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Liuqun Zhao, Jingqi Chen, Jibin Sun, Dawei Zhang
Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis...
March 9, 2017: Scientific Reports
Jingqi Chen, Zhaoxia Jin, Yuanming Gai, Jibin Sun, Dawei Zhang
BACKGROUND: Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis. RESULTS: In this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host...
2017: Bioresources and Bioprocessing
Patrick Hossler, Christopher Racicot, Christopher Chumsae, Sean McDermott, Keith Cochran
Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized...
March 2017: Biotechnology Progress
Wenli Zhang, Hao Li, Bo Jiang, Tao Zhang, Wanmeng Mu
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...
December 23, 2016: Journal of the Science of Food and Agriculture
Jiangang Yang, Yueming Zhu, Yan Men, Shangshang Sun, Yan Zeng, Ying Zhang, Yuanxia Sun, Yanhe Ma
Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered...
December 21, 2016: Journal of Agricultural and Food Chemistry
Yoshitaka Ano, Roque A Hours, Yoshihiko Akakabe, Naoya Kataoka, Toshiharu Yakushi, Kazunobu Matsushita, Osao Adachi
A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca(2+). D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4...
February 2017: Bioscience, Biotechnology, and Biochemistry
Younho Song, Quynh Anh Nguyen, Seung Gon Wi, Jianming Yang, Hyeun-Jong Bae
In this study, fermentable sugars and d-fructose were produced from cruciferous vegetable residue by enzymatic method without the use of either chemical or mechanical mechanisms. Production of d-psicose was effectively converted from hydrolyzed d-fructose in cabbage residue by d-psicose-3 epimerase; the presence of the borate increased the conversion rate by about two fold, and ethanol production yield was 85.7% of the theoretical yield. Both products, bioethanol and d-psicose, were successfully separated and purified by pervaporation and cation exchange chromatography, and their recovery yields were approximately 87% and 86...
January 2017: Bioresource Technology
Celina Bermúdez, Isabel Peña, Santiago Mata, José L Alonso
The conformational behaviour of naturally occurring ketohexoses has been revealed in a supersonic expansion by Fourier transform microwave spectroscopy coupled with a laser ablation source. Three, two and one conformers of d-tagatose, d-psicose and l-sorbose, respectively, have been identified by their rotational constants extracted from the analysis of the spectra. Singular structural signatures involving the hydroxyl groups OH(1) and OH(2) have been disentangled from the intricate intramolecular hydrogen bond networks stabilising the most abundant conformers...
November 14, 2016: Chemistry: a European Journal
Weiwei He, Bo Jiang, Wanmeng Mu, Tao Zhang
The production of d-allulose is usually conducted via isolated-enzyme or whole-cell biocatalysis reactions. In the present study, an innovative biocatalyst, d-psicose 3-epimerase (DPEase) from Clostridium scindens ATCC 35704, presented on the surface of Bacillus subtilis spores, was applied for d-allulose production. DPEase was fused at the C-terminus of the anchoring protein, CotZ, via a peptide linker, and trophic genes were used as selection markers during the chromosomal integration. The optimal temperature and pH of the fusion protein CotZ-DPEase were 55 °C and pH 7...
September 28, 2016: Journal of Agricultural and Food Chemistry
Liuqing Wen, Kenneth Huang, Yuan Zheng, Junqiang Fang, Shukkoor Muhammed Kondengaden, Peng George Wang
Rare sugars offer a plethora of applications in the pharmaceutical, medicinal, and industries, as well as in synthetic chemistry. However, studies of rare sugars have been hampered by their relative scarcity. In this work, we describe a two-step strategy to efficiently and conveniently prepare 6-deoxy-L-psicose from L-rhamnose. In the first reaction step, the isomerization of L-rhamnose (6-deoxy-L-mannose) to L-rhamnulose (6-deoxy-L-fructose) catalyzed by L-rhamnose isomerase (RhaI), and the epimerization of L-rhamnulose to 6-deoxy-L-psicose catalyzed by D-tagatose 3-epimerase (DTE) were coupled with selective phosphorylation reaction by fructose kinase from human (HK), which selectively phosphorylate 6-deoxy-L-psicose at C-1 position...
August 24, 2016: Tetrahedron Letters
Jingqi Chen, Yueming Zhu, Gang Fu, Yafeng Song, Zhaoxia Jin, Yuanxia Sun, Dawei Zhang
D-Psicose 3-epimerase (DPEase) converts D-fructose into D-psicose which exists in nature in limited quantities and has key physiological functions. In this study, RDPE (DPEase from Ruminococcus sp. 5_1_39BFAA) was successfully constitutively expressed in Bacillus subtilis, which is the first report of its kind. Three sugar-inducible promoters were compared, and the xylose-inducible promoter P xylA was proved to be the most efficient for RDPE production. Based on the analysis of the inducer concentration and RDPE expression, we surmised that there was an extremely close correlation between the intracellular RDPE expression and xylose accumulation level...
November 2016: Journal of Industrial Microbiology & Biotechnology
Manisha Sharma, Satya Narayan Patel, Kusum Lata, Umesh Singh, Meena Krishania, Rajender S Sangwan, Sudhir P Singh
In this work, the sugar industry by-product cane molasses was investigated as feedstock for acceptor reactions by dextransucrase from Leuconostoc mesenteroides MTCC 10508, leading to the biosynthesis of oligosaccharides. The starch industry corn fiber residue was used as a source for acceptor molecules, maltose, in the reaction. Production of approximately 124g oligosaccharides (DP3-DP6) per kg of fresh molasses was achieved. Further, cane molasses based medium was demonstrated as a sole carbon source for L...
November 2016: Bioresource Technology
Dong-Ho Chang, Moon-Soo Rhee, Ji-Sun Kim, Yookyung Lee, Mi Young Park, Haseong Kim, Seung-Goo Lee, Byoung-Chan Kim
Two bacterial strains, 46-1 and 46-2(T), were isolated from garden soil. These strains were observed to be aerobic, Gram-stain negative, rod-shaped, non-spore-forming, motile and catalase and oxidase positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two strains shared 100 % sequence similarity with each other and belong to the genus Pseudomonas in the class Gammaproteobacteria. The concatenated 16S rRNA, gyrB, rpoB and rpoD gene sequences further confirmed that the isolates belong to the Pseudomonas koreensis subgroup (SG), with P...
November 2016: Antonie Van Leeuwenhoek
Hiromi Yoshida, Akihide Yoshihara, