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Carbohydrate-Active Enzymes
Published in Jean-Luc Wertz, Magali Deleu, Séverine Coppée, Aurore Richel, Hemicelluloses and Lignin in Biorefineries, 2017
Jean-Luc Wertz, Magali Deleu, Séverine Coppée, Aurore Richel
A carbohydrate-binding module (CBM) is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity.49 A few exceptions are CBMs of cellulosomal scaffoldin proteins and rare instances of independent putative CBMs. Most cellulases have a catalytic module and a CBM joined by a highly glycosylated and presumably flexible linker peptide.50 Removal of the CBM results in a significantly reduced enzymatic activity on crystalline cellulose probably due to a decreased binding capacity, but the activity on soluble cellulose oligomers is retained. Like the catalytic modules, the CBMs also form distinct families of related amino acid sequences (Table 2.3).35 Representative structures for each CBM family have been elucidated either by crystallography or by NMR spectroscopy.37
Role of carbohydrate active enzymes (CAZymes) in production of marine bioactive oligosaccharides and their pharmacological applications
Published in Antonio Trincone, Enzymatic Technologies for Marine Polysaccharides, 2019
CAZymes are implicated in synthesis, breakdown, and modification of complex polysaccharides (CPs) or glycoconjugates. CAZy classification is a sequence-based classification of CAZymes and is recorded in the CAZy database. On the basis of the amino acid sequence, CAZymes are classified under five different families: glycoside hydrolase (GHs), polysaccharide lyase (PLs), glycosyl transferase (GTs), carbohydrate esterase (CEs), and enzymes with auxiliary activities (AAs) (Cantarel et al. 2009). GHs and PLs are class of CAZymes that are involved in degradation of CPs. The GHs are the largest family of CAZymes that hydrolyze the glycosidic bond between two or more carbohydrate or between carbohydrate and noncarbohydrate moieties via overall retention or by overall inversion of an anomeric carbon mechanism (Figure 16.1). On the contrary, PLs cleave uronic acid–containing polysaccharide chains via a β-elimination mechanism to generate an unsaturated hexenuronic acid residue and a new reducing end. Currently, in the CAZy database, GHs are classified in a total of 152 families whereas PLs are reported in 28 different families. CAZymes often display noncatalytic carbohydrate-binding modules (CBMs) that reportedly enhance the activity of a cognate catalytic module (Cantarel et al. 2009). Because of the high degree of polymerization and presence of substituted groups such as sulfate, CPs are often recalcitrant and their glycosidic bonds are inaccessible to the active site of CPs-degrading CAZymes. Noncatalytic CBMs appended to the catalytic modules assists the CPs-degrading CAZymes in accessing the target glycosidic bonds and promotes the association of the enzyme with the substrate (Boraston et al. 2004).
Two β-glucanases from bacterium Cellulomonas flavigena: expression in Pichia pastoris, properties, biotechnological potential
Published in Preparative Biochemistry & Biotechnology, 2023
Alexander Lisov, Oksana Belova, Zoya Lisova, Alexey Nagel, Andrey Shadrin, Zhanna Andreeva-Kovalevskaya, Maxim Nagornykh, Marina Zakharova, Alexey Leontievsky
In the genome of C. flavigena, were identified two genes encoding glucohydrolases of the GH 6 family and Cfla_2913 (GenBank Ass. No. ADG75796.1) and Cfla_2912 (GenBank Ass. No. ADG75795.1). These genes were amplified by PCR and cloned into the pPic9m vector. The sequences of the cloned Cfla_2912 and Cfla_2913 genes were completely identical to the sequences of the corresponding genes in the GenBank, based on which we designed the primers for PCR. Both proteins contain a TAT signal peptide (a.a. 1–33 for Cfla_2913 and a.a. 1–36 for Cfla_2912), indicating that they may be secreted enzymes; both of them also contain a carbohydrate binding module (CBM) from family 2 (a.a. 36–145 for both Cfla_2912 and Cfla_2913). At the C-terminus, both Cfla_2912 and Cfla_2913 contain the GH6 catalytic domain. Cfla_2912 and Cfla_2913 have a high homology of 76%; 68% of amino acids in their sequences are identical (Figure 1). Cfla_2913 is shorter than Cfla_2912 due to a 22 a.a. deletion in the region of the molecule located between the CBM2 and GH6 domains.
Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus
Published in Preparative Biochemistry & Biotechnology, 2021
María Teresa Alvarez-Zúñiga, Diana Castañeda García, Guillermo Aguilar Osorio
Since understanding the genetic composition of a microorganism can reveal its plant biomass-degrading abilities, we analyzed the potential genomic capacity of A. flavus through its CAZymes. It has been reported that both morphotypes of A. flavus share the same ancestor.[16] According to Ohkura et al.[7] the differences in copy numbers of CAZymes between morphotypes (S and L) are minimal. Therefore, to analyze the potential genomic capacity of A. flavus, the reference genome NRRL was used. A total of 624 enzymes were identified in the A. flavus NRRL 3357 genome. This value was similar to the number of CAZymes reported by Benoit (579) and de Vries (600) for the same organism.[11,12] The ring graph (Fig. 1) shows the distribution of the different CAZy families, glycoside hydrolases (GH), glycoside transferase (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), carbohydrate-binding module (CBM) and, auxiliary activity (AA) in the three studies. The variation between analyses of the genome is very small; in GH the variation was of 25 proteins and, 10 proteins for AA, a consequence of the pipelines employed not being identical.
Polar residues lining the binding cleft of a Serratia marcescens family 18 chitinase position the substrate for attack and stabilize associative interactions
Published in Molecular Physics, 2019
Suvamay Jana, Anne Grethe Hamre, Vincent G. H. Eijsink, Morten Sørlie, Christina M. Payne
To further explore the role of polar residues in GH carbohydrate recognition, activity, and processivity, in this study, we examined the function of polar residues along the binding cleft of Serratia marcescens family 18 chitinase B (ChiB). ChiB is an exo-acting, non-reducing end-specific GH when acting on crystalline chitin [22]. Notably, the mode of action changes from exo- to endo- when acting on soluble chitosan substrate [23]. The deep catalytic cleft of ChiB has four substrate subsites (+4 to+1) and three product subsites (−1 to −3) within the catalytic domain (CD), as partially illustrated in Figure 1 [7]. The carbohydrate binding module (CBM), fused with the CD, provides additional substrate binding sites beyond the four substrate subsites of the CD. The roles of aromatic residues across the binding sites of ChiB, including in the CD and CBM, have previously been examined. In the ChiB CD, Trp-97 (+1 site) and Trp-220 (+2 site) were found to be critical for substrate binding and processivity, while Phe-190 at the +3 site seems to be less important in ligand binding (Figure 1) [14,24]. Similarly, several tryptophan and tyrosine residues in the ChiB CBM were found to be crucial in crystalline substrate recruitment and hydrolysis [13]. A comparable molecular-level understanding of polar residue function in the ChiB substrate binding cleft does not yet exist.