Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd.

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other ß-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters Km of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the Kcat/Km were 181.5 s-1 mM-1 and 197.8 s-1 mM-1, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C20 position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.

CaCO3 blowing agent mixing method for biomass composites improved buffer packaging performance.

Biodegradable composites with an open-cell structure were developed to replace petroleum-based buffer packaging materials. To overcome the problem of uneven and insufficient foam in the composites, CaCO3 was used as a nucleating agent to prepare porous composites. At 5 wt% CaCO3, more uniform and dense composite cells with better cushioning performance were obtained. A further increase in the CaCO3 content caused the density of the cells and the cushioning properties of the composites to decrease. The addition of CaCO3 improved the thermal stability and water barrier properties. The moisture absorption was reduced by 15%. X-ray diffraction analysis indicated that the addition of CaCO3 destroyed the crystalline structure of the starch and produced a new crystalline peak, resulting in a significant reduction in the crystallinity. The decrease in the crystallinity of the starch resulted in the formation of a homogeneous slurry that produced a uniform foam in the composites.