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XYLITOL PRODUCTION FROM D-XYLOSE BY FACULTATIVE ANAEROBIC BACTERIA by Sendil Rangaswamy Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Biological Systems Engineering _________________________ Dr. Foster A. Agblevor, Chair ___________________ __________________ Dr. Jiann-Shin Chen Dr. Brenda Winkel-Shirley ___________________ __________________ Dr. Richard Helm Dr. Gene Haugh Dr. John V. Perumpral, Department Head January 27, 2003 Blacksburg, Virginia Keywords: xylitol, D-xylose, Corynebacterium sp., xylose reductase, Candida tropicalis XYLITOL PRODUCTION FROM D-XYLOSE BY FACULTATIVE ANAEROBIC BACTERIA by Sendil Rangaswamy Committee Chairman: Foster Agblevor Biological Systems Engineering ABSTRACT Seventeen species of facultative anaerobic bacteria belonging to three genera (Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol; a sugar alcohol used as a sweetener in the pharmaceutical and food industries. A chromogenic assay of both solid and liquid cultures showed that 10 of the 17 species screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation. The ten bacterial species were studied for the effect of environmental factors, such as temperature, concentration of D-xylose, and aeration, on xylitol production. Under most conditions, Corynebacterium sp. NRRL B 4247 produced the highest amount of xylitol. The xylitol produced by Corynebacterium sp. NRRL B 4247 was confirmed by mass spectrometry. Corynebacterium sp. NRRL B 4247 was studied for the effect of initial D-xylose concentration, glucose, glyceraldehyde, and gluconate, aeration, and growth medium. Corynebacterium sp. NRRL B 4247 produced xylitol only in the presence of xylose, and did not produce xylitol when gluconate or glucose was the substrate. The highest yield of xylitol produced in 24 h (0.57 g/g xylose) was using an initial D-xylose concentration of 75 g/l. Under aerobic conditions the highest xylitol yield was 0.55 g/g while under anaerobic conditions the highest yield was 0.2 g/g. Glyceraldehyde in concentrations greater than 1 g/l inhibited Corynebacterium sp. B 4247 growth and xylitol production. Corynebacterium sp. NRRL B 4247 culture grown in the presence of potassium gluconate (96 g/l) for 48 h and on addition of D-xylose to the media increased accumulation to 10.1 g/l of xylitol after 150 h. Corynebacterium sp. NRRL B 4247 exhibited both NADH and NADPH-dependent xylose reductase activity in cell-free extracts. The NADPH-dependent activity was substrate dependent. The activity was 2.2-fold higher when DL-glyceraldehyde was used as substrate than with D-xylose. In cell-free extracts the difference in xylose reductase and xylitol dehydrogenase activity was highest at 24 h, whereas for cell cultures that were grown in gluconate and xylose, the difference in the reductase and dehydrogenase activities was highest at 12 h after xylose addition. The NAD+ dependent xylitol dehydrogenase activity was low compared to the cells grown without gluconate. The molecular weight of NADPH-dependent xylose reductase protein obtained by gel filtration chromatography was 58 kDa. Initial purification was performed on a DE-52 anion exchange column. Purification using Red Sepharose affinity column resulted in a 58 kDa protein on the SDS PAGE gel and was further purified on a Mono-Q column. The activity stained band on the native gel yielded 58, 49, 39 and 30 kDa bands on the denaturing gel. The peptides of the 58 kDa protein of Corynebacterium sp. B 4247 sequenced by mass spectrometry, identified with E2 and E3 (Bacillus subtilis) components of multi-enzyme system consisting of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and oxo-acid dehydrogenase complex. A 75% match was shown by the peptide “QMSSLVTR” with E-value of 8e-04 to the keto-aldose reductase Gre3p (Saccharomyces cerevisiae) that is induced by osmotic stress and is similar to xylose reductases from other fungi. The peptide “LLNDPQLILMEA” had conserved match “LL + DP” over several aldose reductases. The xylose reductase of the yeast Candida tropicalis ATCC 96745 was also purified. The molecular weight of the yeast NADPH-dependent xylose reductase by SDS PAGE was about 37 kDa. iv ACKNOWLEDGEMENTS I thank my major advisor, Dr. Foster A. Agblevor for his advice and guidance. I thank him for the opportunity provided and the facilities provided for this research. In addition I would like to thank him for the valuable hours spent in correcting my writing and for timely help at critical moments. I thank Dr. Jiann-Shin Chen for his suggestions and guidance. I thank Dr. Brenda Winkel-Shirley for the valuable suggestions and inspiration. I thank Dr. Richard Helm and for his time and advice. I thank Dr. Gene Haugh for his guidance and mentorship. I am sincerely grateful to Dr. Dennis Dean (Fralin Biotechnology Center), Dr. Sriranganathan (CMMID), Dr. Peter Kennelly (Biochemistry Dept.), Dr. Chenming Zhang (Biological Systems Engineering Dept.) and Dr. Kumar Mallikarjunan (Biological Systems Engineering Dept.) for permitting me to use their laboratories and instruments. I am grateful to Harriet Williams, Kim Harich, Dr. Kay Carlson (VMRC), Valerie Cash, Dr. Brian Lower, Ben Potters, Dr. Murat Kasap and Dr. Adnan Ismaiel for their timely assistance and suggestions. I thank Dr. John Perumpral, Head of the Department, Dr. Saied Mostaghimi, chair graduate studies and Linda Altizer, Joyce Longrie, Terassa Cox, Diane, Dana Partin, Leon Alley, Julie Jordan, Jack Davis, Aubrey Murden of Biological Systems Engineering Department for their time and assistance. I sincerely thank the Biological Systems Engineering Department and Hatch and ASPIRES programs for providing the research funding. Most of all, I thank my parents and God for making it possible. iv TABLE OF CONTENTS ABSTRACT................................................................................................................. II ACKNOWLEDGEMENTS........................................................................................ IV TABLE OF CONTENTS............................................................................................. V LIST OF FIGURES ..................................................................................................... X LIST OF TABLES....................................................................................................XIV LIST OF ABBREVIATIONS....................................................................................XV LIST OF CULTURES ..............................................................................................XVI CHAPTER 1 INTRODUCTION..................................................................................... 1 Research Objectives: .................................................................................................. 2 CHAPTER 2 REVIEW OF LITERATURE .................................................................. 3 Xylitol: The sweetener............................................................................................ 3 Xylitol occurrence................................................................................................... 3 Xylitol Applications................................................................................................ 3 Food and confectionery....................................................................................... 3 Pharmaceutical Industry...................................................................................... 6 Yeasts................................................................................................................ 11 Fungi ................................................................................................................. 14 Bacteria ............................................................................................................. 14 Factors influencing microbial production of xylitol ............................................. 15 Inoculum age..................................................................................................... 15 Cell density ....................................................................................................... 15 Effect of pH....................................................................................................... 16 Effect of temperature ........................................................................................ 16 Inhibition by xylitol .......................................................................................... 17 Nitrogen source................................................................................................. 17 Carbon source ................................................................................................... 18 Effect of the initial substrate concentration ...................................................... 19 v Process Strategies.............................................................................................. 21 Effect of vitamins.............................................................................................. 21 Aeration............................................................................................................. 21 Metabolism ........................................................................................................... 22 Yeast

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