2-Keto-d-gluconic acid (2KGA) is an important organic acid derived from d-glucose and is used to produce the food antioxidant erythorbic acid. To improve the 2KGA production performance and cell reusability, various carriers such as calcium alginate, k-carrageenan, chitosan, and poly(vinyl alcohol)-alginate were evaluated to immobilize Pseudomonas plecoglossicida JUIM01 resting cells. Calcium alginate was shown to be a suitable carrier since the immobilized cells had the highest number of reuse times and produced the highest 2KGA concentration of 171.77 g/L, with a productivity of 3.58 g/L·h and conversion ratio of 98.38%. The cell concentration, cultivation temperature, aeration rate and initial glucose concentration were further optimized in a 5-L airlift bioreactor to obtain the best 2KGA production performance by calcium alginate-immobilized P. plecoglossicida cells. Under the optimal conditions including a cell concentration of 4.0 g/L, glucose concentration of 126.0 g/L, temperature of 34 °C and aeration rate of 2.8 L/min, 134.45 g/L 2KGA was produced by alginate-immobilized P. plecoglossicida cells within 30 h, with a total productivity of 4.48 g/L·h and yield of 1.07 g/g (conversion ratio of over 99.0%). The immobilized cells maintained a stable conversion capacity after nine reuses and 25 days of storage at 4 °C, which indicated that calcium alginate immobilization of P. plecoglossicida cells had industrial practicability for 2KGA production.

The full-length sequence of 2-ketegluconate kinase gene (kguK) from was cloned from an industrial 2KGA producer of Pseudomonas plecoglossicida JUIM01 and expressed in the recombinant strain Escherichia coli BL21(DE3)/ pET-28a(+)-kguK with isopropyl β-D-1-thiogalactopyranoside induction.The specific fusion protein KguK had a molecular weight of about 36.0 ku,which was confirmed by Western-Blot.The bioinformatics analysis showed that KguK in P.plecoglossicida was a hydrophilic protein with 305 amino acid residues.It was located in the cytoplasm,having a conserved domain similar to that of the pfkB family.The predicted secondary structure contained 35.73％ of α-helixes,12.79％ of extended strand and 51.48％ of random coil.

A non-sterile and buffer-free 2-keto-gluconic acid (2KGA) production method was proposed by using resting cells from an industrial strain of Pseudomonas fluorescens AR4. 2KGA achieved a maximum production performance of 195.00 g/L, which corresponded to 3.05 g/L h of total productivity and 1.07 g/g of yield under optimal conditions, including resting cell aged 20 h, a cell concentration of 3.0 g/L, a reaction temperature of 30 degrees C, a glucose concentration of 181.82 g/L, and a reaction volume of 20 mL during the 64-h bioconversion process. P. fluorescens AR4 resting cells showed stable bioconversion activity after reuse 3 times or storage for less than 28 d at 4 degrees C. These findings suggested that the proposed non-sterile and buffer-free bioconversion of glucose to 2KGA using P. fluorescens AR4 resting cells had remarkable advantages, including ease handling and low cost, and they are a potent application for industrial production of 2KGA. (C) 2015 Elsevier Ltd. All rights reserved.

Glucose dehydrogenase (GlcDH) is the rate-limiting catalyst for microbial conversion of glucose to the important organic acid 2-ketogluconic acid (2KGlcA). In this study, a D-glucose dehydrogenase was purified from the industrial 2KGlcA producer Arthrobacter globiformis C224. After four purification steps, the GlcDH was successfully purified over 180 folds and specific activity of 88.1 U/mg. A single protein band of 87 kDa was detected by SDS-PAGE. The purified GlcDH had the broad substrate specificity with the Km values for D-glucose, D-xylose, D-galactose and maltose of 0.21 mM, 0.34 mM, 0.46 mM and 0.59 mM, respectively. The kinetic studies proved that A. globiformis GlcDH followed the ping-pong kinetic mechanism. The GlcDH showed an optimum catalytic activity at pH 5.0 and 45 °C with the stable activity at temperature of 20–40 °C and pH of 6.0–7.0. Organic solvents, metal ions or EDTA could significantly influence the GlcDH activity to different degrees.

