Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-ß-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose. Glycosylation using the intermolecular aglycon delivery (IAD) method was employed to selectively form 1,2-cis α-glycosidic linkages of the allopyranose residues. Moreover, ß-selective psicofuranosylation was performed using a psicofuranosyl acceptor with 1,3,4,6-tetra-O-benzoyl groups. This is the first report on the synthesis of non-reducing disaccharides comprising only rare d-sugars by IAD using protected ketose as a unique acceptor; additionally, this approach is expected to be applicable to the synthesis of functional sweeteners.

A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water.

D-Psicose is a rare, low-calorie sugar that is found in limited quantities in national products. Recently, D-psicose has gained considerable attention due to its potential applications in the food, nutraceutical, and pharmaceutical industries. In this study, a novel D-psicose 3-epimerase (a group of ketose 3-epimerase) from an extremely halophilic, anaerobic bacterium, Iocasia fonsfrigidae strain SP3-1 (IfDPEase), was cloned, expressed in Escherichia coli, and characterized. Unlike other ketose 3-epimerase members, IfDPEase shows reversible epimerization only for D-fructose and D-psicose at the C-3 position but not for D-tagatose, most likely because the Gly218 and Cys6 at the substrate-binding subsites of IfDPEase, which are involved in interactions at the O-1 and O-6 positions of D-fructose, respectively, differ from those of other 3-epimerases. Under optimum conditions (5 µM IfDPEase, 1 mM Mn2+, 50 °C, and pH 7.5), 36.1% of D-psicose was obtained from 10 mg/mL D-fructose. The IfDPEase is highly active against D-fructose under NaCl concentrations of up to 500 mM, possibly due to the excessive negative charges of acidic amino acid residues (aspartic and glutamic acids), which are localized on the surface of the halophilic enzyme. These negative charges may protect the enzyme from Na+ ions from the environment and result in the lowest pI value compared to those of other 3-epimerase members. Moreover, without adjusting any ingredients, IfDPEase could improve coconut water quality by converting D-fructose into D-psicose with a yield of 26.8%. Therefore, IfDPEase is an attractive alternative to enhancing the quality of fructose-containing foods.

High production of d-psicose from d-fructose by immobilized whole recombinant Bacillus subtilis cells expressing d-psicose 3-epimerase from Agrobacterium tumefaciens.

d-Psicose 3-epimerase (DPEase) can catalyze the isomerization of d-fructose to be rare sugar d-psicose, which has wide application prospects in the food and medical fields. In this study, the DPEase gene from Agrobacterium tumefaciens was constructed into plasmid pMA5, and was successfully expressed in the host Bacillus subtilis WB600 (B. subtilis). After optimization of the fermentation conditions, whole recombinant B. subtilis WB600/pMA5-At-DEPase(O) cells produced d-psicose from d-fructose with a conversion rate of 29.01 ± 0.19%, which could be used for the efficient synthesis of d-psicose. To further improve the whole recombinant B. subtilis application, B. subtilis cells were immobilized onto a gel bead biocatalyst by Ca-alginate. After optimization of the biotransformation conditions, the conversion rate of the immobilized biocatalyst reached 20.74 ± 0.39%, which was lower than the free cells. However, the results showed that the immobilized biocatalyst had higher thermal/pH stability and storability, and the gel beads could be recycled for at least six batches. The results showed that the amount of d-psicose generated reached 32.83 ± 2.56 g/L with the immobilized biocatalyst after six times biotransformation, whereas the free cells produced only approximately 10.44 ± 0.07 g/L. The results showed that immobilized recombinant B. subtilis cells are promising to use for the efficient synthesis of d-psicose.

Rare sugars: metabolic impacts and mechanisms of action: a scoping review.

