RESUMO
Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.
RESUMO
Non-specific endonucleases can be used for the digestion of nucleic acids because they hydrolyze DNA/RNA into 3-5 base pairs (bp) length oligonucleotide fragments without strict selectivity. In this work, a novel non-specific endonuclease from Pseudomonas fluorescens (PfNuc) with high activities for both DNA and RNA was successfully cloned and expressed in Escherichia coli. The production of PfNuc in flask scale could be achieved to 1.73 × 106 U/L and 4.82 × 106 U/L for DNA and RNA by investigation of the culture and induction conditions. The characterization of PfNuc indicated that it was Mg2+-dependent and the catalytic activity was enhanced by 3.74 folds for DNA and 1.06 folds for RNA in the presence of 5 mM Mg2+. The specific activity of PfNuc for DNA was 1.44 × 105 U/mg at pH 8.0 and 40 °C, and 3.93 × 105 U/mg for RNA at pH 8.5 and 45 °C. The Km of the enzyme for both DNA and RNA was close to 43 µM. The Vmax was 6.40 × 105 U/mg and 1.11 × 106 U/mg for DNA and RNA, respectively. There was no observed activity loss when PfNuc was stored at 4 °C and - 20 °C after 28 days or 10 repeated freeze-thaw cycles at - 80 °C. Molecular docking revealed that PfNuc formed 17 and 19 hydrogen bonds with single-stranded RNA and double-stranded DNA, respectively. These results could explain the high activity and stability of PfNuc, suggesting its great potential applications in the industry and clinic.
Assuntos
Pseudomonas fluorescens , Pseudomonas fluorescens/genética , Simulação de Acoplamento Molecular , RNA , Endonucleases/genética , Escherichia coli/genética , DNA , Clonagem MolecularRESUMO
A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like â4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 â 4)ΔUA/ΔUA2S[1 â and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.
Assuntos
Heparina , Polissacarídeo-Liases , Heparina/análise , Heparina/química , Heparina/metabolismo , Heparina Liase/genética , Heparina Liase/química , Heparina Liase/metabolismo , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Sítios de LigaçãoRESUMO
Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (km and Vmax). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze-thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.
Assuntos
Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Engenharia de Proteínas/métodos , Rúmen/enzimologia , Animais , Catálise , Dicroísmo Circular , Ciclização , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Ovinos , TermodinâmicaRESUMO
Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.