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The applications of heparinase I in the preparation of low molecular weight heparin were limited due to the poor thermostability. In this work, the Foldx was used to calculate the folding free energy to find the possible thermal stable conformation. In the calculation results, the mutant Q157H had the lowest folding free energy in 17 possible mutants indicating that the Q157H was more stable than the wild-type heparinase I. The mutant Q157H exhibited a 6.0-fold and 2.1-fold half-life time than that of the wild type (WT). Notably, the enzyme activity of Q157H was also increased 1.88-fold compared with the WT. To explore the role of the mutation Q157H on the structure of heparinase I, molecular dynamic (MD) simulation and docking were performed. The MD simulation revealed that the Q157H had lower root-mean-square deviation (RMSD) value than the WT. Moreover, the model showed 11 hydrogen bonds formed between the mutant with the heparin, and there were 4 more hydrogen bonds than that of the WT. Based on the molecule docking results, the Q157H had a higher affinity than the WT. This work will expand the applications of heparinase in the industry and clinic. [ABSTRACT FROM AUTHOR]

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg−1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL−1 and 647.93 U·mg−1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K−1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability. [ABSTRACT FROM AUTHOR]

• A novel water-forming flavin oxidoreductase was first expressed and characterized. • A rate-limiting Thr29 was identified and confirmed to be employed to improve its activity. • The distance between FMN and NADH was shortened by introducing a small side chain containing residue into position 29. Enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H 2 O 2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg−1 with the optimal pH and temperature of 5.6 and 35 °C and produces H 2 O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration. [ABSTRACT FROM AUTHOR]