Food manufacturers are under increasing pressure to limit the amount of free sugars in their products. Many have reformulated products to replace sucrose, glucose and fructose with alternative sweeteners, but some of these have been associated with additional health concerns. Rare sugars are 'monosaccharides and their derivatives that hardly exist in nature', and there is increasing evidence that they could have health benefits. This review aimed to scope the existing literature in order to identify the most commonly researched rare sugars, to ascertain their proposed health benefits, mechanisms of action and potential uses and to highlight knowledge gaps. A process of iterative database searching identified fifty-five relevant articles. The reported effects of rare sugars were noted, along with details of the research methodologies conducted. Our results indicated that the most common rare sugars investigated are d-psicose and d-tagatose, with the potential health benefits divided into three topics: glycaemic control, body composition and CVD. All the rare sugars investigated have the potential to suppress postprandial elevation of blood glucose and improve glycaemic control in both human and animal models. Some animal studies have suggested that certain rare sugars may also improve lipid profiles, alter the gut microbiome and reduce pro-inflammatory cytokine expression. The present review demonstrates that rare sugars could play a role in reducing the development of obesity, type 2 diabetes and/or CVD. However, understanding of the mechanisms by which rare sugars may exert their effects is limited, and their effectiveness when used in reformulated products is unknown.

A Possible Mechanism of Graphene Oxide to Enhance Thermostability of D-Psicose 3-Epimerase Revealed by Molecular Dynamics Simulations.

Thermal stability is a limiting factor for effective application of D-psicose 3-epimerase (DPEase) enzyme. Recently, it was reported that the thermal stability of DPEase was improved by immobilizing enzymes on graphene oxide (GO) nanoparticles. However, the detailed mechanism is not known. In this study, we investigated interaction details between GO and DPEase by performing molecular dynamics (MD) simulations. The results indicated that the domain (K248 to D268) of DPEase was an important anchor for immobilizing DPEase on GO surface. Moreover, the strong interactions between DPEase and GO can prevent loop α1'-α1 and ß4-α4 of DPEase from the drastic fluctuation. Since these two loops contained active site residues, the geometry of the active pocket of the enzyme remained stable at high temperature after the DPEase was immobilized by GO, which facilitated efficient catalytic activity of the enzyme. Our research provided a detailed mechanism for the interaction between GO and DPEase at the nano-biology interface.

Bioproduction of D-allulose: Properties, applications, purification, and future perspectives.

D-allulose is the C-3 epimer of D-fructose, which rarely exists in nature, and can be biosynthesized from D-fructose by the catalysis of D-psicose 3-epimerase. D-allulose is safe for human consumption and was recently approved by the United States Food and Drug Administration for food applications. It is not only able be used in food and dietary supplements as a low-calorie sweetener, but also modulates a variety of physiological functions. D-allulose has gained increasing attention owing to its excellent properties. This article presents a review of recent progress on the properties, applications, and bioproduction progress of D-allulose.

Enhanced production of d-psicose 3-epimerase in Bacillus subtilis by regulation of segmented fermentation.

d-Psicose 3-epimerase is an enzyme that catalyzes the synthesis of d-psicose from d-fructose. We cloned the d-psicose 3-epimerase from Ruminococcus sp. (RDPE) and expressed it in Bacillus subtilis A311. By a two-step pH regulation of segmented fermentation, we significantly improved the RDPE production and decreased the fermentation cost. The two-step regulation consisted of the first step maintained the pH value at 7.0 for 24 H and the second step adjusted the pH value up to 7.5 slowly for another 24 H. Finally, the RDPE production was increased to 74 U/mL, which was about 2.5-fold compared with the control. Our segmented fermentation strategy provides an important experimental basis for the industrial-scale production of RDPE.

Production of D-allose from D-fructose using immobilized L-rhamnose isomerase and D-psicose 3-epimerase.

D-Allose is a rare sugar, can be used as an ingredient in a range of foods and dietary supplements, has alimentary activities, especially excellent anti-cancer effects and used in assisting cancer chemotherapy and radiotherapy, etc. To develop a simple and low-cost process for D-allose production, a one-pot enzymatic process using the substrate of D-fructose, and the recombinant enzymes of D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) was developed. These enzymes were cloned from Ruminococcus sp. and B. subtilis, respectively, successfully expressed in E. coli, extracted and immobilized using anion exchange resin and amino resin, respectively. The mass ratio of D-fructose, D-psicose and D-allose was 6.6:2.4:1.0 when the reaction reached equilibrium after 5 h of reaction. Using the low-cost substrate of D-fructose, the reusable immobilized enzymes and the one-pot reaction, the production process is simplified and the production cost is decreased. In addition, to simplify the enzyme extraction and immobilization processes, new methods for enzyme capture and immobilization were developed especially for DPE immobilization. This is the first report for one-pot D-allose production using immobilized L-RhI and DPE.

