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A computer subroutine has been developed in Visual Basic for Applications (VBA) 6.3 and implemented on Microsoft ® EXCEL 2003 to simulate and select the best feeding strategy that maximise the production of R-phenylacetylcarbinol (PAC) from pyruvate decarboxylase (PDC). Based on the optimal simulation results, the PAC level of 705 mM could be achieved with in 60 h when the benzaldehyde level was maintained at 90 mM (with pyruvate to benzaldehyde concentration ratio of 1.2:1.0). Experimental verification of the simulated results was performed with initial reaction volume of 30 ml. The PAC production reached the maximum value of 293 ± 9 mM at 60 h. The experimental PAC production was lower than the predicted value by 2.4 times. This might be the indication of PAC and by-products (acetaldehyde and acetoin) inhibitions has yet to be incorporated in the mathematical model describing the production of PAC. 1 Department of Food Engineering, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, Thailand Fed Batch Enzymatic Biotransformation Of Benzaldehyde And Pyruvate To R-phenylacetylcarbinol Using Pyruvate Decarboxylase Noppol Leksawasdi 1 Abstract 1. Introduction 2. Materials and Methods 3. Results, Discussion & Conclusion 4. References 5. Keywords Corresponding author: E-mail: noppol@hotmail.com Research on the biotransformation of pyruvate and benzaldehyde by pyruvate decarboxylase (PDC) to produce R-phenylacetylcarbinol (PAC), a precursor for production of R-ephedrine which is used principally in the temporary treatment of hypotension and bronchial asthma, has been considered as one of the earliest (since 1932) and most intensively investigated in terms of yield improvement and biocatalyst stability. This process has shown significant improvements when compared to the traditional yeast-based fermentation process. Recent publications of a comprehensive mathematical model describing PDC deactivation by benzaldehyde and batch biotransformation of PAC (up to 140 mM) and its by-products using partially purified PDC (Leksawasdi et al. 2003, 2004, 2005) have prompted an interest in the development of substrates feeding profiles that can be used to optimize the production of PAC or its productivity in a fed batch biotransformation system. Ephedra sp., a natural source of ephedrine Products from R-PAC Based on the optimal simulation results, the PAC level of 705 mM could be achieved within 60 h when the benzaldehyde level in the biotransformation reactor was maintained at 90 mM (with pyruvate to benzaldehyde concentration ratio of 1.2:1). The predicted corresponding PAC productivity was 11.8 mM h -1 with remaining PDC activity of 10%. However, PAC production began to level off during 45 - 54 h of the biotransformation time course in the actual experiment and reached the maximum value of 293 ± 9 mM at 60 h with PAC productivity and remaining PDC activity of 4.9 ± 0.2 mM h -1 and 28 ± 1 %, respectively. The cessation of PAC production during the final 20 h of biotransformation in the presence of significant level of PDC activity (19-37%) and substrates (90 ± 12 mM benzaldehyde and 206 ± 6 mM pyruvate) might be the indication of PAC and by-products (acetaldehyde and acetoin) inhibitions has yet to be incorporated in the model. บทคัดย่อ โปรแกรมคอมพิวเตอร์ได้ถูกพัฒนาขึ้นในวิช่วลเบสิค 6.3 และนำไปประยุกต์ใช้ใน โปรแกรมไมโครซอฟท์เอ็กซ์เซล ® 2003 เพื่อซิมมิวเลทและเลือกรูปแบบการป้อนเข้าที่ดีที่สุด สำหรับเพิ่มการผลิต R-phenylacetylcarbinol (PAC) ด้วยเอนไซม์ไพรูเวตดีคาร์บอกซิเลส (PDC) ให้ อยู่ในระดับที่สูงสุด ผลซิมมิวเลชั่นที่ดีที่สุดทำนายว่าความเข้มข้นของ PAC อาจเพิ่มขึ้นสูงถึง 705 mM ภายในระยะเวลา 60 ชั่วโมง เมื่อรักษาระดับความเข้มข้นของเบนซาลดีไฮด์ไว้ที่ 90 mM ( โดยมีอัตราส่วนความเข้มข้นของไพรูเวตและเบนซาลดีไฮด์เท่ากับ 1.2:1.0 ) เมื่อทำการ ทดลองเพื่อตรวจสอบผลการซิมมิวเลทจากโปรแกรมด้วยปริมาตรปฏิกิริยาเริ่มต้นที่ 30 ml พบว่าความเข้มข้นสูงสุดของ PAC ขึ้นไปถึงค่าสูงสุดที่ 293 ± 9 mM หลังจากเวลาผ่านไป 60 ชั่วโมง ผลการผลิต PAC จากการทดลองซึ่งต่ำกว่าค่าที่ทำนายไว้ 2.4 เท่าอาจมีเหตุจาก ปฏิกิริยายับยั้งการทำงานของเอนไซม์ PDC ด้วย PAC หรือผลิตภัณฑ์ข้างเคียงอย่างอะเซตาล ดีไฮด์และอะเซโตอิน ซึ่งยังไม่ได้นำมารวมเข้าไว้ในโมเดลคณิตศาสตร์ที่ใช้อธิบายการผลิต PAC The volumes of benzaldehyde stock (10.6 M) and concentrated pyruvate (1.4 M) to be added at a particular time point to maintain a constant level of substrates concentration were computed based on a mathematical model published previously (Leksawasdi et al. 2003, 2004, 2005) for a biotransformation system containing 2.5 M MOPS, 1 mM Mg 2+, 1 mM TPP at 6 C with initial PDC activity of 4 U ml -1 carboligase and pH of 7.0. Experimental verification of the simulated results was performed with initial reaction volume of 30 ml using feeding strategy obtained from the program. The sample was obtained from the biotransformation reactor at regular intervals and analysed for pyruvate, benzaldehyde, PAC, acetaldehyde, acetoin and remnant PDC activity. PAC and benzaldehyde concentrations were determined by HPLC with UV detection at 283 nm as described by Rosche et al. (2001). Concentrations of pyruvate and acetaldehyde were determined by enzymatic NADH coupled assay with alcohol dehydrogenase and lactate dehydrogenase, respectively (Rosche et al., 2002). Quantification of acetoin was done by GC with flame ionisation detector as described previously (Rosche et al., 2002). To determine changes in PDC activity, gel filtration columns (Micro Bio-Spin ® 6, Cat. No. 732-6200) were used to remove low molecular weight compounds from the reaction solutions (Rosche et al., 2002). PDC was recovered and mixed with collection buffer at the lower end of the column, incubated on ice for 20 min and analysed for carboligase as described by Rosche et al. (2002). R-ephedrine and Related AlkaloidsEnzyme Production in 5 L Bioreactor R-PAC Biotransformation Predicted Benzaldehyde Feeding Profile Predicted Biotransformation Profile E: Enzyme, A: Pyruvate, B: Benzaldehyde, Q: Acetaldehyde, R: Acetoin, P: R-PAC Experimental Fed Batch Biotransformation Profile Product Reactants By-products Equations for the Prediction of R-PAC Biotransformation PDC (1) Noppol Leksawasdi, Michael Breuer, Bernhard Hauer, Bettina Rosche and Peter L. Rogers (2003) Biocatalysis and Biotransformation, 21, 315. (2) Noppol Leksawasdi, Yvonne Chow, Michael Breuer, Bernhard Hauer, Bettina Rosche and Peter L. Rogers (2004) Journal of Biotechnology, 111, 179. (3) Noppol Leksawasdi, Bettina Rosche and Peter L. Rogers (2005) Biochemical Engineering Journal, 23, 211. (4) Bettina Rosche, Vanessa Sandford, Michael Breuer, Bernhard Hauer and Peter L. Rogers (2001) Applied Microbiology and Biotechnology, 57, 309. (5) Bettina Rosche, Noppol Leksawasdi, Vanessa Sandford, Michael Breuer, Bernhard Hauer and Peter L. Rogers (2002) Applied Microbiology and Biotechnology, 60, 94. Digital pH controlling system Deactivation Effect of Benzaldehyde on PDC Biotransformation, R-phenylacetylcarbinol, pyruvate decarboxylase, enzyme catalysis, feeding profiles, fed batch
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