Enzymatic Synthesis of Maltitol and Its Inhibitory Effect on the Growth of Streptococcus mutans DMST 18777.

This study aimed to synthesize maltitol using recombinant CGTase from Bacillus circulans A11 with ß-cyclodextrin (ß-CD) and sorbitol as a glucosyl donor and acceptor, respectively, and assess its antibacterial activity. Optimal conditions for producing the highest yield, 25.0% (w/w), were incubation of 1% (w/v) ß-CD and sorbitol with 400 U/mL of CGTase in 20 mM phosphate buffer at pH 6.0 and 50 °C for 72 h. Subsequently, maltitol underwent large-scale production and was purified by HPLC. By mass spectrometry, the molecular weight of the synthesized maltitol was 379.08 daltons, corresponding exactly to that of standard maltitol. The relative sweetness of synthesized and standard maltitol was ~90% of that of sucrose. Spot assay on the agar plate showed that maltitol inhibited the growth of Streptococcus mutans DMST 18777 cells. In addition, the MIC and MBC values of synthesized and standard maltitol against S. mutans were also determined as 20 and 40 mg/mL, respectively. These results show that the synthesized maltitol can be produced at high yields and has the potential to be used as an anticariogenic agent in products such as toothpaste.

Optimization of amylomaltase for the synthesis of α-arbutin derivatives as tyrosinase inhibitors.

α-Arbutin is widely used as a skin-whitening agent in the pharmaceutical and cosmetic industries because of its inhibitory effect on tyrosinase, an important enzyme for generating melanin pigments. Given the increasing demand for such products, we synthesized α- and ß-arbutin-α-D-glycosides through transglycosylation reactions catalyzed by a recombinant amylomaltase, using tapioca starch and α- and ß-arbutin as donor and acceptor molecules, respectively. The catalytic yield of products by the amylomaltase was greater with α-arbutin than with ß-arbutin. The highest glycoside yield (83%) was achieved by adjusting the following six parameters: starch and α-arbutin concentration, enzyme concentration, pH, temperature, and incubation time. The glycoside products were isolated and analyzed by HPLC, and two major products were identified by mass spectrometry and nuclear magnetic resonance spectroscopy, namely, α-arbutin-α-d-glucopyranoside (α-Ab-α-G1) and α-arbutin-α-d-maltopyranoside (α-Ab-α-G2). Both α-Ab-α-G1 and α-Ab-α-G2 are more water soluble than α-arbutin. Like α-arbutin, α-Ab-α-G1 and α-Ab-α-G2 showed competitive inhibition of human tyrosinase. However, their Ki values were 0.53 and 1.40 mM, respectively, which are slightly higher than that of α-arbutin (0.25 mM). The addition of glucosyl residues to α-arbutin improved its water solubility. Therefore, α-Ab-α-G1 and α-Ab-α-G2 could be easily absorbed by the skin and used as skin-whitening agents in pharmaceutical and cosmetic industries.

Application of amylomaltase for the synthesis of salicin-α-glucosides as efficient anticoagulant and anti-inflammatory agents.

The focus of this study was the synthesis of α-glucosyl derivatives of salicin by a transglucosylation reaction. The reaction was catalyzed by recombinant amylomaltase using tapioca starch as a glucosyl donor. Several reaction parameters, such as the enzyme-substrate concentrations, pH, temperature and incubation time, were optimized. Using the optimum conditions, at least three products with retention times (Rt) of 6.2, 9.2 and 14.1 were observed. The maximum yield of glucosylated salicin derivatives was 63% (w/w) of the total products. The structures of the glucosylated salicin derivatives were confirmed to be salicin-α-D-glucopyranoside, salicin-α-D-maltopyranoside and salicin-α-D-maltotriopyranoside through a combination of enzyme treatments, mass spectrometry and NMR analyses. The glycosidic bond between glucose units consisted of an α-1,4-configuration. The water solubility of salicin-α-D-glucopyranoside, salicin-α-D-maltopyranoside and salicin-α-D-maltotriopyranoside was 3-, 5- and 8-fold higher, respectively, than that of salicin, whereas their relative sweetness values were lower than that of sucrose. Interestingly, the long-chain salicin-α-D-glucosides showed greater anticoagulant and anti-inflammatory activities than salicin. In addition, the synthesized salicin-α-D-glucosides were able to tolerate acidic and high temperature conditions, but not α-glucosidase or human digestive enzymes. Therefore, these salicin-α-D-glucosides should be applied by the injection route to achieve greater bioavailability than is possible by the oral route.

