High molecular weight chitosan oligosaccharide exhibited antifungal activity by misleading cell wall organization via targeting PHR transglucosidases.

The fungal cell wall is an ideal target for the design of antifungal drugs. In this study we used an analog of cell wall polymer, a highly deacetylated high molecular-weight chitosan oligosaccharide (HCOS), to test its effect against pathogenic Candida strains. Results showed that HCOS was successfully incorporated into the dynamic cell wall organization process and exhibited an apparent antifungal activity against both plankton and mature fungal biofilm, by impairing the cell wall integrity. Unexpectedly, mechanistic studies suggested that HCOS exerts its activity by interfering with family members of PHR ß-(1,3)-glucanosyl transferases and affecting the connection and assembly of cell wall polysaccharides. Furthermore, HCOS showed great synergistic activity with different fungicides against Candida cells, especially those in biofilm. These findings indicated HCOS has a great potential as an antifungal drug or drug synergist and proposed a novel antifungal strategy with structure-specific oligosaccharides mimicking cell wall polysaccharide fragments.

Glucosamine Ameliorates Symptoms of High-Fat Diet-Fed Mice by Reversing Imbalanced Gut Microbiota.

Glucosamine (GlcN) is used as a supplement for arthritis and joint pain and has been proved to have effects on inflammation, cancer, and cardiovascular diseases. However, there are limited studies on the regulatory mechanism of GlcN against glucose and lipid metabolism disorder. In this study, we treated high-fat diet (HFD)-induced diabetic mice with GlcN (1 mg/ml, in drinking water) for five months. The results show that GlcN significantly reduced the fasting blood glucose of HFD-fed mice and improved glucose tolerance. The feces of intestinal contents in mice were analyzed using 16s rDNA sequencing. It was indicated that GlcN reversed the imbalanced gut microbiota in HFD-fed mice. Based on the PICRUSt assay, the signaling pathways of glucolipid metabolism and biosynthesis were changed in mice with HFD feeding. By quantitative real-time PCR (qPCR) and hematoxylin and eosin (H&E) staining, it was demonstrated that GlcN not only inhibited the inflammatory responses of colon and white adipose tissues, but also improved the intestinal barrier damage of HFD-fed mice. Finally, the correlation analysis suggests the most significantly changed intestinal bacteria were positively or negatively related to the occurrence of inflammation in the colon and fat tissues of HFD-fed mice. In summary, our studies provide a theoretical basis for the potential application of GlcN to glucolipid metabolism disorder through the regulation of gut microbiota.

Expression and Biochemical Characterization of a Novel Marine Chitosanase from Streptomyces niveus Suitable for Preparation of Chitobiose.

It is known that bioactivities of chitooligosaccharide (COS) are closely related to the degree of polymerization (DP); therefore, it is essential to prepare COS with controllable DP, such as chitobiose showing high antioxidant and antihyperlipidemia activities. In this study, BLAST, sequence alignment and phylogenetic analysis of characterized glycoside hydrolase (GH) 46 endo-chitosanases revealed that a chitosanase Sn1-CSN from Streptomyces niveus was different from others. Sn1-CSN was overexpressed in E. coli, purified and characterized in detail. It showed the highest activity at pH 6.0 and exhibited superior stability between pH 4.0 and pH 11.0. Sn1-CSN displayed the highest activity at 50 °C and was fairly stable at ≤45 °C. Its apparent kinetic parameters against chitosan (DDA: degree of deacetylation, >94%) were determined, with Km and kcat values of 1.8 mg/mL and 88.3 s-1, respectively. Cu2+ enhanced the activity of Sn1-CSN by 54.2%, whereas Fe3+ inhibited activity by 15.1%. Hydrolysis products of chitosan (DDA > 94%) by Sn1-CSN were mainly composed of chitobiose (87.3%), whereas partially acetylated chitosan with DDA 69% was mainly converted into partially acetylated COS with DP 2-13. This endo-chitosanase has great potential to be used for the preparation of chitobiose and partially acetylated COS with different DPs.

