Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.
Introduction Perfluorinated compounds (PFC) are toxic and bioaccumulative compounds that are ubiquitous in the environment. It is important to develop effective techniques to remove PFC from water. This study is the first to investigate sorption of PFC to activated carbon (AC) at environmentally relevant nanogram per liter concentrations. Methods Batch AC sorption isotherms were measured for water from a contaminated groundwater well, for three perfluorosulfonates and five perfluoroacetic acids. Results For perfluorooctane sulfonate and perfluorooctanoic acid Freundlich sorption coefficients, log K iF , for powdered activated carbon (PAC) were 4.0 and 3.8 (ng/g)(ng/L) -n , respectively, and for granular activated carbon (GAC) were 2.7 and 2.3 (ng/g)(ng/L) -n , respectively. Sorption was nonlinear, with Freundlich n coefficients generally around 0.5. The K iF on both GAC and PAC were PFC chain-length dependant, with increasing number of carbon yielding increasing K iF . This chain-length dependence appeared stronger for perfluorosulfonates than for perfluoroacetic acids. Tests with short (10 min) adsorption times still yielded substantial PFC removal (20-40% for GAC, 60-90% for PAC) and revealed that AC is probably suitable for PFC removal in flow-through systems. A perfluorinated polymer, Teflon, was also tested as a PFC removal agent but proved not to be effective for PFC-contaminated water purification.
Urbanization has resulted in increased contaminant run-off in densely populated areas. Climate change is expected to result in a higher frequency of extreme weather events including torrential rainfall and storms. The contaminant levels of polycyclic aromatic hydrocarbons (PAH), chlorinated paraffins (CPF) and selected metals, in a small urban river were monitored during snow-melting and rainfall events to quantify the contribution to the contamination load of receiving waters of the inner Oslo fjord, Norway. Suspended particulate matter (SPM) was characterized with respect to levels of contaminants as well as toxic response using a battery of bioassays. The contaminant flux from the river to the fjord was quantified and assessed relative to sediment data. Historic data for near-shore sediment samples from the fjord were used to document urban input. The results show a clear episodic response in contaminant load emitted from the river to the fjord. The main historic input to the fjord was found to be PAH from pyrogenic sources like coal and wood burning as well as traffic. A significant reduction in the level of PAH was observed since the 1980s. The measured flux of CPF is consistent with on-going societal use despite a ban on the use of short chain CPF imposed in Norway from 2002.
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