We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin-remazol-brilliant violet) and a small, presumably amorphous, subfraction of alpha-chitin and beta-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472-484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (- 2 to + 2), as opposed to six (- 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study.
Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chitooligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4).
We have studied the degradation of well-characterized soluble heteropolymeric chitosans by a novel family 46 chitosanase, ScCsn46A from Streptomyces coelicolor A3(2), to obtain insight into the enzyme's mode of action and to determine its potential for production of different chitooligosaccharides. The degradation of both a fully deacetylated chitosan and a 32% acetylated chitosan showed a continuum of oligomeric products and a rapid disappearance of the polymeric fraction, which is diagnostic for a nonprocessive endomode of action. The kinetics of the degradation of the 32% acetylated chitosan demonstrated an initial rapid phase and a slower second phase, in addition to a third and even slower kinetic phase. The first phase reflects the cleavage of the glycosidic linkage between two deacetylated units (D-D), the primary products being fully deacetylated dimers, trimers, and tetramers, as well as longer oligomers with increasing degrees of acetylation. In the subsequent slower kinetic phases, oligomers with a higher degree of acetylated units (A) appear, including oligomers with A's at the reducing or nonreducing end, which indicate that there are no absolute preferences for D in subsites -1 and +1. After maximum degradation of the chitosan, the dimers DA and DD were the dominant products. The degradation of chitosans with varying degrees of acetylation to a maximum degree of scission showed that ScCsn46A could degrade all chitosan substrates extensively, although to decreasing degrees of scission with increasing F(A). The potential use of ScCsn46A to prepare fully deacetylated oligomers and more highly acetylated oligomers from chitosan substrates with varying degrees of acetylation is discussed.
Microplastics are globally recognized as contaminants in freshwater and marine aquatic systems. To date there is no universally accepted protocol for isolation and quantification of microplastics from aqueous media. Various methodologies exist, many of which are time consuming and have the potential to introduce contaminants into samples, thereby obscuring characterization of the environmental microplastic load. Here, we present first steps in the detection of microplastics in liquid samples, based on their fluorescent staining followed by high throughput analysis and quantification using Flow Cytometry. Using controlled laboratory settings nine polymer types [polystyrene (PS); polyethylene (PE); polyethylene terephthalate (PET/PETE); high density polyethylene (HDPE); low density polyethylene (LDPE); polyvinyl chloride (PVC); polypropylene (PP); nylon (PA); polycarbonate (PC)] were tested for identification and quantification in freshwater. All nine plastic types were stained with 10 µg/mL Nile Red in 10% dimethyl sulfoxide with a 10 min incubation time. The lowest spatial detectable limit for plastic particles was 200 nm. Out of the nine polymer types chosen for the study PS, PE, PET, and PC were well-identified; however, results for other plastic types (PVC, PP, PA, LDPE, and HDPE) were masked to certain extent by Nile Red aggregation and precipitation. The methodology presented here permits identification of a range of particle sizes and types. It represents a significant step in the quantification of microplastics by replacing visual data interpretation with a sensitive and automated method.
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