Keratin is an insoluble and protein-rich epidermal material found in e.g. feather, wool, hair. It is produced in substantial amounts as co-product from poultry processing plants and pig slaughterhouses. Keratin is packed by disulfide bonds and hydrogen bonds. Based on the secondary structure, keratin can be classified into α-keratin and β-keratin. Keratinases (EC 3.4.-.- peptide hydrolases) have major potential to degrade keratin for sustainable recycling of the protein and amino acids. Currently, the known keratinolytic enzymes belong to at least 14 different protease families: S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, M55 (MEROPS database). The various keratinolytic enzymes act via endo -attack (proteases in families S1, S8, S16, M4, M16, M36), exo -attack (proteases in families S9, S10, M14, M28, M38, M55) or by action only on oligopeptides (proteases in families M3, M32), respectively. Other enzymes, particularly disulfide reductases, also play a key role in keratin degradation as they catalyze the breakage of disulfide bonds for better keratinase catalysis. This review aims to contribute an overview of keratin biomass as an enzyme substrate and a systematic analysis of currently sequenced keratinolytic enzymes and their classification and reaction mechanisms. We also summarize and discuss keratinase assays, available keratinase structures and finally examine the available data on uses of keratinases in practical biorefinery protein upcycling applications.

Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. --1,3 arabinofuranosyl specific -L-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release -L-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies -L-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively -L-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 -L-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.3

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We set out to investigate the genetic adaptations of the marine fungus Paradendryphiella salina CBS112865 for degradation of brown macroalgae. We performed whole genome and transcriptome sequencing and shotgun proteomic analysis of the secretome of P . salina grown on three species of brown algae and under carbon limitation. Genome comparison with closely related terrestrial fungi revealed that P . salina had a similar but reduced CAZyme profile relative to the terrestrial fungi except for the presence of three putative alginate lyases from Polysaccharide Lyase (PL) family 7 and a putative PL8 with similarity to ascomycete chondroitin AC lyases. Phylogenetic and homology analyses place the PL7 sequences amongst mannuronic acid specific PL7 proteins from marine bacteria. Recombinant expression, purification and characterization of one of the PL7 genes confirmed the specificity. Proteomic analysis of the P . salina secretome when growing on brown algae, revealed the PL7 and PL8 enzymes abundantly secreted together with enzymes necessary for degradation of laminarin, cellulose, lipids and peptides. Our findings indicate that the basic CAZyme repertoire of saprobic and plant pathogenic ascomycetes, with the addition of PL7 alginate lyases, provide P . salina with sufficient enzymatic capabilities to degrade several types of brown algae polysaccharides.

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1–CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

Cereal brans and grain endosperm cell walls are key dietary sources of different types of arabinoxylan. Arabinoxylan is the main group of hemicellulosic polysaccharides that are present in the cell walls of monocot grass crops and hence in cereal grains. The arabinoxylan polysaccharides consist of a backbone of β-(1→4)-linked xylopyranosyl residues, which carry arabinofuranosyl moieties, hence the term arabinoxylan. Moreover, the xylopyranosyl residues can be acetylated or substituted by methyl-glucuronic acid. The arabinofuranosyls may be esterified with a feruloyl group. Feruloylated arabinoxylo-oligosaccharides exert beneficial bioactivities via prebiotic, immunomodulatory, and/or antioxidant effects. New knowledge on microbial enzymes that catalyze specific structural modifications of arabinoxylans can help us understand how these complex fibers are converted in the gut and provide a foundation for the production of feruloylated arabinoxylo-oligosaccharides from brans or other cereal grain processing sidestreams as functional food ingredients. There is a gap between the structural knowledge, bioactivity data, and enzymology insight. Our goal with this review is to present an overview of the structures and bioactivities of feruloylated arabinoxylo-oligosaccharides and review the enzyme reactions that catalyze specific changes in differentially substituted arabinoxylans. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 12 is March 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

BackgroundImproved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry.ResultsEnzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs.ConclusionsOur findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0840-y) contains supplementary material, which is available to authorized users.