Production Of 2-O-α-D-glucopyranosyl-L-Ascorbic Acid Using Ultrasonic Radiation

L-ascorbic acid(vitamin C), a well-known antiscorbutic agent, has multiple functions and has been used for curing many diseases. However, the major drawback of using L-ascorbic acid is its extreme instability under oxidative conditions. Therefore, different attempts have been made chemically or enzymatically to synthesize L-ascorbic acid derivatives possessing its biologic functions. 2-O-α-D-glucopyranosyl-L-ascorbic acid(AA-2G) is L-ascorbic acid glycoside, enzymatically synthesized and it has been found more stable and possess all Lascorbic acid biological functions after hydrolysis by α-glucosidase into L-ascorbic acid and glucose either in vitro or in vivo. Therefore, AA-2G is considered a superior among all known L-ascorbic acid derivatives.Cyclodextrin glucanotransferase(CGTase) has been found superior for AA-2G production because of its high product specificity. Many substrates, including maltodextrin and maltose have been used as glucose donors to L-ascorbic acid for AA-2G production by CGTases. Maltodextrin, maltose, and sucrose with their low-cost and high solubility in aqueous solution, have great potentials in production of AA-2G, but the yield is too low when using maltose and maltodextrin. Meanwhile, this research aimed to select CGTase using maltodextrin and maltose as glucose donors for producing AA-2G in large quantities, using amylosucrase with sucrose as glucose donor for AA-2G production and improving AA-2G production by ultrasonic radiation treatment.CGTase from Bacillus sp SK13.002 has used maltodextrin and maltose as glucose donors for AA-2G production and productions were confirmed by LC/MS/MS. The optimum conditions were found as follows; p H 8.0, at 37 oC for 24 h with 1:1(0.016 g/m L: 0.016 g/m L) maltodextrin/maltose to the L-ascorbic acid ratio. At concentration of 400 U/m L and 200 U/m L of partially purified Cyclodextrin glucanotransferase from Bacillus sp. SK13.002 the productionsof AA-2G were 6.3 and 5.5 g/L by using maltodextrin and maltose respectively as glucose donors.Amylosucrase from Neisseria polysaccharea(NPAS) has produced AA-2G from sucrose as a glucose donor and the result was confirmed by LC/MS/MS. The optimum reaction parameters were 2.75 sucrose/L-ascorbic acid(0.04 g/m L / 0.008 g/m L) molar ratio, p H 8.0, 40 oC and 2 U/m L of NPAS for 21 h. At these optimum conditions the production of AA-2G was reached 4.92 g/L. For the first-time, AA-2G has produced via whole-cell biotransformation by E coli harboring amylosucrase from N. polysaccharea ATCC 43768. By using a fixed sucrose to L-AA molar ratio of 2.75, temperature of 37 oC and 200 rpm the optimal reaction conditions: p H 7.5, cell concentration 0.8(absorbance at 600 nm), 1.5% glucose for 72 h. At the optimal transformation conditions, the AA-2G yield was 6.93 g/L.Ultrasonic water bath cleaner working at 40 KHz and generating a maximum power of 500 W has used for AA-2G production using maltodextrin as a glucose donor. The results shown that at maximum condition of 40 KHz, 400 W and 37 oC for 18 h the yield of AA-2G was 5.69 g/L which represents an 87.54% of that obtained by shaking water bath. Ultrasonic radiation combined with shake(ultrasonic/shake) at 500 W/150 rpm and 37 oC for 14 h was yielded a 7.05 g/L of AA-2G which represents a significant(p < 0.05) increase of 11.90% compared with that obtained by conventional shaking water bath. Ultrasonic/ shake at 500 W/150 rpm was shifted the optimum temperature for AA-2G from 37 to 55 oC and yielded in 6 h a 6.6 g/L of AA-2G. This yield represents a significant(p < 0.05) increase of 4.76% % compared with that obtained by conventional shaking water bath.Conformational studies on CGTase were performed after pretreatment with ultrasonic/ shake(500 W/150 rpm) at 37 oC and 55 oC for 2 h. Study of tertiary structure study by fluorescence spectroscopy was shown that treatment at 37 oC induced slight reduction in fluorescence intensity indicting that some Trp residues were buried and the CGTase was slightly folded. On the other hand, fluorescence spectroscopy was shown that treatment at 55 oC induced a clear increase in fluorescence intensity indicting that some Trp residues were come to CGTase surface. Study of secondary structure by circular dichroism(CD) spectroscopy analysis was shown that treatment at 37 oC was slightly reduced α-helix, β-Sheet and random coil in CGTase secondary structure. On the other hand, CD spectroscopy analysis was shown that treatment at 55 oC was remarkably reduced α-helix is by a mean of 34.36%, β-Sheet and random coils were increased by a mean of 21.46% and 26.30%, respectively. Slight changes were induced in turn for both treatments.Hydrolytic and cyclization activities of CGTase were directly measured under ultrasonic/shake(500 W/150 rpm) or were measured after ultrasonic/shake(500 W/150 rpm) pretreatments at 37 oC and 55 oC for 2 h. The results were shown that both activities were significantly increased compared to the control.Improvement and acceleration of AA-2G yield by ultrasonic/shake were a result of conformational changes and activation of CGTase from Bacillus sp SK13.002.