Gene knockout of glutathione reductase results in increased sensitivity to heavy metals in Acidithiobacillus caldus.

Acidithiobacillus caldus plays an important role in bioleaching of low-grade metal ore. It can promote the release of heavy metals in mining-associated habitats and survive in high concentrations of heavy metals. Functions of glutathione reductase (GR) in cell defense against reactive oxygen species caused by heavy metals have been elucidated in some eukaryotic cells and bacteria; however, no information is available in A. caldus. In this research, the methods of bioinformatics, gene expression, GR activity assays were used to detect and characterize the glutathione reductase gene from the A. caldus MTH-04 strain. Then, A. caldus gr knockout mutant and gr overexpression strain were constructed, and the heavy metal tolerant properties and transcriptional levels of ROS related genes of them were compared to study the function of GR. The results showed that, a putative gr gene F0726_RS04210 was detected in the genome of A. caldus MTH-04. The purified recombinant protein of F0726_RS04210 showed remarkable GR activity at optimal pH 7.0 and 30°C using in vitro assay. The evolutionary relationship of GR from A. caldus MTH-04 was close to that from Escherichia coli K12. Gene knockout or overexpression of gr in A. caldus did not affect the growth rate on S0 medium, suggesting that GR did not play a key role in the activation of sulfur. Deletion of gr resulted in increased sensitivity to heavy metals (Cu2+ and Zn2+) in A. caldus, and the gr overexpression strain showed enhanced tolerance to heavy metals. Furthermore, transcription analysis also revealed strong correlations between GR and the antioxidant pathway. The above results suggest that GR can play an important role in heavy metal tolerance in A. caldus.

Application of logistic regression and machine learning methods for idiopathic inflammatory myopathies malignancy prediction.

OBJECTIVES: Malignancy is related to idiopathic inflammatory myopathies (IIM) and leads to a poor prognosis. Early prediction of malignancy is thought to improve the prognosis. However, predictive models have rarely been reported in IIM. Herein, we aimed to establish and use a machine learning (ML) algorithm to predict the possible risk factors for malignancy in IIM patients. METHODS: We retrospectively reviewed the medical records of 168 patients diagnosed with IIM in Shantou Central hospital, from 2013 to 2021. We randomly divided patients into two groups, the training sets (70%) for construction of the prediction model, and the validation sets (30%) for evaluation of model performance. We constructed six types of ML algorithms models and the AUC of ROC curves were used to describe the efficacy of the model. Finally, we set up a web version using the best prediction model to make it more generally available. RESULTS: According to the multi-variable regression analysis, three predictors were found to be the risk factors to establish the prediction model, including age, ALT<80U/L, and anti-TIF1-γ, and ILD was found to be a protective factor. Compared with five other ML algorithms models, the traditional algorithm logistic regression (LR) model was as good or better than the other models to predict malignancy in IIM. The AUC of the ROC using LR was 0.900 in the training set and 0.784 in the validation set. We selected the LR model as the final prediction model. Accordingly, a nomogram was constructed using the above four factors. A web version was built and can be visited on the website or acquired by scanning the QR code. CONCLUSIONS: The LR algorithm appears to be a good predictor of malignancy and may help clinicians screen, evaluate and follow up high-risk patients with IIM.

Predictive model of risk and severity of enteritis in systemic lupus erythematosus.

INTRODUCTION: To describe the clinical and laboratory features of systemic lupus erythematosus (SLE) enteritis and to establish a predictive model of risk and severity of lupus enteritis (LE). METHODS: Records of patients with SLE complaining about acute digestive symptoms were reviewed. The predictive nomogram for the diagnosis of LE was constructed by using R. The accuracy of the model was tested with correction curves. The receiver operating characteristic curve (ROC curve) program and a Decision curve analysis (DCA) were used for the verification of LE model. Receiver operating characteristic curve was also employed for evaluation of factors in the prediction of severity of LE. RESULTS: During the eight year period, 46 patients were in the LE group, while 32 were in the non-LE group. Abdominal pain, emesis, D-dimer >5 µg/mL, hypo-C3, and anti-SSA positive remained statistically significant and were included into the prediction model. Area under the curve (AUC) of ROC curve in this model was 0.909. Correction curve indicated consistency between the predicted rate and actual diagnostic rates. The DCA showed that the LE model was of benefit. Forty-four patients were included in developing the prediction model of LE severity. Infection, SLE disease activity index (SLEDAI), CT score, and new CT score were validated as risk factors for LE severity. The AUC of the combined SLEDAI, infection and new CT score were 0.870. CONCLUSION: The LE model exhibits good predictive ability to assess LE risk in SLE patients with acute digestive symptoms. The combination of SLEDAI, infection, and new CT score could improve the assessment of LE severity.