Arthrobacter globiformis C224 is an industrial 2-keto-gluconic acid (2KGA) producer and currently used for erythorbic acid production in China. In the present study, a membrane-bound gluconate dehydrogenase (GADH) with specific activity of 326.06U/mg and purification fold of 412 was purified from A. globiformis using a five-step procedure including ultrasonication, phase separation with Triton X-114, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The GADH was identified as to be flavin adenine dinucleotide (FAD)-dependent and consisted of three subunits with molecular mass of 66,000Da, 44,000Da and 23,000Da. The optimal pH and temperature of A. globiformis GADH were 5.0 and 40°C, respectively. It had the stable activity at pH of 5.0–7.0 or below 50°C, and the strict substrate specificity for d-gluconate with the Km of 3.15mmolL−1, 1.04mmolL−1 at pH 5.0 and pH 6.0, respectively. The GADH activity also was significantly influenced by metal ions, organic solvents, and organic acids. Our study will benefit for better understanding of 2KGA production process by A. globiformis C224.

6-D-Isoascorbyl trans-β-n-alkoxycarbonyl( C1-C8 n-alkoxycarbonyl) acrylates were synthesized by the reaction of D-isoascorbic acid with trans-β-n-alkoxycarbonyl acryloyl chloride which from the chlorization of trans-β-n-alkoxycarbonyl acrylic acid with thionyl chloride. The structures of target compounds were confirmed by IR,1HNMR,13CNMR and EI-MS. The scavenging activity of trans-β-n-hexyl acrylate oxycarbonyl-6-D-iso-ascorbate on DPPH free-radical was tested by UV-Vis spectrophotometry,and the anti-microbial activities of target compounds were tested by peptide-agarose diffusion assay. The results showed that the same molar concentration of D-isoascorbic acid and trans-β-n-hexyl acrylate oxycarbonyl-6-D-iso-ascorbate exhibited almost the same scavenging DPPH free radical activity. The diameters of antibacterial active ring of target compounds against Staphyloccocus aureus, Escherichia coli and Bacillus subtilis were between 5. 00 ~ 7. 25 mm,and those of target compounds against Streptomyces microflavus were between 15. 00 ~ 17. 50 mm,when the concentration of tested samples were in 2 mg / mL.

HEG-5, a novel glycoprotein with hemagglutinating activity, was firstly isolated and purified from the cultured mycelia of Hericium erinaceus CZ-2. SDS-PAGE, Native-PAGE and MALDI-TOF-MS proved that HEG-5 was a single band with the molecular weight of approximately 14.4 kDa. HEG-5 had the protein: polysaccharide ratio of approximately 10:1 (%/%) and contained ID-glucose, L-rhamnose, D-galactose and D-mannose with a molar ratio of 1.00:1.09:2.45:7.14 in polysaccharide fraction. HEG-5 was an acidic glycoprotein with a PI value of 6.3 and the higher content of acidic amino acids (Asp, 12.42 +/- 0.25% and Glu, 12.24 +/- 0.26%) in protein fraction. FT-IR and NMR spectra revealed that HEG-5 contained the protein and carbohydrate portions with (1 -> 4)-linked beta-galactose residues and beta-linked glucose residues. Circular dichroism (CD) demonstrated that HEG-5 was a beta-sheet predominant glycoprotein. Hemagglutination assay proved it was a thermo-unstable glycoprotein. The HEG-5 structural novelty was finally presented by protein sequencing and modeling by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODEL Workspace service. (C) 2014 Elsevier Ltd. All rights reserved.

A novel glycoprotein GFG-3a with the molecular weight of 88.01kDa and potent anti-tumor activity was isolated from the cultured mycelia of Grifola frondosa GF9801. GFG-3a was heat-sensitive with the decreasing anti-proliferative activity after treated from 56°C to 100°C for 10-120min. GFG-3a was a glycoprotein with O-glycosylation and contained 6.20% carbohydrate composed of d-arabinose, d-fructose, d-mannose, and d-glucose with a molar ratio of 1.33:4.51:2.46:1.00. FT-IR and NMR spectra proved that GFG-3a contained protein and carbohydrate portions with 3-O-methyl-galactose residues, (1→4)-linked β-galactose residues, and β-linked glucose residues. Circular dichroism (CD) revealed that GFG-3a was a predominantly β-sheet glycoprotein with a relatively small α-helical content. Protein sequencing and 3D model of GFG-3a were finally predicted by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODLE Workspace service. Our findings will be a reference for the further structure-activity relationship analysis of the mushroom glycoproteins.

