Pectinolytic Enzymes of Aspergillus Sojae ATCC 20235: the Impact of Bioprocessing Strategy on Solid-State Production and Downstream Processing of Polygalacturonase
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Pectinolytic enzymes of Aspergillus sojae ATCC 20235: The impact of bioprocessing strategy on solid-state production and downstream processing of polygalacturonase by Doreen Heerd A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemical Engineering Approved, Thesis Committee Prof. Dr. Marcelo Fernández-Lahore Jacobs University Bremen Prof. Dr. Matthias Ullrich Jacobs University Bremen Dr.-Ing. Dirk Holtmann DECHEMA Research Institute Dr. Sonja Diercks-Horn Jacobs University Bremen Date of Defense: June 14, 2013 School of Engineering and Sciences Summary Since antiquity up to the present Aspergillus spp. like A. oryzae or A. sojae have been used in traditional Japanese fermented food production. The long history of safe use in the production of oriental fermented food favors these microorganisms for their application in industrial enzyme production that are applied in the food industry. This thesis deals with the investigation of A. sojae ATCC 20235 as potential pectinolytic enzyme production organism with focus on polygalacturonase (PG) production under solid-state conditions. Pectinolytic enzymes have been exploited for many industrial applications, e.g. the largest industrial application of these enzymes is in juice and wine production. PGs belong to the pectinolytic enzyme group and are an inherent part of commercial enzyme preparations used for food processing. Recent articles reported about the potential of A. sojae ATCC 20235 to produce PG enzyme in submerged fermentation and via surface cultivation methods. These studies have triggered an interest on the investigation of the potential of this strain for pectinolytic enzyme production in solid-state fermentation (SSF). For this, a microbial screening between A. sojae ATCC 20235, a know pectinase producer A. niger IMI 91881, and two further A. sojae strains was performed, which revealed the potential of A. sojae ATCC 20235 as pectinase producer under solid- state culture conditions. Media design and SSF process optimization applying advanced statistical design tools yielded in 10.9 times increased PG production under optimized SSF conditions. Moreover, SSF facilitated the utilization of agricultural and agro-industrial by-products as cultivation medium, which provided the establishment of an economical process for enzyme production. Simultaneously, an enhancement in PG production was traced by microbial strain improvement. Therefore, a classical mutation and selection strategy was developed in order to generate mutants showing increased exo-PG activity. Mutation was induced by ultraviolet irradiation, and via the chemical agent N-methyl-N’-nitro-N- nitrosoguanidine (NTG). The selection of desired mutants based on a three-step strategy. The first pre-screening focused on morphological parameters regarding the sporulation. The second screening based on the selection of “zone mutants”, which enabled the detection of desired mutants with enhanced pectinase activity measured as clear zones on an agar screening medium containing polygalacturonic acid (PGA) sodium salt as substrate. These mutants were screened in the third step for enhanced exo-PG production in SSF. Repeated cycles of mutagenesis by UV rays and sequential mutagenesis employing both methods generated stable mutants iii showing enhanced pectinase activities. However, highest improvement of 72 % was achieved after 3 cycles of exposure to UV irradiation. PG production by the generated mutant under optimized process conditions was successfully transferred into a rotating drum type bioreactor at a scaling ratio of 100, without losses in the enzyme activity. Besides the enzyme production focus was also set on efficient enzyme recovery and PG purification. Optimization of enzyme leaching conditions facilitated sufficient PG recovery in the crude extract. PG purification was traced by combination of various chromatographic techniques. Apparently purity of PG enzyme by means of a single band on SDS-polyacrylamide gel was achieved after separating A. sojae proteins on the basis of their charge by ion exchange chromatography, followed by separation on the basis of their size and shape (size exclusion chromatography), and finally on the degree of hydrophobicity (hydrophobic interaction chromatography). Mass spectrometric characterization of A. sojae proteins led to the identification of a broad enzyme spectrum. Several application studies related to fruit juice production and wine making were performed exploring the efficiency of A. sojae enzyme extracts in comparison to commercial pectinolytic enzyme preparations. Results of this investigation revealed the potential of A. sojae ATCC 20235 as promising enzyme producer in SSF, because of its broad enzyme set and this study showed that the fungal strain is capable of secreting high levels of proteins after optimizing the culture conditions for the specific enzyme production. iv Acknowledgements I am very grateful for given the chance to purse a PhD, as well as for all other things I learned during my PhD time. For this, I am especially indebted to Prof. Dr. Marcelo Fernández-Lahore for his trust in me beyond my expertise. Special thanks to Dr. Sonja Diercks-Horn, who gave me support throughout the work, kind supervision, and thanks for her encouragement. Thanks to Prof. Dr. Matthias Ullrich and Dr.