D-Allulose (D-Psicose) Biotransformation From Allitol by a Newly Found NAD(P)-Dependent Alcohol Dehydrogenase From Gluconobacter frateurii NBRC 3264 and the Enzyme Characterization.

The NAD(P)-dependent alcohol dehydrogenase (ADH) gene was cloned from Gluconobacter frateurii NBRC 3264 and expressed in Escherichia coli BL21 star (DE3). The expressed enzyme was purified and the characteristics were investigated. The results showed that this ADH can convert allitol into D-allulose (D-psicose), which is the first reported enzyme with this catalytic ability. The optimum temperature and pH of this enzyme were 50°C and pH 7.0, respectively, and the enzyme showed a maximal activity in the presence of Co2+. At 1 mM Co2+ and allitol concentrations of 50, 150, and 250 mM, the D-allulose yields of 97, 56, and 38%, respectively, were obtained after reaction for 4 h under optimal conditions, which were much higher than that obtained by using the epimerase method of about 30%.

Serum B-cell activating factor and lung ultrasound B-lines in connective tissue disease related interstitial lung disease.

Objective: To investigate the role of serum B-cell activating factor (BAFF) and lung ultrasound (LUS) B-lines in connective tissue disease related interstitial lung disease (CTD-ILD), and their association with different ILD patterns on high resolution computed tomography (HRCT) of chest. Methods: We measured the levels of BAFF and KL-6 by ELISA in the sera of 63 CTD-ILD patients [26 with fibrotic ILD (F-ILD), 37 with non-fibrotic ILD (NF-ILD)], 30 CTD patients without ILD, and 26 healthy controls. All patients underwent chest HRCT and LUS examination. Results: Serum BAFF levels were significantly higher in CTD patients compared to healthy subjects (617.6 ± 288.1 pg/ml vs. 269.0 ± 60.4 pg/ml, p < 0.01). BAFF concentrations were significantly different between ILD group and non-ILD group (698.3 ± 627.4 pg/ml vs. 448.3 ± 188.6 pg/ml, p < 0.01). In patients with ILD, BAFF concentrations were significantly correlated with B-lines number (r = 0.37, 95% CI 0.13-0.56, p < 0.01), KL-6 level (r = 0.26, 95% CI 0.01-0.48, p < 0.05), and Warrick score (r = 0.33, 95% CI 0.09-0.53, p < 0.01), although all correlations were only low to moderate. B-lines number correlated with Warrick score (r = 0.65, 95% CI 0.48-0.78, p < 0.01), and KL-6 levels (r = 0.43, 95% CI 0.21-0.61, p < 0.01). Patients with F-ILD had higher serum BAFF concentrations (957.5 ± 811.0 pg/ml vs. 516.1 ± 357.5 pg/ml, p < 0.05), KL-6 levels (750.7 ± 759.0 U/ml vs. 432.5 ± 277.5 U/ml, p < 0.05), B-lines numbers (174.1 ± 82 vs. 52.3 ± 57.5, p < 0.01), and Warrick score (19.9 ± 4.6 vs. 13.6 ± 3.4, p < 0.01) vs. NF-ILD patients. The best cut-off values to separate F-ILD from NF-ILD using ROC curves were 408 pg/ml for BAFF (AUC = 0.73, p < 0.01), 367 U/ml for KL-6 (AUC = 0.72, p < 0.05), 122 for B-lines number (AUC = 0.89, p < 0.01), and 14 for Warrick score (AUC = 0.87, p < 0.01) respectively. Conclusion: Serum BAFF levels and LUS B-lines number could be useful supportive biomarkers for detecting and evaluating the severity and/or subsets of CTD-ILD. If corroborated, combining imaging, serological, and sonographic biomarkers might be beneficial and comprehensive in management of CTD-ILD.

Rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren's syndrome shared megakaryocyte expansion in peripheral blood.

OBJECTIVES: Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) share many clinical manifestations and serological features. The aim of this study was to identify the common transcriptional profiling and composition of immune cells in peripheral blood in these autoimmune diseases (ADs). METHODS: We analysed bulk RNA-seq data for enrichment of biological processes, transcription factors (TFs) and deconvolution-based immune cell types from peripheral blood mononuclear cells (PBMCs) in 119 treatment-naive patients (41 RA, 38 pSS, 28 SLE and 12 polyautoimmunity) and 20 healthy controls. The single-cell RNA-seq (scRNA-seq) and flow cytometry had been performed to further define the immune cell subsets on PBMCs. RESULTS: Similar transcriptional profiles and common gene expression signatures associated with nucleosome assembly and haemostasis were identified across RA, SLE, pSS and polyautoimmunity. Distinct TF ensembles and gene regulatory network were mainly enriched in haematopoiesis. The upregulated cell-lineage-specific TFs PBX1, GATA1, TAL1 and GFI1B demonstrated a strong gene expression signature of megakaryocyte (MK) expansion. Gene expression-based cell type enrichment revealed elevated MK composition, specifically, CD41b+CD42b+ and CD41b+CD61+ MKs were expanded, further confirmed by flow cytometry in these ADs. In scRNA-seq data, MKs were defined by TFs PBX1/GATA1/TAL1 and pre-T-cell antigen receptor gene, PTCRA. Cellular heterogeneity and a distinct immune subpopulation with functional enrichment of antigen presentation were observed in MKs. CONCLUSIONS: The identification of MK expansion provided new insights into the peripheral immune cell atlas across RA, SLE, pSS and polyautoimmunity. Aberrant regulation of the MK expansion might contribute to the pathogenesis of these ADs.

The role of lung ultrasound B-lines and serum KL-6 in the screening and follow-up of rheumatoid arthritis patients for an identification of interstitial lung disease: review of the literature, proposal for a preliminary algorithm, and clinical application to cases.

Screening and follow-up of interstitial lung disease associated with rheumatoid arthritis (RA-ILD) is a challenge in clinical practice. In fact, the majority of RA-ILD patients are asymptomatic and optimal tools for early screening and regular follow-up are lacking. Furthermore, some patients may remain oligosymptomatic despite significant radiological abnormalities. In RA-ILD, usual interstitial pneumonia (UIP) is the most frequent radiological and pathological pattern, associated with a poor prognosis and a high risk to develop acute exacerbations and infections. If RA-ILD can be identified early, there may be an opportunity for an early treatment and close follow-up that might delay ILD progression and improve the long-term outcome.In connective tissue disease-associated interstitial lung disease (CTD-ILD), lung ultrasound (LUS) with the assessment of B-lines and serum Krebs von den Lungen-6 antigen (KL-6) has been recognized as sensitive biomarkers for the early detection of ILD. B-line number and serum KL-6 level were found to correlate with high-resolution computed tomography (HRCT), pulmonary function tests (PFTs), and other clinical parameters in systemic sclerosis-associated ILD (SSc-ILD). Recently, the significant correlation between B-lines and KL-6, two non-ionizing and non-invasive biomarkers, was demonstrated. Hence, the combined use of LUS and KL-6 to screen and follow up ILD in RA patients might be useful in clinical practice in addition to existing tools. Herein, we review relevant literature to support this concept, propose a preliminary screening algorithm, and present 2 cases where the algorithm was used.

[Research of progress of pyruvate dehydrogenase complex in sepsis metabolism].

