Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell.

Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with ß-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.

Green and efficient extraction of rutin from tartary buckwheat hull by using natural deep eutectic solvents.

In this study, an efficient extraction technique using a combination of ultrasound and natural deep eutectic solvents (NADESs) was developed. Some basic physical properties, including viscosity, polarity, and solubility, of thirteen NADESs prepared from natural components were investigated systematically. Results show that the solubility of rutin increased in choline chloride- and glycerol-based NADESs by 660-1577times compared to water. NADESs with high rutin extractability can be designed by combining NADESs components. A maximum of 9.5mg/g rutin was extracted from tartary buckwheat hull with extraction efficiencies of 95%. NADESs can be recovered and recycled. In addition, the biocompatibility and biodegradability of the tested NADESs were also evaluated. The results demonstrated that these NADESs were excellent solvents with extremely low toxicities and favorable biodegradabilities. Our findings suggest that NADESs can be used as green solvents for the extraction of bioactive ingredients.

Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila.

Since the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu(2+) and Zn(2+), and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.