Two laccase isoenzymes, Lacc A and Lacc B, were purified and characterized from Pleurotus ostreatus NRC 620, and Lacc A was subsequently evaluated for apple juice treatment. The two isoenzymes exhibited distinct thermal profiles, where after 120 min, Lacc B retained about 92% and 82% of its activity at 40 and 50 °C, respectively, whereas Lacc A retained at least 95% at 30 °C but lost most of its activity within 60 min at 50 °C and within 15 min at 60–70 °C. pH stability profiling revealed a complementary pattern, with Lacc A showing greater acid tolerance (70% residual activity at pH 3.0) and Lacc B exhibiting pronounced alkaline stability (85–90% residual activity at pH 9.0). Kinetic analysis using ABTS as substrate showed that Lacc A had higher substrate affinity (Km, 0.2728 mM) than Lacc B (Km, 0.7431 mM), while Lacc B displayed a higher maximum catalytic rate (Vmax, 0.3845 μmol·min−1·mL−1) than Lacc A (0.1734 μmol·min−1·mL−1). Metal ion sensitivity assays further differentiated the two isoenzymes, where Lacc A was activated by CuSO₄ (170%), MgSO₄, and CoSO₄, whereas Lacc B was unaffected by these ions and was completely inhibited by ZnSO₄. Application of purified Lacc A to apple juice induced compound-specific phenolic modifications; chlorogenic acid, the predominant phenolic compound, was only slightly reduced, whereas gallic acid, caffeic acid, vanillic acid, and catechin decreased substantially, with several becoming undetectable after storage. HPLC/GC analysis identified 27 volatile compounds, with laccase treatment enhancing key aroma-active esters, including ethyl acetate and ethyl butyrate. Sensory evaluation confirmed acceptable product quality over two weeks of refrigerated storage, supporting the potential of Lacc A as a biocatalyst for enzymatic apple juice processing.
A novel GH16 β-1,3-1,4-glucanase, SlGH16, was identified from Streptomyces lividans and exhibits considerable thermostability and a broad pH tolerance, retaining appreciable activity under industrially relevant high-temperature and extreme pH conditions. SlGH16 preferentially hydrolyzes barley β-glucan to produce low-molecular-weight oligosaccharides (DP3–DP5), offering some potential for functional feed and oligosaccharide production. Integrative structural analyses, including AlphaFold3 modeling, molecular docking, and molecular dynamics simulations, revealed that two disulfide bonds (Cys161-Cys169, Cys181-Cys186) and the central aromatic residue Phe248 act as important stabilizing elements, preserving substrate-binding integrity and overall protein rigidity. Site-directed mutagenesis confirmed the important roles of these residues in catalytic efficiency and thermal resilience. The combination of moderate stability, precise substrate recognition, and efficient oligosaccharide generation positions SlGH16 as a promising candidate biocatalyst for high-temperature industrial applications. This study not only elucidates the structural determinants of GH16 enzyme thermostability but also provides a mechanistic reference for rational protein engineering aimed at enhancing enzyme performance under harsh processing conditions.
Conjugated linoleic acid (CLA) is an important functional compound in the food and pharmaceutical industries, and its biosynthesis depends on linoleic acid isomerase (LAI). The natural LAI, however, suffers from low catalytic efficiency and substrate affinity, which limits CLA synthesis and industrial application. To address this, a semi-rational engineering strategy was applied to modify LAI from Propionibacterium acnes (P. acnes), constructing a soluble single-enzyme system using P229 as the parent strain. Five promising mutation sites (G36R, G7Q, Y319W, E392R, F399W) were identified and experimentally validated through overlap extension PCR. The pCold-lai-pG36R variant showed 4.56-fold higher gene expression and 1.95-fold increased enzymatic activity compared to the wild type P229 (WT). Structural analysis revealed that the G36R mutation introduced salt bridges and shorter hydrogen bonds. The purified mutant retained > 60% of its CLA productivity across 20–40 ℃ and pH 7–8, achieving optimal activity at 30 ℃ and pH 7.0 (Vmax = 21.41 μg·mL−1·h−1 and Km = 0.22 mg·mL−1). These findings highlight the potential of semi-rational engineering to enhance LAI performance, offering valuable insights for industrial applications.
Laccase-catalyzed repolymerization of technical lignins is an emerging biocatalytic strategy for upgrading lignin streams into polymeric systems and biomaterial precursors under mild reaction conditions. Lignosulfonates are attractive substrates for enzymatic polymerization due to their high water-solubility; however, their relatively low reactivity can limit their application. In contrast, organosolv lignin typically presents low-molecular weight and high phenolic content, which increase its susceptibility to laccase-mediated oxidation; however, its limited water solubility restricts enzymatic processing. In this work, lignin blending was evaluated as a strategy to modulate laccase-catalyzed polymerization by combining lignosulfonates (LS) with organosolv lignin from vine shoots (LVS). The effect of LVS incorporation on enzymatic oxidation was investigated through oxygen consumption measurements, viscosity evolution, molecular weight analysis and characterization of LS-based films. The presence of LVS modified oxidation kinetics, leading to prolonged oxidative activity, accelerated viscosity development and higher apparent molecular weight fractions at shorter reaction times compared to LS alone. LS-LVS films were produced to evaluate whether blending affected material surface properties. FTIR analysis supported structural changes consistent with oxidative coupling, while water contact angle measurements showed increased surface hydrophobicity in LS/LVS systems. Overall, the results demonstrate that blending lignins with different structural features can modulate enzymatic polymerization and influence the properties of LS films. This work highlights lignin blending as a simple biocatalytic strategy to modulate laccase-catalyzed polymerization and upgrade heterogeneous lignin streams toward lignin-based biomaterials within biorefinery concepts.
Deacetylases can directly hydrolyze amide bonds for the environmentally friendly synthesis of the herbicide intermediate O-(3-chloro-2-propenyl)hydroxylamine. However, the rigid halogenated chain of N-[(E)-3-chloroprop-2-enoxy]acetamide (NECA) may hinder productive substrate positioning and limit catalytic efficiency. Accordingly, 20 deacetylases were initially screened, and a deacetylase from Marinobacter subterrani (MsDeac) was identified as the optimal starting scaffold. To improve its catalytic performance toward NECA, a synergistic pocket and tunnel reshaping strategy was subsequently implemented to modify the substrate-binding environment and predicted access pathway. Molecular docking suggested a putative halogen-bonding interaction in M3 that may contribute to substrate positioning. M3 also exhibited an expanded predicted pocket volume (from 777 to 1055 Å3) and an increased number of predicted access channels. After stepwise mutagenesis, a triple mutant (Y325H-M321H-Y170H, M3) was successfully obtained, demonstrating a 68-fold increase in catalytic efficiency (kcat/Km), with an increased conversion of 80 mM NECA from approximately 10% to > 99.5% within 12 h. The results suggest that improved spatial complementarity and a potential halogen-bonding interaction may contribute to enhanced catalytic efficiency, providing structural hypotheses for the engineering of deacetylases toward sterically challenging substrates.
