Heparinase III with High Activity and Stability: Heterologous Expression, Biochemical Characterization, and Application in Depolymerization of Heparin.

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 →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → 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.

Recent Strategies for the Immobilization of Therapeutic Enzymes.

Therapeutic enzymes play important roles in modern medicine due to their high affinity and specificity. However, it is very expensive to use them in clinical medicine because of their low stability and bioavailability. To improve the stability and effectiveness of therapeutic enzymes, immobilization techniques have been employed to enhance the applications of therapeutic enzymes in the past few years. Reported immobilization techniques include entrapment, adsorption, and covalent attachment. In addition, protein engineering is often used to improve enzyme properties; however, all methods present certain advantages and limitations. For carrier-bound immobilization, the delivery and release of the immobilized enzyme depend on the properties of the carrier and enzyme. In this review, we summarize the advantages and challenges of the current strategies developed to deliver therapeutic enzymes and provide a future perspective on the immobilization technologies used for therapeutic enzyme delivery.

Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

[Berberine promotes osteogenic differentiation of rat adipose-derived stem cells through JNK signaling pathway].

PURPOSE: This study aimed at exploring the effect of berberine (C20H18NO4) on osteogenic differentiation of rat adipose-derived stem cells(ADSCs) and clarifying the related mechanism. METHODS: ADSCs were subjected to 5, 10, 20 µmol/L berberine culture solution. The untreated ADSCs were set as the control group. Cell proliferation activity was determined by MTT method. Alkaline phosphatase (ALP) staining, semi-quantitative assay and alizarin red staining (ARS) were applied to analyze the effect of berberine on osteogenic differentiation of ADSCs. The phosphorylation level of c-Jun amino terminal kinase (JNK) protein was tested by Western blot. Runx2, OCN were tested by Western blot before and after application of JNK pathway inhibitor SP600125. SPSS 22.0 software package was used for statistical analysis. RESULTS: There was no significant difference on cell proliferation activity of ADSCs treated with 5, 10 and 20 µmol/L berberine at 1, 3 and 7 day(P>0.05). ALP staining and ARS staining in groups treated by berberine were significantly darker than those of the control group, and ALP protein secretion in the experimental group was significantly up-regulated (P<0.05). The phosphorylation level of JNK was increased after treated with 10 µmol/L berberine culture medium. The expression of osteogenic related proteins Runx2 and OCN was up-regulated in the experimental group. After inhibition of JNK signaling pathway, the expression of Runx2 and OCN was down-regulated. CONCLUSIONS: Berberine has no effect on cell proliferation of ADSCs, and can up-regulate osteogenic differentiation of ADSCs through activation of JNK signaling pathway.

A novel chondroitin AC lyase from Pedobacter xixiisoli: Cloning, expression, characterization and the application in the preparation of oligosaccharides.

Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.