90-Day repeated oral toxicity test of D-allulose produced from Microbacterium foliorum.

D-allulose is considered an ideal substitute for sucrose, because it has 70% of the sweetness of sucrose and ultra-low energy. Chemical and biotechnological methods have been developed to produce Dallulose from D-fructose because D-allulose exists in extremely small quantities in nature. In this study, we performed a 90-day repeated oral dose toxicity test on rats using D-allulose produced from Microbacterium foliorum-a non-GMO species isolated from salad ginseng-in dosages of 0, 1250, 2500 and 5000 mg/kg/day. We developed a toxicity determination criterion based on the significant change caused by the administration of the substance to estimate the NOEL, NOAEL, and LOAEL of the substance applied in this study. This test found only minor compound-related changes in both male and female rats in the high dose group and no important compound-related changes. Thus, we determined the NOAEL of Dallulose in both sexes to be 5,000 mg/kg/day. This study's finding of a NOAEL of 5,000 mg/kg/day should ensure that D-allulose produced from Microbacterium foliorum is classified as a safe and ordinary substance.

Effect of d-psicose substitution on gelatin based soft candies: A TD-NMR study.

Confectionary gels are considered as composite gel systems composed of high amount of sugar and gelling agent such as gelatin or starch. d-Psicose is classified as a type of rare sugar, which is a C-3 epimer of fructose and has 70% of the sweetness of sucrose with a caloric value of 0.39 kcal/g. Utilization of d-psicose in food products is gaining particular interest due to its low caloric value. In this study, gelatin-based soft candies were formulated, and the effect of d-psicose substitution was explored on the quality of the products. For characterization of the soft candies, moisture content, water activity, color, hardness, and glass transition temperature of samples were investigated. X-ray diffraction analysis was also performed to explain the crystallization tendency of jelly candies. Results showed that, the softest sample with the highest moisture content and the smallest crystallization tendency was the sample that included the highest amount of d-psicose. Time domain (TD) NMR relaxometry experiments were also conducted on gel samples, and three distinct proton populations were observed in the relaxation spectrum for all formulations. Spin-lattice relaxation times obtained through monoexponential fitting (T1 ) were also obtained to explain some quality parameters.

Towards the recovery of a sense of self: An interpretative phenomenological analysis of patients' experience of body-oriented psychotherapy for schizophrenia.

OBJECTIVE: Increasing evidence supports the efficacy of body-oriented psychotherapy (BPT) for schizophrenia. Yet, so far no research has investigated outcome in relation to therapy process: Why and how BPT is effective. In this study, we qualitatively explore participants' experience of a manualized BPT for schizophrenia to shed light on the process of therapeutic change. METHOD: We conducted in-depth interviews with 6 participants who completed a 10-week BPT group intervention. Interviews explored participants' experience of change and helpful aspects of therapy and were analysed using interpretative phenomenological analysis. FINDINGS: We identified six master themes across the interviews: (i) Being a whole: body-mind connection; (ii) Being agentic and being able; (iii) Being unique and worthy: Being accepted for who one is; (iv) Changing interactions: Engaging in authentic interpersonal contact; (v) Being part of a group: Feeling integrated; and (vi) Hope and investing in the future. CONCLUSION: We discuss the clinical implications for each theme and bring the findings together by describing therapeutic change in schizophrenia as a recovery of sense of self at different but interlocked levels. Moreover, we put forward recommendations for both specific and common factors for schizophrenia therapy. Clinical or methodological significance of this article: The clinical significance of this study is twofold. On the one hand, the findings of this analysis might inform the theory and practice of BPT and might directly feedback into a further development of the manual guidelines. On the other hand, common helpful factors have been identified thatmight also be relevant for the more general clinical practice concerning patients with schizophrenia. Here, we summarize our key messages for the clinical practitioner emerging from the findings: The inclusion of bodily aspects and a focus on pre-reflective experience in psychotherapy can help persons with schizophrenia recover the sense of being a body-mind unity. Empowering persons with schizophrenia, instead of instructing them, seems to foster a sense of agency and self-confidence, which are crucial to the recovery process. A twofold therapeutic stance characterized by openness towards the other and authenticity was experienced by patients as facilitating the rapport building. This might help persons with schizophrenia engage in the relation. Social inclusion might enhance therapeutic change and recovery in schizophrenia. Group therapy might be helpful for fostering a feeling of social belonging, but the inclusion in the wider social and community context remains a critical issue. The experience of joyful moments in psychotherapy might positively contribute to therapeutic change in that it fosters a sense of hope for the future. Overall our paper contributes to the literature aiming at expanding the range of therapeutic modalities, focussing on the creation and use of mixed models of therapy within and beyond talking practices.