L-ascorbyl cinnamate 1 and D-isoascorbyl cinnamate 2 were synthesized by the reaction of cinnamoyl chloride with L-ascorbic acid and D-isoascorbic acid,respectively.The structures of 1 and 2 were confirmed by IR、1H NMR、13C NMR and EIMS.The scavenging activity of 1 and 2 on DPPH free-radical,hydroxyl free-radical and superoxide anion free-radical was tested by UV-Vis spectrophotometry,and the anti-microbial activity of 1 and 2 against Staphyloccocus aureus,Escherichia coli,Bacillus subtilis and Streptomyces microflavus was tested by peptide-agarose diffusion assay.It was found that 1 and 2 had higher scavenging activity than that of the corresponding L-ascorbic acid and D-isoascorbic acid,respectively,when the molar concentration of tested samples was the same.It was also found that both 1 and 2 had the anti-microbial activity similar to that of cinnamic acid,potassium sorbate and sodium benzoate when the concentration of tested samples was 2 mg·mL-1.

Kinetic models are proposed for the 2KGA batch production from rice starch hydrolysate containing 162 g/L of glucose by Pseudomonas fluorescens AR4. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from the batch fermentations were used to estimate parameters and also to validate the models proposed. The growth of Ps. fluorescens could be expressed by a Logistic model wihout incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the 2KGA formation as the fermentation proceeded with a mixed-growth-associated pattern. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of Ps. fluorescens AR4 during efficient 2KGA production from glucose.

D-isoascorbyl mixed-fatty acid esters were synthesized with Lipozyme 435 as catalyst by the transesterification of D-isoascorbic acid with camellia oil,lard,sesame oil and canola oil,respectively.The effect of factors such as reaction solvent,reaction time,the ratio of reactants,the amount of lipase,and the recycled oils and fats on the reaction conversion were explored with the synthesis of D-isoascorbyl camellia oil fatty acid esters as reaction model to optimize the reaction conditions.Under the optimized conditions,the reaction conversion could reach 48 %.The antioxidation activities of D-isoascorbyl camellia oil fatty acid esters,D-isoascorbyl lard fatty acid esters,D-isoascorbyl sesame oil fatty acid ester and D-isoascorbyl rapeseed oil fatty acid ester in camellia oil was tested by POV determination,respectively.The results showed that all the synthesized D-isoascorbyl mixed-fatty acid esters were excellent fat-soluble antioxidants and that their antioxidation activities increased with the applied concentration.

D-isoascorbic acid is a food antioxidant additive and used in accordance with Good Manufacturing Practice (GMP). High solubility in water (about 150 g/L at 25°C) reduces its effectiveness in stabilizing fats and oils. Our research group had successfully synthesized D-isoascorbyl palmitate using immobilized lipase Novozym 435 as a biocatalyst. Low production efficiency of D-isoascorbyl palmitate is still a problem for industrial production due to the long reaction time of over 24 h. In the present work, ultrasonic treatment was applied for accelerating the reaction process. The operation parameters were optimized to obtain the maximum D-isoascorbyl palmitate conversion rate by using a 5-level-4-factor Central Composite Design (CCD) and Response Surface Methdology (RSM). The reaction apparent kinetic parameters under the ultrasound treatment and mechanical shaking conditions were also determined and compared.

The lipase-catalyzed synthesis of D-isoascorbyl laurate via direct esterification of D-isoascorbic acid and lauric acid was studied.The factors affecting synthetic reaction,such as reaction temperature,the type of organic solvent,the molar ratio of reactants,the amount of lipase,reaction time,the concentration of lauric acid,the amount of molecular sieves and the shaker speed,were investigated.It was found that when the reaction of 0.88 g D-isoascorbic acid and 2.50 g lauric acid was catalyzed by 0.10 g lipozyme 435 in 20 mL tert-amyl alcohol with 2.0 g 4A molecular sieves added and shaker speed of 170 r/min for 36h at 55℃,the yield of D-isoascorbyl laurate could reach 68%.The product structure was confirmed by IR,1H NMR,13C NMR and MS.

