Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).¹.

Cellulose is one of the most abundant renewable resources in nature. However, its recalcitrant crystalline structure hinders efficient enzymatic depolymerization. Unlike cellulases, lytic polysaccharide monooxygenases (LPMOs) can oxidatively cleave glycosidic bonds in the crystalline regions of cellulose, playing a crucial role in its enzymatic depolymerization. An AA9 LPMO from Myceliophthora thermophila was previously identified and shown to exhibit a highly efficient catalytic performance. To further enhance its catalytic efficiency, consensus mutagenesis was applied. Compared with the wild-type enzyme, the oxidative activities of mutants A165S and P167N increased by 1.8-fold and 1.4-fold, respectively, and their catalytic efficiencies (k_cat/K_m) improved by 1.6-fold and 1.2-fold, respectively. The mutants also showed significantly enhanced activity in the synergistic degradation of cellulose with cellobiohydrolase. Additionally, the P167N mutant exhibited better H₂O₂ tolerance. A molecular dynamics analysis revealed that the increased activity of mutants A165S and P167N was due to the closer proximity of the active center to the substrate post-mutation. This study demonstrates that selecting appropriate mutation sites via a semi-rational design can significantly improve LPMO activity, providing valuable insights for the protein engineering of similar enzymes.

Recently, a novel two‐domain alginate lyase (AlyAL01) was cloned from Algibacter sp. and successfully overexpressed in Escherichia coli BL21(DE3). Biochemical analysis showed that the N‐terminal carbohydrate‐binding module (CBM) of AlyAL01 had no effect on the enzyme activity and product distributions. Therefore, the N‐terminal CBM was substituted with CBM3 to confer the designed chimeric protein with the ability to specifically bind to regenerated amorphous cellulose. As anticipated, the designed chimeric protein (CBM3‐L1) exhibited a similar enzyme activity. Moreover, it was found that the CBM3‐L1 combined the purification and immobilization in one step with high immobilized efficiency of 65.8%. The immobilized enzyme exhibited good storage stability and moderate reusability. The immobilized enzyme could keep 85% activity when incubated at 4°C for 60 days and 70% activity when incubated at 25°C for 30 days. Furthermore, the immobilized CBM3‐L1 kept about 50% of its initial activity after being reused five times. Finally, immobilized CBM3‐L1 successively produced well‐defined alginate oligosaccharides (AOS) with DPs of 2–6 by controlling reaction time. In sum, our current study provided a feasible strategy for well‐defined AOS production.