Biotransformation Kinetics of Benzaldehyde to L-Phenylacetylcarbinol (L-Pac) by Immobilized Candida Utius and Its Pyruvate Decarboxylase
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BIOTRANSFORMATION KINETICS OF BENZALDEHYDE TO L-PHENYLACETYLCARBINOL (L-PAC) BY IMMOBILIZED CANDIDA UTIUS AND ITS PYRUVATE DECARBOXYLASE by HYOUN SEUNG SHIN A thesis submitted for the Degree of Doctor of Philosophy at the University of New South Wales Department of Biotechnology The University of New South Wales September, 1994 Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the University or other Institute of higher learning, except where due acknowledgment is made in the text. Hyoun Seung Shin September, 1994 I dedicate this work to my parents, wife and family. TO THE GLORY OF GOD We know that in all things God works for the good of those who love him, who have been called according to his purpose. -Romans 8:28. 1 ACKNOWLEDGMENTS I wish to thank my supervisor, Professor Peter L. Rogers, for his excellent advice, thoughtful guidance and encouragement throughout this study. I am grateful also to Professor Kye Joon Lee, Department of Microbiology, Seoul National University who kindly recommended me to undertake my Ph.D. course in Australia. I would like to thank Professor Peter. P. Gray, Head of Department, Professor Noel W. Dunn, Dr. R. Cail, all other staff members and postgraduate students of the Dept. of Biotechnology, in particular G17 members including Prof. Y. H. Rhee, Wang, Anka, Yaowaluk, Jang, Michael, Keith, Kanda, Somchit and Yvonne for discussions, suggestions and technical assistance. It is my pleasure to thank the Department of Employment, Education and Training (DEET) of the Government of Australia for the award of a Korea/ Australia Postgraduate Research Scholarship, and I would like to extend my appreciation also to Mrs. Rosemary Plain, Scholarships Unit of UNSW for her advice and assistance. I wish to extend my thanks to the good fellows at the Sydney Korean Parish, the Uniting Church in Australia, for their sincere encouragement. My heartfelt appreciation goes to my family, in particular my wife, Ae Hee and my daughter, Hyo Eun (Sharon) for their encouragement, endurance and understanding during the years. 11 ABSTRACT Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-P AC) as a key intermediate for L-ephedrine synthesis has been evaluated using immobilized Candida utilis and its free and immobilized pyruvate decarboxylase (PDC). PDC catalyzes the condensation reaction of benzaldehyde (BZ) and 'active acetaldehyde'. Enhancement of PDC by induction of fermentative conditions and pulse feeding of glucose was conducted prior to immobilization (entrapment in 3 % calcium alginate). Although the immobilized cells have a higher resistance towards the toxic or inhibitory effects of benzaldehyde, benzyl alcohol (BA) production was higher than for the free cell process. During biotransformation, the BZ level and RQ (respiratory quotient) significantly affected both L-PAC and BA formation. By controlling the BZ level at 2 g/L, maintaining RQ=5-7 and pulse feeding glucose, a concentration of 15.6 g/L L p AC was achieved in batch culture. In a single stage continuous process, the steady state L-PAC concentration was reduced, however, 0.33 g/L/h L-PAC productivity could be sustained for more than 110 hat optimal conditions. With purified PDC, several catalytic reactions occurred simultaneously and gave rise to acetaldehyde and acetoin as by-products. Various modifications of the reaction conditions involving pH, temperature, addition of ethanol, optimal PDC activity and substrate ratios, led to an increase in L p AC and minimization of by-products. The highest L-P AC concentration of 28.6 g/L was achieved at 7 unit/mL PDC activity and 200 mM benzaldehyde with 2.0 molar ratio of pyruvate to benzaldehyde in 40 mM KH2PO4 reaction buffer (pH 7.0) at 4°C. In an evaluation of the immobilized enzyme process, entrapment of PDC into spherical polyacrylamide beads was successfully performed with 12.5 % activity retention, longer half life and modified kinetic parameters compared to free enzyme. In a batch process, the immobilized PDC generally produced lower L-P AC at the same concentrations of substrates. However, in this latter case, L-P AC formation could occur at higher benzaldehyde concentrations (e.g. 300 mM BZ) with the highest L-PAC being 27.1 g/L. With the continuous process, when 50 mM BZ and 100 mM sodium pyruvate were 111 fed into a packed-bed reactor, 0.56 g/L/h productivity was obtained at an average L-PAC concentration of 4.5 g/L with 32 days enzyme half life at 4°C. In summary, the present study has resulted in L-PAC concentrations as high as 28.6 g/L, which compare well with fed-batch values from our laboratory of 22 g/L, and previously reported maximum values of 12 g/L from the literature. An economic assessment which takes account of substrate (pyruvate) cost, as well as that of producing purified and/ or immobilized PDC, is needed for a realistic evaluation of the commercial potential of the various modes of L-P AC production. lV Conference Presentations Hyoun S. Shin and Peter L. Rogers (1993) Kinetics of bioconversion of benzaldehyde to L-phenylacetylcarbinol by purified pyruvate decarboxylase (PDC) from Candida utilis. 