Sepsis is a critical illness with high morbidity and mortality. Anaerobic glycolysis plays an important role in the pathogenesis of sepsis. Pyruvate dehydrogenase complex (PDHC) serves as a key regulator during sepsis. With PDHC dephosphorylation and deacetylation, PDHC activity is upregulated, allowing pyruvate translocate to mitochondria in aerobic condition, preceding the production of acetyl-CoA to accelerate aerobic oxidation. Activation of PDHC improves the prognosis of sepsis through regulating the balance of lactate, release of inflammatory factors and energy metabolism. A variety of remedies can improve the prognosis of patients with sepsis by up-regulating the activity of PDHC, including dichloroacetate (DCA), vitamin B1, milrinone, tumor necrosis factor binding protein, and ciprofloxacin.This article reviews the role and the regulatory mechanism of PDHC and signal pathway in the sepsis metabolism, in order to innovate treatment for sepsis and multiple organ dysfunction.

Heterotrophic nitrification and related functional gene expression characteristics of Alcaligenes faecalis SDU20 with the potential use in swine wastewater treatment.

A new heterotrophic nitrifying bacterium was isolated from the compost of swine manure and rice husk and identified as Alcaligenes faecalis SDU20. Strain SDU20 had heterotrophic nitrification potential and could remove 99.7% of the initial NH4+-N. Nitrogen balance analysis revealed that 15.9 and 12.3% of the NH4+-N were converted into biological nitrogen and nitrate nitrogen, respectively. The remaining 71.44% could be converted into N2 or N2O. Single-factor experiments showed that the optimal conditions for ammonium removal were the carbon source of sodium succinate, C/N ratio 10, initial pH 8.0, and temperature 30 °C. Nitrification genes were determined to be upregulated when sodium succinate was used as the carbon source analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Strain SDU20 could tolerate 4% salinity and show resistance to some heavy metal ions. Strain SDU20 removed 72.6% high concentrated NH4+-N of 2000 mg/L within 216 h. In a batch experiment, the highest NH4+-N removal efficiency of 98.7% and COD removal efficiency of 93.7% were obtained in the treatment of unsterilized swine wastewater. Strain SDU20 is promising in high-ammonium wastewater treatment.

Construction of recombinant Escherichia coli expressing xylitol-4-dehydrogenase and optimization for enhanced L-xylulose biotransformation from xylitol.

L-Xylulose is a rare ketopentose which inhibits α-glucosidase and is an indicator of hepatitis or liver cirrhosis. This pentose is also a precursor of other rare sugars such as L-xylose, L-ribose or L-lyxose. Recombinant E. coli expressing xylitol-4-dehydrogenase gene of Pantoea ananatis was constructed. A cost-effective culture media were used for L-xylulose production using the recombinant E. coli strain constructed. Response surface methodology was used to optimize these media components for L-xylulose production. A high conversion rate of 96.5% was achieved under an optimized pH and temperature using 20 g/L xylitol, which is the highest among the reports. The recombinant E. coli cells expressing the xdh gene were immobilized in calcium alginate to improve recycling of cells. Effective immobilization was achieved with 2% (w/v) sodium alginate and 3% (w/v) calcium chloride. The immobilized E. coli cells retained good stability and enzyme activity for 9 batches with conversion between 53 and 92% which would be beneficial for economical production of L-xylulose.

The substrate-dependent regulatory effects of the AfeI/R system in Acidithiobacillus ferrooxidans reveals the novel regulation strategy of quorum sensing in acidophiles.

A LuxI/R-like quorum sensing (QS) system (AfeI/R) has been reported in the acidophilic and chemoautotrophic Acidithiobacillus spp. However, the function of AfeI/R remains unclear because of the difficulties in the genetic manipulation of these bacteria. Here, we constructed different afeI mutants of the sulfur- and iron-oxidizer A. ferrooxidans, identified the N-acyl homoserine lactones (acyl-HSLs) synthesized by AfeI, and determined the regulatory effects of AfeI/R on genes expression, extracellular polymeric substance synthesis, energy metabolism, cell growth and population density of A. ferrooxidans in different energy substrates. Acyl-HSLs-mediated distinct regulation strategies were employed to influence bacterial metabolism and cell growth of A. ferrooxidans cultivated in either sulfur or ferrous iron. Based on these findings, an energy-substrate-dependent regulation mode of AfeI/R in A. ferrooxidans was illuminated that AfeI/R could produce different types of acyl-HSLs and employ specific acyl-HSLs to regulate specific genes in response to different energy substrates. The discovery of the AfeI/R-mediated substrate-dependent regulatory mode expands our knowledge on the function of QS system in the chemoautotrophic sulfur- and ferrous iron-oxidizing bacteria, and provides new insights in understanding energy metabolism modulation, population control, bacteria-driven bioleaching process, and the coevolution between the acidophiles and their acidic habitats.