Vitamin esters are the esterification derivatives produced by a condensation reaction between alcohol-OH orphenol hydroxylof vitamin with carboxylic acid compounds. After esterification modification,the stability of synthesized vitaminsester derivatives in various organic systems will be significantly improved and the new functions from carboxylic acid compounds will also be was discovered,therefore,to extend its vitamins' application was extended. These structure-modified vitamin esters derivatives have been commercialized and widely used as food additives,pharmaceti-cal and healthy products. For example,vitamin C esters are the new safe food additives which have antitumor,ant iagingand anti-oxidationfunctions. Vitamin E esters can substitute vitamin E in medicine,food and cosmetics, and vitamin A esters are the important medicinesand feed additives. This paper reviewed the progress of vitamins ester synthesis methods of( iso) vitamin C ester,vitamin A and vitamin E ester,analyzed the shortcomings of the synthesis method,and finally predicted the development prospects of these vitamin esters.

Isoascorbic acid is a stereoisomer of L-ascorbic acid, and widely used as a food antioxidant. However, its highly hydrophilic behavior prevents its application in cosmetics or fats and oils-based foods. To overcome this problem, D-isoascorbyl palmitate was synthesized in the present study for improving the isoascorbic acid’s oil solubility with an immobilized lipase in organic media. The structural information of synthesized product was clarified using LC-ESI-MS, FT-IR, 1H and 13C NMR analysis, and process parameters for high yield of D-isoascorbyl palmitate were optimized by using One–factor-at-a-time experiments and response surface methodology (RSM).

The lipase-catalyzed synthesis of L-ascorbyl fatty acid esters and D-isoascorbyl fatty acid esters were studied by direct esterification and transesterification,respectively.The structures of products were confirmed by IR,1H NMR,13C NMR and MS.It was found that the yield of the direct esterification were higher than that of the corresponding transesterification under the similar synthetic conditions.The raw material fatty acids and fatty acid methyl esters could be recycled and reused.

2-Keto-D-gluconic acid(2-KGA) in fermentation broth was directly adsorbed on the anion exchange resin D318,and was consequently readsorbed by concentrated sulfuric acid-methanol mixture.The readsorbed elution was directly esterified by heat to give methyl 2-Keto-D-gluconate.The above procedures could simplify the process of sodium D-isoascorbate production.The effect of factors such as the concentration of 2-KGA,the flow rate of feed,the ratio of height to dimmer of resin bed on the adsorption capacity of resin was explored.Furthermore,the relevant experimental results were discussed based on the principle of ion exchange.

D-isoascorbyl camellia oil fatty acid esters was added in lard and peroxide value(POV)in lard sample was tested according to GB/T5009.37-2003.The scavenging activity(SA)of D-isoascorbyl camellia oil fatty acid esters on DPPH free-radical was tested by spectrophotometry.The POV data showed that the antioxidation activity of D-isoascorbyl camellia oil fatty acid esters was better than that of L-ascorbyl palmitate,and was almost the same as that of D-isoascorbyl palmitate and TBHQ at the highest permitted amount by China food additives regulation.The SA data showed that when the concentration of samples was higher than 1.666710-4mol·L-1,the activity of scavenging DPPH· of D-isoascorbyl camellia oil fatty acid esters was higher than that of D-isoascorbyl palmitate,L-ascorbyl palmitate and TBHQ.

The 2-keto-D-gluconic acid(2-KGA) in fermentation broth was determined by polarimetry.Some factors affecting the determination of 2-KGA were investigated in detail,such as the concentration of the test solution,the temperature of the test solution and the pH of the test solution.It was found that the test solution content in 2.5~5.00 g 2-KGA per 100 mL,pH in the acidic range,more accurate measurement results could be achieved.The relevant phenomena were discussed according to the relevant principles of organic chemistry.

Bacteriophages have the destructive damage on the industrial bioprocess. 2-Keto-gluconic acid (2KGA) producing bacteria had also been attacked and lysed by bacteriophages which lowered the glucose consumption and 2KGA yield and even stopped the fermentation process. In this study, we presented the characteristics of a novel virulent bacteriophage specifically infecting Pseudomonas fluorescens K1005 and proposed an efficient remedial action for this phage infection to reduce the production loss.