•A combined heparinases depolymerized heparin libraries were built and systematically screened for the anti-pulmonary injury and fibrosis efficacy directly with Bleomycin challenged mice model.•Structural properties of the efficient components have been characterized by combination use of chip-based amide hydrophilic interaction chromatography HILIC- FT-ESI–MS, PAGE, and High performance liquid chromatography (HPLC).•The possible mechanisms that heparins exert their effects on have been primarily evaluated.Heparin has recently been shown to slow down idiopathic pulmonary fibrosis (IPF) process and improve survival of patients in some cases. To improve the anti-IPF function while minimizing their side effects, we developed heparin libraries with different structures depolymerized by single or combined heparinases, and systematically screened the efficacy of the different heparins for treatment of Bleomycin-induced pulmonary injury and fibrosis using mice model. Then we characterized the structural properties of the components capable of treating pulmonary injury and fibrosis by use of chip-based amide hydrophilic interaction chromatography (HILIC)-fourier transform (FT)-ESI–MS, polyacrylamide gel electrophoresis (PAGE), and high performance liquid chromatography (HPLC). Our results showed that the depolymerized heparins with relative higher molecular weight (I-2 and III-2) by the respective heparinase I and III protected mice from the induced pulmonary injury and fibrosis. In addition, the selected depolymerized heparins inhibited high-mobility group protein B1 (HMGB-1) expression, prevented E-cadhesin from downregulation, and reduced fibroblasts accumulation in the mouse lung tissue. Our study suggested that the depolymerized heparins of I-2 and III-2 with the most significant efficacy might target several pathways in alleviating the induced pulmonary fibrosis.

•Chondroitin B lyase-based analytical methods selectively quantify dermatan sulfate in heparin.•Simple enzymatic colorimetric method rapidly detected DS above 0.1 mg mL−1 in heparin.•LOD of the novel enzymatic SAX-HPLC method reached 1.0 μg mL−1.•Both methods exhibited good linearity, repeatability, precision and recovery.Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1 mg mL−1 at 232 nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1 mg mL−1, exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5 mg mL−1.

•Heparin depolymerization characteristics of Hep I/II/III were compared systemically.•Heparin depolymerization by different heparinase combinations were elucidated.•A novel combinatorial enzymatic method for LMWHs production was established.•HepIII and HepI is the best for maintaining high anti-IIa activity (75.7 ± 4.21 IU/mg).•A action pattern of HepI and HepII on heparin was excluded.Enzymatic depolymerization of heparin by heparinases is promising for production of low molecular weight heparins (LMWHs) as anticoagulants, due to its mild reaction conditions and high selectivity. Here, different heparinase combinations were used to depolymerize heparin. Heparinase I and heparinase II can depolymerize heparin more efficiently than heparinase III, respectively, but heparinase III was the best able to protect the anticoagulant activities of LMWHs. Heparinase III and heparinase I/II combinations were able to efficiently depolymerize heparin to LMWHs with higher anticoagulant activity than the LMWHs produced by the respective heparinase I and heparinase II. HepIII and HepI is the best combination for maintaining high anti-IIa activity (75.7 ± 4.21 IU/mg) at the same Mw value. Furthermore, considering both the changes in molecular weight and anticoagulant activity, the action patterns of heparinase I and heparinase II were found not to follow the exolytic and processive depolymerizing mechanism from the reducing end of heparin.

•The yields of hydrogen production were improved in co-culture systems.•Formate acted as the predominant metabolite for carbon transfer in the co-culture.•Lower sugar concentrations were favorable for reaching high hydrogen yield.Hydrogen was produced from steam-exploded corn stover by using a combination of the cellulolytic bacterium Clostridium cellulolyticum and non-cellulolytic hydrogen-producing bacteria. The highest hydrogen yield of the co-culture system with C. cellulolyticum and Citrobacter amalonaticus reached 51.9 L H2/kg total solid (TS). The metabolites from the co-culture system were significantly different from those of the mono-culture systems. Formate, which inhibits the growth of C. cellulolyticum, could be consumed by the hydrogen-evolving bacteria, and transformed into hydrogen. Glucose and xylose were released from corn stover via hydrolysis by C. cellulolyticum and were quickly utilized in dark fermentation with the co-cultured hydrogen-producing bacteria. Because the hydrolysis of corn stover by C. cellulolyticum was much slower than the utilization of glucose and xylose by the hydrogen-evolving bacteria, the sugar concentrations were always maintained at low levels, which favored a high hydrogen molar yield.

•Major advances in the development of anti-inflammatory low molecular weight heparins (LMWHs), preclinical and clinical studies as well as possible underlying molecular mechanisms of actions have been summarized.•The activities of commercially available LMWHs have been compared to highlight different anti-inflammatory effects in LMWHs produced using different manufacturing processes.•The importance of structure-activity relationship (SAR) studies on the non-anticoagulant effects of LMWHs was stressed, and strategies for exploring new clinical indications were discussed.Low molecular weight heparins (LMWHs) are produced by chemical or enzymatic depolymerization of unfractionated heparin (UFH). Besides their well-known anticoagulant effects, LMWHs have also been reported to exhibit numerous anti-inflammatory properties. Previous studies have, however, shown that different production processes result in unique structural characteristics of LMWHs. The structural variations may help explain the different therapeutic spectrums in disease treatment for non-anticoagulant effects. In the present review, we summarize major advances in understanding and exploiting the anti-inflammatory disorder activities of LMWHs, based on mechanistic studies, preclinical experiments and clinical trials. We highlight differences in these activities of commercially available LMWHs produced using different manufacturing processes. We stress the importance of structure-activity relationship (SAR) studies on the non-anticoagulant effects of LMWHs and discuss strategies for exploring new clinical indications.

•Novel method to translate active (αβγ)2 XDH by separate expression of redox domains.•Two (αβγ)2 variants of R. capsulatus XDH constructed by splitting its small subunit.•Both (αβγ)2 R. capsulatus XDH variants increased the thermostability by 11 °C.•A split variant enhanced the turnover number and catalytic efficiency.•The first in vivo biosynthesis of active split (αβγ)2 XDH with improved properties.Post-translational proteolysis is usually necessary for the commercial production of xanthine dehydrogenases (XDHs), such as bovine (αβγ)2 XDH, to increase its catalytic activity. The proteolysis approach suffers from low controllability and inefficient purification. To obviate these disadvantages, we have developed a method that translates active Rhodobacter capsulatus (αβγ)2 XDH by directly expressing the iron-sulfur domain, the flavin adenine dinucleotide domain and the sulfurated molybdenum domain as three separate proteins in Escherichia coli. Two (αβγ)2 XDH variants, Split166 and Split178, which were designed by splitting the small subunit of R. capsulatus CGMCC 1.3366 (αβ)2 XDH at the N- and C-terminal ends of the L167-A178 peptide linking the iron–sulfur clusters and flavin adenine dinucleotide domains, respectively, were expressed in E. coli. Compared to the wild type, both split variants increased the thermostability by 11 °C and Split178 enhanced the turnover number and catalytic efficiency by 1.15-fold and 1.66-fold, while Split166 decreased these parameters by 3.2-fold and 5-fold, respectively. This study is the first successful trial to express an active split (αβγ)2 XDH directly by manipulating genes encoding redox domains, and the enhanced properties of the expressed (αβγ)2 XDH using the in vivo splitting strategy may be a promising technique for the commercial production of XDHs.