Guangya Zhang

The scarcity of a simple, cost-effective, and green method for the immobilization of enzymes severely hampers their application. Herein, a versatile and mild xylanase and lichenase bienzyme (XLBE) immobilization strategy including biofunctionalization of the magnetic particles, enzyme-free purification, and spontaneous covalent bridging based on SpyCatcher "Click Biology" was proposed. Only biocompatible tannic acid (TA), Fe³⁺, and elastin-like polypeptide-SpyCatcher were fed. The biomodified magnetic particles exhibited excellent stability with a loss of only 3.51% EC after 1 h of incubation at pH 7.5. Then, they were applied to immobilize SpyTag fused XLBE directly from the crude solution at a loading of 12.5 mg/g. The retention of XLBE and the xylanase activity were as high as 87.73% and 82.77%, respectively. The half-lives of the immobilized xylanase increased by 1535.75% (50 °C) compared to those of the free xylanase. The immobilized XLBE showed excellent reusability, retaining 70.15% (xylanase) and 78.81% (lichenase) of the initial activity after 8 cycles of recycling. They also showed superior catalytic performance with 202.25% improvement in green production of total reducing sugar and 30.77% improvement in juice clarification. Moreover, the versatility of the immobilization strategy was also demonstrated on inorganic carriers such as silicon dioxide and carbon nanotubes. This innovative all-in-one strategy avoids too many chemical reagents for surface modification and omits the complex enzyme prepurification process for immobilization, which will shed light on the green biocatalytic applications based on time-effective and low-byproduct surface functionalization strategies.

Quorum quenching (QQ) is of interest for potential application as a sustainable strategy for bacterial disease control via communication interruption. The QQ enzyme can be used as a good alternative antagonist to combat antibiotic abuse and bacterial resistance. Here, genomic DNA sequencing was performed on N-acyl homoserine lactonase from the deep-sea strain Bacillus velezensis DH82 with Cluster of Orthologous Groups of proteins (COGs) annotation. The homologous sequences with β-lactamase domain-containing protein were predicted to be potential QQ enzymes and were cloned and expressed to study their quorum quenching properties by comparing them with the reported enzyme AiiA_3DHB. The experimental results of enzyme activity analysis and steady-state kinetics, as well as enzyme structure and substrate docking simulations and predictions, all consistently demonstrated that YtnP_DH82 presented superior enzyme structural stability and higher degradation efficiency of N-acyl homoserine lactones than AiiA_DH82 under the effects of pH, and temperature, and performed better on short -chain and 3-O-substituted AHSLs. The findings revealed the structural and biochemical characterization of YtnP_DH82 from the deep sea, which provide the capacity for further application in sustainable aquaculture as an alternative to antibiotics.