Abstract: 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important industrial derivative of L-ascorbic acid (AA), which has the distinct advantages of non-reducibility, antioxidation, and reproducible decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α-Glucosidase, an enzyme classed into O- glycoside hydrolases, may be used in glycosylation reactions to synthesize AA-2G. Here, one α-glucosidase from Oryza sativa (rAGL) was recombinantly produced in Pichia pastoris GS115 and used for biosynthesis of AA-2G with few intermediates and byproducts. The extracellular rAGL reached 9.11 U/mL after fed-batch cultivation for 102 h in a 5-L fermenter. The specific activity of purified rAGL is 49.83 U/mg at 37 °C and pH 4.0. The optimal temperature of rAGL was 65 °C, and it was stable below 55 °C. rAGL was active over the range of pH 3.0–7.0, with the maximal activity at pH 4.0. Under the condition of 37 °C , pH 4.0, equimolar maltose and AA·Na, 8.7±0.4 g/L of AA-2G was synthesized by rAGL. These studies lay the basis for the industrial application of recombinant α-glucosidase. Keywords: α-Glucosidase; Oryza sativa; 2-O-α-D-glucopyranosyl-L-ascorbic acid; Transglycosylation; Pichia pastoris

Quercetin-4'-O-glucoside is one of the major quercetin derivatives in the mature red onion bulb. It has an adjuvant effect on allergies, asthma, arthritis, and cancer. The present study aimed to use uridine diphosphate glycosyltransferase 88A1 (UGT88A1) from Arabidopsis thaliana to achieve the enzymatic synthesis of quercetin-4'-O-glucoside from quercetin. The results showed that UGT88A1 was most active at pH 9.0. The optimum temperature of UGT88A1 for synthesizing quercetin-4'-O-glucoside was 45°C, which was a little lower than that for synthesizing quercetin-3-O-glucoside (50°C). One mutant, V18R, of UGT88A1 was obtained by site-directed mutation and showed a greater affinity (Km 0.20 mM) and twice the enzyme activity (552.3 mU/mg) towards quercetin compared with the wild-type enzyme (0.36 mM and 227.6 mU/mg, respectively). The possible reason could be attributed to the distance change between the 18th amino-acid residue of UGT88A1 and the substrate quercetin, as deduced by molecular simulation.

Sucrose phosphorylase (SPase) is capable of specifically catalysing transglucosylation reactions and can be employed in the enzymatic synthesis of alpha-D-glycosides. In the present study, a putative Thermobacillus SPase gene (TSPase) was synthesised with optimised codons and overexpressed in Escherichia coli. The 1467 bp gene encodes a 488-amino acid protein with a calculated molecular mass of 55.8 kDa. The specific activity of the recombinant TSPase (rTSPase) was 6.42 U/mg for sucrose, and the optimum temperature and pH were 65 degrees C and pH 7.0. The T-1/2 value of the rTSPase was 212 h at 50 degrees C and 98 h at 60 degrees C. A stimulating effect on the activity of the rTSPase was observed in the presence of 5 mM Co2+. The rTSPase showed increased stability against DMSO as organic co-solvent at 50 degrees C. The K-m and k(cat) of the rTSPase with sucrose were determined as 6.24 mM and 5.73 s(-1) respectively. The rTSPase produced 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid (AA-2 G) from ascorbic acid in both crude extract and whole-cell forms. A maximum yield of 19.7% (39.94 +/- 0.17 g/L) was achieved after incubation of ascorbic acid sodium salt and sucrose (1:2) with 19.76 U/mL of the rTSPase at pH 7.0 and 50 degrees C for 24 h.

本文研究了抗坏血酸葡萄糖苷（2-O-α-D-glucopyranosyl-L-ascorbicacid,简称AA-2G）/β-环糊精包合物的制备工艺,以提高它在应用中的稳定性、生物利用度。选用β-环糊精（β-cyclodextrin,β-CD）对AA-2G进行包合,采用饱和水溶液法研究了AA-2G-β-CD包合物的制备工艺。以包合率为考察指标,通过单因素试验考察了温度、时间、搅拌速度以及β-环糊精和AA-2G的摩尔比对包合物制备效果的影响。进一步运用正交试验研究确定了AA-2G-β-CD包合物的最佳工艺条件为:AA-2G与β-CD的摩尔比为1:3,温度为60℃、搅拌速度为200 r/min,时间为5 h时,包合率为49.55%。影响包合率的因素顺序为:时间＞温度＞转速＞摩尔比。验证试验表明,饱和水溶液法制备AA-2G-β-CD包合物工艺稳定。通过傅里叶红外色谱法对制备的AA-2G-β-CD包合物进行了鉴定,证明了AA-2G-β-CD包合物的形成。通过抗氧化性实验发现,包合物清除氧自由基能力高于AA-2G与β-CD混合物。综上,采用饱和水溶液法制备AA-2G-β-CD包合物,经验证AA-2G-β-CD包合物形成,通过正交实验优化制备工艺后,其包合率达到49.55%,同时包合物的抗氧化性能力高于AA-2G与β-CD混合物。