11th Australian Biotechnology Conference, 20-24 September, Perth. Australia pp. 240-242. Wang, Bin, H. S. Shin and P. L. Rogers (1994) Microbial and Enzymatic biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC), an intermediate in L-ephedrine production. 3rd Asia-Pacific Biochemical Engineering Conference, 13-15, June. Singapore. pp. 249-252. V AIMS OF THE PRESENT STUDY The present study follows from previous evaluations in our laboratory of L-PAC production using batch, fed-batch and continuous modes of operation with Candida utilis. The specific aims of the investigation are: (1) evaluation of fermentative enzyme profiles of free and immobilized Candida utilis to find optimal conditions for high PDC activity; (2) kinetic evaluation of immobilized C. utilis for biotransformation of benzaldehyde to L-PAC; (3) characterization and kinetic evaluation of purified pyruvate decarboxylase (PDC) as the key catalytic enzyme for biotransformation. This evaluation includes an investigation of various PDC sources, partial purification of PDC, evaluation of biotransformation kinetics and subsequent improvement of biotransformation efficiency for L-P AC production; (4) immobilization of pyruvate decarboxylase and evaluation of the resultant kinetics of biotransformation. This includes an investigation of immobilization methods, assessment of the stability of the immobilized enzyme, optimization of reaction conditions, and comparison of kinetic parameters with free enzyme; (5) application of the immobilized enzyme system to continuous L-PAC production and comparison with previous batch data. From these investigations, it is anticipated that a clearer understanding will emerge of L-P AC production from benzaldehyde, as well as the provision of data for a comparative analysis of various reaction modes for the L-PAC biotransformation process. VI TABLE OF CONTENTS Acknowledgment i Abstract ii List of Publication iii Aims of the present study V Table of contents vi List of Figures xii List of Tables xv Chapter 1 Literature Review 1.1 Biotransformations 1 1.1.1 Introduction 1 1.1.2 Characterization of Biotransformations 2 1.1.3 Current Trends of Biotransformation processes 5 1.1.3.1 Introduction 5 1.1.3.2 Biotransformation in homogenous water miscible organic solvents 8 1.1.3.3 Biotransformation in two phase system 9 1.1.3.4 Biotransformation in mono-phasic organic solvents 11 1.1.3.5 Biotransformation in reverse micelles 13 1.1.3.6 Biotransformation of supercritical fluids 16 1.2 L-Ephedrine 18 1.2.1 Introduction 18 1.2.2 Medicinal uses of L-ephedrine 20 1.2.3 Production of Ephedrine 22 1.2.3.1 Ephedrine from natural herbs 'Ma Huang' 23 1.2.3.2 Chemical synthesis of ephedrine 27 1.2.3.3 Ephedrine synthesis via biotransformation of benzaldehyde to L-phenylacetylcarbinol 30 1.3 Biotransformation of benzaldehyde to L-phenylacetylcarbinol 34 1.3.1 Reaction mechanism of pyruvate decarboxylase for acyloin formation 34 1.3.2 Screening of microorganisms for L-P AC 36 1.3.3 Factors affecting the biotransformation with yeast cells 38 vu 1.3.3.1 Factors affecting the growth phase 38 1.3.3.2 Factors affecting the biotransformation phase 39 1.3.4 Kinetics of Biotransformation 41 1.4 Pyruvate decarboxylase (PDC) 45 1.4.1 Characteristics of pyruvate decarboxylase 45 1.4.2 Reaction mechanism of PDC 47 1.4.3 Roles of pyruvate decarboxylase 48 1.4.4 Genetic regulation of PDC 52 CHAPTER 2 Materials and Methods 55 2.1 Microorganisms 55 2.2 Media 55 2.2.1 Stock culture medium 55 2.2.2 Seed culture medium 56 2.2.3 Medium for cultivation in Erlenmeyer flasks and LH fermenter 56 2.2.4 Medium for cultivation in 100 L fermenter 56 2.2.5 Media Sterilization 57 2.3 Culture systems 58 2.3.1 Flask culture 58 2.3.2 LH fermenter 58 2.3.3 Porton-type stirred tank reactor 59 2.3.4 Pilot scale 100 L fermenter 62 2.3.4.1 The vessel 62 2.3.4.2 The control console 64 2.3.5 Sterilization of fermenters 65 2.4 Biotransformation system with immobilized cells 66 2.5 Methods of analysis 70 2.5.1 Procedure of sample preparation for analysis 70 2.5.2 Cell disruption and enzyme extraction 72 2.5.2.1 Extraction of enzymes from free cells 72 2.5.2.2 Extraction of enzymes from