We identified unusual rotavirus strains in fecal specimens from sheltered dogs in Hungary by viral metagenomics. The novel rotavirus species displayed limited genome sequence homology to representatives of the 8 rotavirus species, A–H, and qualifies as a candidate new rotavirus species that we tentatively named Rotavirus I.
An immobilization engineering approach using bioinformatics and experimental design tools was applied to improve the sol-gel enzyme entrapment methodology. This strategy was used for the immobilization of lipase B from Candida antarctica (CaLB), a versatile enzyme widely used even on the industrial scale.The optimized entrapment of CaLB in sol-gel matrices is reported by the response-surface methodology enabling efficient process development. The immobilized CaLBs characterized by functional efficiency and enhanced recovery provided economical and green options for flow chemistry. Various ternary mixtures of sol-gel precursors allowed the creation of tailored entrapment matrices best suited for the enzyme and its targeted substrate. The sol-gel-entrapped forms of CaLB were excellent biocatalysts in the kinetic resolutions of secondary alcohols and secondary amines with aromatic or aliphatic substituents both in batch and continuous-flow biotransformations. † Electronic supplementary information (ESI) available. See
This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower k/K value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.
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