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- Wang, Yang
(författare)
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Discovery and investigation of glycoside hydrolase family 5 enzymes with potential use in biomass conversion
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glycoside hydrolases (GHs) cleave glycosidic bonds in glycoconjugates, oligosaccharides and polysaccharides such as cellulose and various hemicelluloses. Mannan is a major group of hemicelluloses. In higher plants, they usually serve as storage carbohydrates in seeds and tubers or as structural polysaccharides cross-linking with cellulose/lignin in cell walls. In industrial fields, this renewable biomass component can be used in various areas such as production of biofuels and health-benefit manno-oligosaccharides; and mannan degrading enzymes, especially mannanases, are important molecular tools for controlling mannan polysaccharides properties in biomass conversion. In this thesis, the evolution, substrate specificity and subfamily classification of the most important GH family, i.e., glycoside hydrolase family 5 (GH5), are presented providing a powerful tool for exploring GH5 enzymes in search for enzymes with interesting properties for sustainable biomass conversion. Additionally, three GH5_7 mannanases from Arabidopsis thaliana (AtMan5-1, AtMan5-2 and AtMan5-6) were investigated in the present study. Bioinformatics tools, heterologous expression, and enzymology were applied in order to reveal the catalytic properties of the target enzymes, increase understanding of plant mannanase evolution, and evaluate their potential use in biomass conversion. This approach revealed: (1) AtMan5-1 exhibits mannan hydrolase/transglycosylase activity (MHT), (2) AtMan5-2 preferably degrades mannans with a glucomannan backbone, and (3) AtMan5-6 is a relatively thermotolerant enzyme showing high catalytic efficiency for conversion of glucomannan and galactomannan making this plant mannanase an interesting candidate for biotechnological applications of digesting various mannans. Moreover, these studies suggest an evolutionary diversification of plant mannanase enzymatic function.
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| 2. |
- Wang, Yang
(författare)
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Exploring glycoside hydrolase family 5 (GH5) enzymes
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In 1990, the classification of carbohydrate-active enzymes (CAZymes) was introduced by the scientist Bernard Henrissat. According to sequence similarity, these enzymes were separated into families with conserved structures and reaction mechanisms. One interesting class of CAZymes is the group of glycoside hydrolases (GHs) containing more than 138000 modules divided into 131 families as of February 2013. One of the most versatile and the largest of these GH families, containing enzymes with numerous biomass-deconstructing activities, is glycoside hydrolase family 5 (GH5). However, for large and diverse families like the GH5 family, another layer of classification is required to get a better understanding of the evolution of diverse enzyme activities. In Paper I, a new subfamily classification of GH5 is presented in order to sort the family members into distinct groups with predictive power. In total, 51 subfamilies were defined. Despite the fact that several hundred GH5 enzymes have been characterized, 20 subfamilies lacking biochemically characterized enzymes and 38 subfamilies without structural data were identified. These highlighted subfamilies contain interesting targets for future investigation.The GH5 family includes endo-β-mannanases catalyzing the hydrolysis of the β-1,4-linked backbone of mannan polysaccharides, which are common hemicelluloses found as storage and structural polymers in plant cell walls. Mannans are commonly utilized as raw biomaterials in food, feed, paper, textile and cosmetic industries, and mannanases are often applied for modifying and controlling the property of mannan polysaccharides in such applications. The overwhelming majority of characterized mannanases are from microbial origin. The situation for plant mannanases is quite different, as the catalytic properties for only a handful have been determined. Paper II describes the first characterization of a heterologously expressed Arabidopsis β-mannanase.
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| 3. |
- Winzell, Anders, 1978-
(författare)
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Investigation of genes and proteins involved in xylan biosynthesis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates. Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes. By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering.
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