Addition of vancomycin (1, the receptor) to the supramolecular hydrogel of self-assembled pyrene-d-Ala-d-Ala (2, the ligand) increases the storage modulus of the hydrogel of 2 by about 106-fold. Rheology, microscopy, and spectroscopy investigations suggest that the two-dimensional polymers, formed by the ligand-receptor interaction between 1 and 2, and the self-dimerization of 1, are mainly responsible for the observed dramatic increase in elasticity.

Chitosanase (EC3.2.1.132) can be used to selectively produce partially acetylated chitosan oligosaccharides (COSs), which are functional biopolymers that can widely be utilized as environmentally friendly agents, due to their bioactive, nontoxic, nonallergenic, and biodegradable properties. Different chitosanases can give rise to hydrolysates with diverse functions, making the development of novel chitosanases of great importance in applied biotechnologies. Here, we successfully expressed a novel chitosanase derived from Aspergillus fumigatus CJ22-326 in Pichia pastoris GS115, which could controllably produce chitosan oligosaccharides with desirable polymeric degrees. After glycosylation, the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant enzyme Csn75 showed two specific protein bands with sizes of 25.5 and 23.5 kDa individually. The activity of the glycosylated Csn75 was 3.6-fold of that of the deglycosylated Csn75, indicating that glycosylation can greatly increase the activity. K m and V max values of the purified Csn75 were 0.46 mg/mL and 6.03 μmol·mL–1·min–1 separately, revealing a good affinity and high catalytic efficiency to the substrate. Moreover, biochemical tests confirmed that Csn75 had the highest activity from 55 to 65 °C when pH ranged from 5.0 to 6.0 and exhibited 4-fold activity in the presence of 1 mM Mn2+. Interestingly, the hydrolysis rate of the crude Csn75 (30 U/mL) can reach up to 90.65% in 4 h when using 2% chitosan as the substrate, producing a mixture of COS with desirable degrees of polymerization (DP), which significantly inhibited the mycelium growth of phytopathogenic fungi. This study proved Csn75 to be a promising chitosanase for the preparation of desirable chitosan oligosaccharides that are green biocides applicable for sustainable agricultural production and environmental protection.

Herein we report a general and novel strategy for high-throughput photoelectrochemical (PEC) enzymatic bioanalysis on the basis of enzyme-initiated quinone-chitosan conjugation chemistry (QCCC). Specifically, the strategy was illustrated by using a model quinones-generating oxidase of tyrosinase (Tyr) to catalytically produce 1,2-bezoquinone or its derivative, which can easily and selectively be conjugated onto the surface of the chitosan deposited PbS/NiO/FTO photocathode via the QCCC. Upon illumination, the covalently attached quinones could act as electron acceptors of PbS quantum dots (QDs), improving the photocurrent generation and thus allowing the elegant probing of Tyr activity. Enzyme cascades, such as alkaline phosphatase (ALP)/Tyr and β-galactosidase (Gal)/Tyr, were further introduced into the system for the successful probing of the corresponding targets. This work features not only the first use of QCCC in PEC bioanalysis but also the separation of enzymatic reaction from the photoelectrode as well as the direct signal recording in a split-type protocol, which enables quite convenient and high-throughput detection as compared to previous formats. More importantly, by using numerous other oxidoreductases that involve quinones as reactants/products, this protocol could serve as a common basis for the development of a new class of QCCC-based PEC enzymatic bioanalysis and further extended for general enzyme-labeled PEC bioanalysis of versatile targets.

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