Candida antartica lipase B were 52.12 kJ/mol.K and -0.18 kJ/mol.K, respectively. Based on the finding from the batch reaction, kinetic resolution of acebutolol has been successfully conducted in enzymatic membrane reactor (EMR). The effects of enzyme loading, substrate and acyl donor concentration, pH of buffer solution, reaction temperature, and organic phase flow rates, and transmembrane pressure (TMP) were investigated. The optimum operating conditions for the lipase-catalyzed kinetic resolution in an EMR system were pH 7, 40 oC and TMP of 6 psi at organic flow rate of 40 ml/min. This condition gave 40% overall conversion, enentioselectivity of 23 and ees of 84%. The reaction kinetic was found to obey the Ping Pong Bi Bi mechanisms for both free and immobilized lipase from Candida antartica B . The kinetic parameters for the free lipase were: KMace =8.53 mM, KMva =5.19 mM, and Vmax =1.18 mM/h. The apparent kinetic parameters for the immobilized lipase were: Kmace app = 2.13 mM, KMvaapp = 1.23 mM and Vmax app =2.33 mM/h. The kinetics of kinetic resolution accounted for both substrates inhibitions. The inhibition constants were given by KIace=10.72 mM, KIva,= 3.71 mM, KIace app = 11.56 mM and KIva app=3.89 mM. The performance of free and immobilized CALB were compared. The immobilized lipase in EMR gave higher reaction capacity, better thermal stability, higher affinity to the substrates and exhibited higher resistance towards the inhibition effect. The advantages of immobilized enzyme makes it possible for economical industrial production of chiral drugs, particularly beta blockers in the near future.

Item Type:	Thesis (PhD)
Additional Information:	Full text is available at http://irplus.eng.usm.my:8080/ir_plus/institutionalPublicationPublicView.action?institutionalItemId=2791
Subjects:	T Technology T Technology > TP Chemical Technology > TP155-156 Chemical engineering
Divisions:	Kampus Kejuruteraan (Engineering Campus) > Pusat Pengajian Kejuruteraan Kimia (School of Chemical Engineering) > Thesis
Depositing User:	Mr Mohd Jasnizam Mohd Salleh
Date Deposited:	12 Jul 2018 08:35
Last Modified:	14 Aug 2018 08:45
URI:	http://eprints.usm.my/id/eprint/41029

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