Kinetic inhibition of human salivary alpha-amylase by a novel cellobiose-containing tetrasaccharide.

OBJECTIVE: The aim of the study was to evaluate the inhibitory kinetics of a novel cellobiose-containing tetrasaccharide on human salivary alpha-amylase (HSA). MATERIAL AND METHOD: Synthesis of cellobiose-containing tetrasaccharide was catalyzed by Paenibacillus sp. All CGTase using beta-CD as a donor and cellobiose as an acceptor under the optimal conditions. The reaction mixture was analyzed by HPLC and a cellobiose-containing tetrasaccharide obtained was studied for its inhibitory kinetics. RESULTS: In vitro activity of human salivary alpha-amylase showed the optimum pH and temperature at 7.0 and 37 degrees C, respectively. The effects of metal ions, protective chemicals and saccharides on alpha-amylase activity, they were found that 10 mM concentration of CaCl2 and NaCl enhanced the enzyme activity. In contrast, the enzyme activity was significantly inhibited by 10 mM of HgCI2, alpha-cyclodextrin (alpha-CD) and synthetic cellobiose-containing tetrasaccharide. Chemicals often used as protective substance for enzyme such as beta-mercaptoethanol, EDTA or used as fungicide during enzyme purification (NaN3) had no effect on the activity of this enzyme. As a cellobiose-containing tetrasaccharide was shown to have a pronounce inhibition on alpha-amylase activity. Its inhibition kinetic was performed and found that cellobiose-containing tetrasaccharide was a competitive inhibitor with a Ki value of 7.89 microM. CONCLUSION: Inhibition kinetic of a cellobiose-containing tetrasaccharide on alpha-amylase activity was competitive type with Ki value of 7.89 microM. In addition, these results will be a basic knowledge in controlling alpha-amylase actions that have influence on blood glucose level of trial animal and human further

Effect of temperature on cyclodextrin production and characterization of paracetamol/cyclodextrin complexes.

OBJECTIVE: To compare the effect of different reaction temperatures on the cyclization and coupling reactions of the Toruzyme CGTase influencing the yield of cyclodextrins (CDs) and to study the solubility of paracetamol with CDs. MATERIAL AND METHOD: Type and amount of CDs were analyzed by HPAEC-PAD. The stability constants for the inclusion complex formed between CDs and paracetamol were determined using the phase solubility method. The solubility of paracetamol with CDs was measured by UV-spectrophotometer at 240 nm. RESULTS: The result has shown that the reaction temperature has effect on the Toruzyme CGTase reactions in production of CDs. The CDs yield after 30 min of incubation was higher at 60 degrees C than at 80 degrees C. The catalytic efficiency (k(cat)/K(m)) of this enzyme indicated the higher value of the cyclization reaction at 60 degrees C compared to 80 degrees C while the opposite was found for the coupling reaction. Paracetamol is used as an analgesic and antipyretic but it is poorly water-soluble drug. To improve the solubility of paracetamol CDs obtained were used to study for paracetamol/CDs complexes. The phase-solubility diagrams of paracetamol with alpha-, beta- and gamma-CD were A(N) type while that of paracetamol with maltosyl-beta-CD (G2-beta-CD) complex was A(L) type. The stability constants (K(c)) for the inclusion complex of paracetamol with alpha-, beta-, gamma-CD and G2-beta-CD were 5.69, 16.75, 4.73 and 2,223.25 M(-1), respectively. CONCLUSION: The optimum temperature for CDs production was at 60 degrees C and the low solubility of paracetamol was significantly improved by complexation with CDs, where the enhancing effect was in the order of G2-beta-CD > beta-CD > alpha-CD > gamma-CD.