-Ing. Dirk Holtmann for accepting to review this thesis and evaluating, for helpful and productive hints and support. Thanks also to all DSP group members providing congenial atmosphere to work, especially to Dr. Rosa Cabrera for the warm welcome and dedication to my initial lab training. Thanks to Dr. Rami Reddy Vennapusa and to Dr. Poondi Rajesh Gavara for scientific support. Many thanks to Nina Nentwig and Nina Böttcher for the pleasant cooperation, dedicated work in the lab and for sharing their experience. I am thankful to the members of AG Muskhelishvili, AG Ullrich and all people working in Laboratory 2 at Jacobs University Bremen, especially to Dr. Patrick Sobetzko and Rohan L. Shah for scientific support and helpful hints. Many thanks to Ulf Krause for technical support. This project would not have been possible without financial support provided by Jacobs University Bremen through the project PGSYS / ETB-2008-44 and PGSYS EXCHANGE. Finally, I want to express my deepest gratitude to my family and to Stefan Gerlach for their moral support and encouragement. v Table of Contents Summary iii Acknowledgements v Chapter 1 1 General Introduction Chapter 2 18 Pectinase enzyme-complex production by Aspergillus spp. in solid-state fermentation: A comparative study Chapter 3 41 Statistical media design and SSF process optimization for improved PG production by Aspergillus sojae Chapter 4 75 Microbial strain improvement for enhanced PG production Chapter 5 103 Improved PG enzyme bioproducion by Aspergillus sojae mutant M3 and SSF process studies at bioreactor level Chapter 6 126 Separation, purification and partial characterization of polygalacturonase derived from solid-state culture of Aspergillus sojae Chapter 7 163 Characterization of Aspergillus sojae enzyme extracts and application studies vi Chapter 8 205 Concluding remarks and future prospects Appendix A 215 General Materials and Methods Appendix B 220 Bioreactor studies Appendix C 242 Utilization of NW-Q fiber for PG purification vii List of Figures Chapter 1 Figure 1.1 Asexual reproductive structures used in classification of koji molds 3 Figure 1.2 Mode of action of pectinases 7 Figure 1.3 Scheme of solid-state fermentation system 9 Figure 1.4 Schematic drawing of fungal growth in SSF 10 Chapter 2 Figure 2.1 Cultivation profile of A. niger IMI 91881 26 Figure 2.2 Cultivation profile of A. sojae ATCC 20235 27 Figure 2.3 Cultivation profile of A. sojae IMI 191303 28 Figure 2.4 Cultivation profile of A. sojae CBS 100928 29 Figure 2.5 Analysis of crude extracts from the 5th day of SSF by SDS-PAGE 35 Figure 2.6 Native PAGE for enzyme detection on the electrophoretic gel 36 Chapter 3 Figure 3.1 Replicate plot of 2nd screening step 54 Figure 3.2 Contour plot of the 1st optimization step 58 Figure 3.3 Contour plot of the 2nd optimization step 61 Figure 3.4 Solid-state fermentation profile of A. sojae ATCC 20235 62 Figure 3.5 Contour plot of the optimization with A. sojae CBS 100928 66 Figure 3.6 Analysis of crude extracts by SDS-PAGE 67 Figure 3.7 Native PAGE and zymogram 68 Chapter 4 Figure 4.1 Key steps of culture screening and improvement 79 Figure 4.2 Chemical structure of NTG 80 Figure 4.3 Scheme of repeated and sequential mutagenesis 81 Figure 4.4 Sweet spot plot of the screening with A. sojae ATCC 20235 86 Figure 4.5 Sweet spot plot of the screening with A. sojae CBS 100928 86 Figure 4.6 Replicate plot of results of A. sojae ATCC 20235 88 Figure 4.7 Replicate plot of results of A. sojae CBS 100928 88 Figure 4.8 Contour plot of the optimization with A. sojae CBS 100928 89 Chapter 5 Figure 5.1 Loading plot 113 Figure 5.2 Contour plots with the variation of moisture level and temperature 113 Figure 5.3 Contour plots with the variation of moisture level and time 114 viii Figure 5.4 Cultivation profile of mutant M3 at SSF bioreactor 118 Figure 5.5 Cultivation profile of A. sojae ATCC 20235 at SSF bioreactor 120 Chapter 6 Figure 6.1 Scheme of filtration process 131 Figure 6.2 Leaching efficiency of solvents 134 Figure 6.3 Contour plots of PG activity 139 Figure 6.4 Contour plots of specific activity 139 Figure 6.5 Effect of contact time on enzyme leaching 141 Figure