Camilla Maymann Christiansen

The carbohydrate-binding module family 20--diversity, structure, and function.

Starch‐active enzymes often possess starch‐binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate‐binding module (CBM) families, and CBM20 is the earliest‐assigned and best characterized family. High diversity characterizes CBM20s, which occur in starch‐active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch‐phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily relationships. Data on binding to and enzymatic activity towards soluble ligands and starch granules are summarized for wild‐type, mutant and chimeric fusion proteins involving CBM20s. Noticeably, whereas CBM20s in amylolytic enzymes confer moderate binding affinities, with dissociation constants in the low micromolar range for the starch mimic β‐cyclodextrin, recent findings indicate that CBM20s in regulatory enzymes have weaker, low millimolar affinities, presumably facilitating dynamic regulation. Structures of CBM20s, including the first example of a full‐length glucoamylase featuring both the catalytic domain and the SBD, are summarized, and distinct architectural and functional features of the two SBDs and roles of pivotal amino acids in binding are described. Finally, some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented.

A CBM20 low-affinity starch-binding domain from glucan, water dikinase

The family 20 carbohydrate‐binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50‐fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein‐labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein‐tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

An enzyme family reunion — similarities, differences and eccentricities in actions on α -glucans

Abstractα-Glucans in general, including starch, glycogen and their derived oligosaccharides are processed by a host of more or less closely related enzymes that represent wide diversity in structure, mechanism, specificity and biological role. Sophisticated three-dimensional structures continue to emerge hand-in-hand with the gaining of novel insight in modes of action. We are witnessing the “test of time” blending with remaining questions and new relationships for these enzymes. Information from both within and outside of ALAMY_3 Symposium will provide examples on what the family contains and outline some future directions. In 2007 a quantum leap crowned the structural biology by the glucansucrase crystal structure. This initiates the disclosure of the mystery on the organisation of the multidomain structure and the “robotics mechanism” of this group of enzymes. The central issue on architecture and domain interplay in multidomain enzymes is also relevant in connection with the recent focus on carbohydrate-binding domains as well as on surface binding sites and their long underrated potential. Other questions include, how different or similar are glycoside hydrolase families 13 and 31 and is the lid finally lifted off the disguise of the starch lyase, also belonging to family 31? Is family 57 holding back secret specificities? Will the different families be sporting new “eccentric” functions, are there new families out there, and why are crystal structures of “simple” enzymes still missing? Indeed new understanding and discovery of biological roles continuously emphasize value of the collections of enzyme models, sequences, and evolutionary trees which will also be enabling advancement in design for useful and novel applications.

Economics of biodiesel production

EU has determined that 10 % of all fuel used in 2020 should be replaced by a biofuel. That substitute could be biodiesel. This project deals with the possibility of replacing 10 % of the used fuel in Denmark by biodiesel. Furthermore a comparison of the economic evaluation of two different plants was done. The crucial difference between the plants was purchase of the catalyst, enzymatic or chemical, with a difference of 17.3 mil. kr. The residual from both plants is glycerol. A smaller plant was designed to produce arcrolein from glycerol. An economic evaluation of this plant was done.

α-Amylases. Interaction with Polysaccharide Substrates, Proteinaceous Inhibitors and Regulatory Proteins

Abstract α-Amylases occur widely in plants, animals, and microorganisms. They often act in synergy with other related and degradative enzymes and may also be regulated by proteinaceous inhibitors. Open questions exist on how α-amylases interact with polysaccharides. Several enzymes possess secondary carbohydrate binding sites situated on the surface at a certain distance of the active site cleft. The functions of such sites were studied in barley α-amylase isozymes by structure-guided mutational analysis and measurement of activity and binding parameters. Two surface sites were assigned distinct roles. One of the sites seems to participate in hydrolysis of polysaccharides by a multiple attack mechanism. Polysaccharide processing enzymes can also contain carbohydrate binding modules, e.g. starch binding domains that assist in the attack on macromolecular substrates and are useful in engineering of enzyme efficiency. The multidomain nature of these enzymes raises questions on the dynamics and structural properties in solution and in substrate complexes.