Overexpression and biochemical characterization of a truncated endo-α (1 → 3)-fucoidanase from alteromonas sp. SN-1009.

Endo-fucoidanases are important in structural analysis of fucoidans and preparation of fuco-oligosaccharides. However their enzymological properties and analysis of degradation products are scarcely investigated. Truncated endo-α (1 â†’ 3)-fucoidanase Fda1 (tFda1B from Alteromonas sp. was overexpressed and characterized, showing highest activity at pH 7.0, 35 °C, and 1.0 M NaCl. Its Km and kcat were 3.88 ± 0.81 mg/mL and 0.82 ± 0.17 min-1. Fe3+ and Mn2+ enhanced activity by 100% and 19.5% respectively. Co2+ and Cu2+ completely inactivated tFda1B, whereas Ni2+, Mg2+, Zn2+, Pb2+, Ca2+, Ba2+ and Li+ decreased activity by 58.8%, 56.0%, 50.6%, 47.7%, 28.9%, 15.6% and 37.5%, respectively. Catalytic residues were identified through structure and sequence alignment, and confirmed by mutagenesis. Degradation products of Kjellmaniella crassifolia fucoidan by tFda1B were characterized by LC-ESI-MS/MS, confirming tFda1B belongs to endo-(1 â†’ 3)-fucoidanases, and backbone of K. crassifolia fucoidan is 1 â†’ 3 fucoside linkage. This endo-α (1 â†’ 3)-fucoidanase would be useful for elucidating fucoidan structures, and be used as a food enzyme.

Enteromorpha prolifera oligomers relieve pancreatic injury in streptozotocin (STZ)-induced diabetic mice.

The polysaccharides of Enteromorpha prolifera (PEP) displayed various bioactivities such as anti-viral, anti-inflammatory and immune-regulative effects. However, no studies were performed on the biological effect of Enteromorpha prolifera oligomers (EPO). In this study, we prepared EPO and evaluated their anti-diabetic effect. By enzymatic degradation, EPO were produced from PEP, and the average molecular weight was identified to be 44.1 kDa by Gel Permeation Chromatography (GPC) analysis. The major monosaccharide units of EPO were measured to be rhamnose, glucuronic acid, glucose, xylose and galactose by capillary electrophoresis assay. Based on the in vitro studies, EPO presented potent reducing power and antioxidant effect such as the scavenging of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and NO radicals. The in vivo studies show that EPO relieved the symptoms of polydipsia, polyphagia, emaciation and hyperglycemia in streptozotocin (STZ)-induced diabetic mice to a certain extent. Further, by using the quantitative real-time PCR (qPCR) assay and immunofluorescence staining, EPO was proved to promote the insulin secretion by reducing pancreatic inflammation and apoptosis in diabetic mice. In summary, our results indicate that the mitigation of EPO on pancreatic damage might be an effective way to ameliorate the diabetes mellitus.

Enhanced immune response to inactivated porcine circovirus type 2 (PCV2) vaccine by conjugation of chitosan oligosaccharides.

This study aimed to investigate the effect of chitosan oligosaccharide (COS) conjugation on the immunogenicity of porcine circovirus type-2 (PCV2) vaccine. Two conjugates (PCV2-COS-1 and PCV2-COS-2) were designed by covalent conjugation of an inactivated PCV2 vaccine with COS, and administered to C57BL/6 mice three times at two-week intervals. The results indicate that, as compared to PCV2 alone group, the PCV2-COS conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting T lymphocyte proliferation and initiating a mixed Th1/Th2 response, including the elevated production of PCV-2 specific antibodies and up-regulated secretion of inflammatory cytokines. Noticeably, the immunization with PCV2-COS-1 conjugate displayed similar or even better immune-stimulating effects than that by PCV2/ISA206 (a commercialized adjuvant) and showed no infection or pathological signs at injection sites of the mice. Presumably, the covalent linkage of PCV2 vaccine to COS might be a viable strategy to increase the efficacy against PCV2-associated diseases.