A Review: Manufacturing, and Properties of the D-Fructose Epimer D-Allulose (D-Psicose)

D-allulose, also known by the name D-psicose or just allulose, is a rare keto-sugar epimer of D-fructose in the third carbon (C3), and naturally occurs in small quantity in fruits such as grapes and figs. It is low calorie sweetener produced enzymatically from D-fructose by enzymes D-ketose 3-epimerase (DKEase) family. D-allulose has a similar taste, texture and functionality as sweetener comparing to high calorie sweetener sugar table sucrose. D-allulose is poorly metabolized in the body with minimal impact on blood sugar levels making it a natural low-calorie sweetener. This property makes D-allulose an attractive sweetener for diabetes and for body weight management. Laboratory studies on D-allulose intake demonstrated its safety with no significant adverse effects. United States Food and Drug Administration (FDA) has granted D-allulose the status of Generally Recognized as Safe (GRAS). Plus, it is considered safe for human consumption by regulatory organizations in other countries except in European Union due to their request for further laboratory testing. Maximum acceptable daily intake of D-allulose is 0.9 grams per kilogram body weight. Excessive intake for more than the recommended daily intake could lead to some side effects such as gastrointestinal discomfort or laxative effects. In general, D-allulose is considered one of the preferred natural low calories sweeteners for those seeking an alternative to table sugar sucrose.

Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

D-Psicose 3-epimerase(DPEase)is demonstrated to be useful in the bioproduction of D-psicose,a rare hexose sugar,from D-fructose,found plenty in nature.Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase.In this study,the crystal structure of the C.cellulolyticum DPEase was determined.The enzyme assembles into a tetramer and each subunit shows a(β/α)8 TIM barrel fold with a Mn2+metal ion in the active site.Additional crystal structures of the enzyme in complex with substrates/products(D-psicose,D-fructose,D-tagatose and D-sorbose)were also determined.From the complex structures of C.cellulolyticum DPEase with D-psicose and D-fructose,the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues.Accordingly,based on these ketohexosebound complex structures,a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here.These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential sweeteners.At present,the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase(DAE).However,industrial applications of DAE are still limited by its poor thermostability.Herein,directed evolution was applied to improve the thermostability of dAE from Clostridium cellulolyticum H10(CcDAE).Two optimal mutants D281G and C289R,exhibiting 13.80-fold and 13.88-fold t_(1/2 )values as that of wild type at 65℃,respectively,were obtained.To further enhance the thermostability,the triple mutant A107P/D281G/C289R was constructed after combina-tion of mutants D281G,C289R,and previously identified thermostability-enhanced mutant A107P.The T_(m) and optimal temperature of triple mutant were increased by 14.39℃and 5℃,respectively,compared to the wild type,meanwhile,the half-life of triple mutant was 58.85-fold as that of wild type at 65℃.Furthermore,the conversion rate of triple mutant was increased from 24.76%of wild type to 27.53%using 300 g/L d-fructose as substrate at 70℃.The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.