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Selective Separation of Proteins Using Stimuli-Responsive Polymers

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Selective Separation of Proteins Using Stimuli-Responsive Polymers SELECTIVE SEPARATION OF PROTEINS USING STIMULI-RESPONSIVE POLYMERS IN AN AQUEOUS SYSTEM A Thesis by JECORI SHANA JOHNSON Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Chair of Committee, Carmen Gomes Co-Chair of Committee, Zivko Nikolov Committee Member, Stephen Talcott Head of Department, Stephen Searcy May 2018 Major Subject: Biological and Agricultural Engineering Copyright 2018 Jecori Johnson ABSTRACT Biopolymers provide an option for protein purification and are being used due to their biodegradability, cost efficiency, non-toxicity and abundance in nature. This study investigated the use of natural and synthetic polymers for the flocculation of proteins by evaluating two bioprocesses: polyelectrolyte precipitation and inverse transition cycling. The effectiveness of chitosan (CHI, pKa= 6.5), polyetheleneimine (PEI, pKa= 11), sodium alginate (ALG, pKa= 3.2), and poly-N-isopropylacrylamide (PNIPAAm, lower critical solution temperature= 32 oC) were evaluated as flocculating agents of two model proteins, bovine serum albumin (BSA, pI=4.5, acidic protein) and lysozyme (LYZ, pI= 11, basic protein). Natural polymers, CHI and ALG have an advantage due to their stimuli-responsive characteristic and also because they are positively or negatively charged at certain pH values, while PNIPAAm, a synthetic polymer, is sensitive to temperature. Briefly, protein and polymer were mixed at pH values where charges (if applicable) were complementary, ideally triggering flocculation by electrostatic interactions. The maximum precipitation yield achieved by polyelectrolyte precipitation resulted from the flocculation of LYZ using ALG at 87%. Our model system included the use of PEI as a flocculant for BSA. This resulted in a 65% precipitation yield. In comparison, CHI was evaluated as a natural flocculant for BSA, which resulted in a 61% precipitation yield. If flocculation did not occur, a phase transition change of polymer was induced by triggering the appropriate stimulus, i.e., temperature, pH, or ionic strength, to precipitate proteins by sweep flocculation. We attempted to use PNIPAAm to precipitate both BSA and LYZ; however, precipitation yield was less than 10%. A subsequent study addressed the synthesis, characterization, and use of PNIPAAm based copolymers and blends for protein flocculation, and it was concluded that these copolymers and blends were not as effective as the single ii polymers (less than 10% increase in precipitation yield). Lastly, optimized bioprocess for precipitation using PEI resulted in a precipitation yield of 37% of all milk proteins. Overall, this study showed that natural polymers (CHI and ALG) can produce precipitation yields comparable to the standard method provided by PEI and should be considered as an alternative for flocculation of proteins. iii ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Carmen Gomes, and my committee members, Dr. Nikolov and Dr. Talcott, for their patience and support throughout the course of this research. Thanks also go to my friend Ayswarya for the late nights, unlimited favors and teaching me everything needed to successfully precipitate proteins. Finally, thanks to my mother for her encouragement and never allowing me to give up and, also my father, sister and boyfriend for giving me a reason to stay the course even when the road was rough. iv CONTRIBUTORS AND FUNDING SOURCES This work was supported by a thesis committee consisting of Professor Carmen Gomes [advisor] and Professor Zivko Nikolov [co-advisor] of the Department of Biological and Agricultural Engineering and Professor Steven Talcott of the Department of Food Science and Technology. Graduate study was supported by a fellowship from Texas A&M University. All other work conducted for the dissertation was completed by the student independently. v TABLE OF CONTENTS Page ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv CONTRIBUTORS AND FUNDING SOURCES .......................................................................... v TABLE OF CONTENTS ............................................................................................................... vi LIST OF FIGURES ..................................................................................................................... viii LIST OF TABLES ......................................................................................................................... xi CHAPTER I INTRODUCTION AND LITERATURE REVIEW ............................................... 1 1.1 Introduction ........................................................................................................................................ 1 1.2 Literature Review ............................................................................................................................... 2 CHAPTER II RATIONALE, HYPOTHESIS AND OBJECTIVES ............................................ 15 1.1 Rationale ........................................................................................................................................... 15 1.2 Hypothesis ........................................................................................................................................ 15 1.3 Objectives ......................................................................................................................................... 16 CHAPTER III SELECTIVE SEPARATION OF PROTEINS USING STIMULI-RESPONSIVE POLYMERS IN AN AQUEOUS SYSTEM...................................... 17 3.1 Overview .......................................................................................................................................... 17 3.2 Introduction ...................................................................................................................................... 18 3.3 Materials and Methods ..................................................................................................................... 20 3.3.1 Protein Preparation ................................................................................................................. 20 3.3.2 Polymer Preparation ............................................................................................................... 21 3.3.3 Polymer Characterization ....................................................................................................... 23 3.4 Bioseparation Processes ................................................................................................................... 25 3.5 Optimization Factors ........................................................................................................................ 31 3.6 Application of Optimized Polymer Bioseparation Process .............................................................. 32 3.7 Statistical Analysis ........................................................................................................................... 32 CHAPTER IV RESULTS AND DISCUSSSION ......................................................................... 34 4.1 Characterization ................................................................................................................................ 34 4.1.1 Lower Critical Solution Temperature ......................................................................................34 4.1.2 Fourier Transform-Infrared Spectroscopy (FT-IR) ................................................................ 35 4.1.3 Zeta potential .......................................................................................................................... 36 4.2 Optimization of Bioseparation Process ............................................................................................ 43 4.2.1 PEI as a Flocculant .................................................................................................................. 43 vi 4.2.2 Recovery of Precipitated BSA ............................................................................................... 44 4.2.3 Chitosan Used as a Polyelectrolyte Flocculant ...................................................................... 47 4.2.4 Alginate Used as a Polyelectrolyte Flocculant ...................................................................... 52 4.2.5 PNIPAAm used as a Polyelectrolyte Flocculant ................................................................... 56 4.2.6 Protein Capture Using PNIPAAm-co-PEI Copolymer .......................................................... 58 4.2.7 Precipitation of Proteins Using PNIPAAm and Other Polymer Blends ................................ 59 4.2.8 Precipitation of Milk Proteins Using PEI .............................................................................. 60 CHAPTER V CONCLUSIONS AND FUTURE RECOMMENDATIONS ................................ 66 5.1 Conclusions ...................................................................................................................................... 66 5.2. Future Recommendations ................................................................................................................ 69 REFERENCES ............................................................................................................................
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