Structural Characteristics and Antioxidant Activity Analysis of Polysaccharides from Pinelliae Rhizoma and Its Processed Products Before and After Hydrolysis （Enzymolysis） by Sugar Spectrum / 中国实验方剂学杂志

ObjectiveThe glycosidic linkage structural characteristics of polysaccharides from Pinelliae Rhizoma（PR） and its processed products were analyzed by sugar spectrum， high performance thin layer chromatography（HPTLC）， fluorescence-assisted carbohydrate gel electrophoresis（PACE） based on partial acid hydrolysis and specific glycosidase hydrolysis， and the antioxidant activities of polysaccharides before and after hydrolysis（enzymolysis） were compared. MethodPolysaccharides from PR and its processed products were extracted by ultrasound extraction， starch was hydrolyzed by α-amylase， and small molecules below 3 kDa were removed by ultrafiltration. The purified polysaccharides were prepared by hydrolysis of acid and five different specific glycosidases， and the hydrolysates were analyzed by HPTLC and PACE. The antioxidant capacity of polysaccharides was analyzed by 2，2′-azino-bis（3-ethylbenzthiazoline-6-sulfonic acid）（ABTS） and 2，2-diphenyl-1-picrylhydrazyl（DPPH） free radical scavenging experiment before and after different hydrolysis. ResultThrough HPTLC and PACE analysis， it was found that polysaccharides from PR and its processed products could be hydrolyzed by β-galactosidase， β-mannase， cellulase and pectinase， but hardly hydrolyzed by glucanase， indicating that the polysaccharides contained β-galactopyranoside bond， β-1，4-mannoside bond， β-1，4-glucoside bond and α-1，4-galacturonic acid glycosidic bond. In vitro antioxidant experiments showed that the ABTS radical scavenging capacity of the polysaccharides from PR and its processed products was weakened after acid hydrolysis and pectinase enzymatic hydrolysis， while the ABTS radical scavenging capacity was enhanced after enzymatic hydrolysis with cellulase， β-galactosidase， and β-mannase. And after different hydrolysis， the DPPH free radical scavenging capacity of polysaccharides from PR and its processed products was all significantly enhanced. ConclusionThe glycosidic linkage structural characteristics of polysaccharides from PR and its processed products was analyzed by sugar spectrum in this paper， and the relationship between glycosidic bond types and their antioxidant activity was clarified through in vitro antioxidant experiments， which is beneficial for further elucidating the material basis of the related efficacy of PR and its processed products， and providing new ideas and methods for analyzing the structural characteristics of polysaccharides in Chinese medicines.

Combinatorial mutation on the -glycosidase specific to 7--xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

Taxol is a "blockbuster" antitumor drug produced by species with extremely low amount, while its analogue 7--xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1-1 and LXYL-P1-2 can convert 7--xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1-2 is twice more active than LXYL-P1-1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1-1 to LXYL-P1-2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1-2 on -xylosidase and -glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7--xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant , the molecular chaperone gene and the bacterial hemoglobin gene . This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1-2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1-2 for industrial purpose.

Glycosynthases: a Novel Efficient Synthetic Tool for Oligosaccharides / 生物化学与生物物理进展

Oligosaccharides are one of the essential physiological constituents of glycoproteins and glycolipids on mammalian cell surfaces and microbial metabolites. They have considerable potential as therapeutics but are only now slowly assuming this important role. One of the reasons for their slow development has been the considerable difficulty in synthesizing oligosaccharides on the scale necessary for their clinical evaluation. Classical chemical and enzymatic methods both have limitations in synthesizing large-scale oligosaccharides. In recent years, the rapid progress on molecular biotechnology has promoted the development of retaining glycosidases in oligosaccharides synthesis, which led to the production of a novel class of enzymatic activities termed the glycosynthases. These new enzymes are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue,synthesizing oligosaccharides in high yields ( the highest yields reach 99%) without any hydrolysis. Furthermore thioglycoligases and thioglycosynthases have been developed subsequently in the past three years. Glycosynthases can be screened in high-throughput assay by the two-plasmid system and the yeast three-hybid system respectively. Their activity can be significantly enhanced by substituting alternative residues for nucleophile, additional random mutations and optimizing reaction conditions. Their regioselectivity can be modified through changes in receptors.

Activity of glycosidases from freshwater heterotrophic microorganisms on the degradation of extracellular polysaccharide produced by Anabaena spiroides (Cyanobacteria)

The activity of specific glycosidases during the degradation of the extracellular polysaccharide (EPS) produced by Anabaena spiroides was determined using MUF-substrates (MUF-monosaccharides). Polysaccharide degradation was found to occur in a two-phase process. The first consisted of high enzymatic activity that consumed 41% of the EPS at a relatively high rate, while the second consumed the remaining polysaccharide (59%) at a slower rate. A transition phase from the higher to the slower degradation rates was marked by a replacement of bacterial populations from coccoid to bacillus cells. During the degradation process, the bacterial biomass increased with the decrease of EPS, as revealed by bacterial cell counts. The enzymatic activity detected through the substrates MUF-alpha-D- and MUF-beta-D-glucoside was higher than that detected by other substrates tested. The remaining glycosides were MUF-alpha-L-rhamnopyranoside, MUF-beta-D-galactoside, MUF-alpha-D-mannopyranoside, MUF-beta-D-fucoside, MUF-beta-D-mannopyranoside, MUF-alpha-L-arabinopyranoside, and MUF-beta-L-fucoside. The fluorescence emitted by each MUF-substrate was proportional to the concentration of the corresponding monosaccharide in A. spiroides EPS. This demonstrates the susceptibility of EPS produced by A. spiroides to enzymatic attack by bacterial populations.

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41% do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59%). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-alfa-D- e 4-MUF-beta-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-alfa-L-ramnopiranosídeo, MUF-beta-D-galactosídeo, MUF-alfa-D-manopiranosídeo, MUF-beta-D-fucosídeo, MUF-beta-D-manopiranosídeo, MUF-alfa-L-arabinopiranosídeo, e MUF-beta-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.

Novel Mutants of Microbial Glycosidases——Generation and Application of Thioglycoligases / 微生物学通报

Acid/base mutants of glycosidases, namely thioglycoligases, are able to catalyze thioglycosides synthesis. Now, many thioglycoligases, including ?-thioglucoligase, ?-thiomannoligase, ?-thiogalactoligase, ?-thioxyloligase and ?-thioglucoligase, have been developed from bacteria and archaebacteria, and applied in synthesizing various thioglycoligases. Recently, thioglycoligases have been used to glycosylate the glycoprotein and firstly generate the thioglycoprotein. The novel extended synthetic function of glycosidases would promote the development of glycobiology, biotechnology and pharmacy.