Application of Firefly Luciferase (Luc) as a Reporter Gene for the Chemoautotrophic and Acidophilic Acidithiobacillus spp.

Acidithiobacillus spp. are the most active bacteria in bioleaching and bioremediation, because of their remarkable extreme environmental adaptabilities and unique metabolic characteristics. The researches on regulatory mechanisms of energy metabolism and stress resistance are critical for the understanding and application of Acidithiobacillus spp. However, the lack of an ideal reporter gene has become an obstacle for studying genes expression and regulatory mechanism in these chemoautotrophic bacteria. In this study, we reported the firefly luciferase as a reporter gene for Acidithiobacillus caldus (A. caldus) and created a firefly luciferase (Luc) reporter system. The Luc system was applied for the quantitative analysis of the transcription strength of the promoters of tetH gene and the feoA gene in A. caldus. Moreover, the regulating effect of ferric uptake regulator (Fur) on the feoP gene in A. caldus was determined using the Luc system. The Luc reporter system is not only used in the study of regulatory mechanism of A. caldus, but also applied in the researches of other Acidithiobacillus species. Therefore, this study provides a new useful tool for the studies on the molecular biological mechanism and synthetic biological modification of these chemoautotrophic bacteria, which would promote the industrial application of Acidithiobacillus spp.

Ammonium Removal by a Newly Isolated Heterotrophic Nitrification-Aerobic Denitrification Bacteria Pseudomonas Stutzeri SDU10 and Its Potential in Treatment of Piggery Wastewater.

A strain SDU10 was isolated from swine manure compost and identified as Pseudomonas stutzeri SDU10. It demonstrated excellent capability in NH4+-N removal. Optimal conditions of NH4+-N removal were determined, which were sodium acetate as the optimal carbon source, carbon to nitrogen (C/N) ratio of 10, temperature of 30 °C, pH of 7.0. Especially, P. stutzeri SDU10 could remove high concentration NH4+-N of 1500.0 and 2000.0 mg/l in 120 h with the NH4+-N removal rates of 91.1% and 61.6%, respectively. In batch experiments, the highest NH4+-N removal rate of 97.6% and chemical oxygen demand (COD) removal rate of 94.2% were obtained at initial C/N ratio 10 during piggery wastewater treatment using P. stutzeri SDU10. Results showed that P. stutzeri SDU10 had the potential for treatment of wastewater of high NH4+-N concentration.

Efficient Allitol Bioproduction from D-Fructose Catalyzed by Recombinant E. coli Whole Cells, and the Condition Optimization, Product Purification.

Allitol is a kind of rare sugar alcohol with potential application value. An engineered strain, which simultaneously expressed D-psicose-3-epimerase (DPE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) three enzymes, was constructed by cloning above three genes into one plasmid and transformed into the host E. coli strain, and used as the whole-cell catalysts for biotransformation of allitol from the low-cost substrate of D-fructose. The whole cell allitol biotransformation conditions were optimized. The medium, recombinant gene induction conditions, and the substrate feeding rate for cultivation of the catalytic cells were optimized. Then, the fed-batch culture was made and scaled up to 10 L fermentor. Finally, 63.44 g/L allitol was obtained from 100 g/L D-fructose after 3 h of biotransformation, and the allitol crystals of 99.9% purity were obtained by using cooling recrystallization. The allitol production method developed in this research has high product purity, and is highly efficient, easily scaled up, and suitable for large-scale production of highly purified allitol.

Optimization for allitol production from D-glucose by using immobilized glucose isomerase and recombinant E. coli expressing D-psicose-3-epimerase, ribitol dehydrogenase and formate dehydrogenase.

