Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose / 生物工程学报

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Content Comparison of Mannose,Glucose and Narinhenin in Dendrobium officinale and Dendrobium huoshanense / 中国实验方剂学杂志

Objective: To optimize the pre-column derivation high performance liquid chromatography (HPLC) content determination method of D-mannose and D-glucose as well as the content determination method of narinhenin in Dendrobium officinale and D. huoshanense, and compare the contents of D-mannose,D-glucose and narinhenin between D. officinale and D. huoshanense. Method: A pre-column derivation HPLC method modified by Chinese Pharmacopoeia(Ch.P) 2015 was used to simultaneously determine the contents of D-mannose and D-glucose,with acetonitrile-0.02 mol·L-1 ammonium acetate solution as mobile phase for gradient elution. Kromasil 100-5 C18 was performed with the wavelength set at 250 nm,and the flow rate was 1 mL·min-1;column temperature was 30℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C18 with the acetonitrile-methanol-0.4% phosphoric acid solution as mobile phase for gradient elution,and the wavelength was set at 290 nm; the flow rate was 0.8 mL·min-1,and column temperature was 40℃. Result: D-mannose and D-glucose showed a good linear relationship within the range of 0.15-3.0 μg and 0.075-2.25 μg (r=0.999 9); and their average recoveries were 99.01% (RSD 2.1%) and 101.69% (RSD 2.0%) respectively. In addition, the other methodological researches such as repeatability and durability all met the requirements. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them (Cm+Cg) were 12.75%-36.40%,2.93%-18.39% and 19.23%-54.58% in 43 batch of D. officinale. Almost all of the results except very few samples reached the D-mannose standard in Ch.P 2015, and the total content of D-mannose and D-glucose was also up to the total polysccharide standard in Ch.P. The correlation between content and origin was not significant. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them (Cm+Cg) were 14.33%-29.47%,6.64%-15.20%,and 25.73%-44.37% in 12 batch of D. huoshanense. These contents and ratio of peak areas of D-mannose to D-glucose (Am/Ag) were within the scope of D. officinale's; in addition, their average contents were basically the same with those in D. officinale (about 33%).Next,naringenin showed a good linear relationship within the range of 0.020 8-0.832 0 μg (r=0.999 9),and its average recovery was 101.96% (RSD 1.8%). The content of naringenin was 0.053 2-0.122 4 mg·g-1 (average value of 0.081 0 mg·g-1) in 11 batch of D. officinale, slightly higher than 0.040 3-0.090 0 mg ·g-1 (average value of 0.068 3 mg ·g-1) in 7 batch of D. huoshanense. All of these results of narinfenin did not reach the content lower limit in Ch.P. Conclusion: The method used to determinate the content of D-mannose and D-glucose is reproducible, and their sum content is possible to substitute the total polysccaride determination (with higher errors) in D. officinale; monosaccharide content determination can be used for quantitative quality control of D. huoshanense. However, it could not distinguish D. officinale and D. huoshanense by determining the contents of polysccharide,D-glucose,D-mannose and narinhenin, and shall be combined with other specificity methods for further identification.

Comparision two kinds of content determination method of mannatide for injec-tion / 药学实践杂志

Objective To compare two methods in the determination of mannatide for injection .Methods The contents were determined by spectrophotometry and HPLC method .Results In the samples determined by Spectrophotometric Determi‐nation ,soluble in phenol solution and sulfuric acid ,was detected at wavelength 490 nm ,D‐mannose derivatives linear concen‐tration range is 10 .2‐51 μg/ml (r=0 .999 1) ,the average content was 92 .14% ,the average RSD value was 1 .17% .In the HPLC determination , with color spectrum column : Gemini C18 (250 mm × 4 .6 mm , 5 μm ) , mobile phase acetonitrile -0 .02 mol/L ammonium acetate solution (20∶80) ,flow rate:1 ml/min ,the detection wavelength was at 250 nm , column temperature:30 ℃ ,sample size:10 μl detection .The average content ws 83 .47% ,the average RSD value was 0 .65% ,the content of two kinds of methods could effectively determine the D‐mannose .Conclusion The average content for spectrophotometry was higher ,but the phenol was used in detection of special odor ,poisonous ,corrosive ,would cause a cer‐tain risk .HPLC was exclusive ,chromatogram was more intuitive ,on the operator harmfulness and less irritating .A new meth‐od for the determination of D‐mannose was established at the same time .

Inhibitory Effects of D-mannose on Streptococcus mutans in the Presence of Sucrose

This study aimed to examine the inhibitory effect of rare sugars on Streptococcus mutans (S. mutans) in the presence of sucrose. Xylitol and three rare sugars (D-xylose, D-lyxose and D-mannose) were used in this study. S. mutans KCTC 3065 was cultured in Brain Heart Infusion (BHI) medium containing xylitol, D-xylose, D-lyxose, or D-mannose in the presence of sucrose, and the effect on S. mutans growth was assessed by measuring solution turbidity at different time points after inoculation. To assess effects on pH, sucrose was added at different concentrations, and solution pH was measured at different time points after inoculation. All sugars significantly inhibited the growth of S. mutans in the presence of sucrose. Especially, D-lyxose and D-mannose exhibited significantly greater inhibition than that of xylitol. Furthermore, unlike D-lyxose, D-mannose significantly inhibited the decrement of pH, and its effect was greater than that of xylitol. Taken together, D-mannose has strong inhibitory effect on S. mutans in the presence of sucrose.