Highly efficient production of Clostridium cellulolyticum H10 D-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce D-psicose.

BACKGROUND: D-Psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to the rare sugar D-psicose, which may help prevent obesity, reduce blood sugar and blood fat, and inhibit intra-abdominal fat accumulation. RESULTS: In this study, the DPEase of Clostridium cellulolyticum H10 was expressed in the food-grade host Bacillus subtilis. Optimization of the culture medium during shake-flask experiments yielded a DPEase activity of 314 U/mL. The optimal medium included 20 g/L peptone, 15 g/L corn steep powder, 5 g/L glycerol, and 1 mM Ca2+. Controlling the carbon source concentration was important because elevated concentrations can result in catabolite metabolic suppression (CCR). To avoid CCR and increase DPEase expression, we developed a fed-batch strategy in a 3.6-L fermenter. We altered the ratio of carbon source to nitrogen source (C/N) in the feeding medium and employed a constant feeding rate (6 g/L/h). This strategy improved the DPEase activity to 2246 U/mL (7.8 g/L), which is almost 15 times higher than that observed in the original shake-flask cultures. Finally, we used the DPEase-expressing B. subtilis cells to produce D-psicose from D-fructose, and a 28% conversion yield was achieved with these cells, demonstrating their potential use in D-psicose production. CONCLUSIONS: This is the first report to enhance recombinant DPEase production in B. subtilis using efficient and convenient fermentation strategy, and the DPEase yield is three times higher than the highest yield reported to date. The recombinant B. subtilis cells were further used in the efficient synthesis of D-psicose. This study provides a basis for the industrial production of D-psicose.

Characterization of L-rhamnose isomerase from Clostridium stercorarium and its application to the production of D-allose from D-allulose (D-psicose).

OBJECTIVE: To characterize L-rhamnose isomerase (L-RI) from the thermophilic bacterium Clostridium stercorarium and apply it to produce D-allose from D-allulose. RESULTS: A recombinant L-RI from C. stercorarium exhibited the highest specific activity and catalytic efficiency (k cat/K m) for L-rhamnose among the reported L-RIs. The L-RI was applied to the high-level production of D-allose from D-allulose. The isomerization activity for D-allulose was maximal at pH 7, 75 °C, and 1 mM Mn2+ over 10 min reaction time. The half-lives of the L-RI at 65, 70, 75, and 80 °C were 22.8, 9.5, 1.9, and 0.2 h, respectively. To ensure full stability during 2.5 h incubation, the optimal temperature was set at 70 °C. Under the optimized conditions of pH 7, 70 °C, 1 mM Mn2+, 27 U L-RI l-1, and 600 g D-allulose l-1, L-RI from C. stercorarium produced 199 g D-allose l-1 without by-products over 2.5 h, with a conversion yield of 33% and a productivity of 79.6 g l-1 h-1. CONCLUSION: To the best of our knowledge, this is the highest concentration and productivity of D-allose reported thus far.

Deactivation kinetics and the effects of additives on storage stability and structure of D-psicose 3-epimerase.

OBJECTIVE: To explore deactivation kinetics and the effects of some additives on the activity and conformational changes of D-psicose 3-epimerase (DPEase) during its storage. RESULTS: The experimental data of DPEase inactivation during storage at 4-45 °C fitted with the first-order expression model. The inactivation rate constants of DPEase stored at 4, 10, 25, 35 and 45 °C were 0.0076, 0.01, 0.0223, 0.0351 and 0.0605 day, respectively. A regression formula of half-lives as storage temperatures, ln t 1/2 = 4.7396/T × 103 - 12.536, was obtained. MnSO4 at 0.15 g l-1 enhanced the residual activity by 16% after 15 days and 17% after 30 days compared with control, but 2 g ascorbic acid l-1 reduced activity by 69 and 58% at the same time. In addition, 0.15 g MnSO4 l-1 and 20 g ethylene glycol l-1 maintained the secondary and tertiary structure of DPEase. CONCLUSIONS: MnSO4 and ethylene glycol actively promoted the storage and conformational stability of DPEase. In contrast, ascorbic acid was disadvantageous.

