Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System.

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained.IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Construction of engineered Saccharomyces cerevisiae strain to improve that whole-cell biocatalytic production of melibiose from raffinose.

There are excessive by-products in the biocatalysis process of this whole-cell biocatalytic production of melibiose from raffinose with current Saccharomyces cerevisiae strains. To solve this problem, we constructed engineered strains based on a liquor yeast (S. cerevisiae) via gene deletion (mel1 gene), heterologous integration (fsy1 or/and ffzi1 gene from Candida magnoliae), and gene overexpression (gcr1 gene). Functional verification showed that deletion of the mel1 gene led to elimination of the reactions catalyzed by α-galactosidase, as well as elimination of the degradation of melibiose and the formation of galactose by-product. Insertion of the fsy1 or/and ffzi1 gene and overexpression of the gcr1 gene could contribute to fructose transport for enhancing the biopurification rate of the fructose by-product. Compared with the wild-type strain, the optimal engineered strain of MP8 (Δmel1::fsy1 cm ::ffzi1 cm ::gcr1 sc ) had improved about 30% on yield, 31% on productivity, and 36% on purity of the melibiose product.

Application of Chlorine Dioxide in Cell Surface Modification to Enhance Its Mechanical Stability and Metal Ion Adsorption.

There has been a trend toward the use of microorganisms as the biomaterial for removing dyes and metals from wastewater. However, native microorganism cells have low mechanical stability, which limit their further application in industries. In this study, chlorine dioxide (ClO2), a high-efficiency, low-toxicity, and environmentally benign disinfectant, was used for microorganism surface modification to enhance the mechanical stability and metal ion adsorption of the cell. ClO2 can either modify cell walls to improve their metal adsorption capacity or modify cell membranes to improve their mechanical stability. Fourier-transform infrared spectroscopy analysis indicated that several cell surface groups were involved in the cell wall modification of Bacillus sp. Microscopic observation indicated that ClO2 treatment could deter cell membranes from forming vesicles in sodium hydroxide (NaOH) aqueous solution, and freeze-etching showed that ClO2 treatment could alter the erythrocyte membrane proteins which might also contribute to improving the cell stability. The experimental results on Bacillus sp., Pseudomonas aeruginosa, and Mucor rouxii show that ClO2 treatment may increase, or at least not reduce, the ability of microbial cells to adsorb heavy metals, but it can significantly improve the resistance of these cells to NaOH cleavage. It seems ClO2 is a promising auxiliary for biosorption of heavy-metal ions.

Efficiency Analysis and Mechanism Insight of that Whole-Cell Biocatalytic Production of Melibiose from Raffinose with Saccharomyces cerevisiae.

Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker's yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes (suc2, mel1, and fsy1) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.

Structural characterization and immunostimulating activity of a levan-type fructan from Curcuma kwangsiensis.

A fructan designated as CKNP with apparent molecular weight of 5.3kD was isolated from the hot water extract of Curcuma kwangsiensis through a combination of ion-exchange chromatography on DEAE 650M and gel filtration on Superdex G-200. CKNP was characterized by chemical derivatization as well as HPLC, GC, and GC-MS technologies. Structural studies revealed that CKNP is composed predominately of fructose (96.8%) and a small amount of glucose (3.2%) with a degree of polymerization (DP) of 30-31. It was deduced to be a levan-type fructan containing a backbone composed of (2→6)-linked ß-d-Fruf residues and single ß-d-Fruf residues as side chains branched at the O-1 position along the backbone. Preliminary in vitro bioactive tests on RAW 264.7 murine macrophage cells revealed that the levan-type fructan from C. kwangsiensis shows significant immunostimulating activity based on its ability to stimulate macrophage proliferation and enhance phagocytosis.