Reliability factor for identification of amylolytic enzyme activity in the optimized starch-iodine assay

Amylolytic enzymes are a group of proteins degrading starch to its constitutional units. For high-throughput screening, simple yet accurate methods in addition to the reducing ends assays are required. In this article, the iodine assay, a photometric assay based on the intensely colored starch-iodine complex, was adapted to enable accurate and objective differentiation between enzyme and background activity using a newly introduced mathematical factor. The method was further improved by designing a simple setup for multiple time point detection and discussing the applicability of single wavelength measurements

Occurrence and function of enzymes for lignocellulose degradation in commercial Agaricus bisporus cultivation

The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed.Peer reviewe

Alpha-1,4-transglycosylation Activity of GH57 Glycogen Branching Enzymes Is Higher in the Absence of a Flexible Loop with a Conserved Tyrosine Residue

Starch-like polymers can be created through the use of enzymatic modification with glycogen branching enzymes (GBEs). GBEs are categorized in the glycoside hydrolase (GH) family 13 and 57. Both GH13 and GH57 GBEs exhibit branching and hydrolytic activity. While GH13 GBEs are also capable of α-1,4-transglycosylation, it is yet unknown whether GH57 share this capability. Among the four crystal structures of GH57 GBEs that have been solved, a flexible loop with a conserved tyrosine was identified to play a role in the branching activity. However, it remains unclear whether this flexible loop is also involved in α-1,4-transglycosylation activity. We hypothesize that GH57 GBEs with the flexible loop and tyrosine are also capable of α-1,4-transglycosylation, similar to GH13 GBEs. The aim of the present study was to characterize the activity of GH57 GBEs to investigate a possible α-1,4-transglycosylation activity. Three GH57 GBEs were selected, one from Thermococcus kodakarensis with the flexible loop and two beta-strands; one from Thermotoga maritima, missing the flexible loop and beta-strands; and one from Meiothermus sp., missing the flexible loop but with the two beta-strands. The analysis of chain length distribution over time of modified maltooctadecaose, revealed, for the first time, that all three GH57 GBEs can generate chains longer than the substrate itself, showing that α-1,4-transglycosylation activity is generally present in GH57 GBEs.</p

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

| openaire: EC/H2020/648925/EU//BHIVEAcetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono-or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.Peer reviewe

The influence of amylose content on the modification of starches by glycogen branching enzymes

Glycogen branching enzymes (GBEs) have been used to generate new branches in starches for producing slowly digestible starches. The aim of this study was to expand the knowledge about the mode of action of these enzymes by identifying structural aspects of starchy substrates affecting the products generated by different GBEs. The structures obtained from incubating five GBEs (three from glycoside hydrolase family (GH) 13 and two from GH57) on five different substrates exhibited minor but statistically significant correlations between the amount of longer chains (degree of polymerization (DP) 9-24) of the product and both the amylose content and the degree of branching of the substrate (Pearson correlation coefficient of ≤-0.773 and ≥0.786, respectively). GH57 GBEs mainly generated large products with long branches (100-700 kDa and DP 11-16) whereas GH13 GBEs produced smaller products with shorter branches (6-150 kDa and DP 3-10)

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

The Synergistic Effect of GH13 and GH57 GBEs of Petrotoga mobilis Results in α-Glucan Molecules with a Higher Branch Density

Glycogen is a biopolymer consisting of glycosyl units, with a linear backbone connected by α-1,4-linkages and branches attached via α-1,6-linkages. In microorganisms, glycogen synthesis involves multiple enzymes, with glycogen branching enzymes (GBEs) being vital for creating α-1,6-linkages. GBEs exist in two families: glycoside hydrolase (GH) 13 and GH57. Some organisms possess either a single GH13 or GH57 GBE, while others, such as Petrotoga mobilis, have both types of GBEs. In this study, the simultaneous use of a GH13 and GH57 GBE each from Petrotoga mobilis for α-glucan modification was investigated using a linear maltodextrin substrate with a degree of polymerization of 18 (DP18). The products from modifications by one or both GBEs in various combinations were analyzed and demonstrated a synergistic effect when both enzymes were combined, leading to a higher branch density in the glycogen structure. In this cooperative process, PmGBE13 was responsible for creating longer branches, whereas PmGBE57 hydrolyzed these branches, resulting in shorter lengths. The combined action of the two enzymes significantly increased the number of branched chains compared to when they acted individually. The results of this study therefore give insight into the role of PmGBE13 and PmGBE57 in glycogen synthesis, and show the potential use of both enzymes in a two-step modification to create an α-glucan structure with short branches at a high branch density.</p

Long chains and crystallinity govern the enzymatic degradability of gelatinized starches from conventional and new sources

Slowly digestible starches have received interest due to their lower increase of postprandial blood glucose and insulin levels and, hence, modification of starches towards slower digestibility has commercial interest. However, chemical characteristics driving enzymatic (digestive) degradation are not fully unraveled. The digestion properties of starches have been linked to their crystalline type, chain length distribution, amylose content or degree of branching, but content and length of relatively long side-chains in amylopectin has not been paid attention to. Therefore, this research focusses on the unique content and length of amylopectin side-chains from conventional and new starch sources (potato, corn, pea, and tulip) correlated to the enzymatic digestion. The rate of hydrolysis was found to be correlated with the crystalline type of starch, as previously suggested, however, the complete hydrolysis of all starches, independent of the crystalline type and source, was shown to be governed by the content of longer amylopectin chains

The Synergistic Effect of GH13 and GH57 GBEs of Petrotoga mobilis Results in α-Glucan Molecules with a Higher Branch Density

Glycogen is a biopolymer consisting of glycosyl units, with a linear backbone connected by α-1,4-linkages and branches attached via α-1,6-linkages. In microorganisms, glycogen synthesis involves multiple enzymes, with glycogen branching enzymes (GBEs) being vital for creating α-1,6-linkages. GBEs exist in two families: glycoside hydrolase (GH) 13 and GH57. Some organisms possess either a single GH13 or GH57 GBE, while others, such as Petrotoga mobilis, have both types of GBEs. In this study, the simultaneous use of a GH13 and GH57 GBE each from Petrotoga mobilis for α-glucan modification was investigated using a linear maltodextrin substrate with a degree of polymerization of 18 (DP18). The products from modifications by one or both GBEs in various combinations were analyzed and demonstrated a synergistic effect when both enzymes were combined, leading to a higher branch density in the glycogen structure. In this cooperative process, PmGBE13 was responsible for creating longer branches, whereas PmGBE57 hydrolyzed these branches, resulting in shorter lengths. The combined action of the two enzymes significantly increased the number of branched chains compared to when they acted individually. The results of this study therefore give insight into the role of PmGBE13 and PmGBE57 in glycogen synthesis, and show the potential use of both enzymes in a two-step modification to create an α-glucan structure with short branches at a high branch density.</p