Green preparation of covalently co-immobilized multienzymes on silica nanoparticles for clean production of reducing sugar from lignocellulosic biomass

Abstract

Multienzyme co-immobilization has garnered increasing interest in biomass conversion due to the green catalytic properties. However, the conventional strategies for covalent co-immobilization were complicated (including carrier preparation, enzyme purification, and immobilization process) and not clean enough (such as harsh reaction conditions, high reagent and water consumption, and toxic chemical input). Herein, a simple and green strategy was proposed to address these issues by the integration of elastin-like polypeptide (ELP)-mediated green biomimetic silicification and the spontaneous covalent bond formation of SpyCatcher/SpyTag (SC/ST) protein pair. In this strategy, the SC was genetically fused to ELP and the resulting SC-ELP (SCE) was purified by inverse transition cycling (ITC) method. The nanocarrier (SCE@SiO2) was prepared rapidly via one-step biomimetic silicification at neutral pH and room temperature under ambient pressure. The obtained nanocarriers could be directly used for covalent co-immobilization without additional surface functionalization and activation. The ST-tagged recombinant bi-enzyme xylanase-ST-lichenase (XSTL) covalently self-conjugated onto SCE@SiO2 through the SC and ST reaction, which eliminated the use of cross-linking reagents. Notably, target-specific immobilization from cell crude extracts with excellent activity recovery (over 90%) eliminated reagent- and time-consuming purification process. Furthermore, the co-immobilized XSTL showed very good reusabilities, which retained 83.9% (lichenase) and 81.4% (xylanase) of the original activities in the seventh cycle, respectively. XSTL could accelerate the production of fermentable sugars from renewable biomass polysaccharide. Overall, this unique ELP-mediated silicification-based Spy-assisted enzyme self-immobilization (ESSESI) offered great potentials towards multienzyme co-immobilization for diverse applications in a cleaner manner.