Metal-Tannin and SpyCatcher Functionalized Magnetic Carriers for Xylanase-Lichenase Chimera Immobilization via “Click Biology”

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.