Rare sugars, d-allulose, d-tagatose and d-sorbose, differently modulate lipid metabolism in rats.

BACKGROUND: Rare sugars including d-allulose, d-tagatose, and d-sorbose are present in limited quantities in nature; some of these rare sugars are now commercially produced using microbial enzymes. Apart from the anti-obesity and anti-hyperglycaemic activities of d-allulose, effects of these sugars on lipid metabolism have not been investigated. Therefore, we aimed to determine if and how d-tagatose and d-sorbose modulate lipid metabolism in rats. After feeding these rare sugars to rats, parameters on lipid metabolism were determined. RESULTS: No diet-related effects were observed on body weight and food intake. Hepatic lipogenic enzyme activity was lowered by d-allulose and d-sorbose but increased by d-tagatose. Faecal fatty acid excretion was non-significantly decreased by d-allulose, but significantly increased by d-sorbose without affecting faecal steroid excretion. A trend toward reduced adipose tissue weight was observed in groups fed rare sugars. Serum adiponectin levels were decreased by d-sorbose relative to the control. Gene expression of cholesterol metabolism-related liver proteins tended to be down-regulated by d-allulose and d-sorbose but not by d-tagatose. In the small intestine, SR-B1 mRNA expression was suppressed by d-sorbose. CONCLUSION: Lipid metabolism in rats varies with rare sugars. Application of rare sugars to functional foods for healthy body weight maintenance requires further studies. © 2017 Society of Chemical Industry.

Cell regeneration and cyclic catalysis of engineered Kluyveromyces marxianus of a D-psicose-3-epimerase gene from Agrobacterium tumefaciens for D-allulose production.

D-Allulose as a low-energy and special bioactive monosaccharide sugar is essential for human health. In this study, the D-psicose-3-epimerase gene (DPEase) of Agrobacterium tumefaciens was transferred into thermotolerant Kluyveromyces marxianus to decrease the production cost of D-allulose and reduce the number of manufacturing procedures. The cell regeneration of K. marxianus and cyclic catalysis via whole-cell reaction were investigated to achieve the sustainable application of K. marxianus and the consumption of residual D-fructose. Results showed that DPEase, encoding a 33 kDa protein, could be effectively expressed in thermotolerant K. marxianus. The engineered K. marxianus produced 190 g L-1 D-allulose with 750 g L-1 D-fructose as a substrate at 55 °C within 12 h. Approximately 100 g of residual D-fructose was converted into 34 g of ethanol, and 15 g of the engineered K. marxianus cells was regenerated after fermentation at 37 °C for 21 h. The purity of D-allulose of more than 90% could be obtained without isolating it from D-allulose and D-fructose mixture through residual D-fructose consumption. This study provided a valuable pathway to regenerate engineered K. marxianus cells and achieve cyclic catalysis for D-allulose production.

Biochemical analysis and the preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides.

BACKGROUND: D-Tagatose 3-epimerase epimerizes D-fructose to yield D-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A D-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a D-tagatose 3-epimerase that can epimerize D-fructose to yield D-psicose with a high conversion rate. RESULTS: The purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris-HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, D-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of D-fructose which indicates RsDTE's preference of D-fructose more than any other family enzymes. CONCLUSIONS: RsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of D-fructose, regulates the substrate recognition. The research on D-tagatose 3-epimerase or D-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

Production of d-allulose from d-glucose by Escherichia coli transformant cells co-expressing d-glucose isomerase and d-psicose 3-epimerase genes.

