Exploring the pH dependence of an improved PETase.

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His.

Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications.

In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes' performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used.

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes that enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization to precise Michaelis-Menten analysis.

Mixed Linkage β-1,3/1,4-Glucan Oligosaccharides Induce Defense Responses in and .

Plants detect conserved microbe-associated molecular patterns (MAMPs) and modified "self" molecules produced during pathogen infection [danger associated molecular patterns (DAMPs)] with plasma membrane-resident pattern recognition receptors (PRRs). PRR-mediated MAMP and/or DAMP perception activates signal transduction cascades, transcriptional reprogramming and plant immune responses collectively referred to as pattern-triggered immunity (PTI). Potential sources for MAMPs and DAMPs are microbial and plant cell walls, which are complex extracellular matrices composed of different carbohydrates and glycoproteins.

N-Glycan Degradation Pathways in Gut- and Soil-Dwelling Actinobacteria Share Common Core Genes.

N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzymes responsible for N-glycan degradation, the enzymes involved in cleaving the N-glycan core have only recently been discovered.

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74.

Glycoside hydrolase family 74 (GH74) is a historically important family of -β-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: , -dissociative, and -processive.

Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 -xyloglucanase.

produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (GH74) revealed a highly specific, processive -xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform - the first in GH74 extending from subsites -4 to +6 - and unique mobile active-site loops.

Structural basis for the flexible recognition of α-glucan substrates by Bacteroides thetaiotaomicron SusG.

Bacteria that reside in the mammalian intestinal tract efficiently hydrolyze dietary carbohydrates, including starch, that escape digestion in the small intestine. Starch is an abundant dietary carbohydrate comprised of α1,4 and α1,6 linked glucose, yet mammalian intestinal glucoamylases cannot effectively hydrolyze starch that has frequent α1,6 branching as these structures hinder recognition and processing by α1,4-specific amylases. Here we present the structure of the cell surface amylase SusG from Bacteroides thetaiotaomicron complexed with a mixed linkage amylosaccharide generated from transglycosylation during crystallization.

Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC).

High-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) is a powerful analytical technique enabling the high-resolution separation and sensitive quantification of oligosaccharides. Here, we describe a general method for the determination of glycoside hydrolase kinetics that harnesses the intrinsic power of HPAEC-PAD to simultaneously monitor the release of multiple products under conditions of low substrate conversion. Thus, the ability to track product release under initial-rate conditions with substrate concentrations as low as 5 μM enables the determination of Michaelis-Menten kinetics for glycosidase activities, including hydrolysis and transglycosylation.

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here, we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes which enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization, to precise Michaelis-Menten analysis.

CAZyChip: dynamic assessment of exploration of glycoside hydrolases in microbial ecosystems.

Background: Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans.

Results: This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database.

A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes.

Investigating the function of an arabinan utilization locus isolated from a termite gut community.

Biocatalysts are essential for the development of bioprocesses efficient for plant biomass degradation. Previously, a metagenomic clone containing DNA from termite gut microbiota was pinpointed in a functional screening that revealed the presence of arabinofuranosidase activity. Subsequent genetic and bioinformatic analysis revealed that the DNA fragment belonged to a member of the genus Bacteroides and encoded 19 open reading frames (ORFs), and annotation suggested the presence of hypothetical transporter and regulator proteins and others involved in the catabolism of pentose sugar.

Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics.

Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.

Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes.