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Microfluidic biocatalytic reactors have attracted widespread attention in recent years, but the production cost is generally high, which makes it difficult to meet the needs of large-scale industrial production. Developing a low-cost, green fluid reactor has become an urgent requirement. Here, inspired by the three-dimensional hierarchical structure of natural wood, a cellulose-based bionic microfluidic biocatalytic reactor with vertically aligned channels and a porous inner wall is reported for the continuous fluid catalytic conversion of polydatin to resveratrol. NiO nanoparticles were immobilized on cellulose-based bionic materials as metal arms to adsorb poly-histidine-tagged enzymes to form a stable and efficient biocatalytic reactor. Due to the unidirectionally aligned channels and porous inner wall, the fluid in different channels of the bionic xylem reactor is disturbed through the porous inner wall, which greatly prolongs the residence time for the fluid inside the reactor and increases the probability for contact with the enzyme, improving the internal mass and heat transfer efficiency to achieve high catalytic activity (98.69% treatment efficiency) and excellent stability (maintain a conversion rate of more than 90% within 15 days). Because of the good and stable enzyme immobilization and high catalytic activity of the reactor, it can greatly simplify industrial upstream and downstream production and has broad application prospects for catalytic reactions. [ABSTRACT FROM AUTHOR]

Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency. [ABSTRACT FROM AUTHOR]

Isoquercitrin of great pharmaceutical value can be transformed from plant-derived rutin by the key enzyme α - L -rhamnosidase. However, the disposable hydrolyzation process and the unsatisfactory properties of α - L -rhamnosidase limit the production of isoquercitrin. In this study, we obtained a mutant of GH78 family RhaB1, RhaB1-ΔN, with improved thermostability and catalytic activity by deleting the domain N. Concretely speaking, the T 50 for 30 min and melting temperature (T m) raised 5.0 °C and 6.2 °C compared with those of RhaB1. Meanwhile, the specific activities were 103 %, 44 %, and 74 % higher than those of the wild type with pNPR, rutin, and naringin as substrates, respectively. With RhaB1-ΔN as a whole-cell catalyst, conversion of isoquercitrin reached 96.6 % within 1.5 h, 8.5 h less than that for free RhaB1. In the 6th recovery, the yield of whole-cell catalyst reached 62.3 % of the maximum, 18.9 times that of the free enzyme (3.3 %). The combined strategies of enzyme modification and whole-cell catalyst helped to realize more recyclable and efficient isoquercitrin production. [Display omitted] • The catalytic efficiency and thermostability of RhaB1 are enhanced by removing its domain N. • The domains D, E, and C of RhaB1 are required for its catalytic activity. • Removal of domain N improves the enzyme's binding to the substrate. • The whole-cell catalyst retained > 60 % activity after six repeated use. • Isoquercitrin yield reached 96.6 % within 1.5 h for the truncated enzyme. [ABSTRACT FROM AUTHOR]

Background: Xylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy. Results: Using fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T50 (3–4.7 °C) and Tm (1.1–4.7 °C), and extended t1/2 (by 1.8–2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase. Conclusions: This study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers. [ABSTRACT FROM AUTHOR]

β-1,3–1,4-Glucanase is an indispensable biocatalyst in barley brewing industry for its crucial effect in reducing the viscosity of mash. However, the unsatisfactory thermostability greatly limited its application performance. In this study, structure-based surface charge engineering was conducted aiming at thermostability improvement of BisGlu16B, a highly active β-1,3–1,4-glucanase from Bispora sp. MEY-1. By applying the enzyme thermal stability system (ETSS), residues D47, D213, and D253 were inferred to be critical sites for thermal properties. Single (D47A, D213A, and D253A) and combination (D47A/D213A/D253A) mutants were generated and compared with BisGlu16B. Among all improved mutants, D47A/D213A/D253A outstanded in thermostability. In comparison with BisGlu16B, its T 50 and T m were respectively increased by 7.0 °C and 4.3 °C, while the t 1/2 at 70 °C was 8.1 times that of the wild type. Furthermore, the catalytic activity of D47A/D213A/D253A also increased by 42.5%, compared with BisGlu16B (42,900 ± 300 U/mg vs. 30,100 ± 800 U/mg). Comparing with BisGlu16B and commercial enzyme treatment groups, under simulated malting conditions, efficiency improvement was observed in decreasement of viscosity (35.5% and 90.7%) and filtration time (30.9% and 34.6%) for D47A/D213A/D253A treatment group. Molecular dynamics simulation showed that mutation sites A47, A213, and A253 increased the protein rigidity by lowering the overall root mean square deviation (RMSD). This study may bring optimization of technology and improvement of producing efficiency to the present brewing industry. [Display omitted] • Surface charge engineering protects the catalytic core during modification. • The β-glucanase activity was improved besides the intended higher thermostability. • Reduced mash viscosity and filtration time was observed during barley malting. • The study provided a more efficient catalyst for brewing industry. [ABSTRACT FROM AUTHOR]