OBJECTIVE: To develop a method combining enzymatic catalysis and resting-cell biotransformation to produce allitol from low cost substrate D-glucose. RESULTS: The recombinant E. coli expressing D-psicose-3-epimerase (DPE), ribitol dehydrogenase (RDH) and formate dehydrogenase (FDH) for allitol production from D-fructose was constructed. The optimizations of the cell catalytic conditions and the cell cultivation conditions were made. Then, 63.4 g allitol L-1 was obtained from 100 g D-fructose L-1 in 4 h catalyzed by the recombinant E. coli cells. In order to decrease the substrate cost, D-glucose was used as the substrate instead of D-fructose and immobilized glucose isomerase was used to convert D-glucose into D-fructose. In order to simplify allitol production process from D-glucose, one-pot reaction using the mixed catalysts was used and the reaction conditions were optimized. Finally, 12.7 g allitol L-1 was obtained from 50 g D-glucose L-1 catalyzed by the mixed catalysts of immobilized glucose isomerase and the recombinant E. coli cells. CONCLUSIONS: Allitol can be efficiently produced from low cost substrate D-glucose by using the method combining enzymatic catalysis and resting-cell biotransformation, which is the first report.

Ferric Uptake Regulator Provides a New Strategy for Acidophile Adaptation to Acidic Ecosystems.

Acidophiles play a dominant role in driving elemental cycling in natural acid mine drainage (AMD) habitats and exhibit important application value in bioleaching and bioremediation. Acidity is an inevitable environmental stress and a key factor that affects the survival of acidophiles in their acidified natural habitats; however, the regulatory strategies applied by acidophilic bacteria to withstand low pH are unclear. We identified the significance of the ferric uptake regulator (Fur) in acidophiles adapting to acidic environments and discovered that Fur is ubiquitous as well as highly conserved in acidophilic bacteria. Mutagenesis of the fur gene of Acidithiobacillus caldus, a prototypical acidophilic sulfur-oxidizing bacterium found in AMD, revealed that Fur is required for the acid resistance of this acidophilic bacterium. Phenotypic characterization, transcriptome sequencing (RNA-seq), mutagenesis, and biochemical assays indicated that the Acidithiobacillus caldus ferric uptake regulator (AcFur) is involved in extreme acid resistance by regulating the expression of several key genes of certain cellular activities, such as iron transport, biofilm formation, sulfur metabolism, chemotaxis, and flagellar biosynthesis. Finally, a Fur-dependent acid resistance regulatory strategy in A. caldus was proposed to illustrate the ecological behavior of acidophilic bacteria under low pH. This study provides new insights into the adaptation strategies of acidophiles to AMD ecosystems and will promote the design and development of engineered biological systems for the environmental adaptation of acidophiles.IMPORTANCE This study advances our understanding of the acid tolerance mechanism of A. caldus, identifies the key fur gene responsible for acid resistance, and elucidates the correlation between fur and acid resistance, thus contributing to an understanding of the ecological behavior of acidophilic bacteria. These findings provide new insights into the acid resistance process in Acidithiobacillus species, thereby promoting the study of the environmental adaptation of acidophilic bacteria and the design of engineered biological systems.

Isolation and Identification of Chromium Reducing Bacillus Cereus Species from Chromium-Contaminated Soil for the Biological Detoxification of Chromium.

Chromium contamination has been an increasing threat to the environment and to human health. Cr(VI) and Cr(III) are the most common states of chromium. However, compared with Cr(III), Cr(VI) is more toxic and more easily absorbed, therefore, it is more harmful to human beings. Thus, the conversion of toxic Cr(VI) into Cr(III) is an accepted strategy for chromium detoxification. Here, we isolated two Bacillus cereus strains with a high chromium tolerance and reduction ability, named B. cereus D and 332, respectively. Both strains demonstrated a strong pH and temperature adaptability and survival under 8 mM Cr(VI). B. cereus D achieved 87.8% Cr(VI) removal in 24 h with an initial 2 mM Cr(VI). Cu(II) was found to increase the removal rate of Cr(VI) significantly. With the addition of 0.4 mM Cu(II), 99.9% of Cr(VI) in the culture was removed by B. cereus 332 in 24 h. This is the highest removal efficiency in the literature that we have seen to date. The immobilization experiments found that sodium alginate with diatomite was the better method for immobilization and B. cereus 332 was more efficient in immobilized cells. Our research provided valuable information and new, highly effective strains for the bioremediation of chromium pollution.

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.