Cellulase engineering to alleviate non-productive enzyme binding to pretreated lignocellulosic biomass
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Nemmaru, Bhargava.
Cellulase engineering to alleviate non-productive enzyme binding to pretreated lignocellulosic biomass. Retrieved from
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TitleCellulase engineering to alleviate non-productive enzyme binding to pretreated lignocellulosic biomass
Date Created2022
Other Date2022-01 (degree)
Extent205 pages : illustrations
DescriptionThe production of fuels and chemicals from lignocellulosic biomass is hinged upon cost-effective production of reactive intermediates such as glucose and xylose from cellulose and hemicellulose, respectively. In nature, hydrolysis of lignocellulosic biomass to produce these intermediates is mediated by cellulases, hemicellulases, and other accessory enzymes secreted by cellulolytic microbes like Trichoderma reesei, Clostridium thermocellum, or Thermobifida fusca. However, enzymatic hydrolysis is impeded due to various factors associated with the structural and chemical complexity of lignocellulosic biomass, collectively termed as ‘biomass recalcitrance’. The key contributors to biomass recalcitrance are: (i) the highly crystalline structure of cellulose nanofibrils held together by inter- and intra-sheet hydrogen bonding, and (ii) restricted access to cellulose and hemicellulose due to protective lignin polymer sheathing. Thermochemical pretreatment of biomass prior to enzymatic hydrolysis can improve accessibility of enzymes to cellulose and hemicellulose. However, most pretreatments also deposit lignin-enriched residues on the outer surface of cell walls, leading to enzyme inhibition due to non-specific binding to lignin. In addition, prior studies have also shown that rate-retardation of cellulase enzymes acting on pure cellulose arises from a phenomenon widely termed as ‘non-productive binding’. This term is used to refer to enzyme molecules that are bound to cellulose but not productively engaged with the substrate via the enzyme active site. Preliminary studies suggest that alleviation of non-productive binding to cellulose and/or lignin can dramatically improve the hydrolytic activity of bound enzymes towards pretreated biomass. This dissertation is focused on the development and implementation of rational engineering strategies for cellulolytic enzymes to reduce non-productive binding to various cell wall polymers, thereby improving hydrolytic activity towards pretreated biomass.This dissertation is divided into three research objectives, which collectively provide insight into the phenomenon of non-productive enzyme binding to pretreated biomass. Objective 1 is focused on understanding whether non-productive binding of carbohydrate-binding modules (CBMs) to various allomorphs of cellulose, such as cellulose-I and cellulose-III, can be simply alleviated by alanine mutations of planar binding motif aromatic residues. CBMs are critical accessory substrate binding domains tethered to catalytic domains of most native cellulolytic enzymes. These mutant CBMs were next fused with a model glycosyl hydrolase family 5 (GH5) cellulase catalytic domain (called CelE from Clostridium thermocellum) followed by detailed characterization of enzymatic hydrolysis activity and binding affinities on various cellulosic substrates. This work led to generation of mutant enzymes with up to 80% higher hydrolytic activity towards cellulose-I. Objective 2 is focused on implementation of a computational protein design strategy called ‘supercharging’ which was used to alter the net surface charge on CBMs and hence altering the strength and nature of electrostatic vs. hydrophobic stacking interactions with biomass polymers such as lignin and cellulose. A small library of positively and negatively supercharged mutant CBMs were fused with a GH5 cellulase catalytic domain (Cel5A) from Thermobifida fusca and these fusion constructs were first tested against various forms of industrially relevant pretreated lignocellulosic biomass (e.g., Ammonia Fiber Expansion or AFEX, Extractive Ammonia or EA, and Dilute Acid or DA treated corn stover). To understand the behavior of these enzymes on pretreated biomass, detailed enzymatic hydrolysis and binding assays were performed on cellulose and lignin. Finally, the work performed under Objective 3 seeks to extend the supercharging approach developed previously, to eleven unique families of cellulolytic enzymes from the biomass-degradation synergistic enzyme cocktail secreted by Thermobifida fusca. A high-throughput screening approach to measure the heterologous expression potential using an ELISA toolkit and specific hydrolytic activities of these enzymes within the soluble cell lysate was developed. Overall, this work highlights the potential of rational enzyme engineering strategies to improve the hydrolytic activity of cellulolytic enzymes towards pretreated cellulosic biomass by reducing non-productive enzyme binding and development of cost-effective approaches for biologically catalyzed conversion of lignocellulosic biomass into fuels and products.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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