BACKGROUND: d-Allulose is a novel and low-calorie rare monosaccharide that is a C-3 epimer of d-fructose. Because of its excellent physiological properties and commercial potential, d-allulose has attracted researchers' interests. Based on the Izumoring strategy, d-allulose is converted from d-fructose by d-psicose 3-epimerase (DPEase), while d-fructose is converted from d-glucose by d-glucose isomerase (GIase). In this study, we created a cellular system capable of converting d-glucose to d-allulose in a one-step process that co-expressed the GIase from Acidothermus cellulolyticus and the DPEase from Dorea sp. CAG. RESULTS: The co-expression plasmid pETDuet-Dosp-DPE/Acce-GI was generated and transformed into Escherichia coli BL21(DE3) cells. The recombinant co-expression cells exhibited maximum catalytic activity at pH 6.5 and 75 °C. These cells were thermostable at less than 60 °C. The addition of Co2+ significantly increased the catalytic activity by 10.8-fold. When the reaction equilibrium was reached, the ratio of d-glucose, d-fructose and d-allulose was approximately 6.5:7:3, respectively. CONCLUSION: A recombinant co-expression strain that catalysed the bioconversion of d-allulose from d-glucose in a one-step process was created and characterised. When adding 500 g L-1 d-glucose as a substrate, 204.3 g L-1 d-fructose and 89.1 g L-1 d-allulose were produced. © 2016 Society of Chemical Industry.

A novel strategy for protein production using non-classical secretion pathway in Bacillus subtilis.

BACKGROUND: The Gram-positive bacterium Bacillus subtilis has been widely used as a cell factory for the production of proteins due to its generally regarded as safe (GRAS) nature and secretion capability. Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by Sec pathway, Tat pathway and ABC transporters, etc. However, the production of heterologous proteins by B. subtilis is unfortunately not that straight forward because of the bottlenecks in classical secretion pathways. The aim of this work is to explore a new method for protein production based on non-classical secretion pathway. RESULTS: One D-psicose 3-epimerase (RDPE) which converts D-fructose into D-psicose from Ruminococcus sp. 5_1_39BFAA was successfully and substantially secreted into the extracellular milieu without the direction of signal peptide. Subsequently, we demonstrated that RDPE contained no native signal peptide, and the secretion of RDPE was not dependent on Sec or Tat pathway or due to cell lysis, which indicated that RDPE is a non-classically secreted protein. Then, we attempted to evaluate the possibility of using RDPE as a signal to export eighteen reporter proteins into the culture medium. Five of eleven homologous proteins, two of five heterologous proteins from other bacterium and two heterologous proteins of eukaryotic source were successfully secreted into the extracellular milieu at different secretion levels when they were fused to RDPE mediated by a flexible 21-bp linker to keep a distance between two single proteins. Furthermore, the secretion rates of two fusion proteins (RDPE-DnaK and RDPE-RFP) reached more than 50 %. In addition, most of the fusion proteins retained enzyme or biological activity of their corresponding target proteins, and all of the fusions still had the activity of RDPE. CONCLUSIONS: We found and identified a heterologous non-classically secreted protein RDPE, and showed that RDPE could direct proteins of various types into the culture medium, and thus non-classical protein secretion pathway can be used as a novel secretion pathway for recombinant proteins. This novel strategy for recombinant protein production is helpful to make B. subtilis as a more ideal cell factory for protein production.

A novel rare sugar inhibitor of murine herpes simplex keratitis.

PURPOSE: To determine the therapeutic efficacy of a novel rare sugar, l-psicose, for the treatment of HSV-1 induced herpetic stromal keratitis (HSK) in a mouse eye model. METHODS: One rare sugar l-psicose was assayed for HSV-1 inhibition of in vitro virus adsorption. The IC50 and IC90 values of l-psicose were determined using plaque reduction assay (PRA) in CV-1 cell. Female Balb/c mice were corneally infected with HSV-1, strain KOS-GFP; A topical eye drop treatment of l-psicose was started 24 h after infection and continued four times daily for ten consecutive days. The severity of HSK was monitored by slit lamp examination in a masked fashion and Infectious HSV-1 shedding was determined by PRA. RESULTS: l-psicose was found to have anti-viral activity in vitro at an IC50 dose of 99.5 mM and an IC90 dose of 160 mM. Topical eye drop treatment with 200 mM l-psicose in PBS solution significantly reduced the severity of HSK compared to the mock treatment group. The in vivo mouse ocular model results of l-psicose therapy correlated with accelerated clearance of virus from eye swabs. CONCLUSION: The results suggest that topical treatment with rare sugar l-psicose has efficacy against HSK through inhibition of HSV-1.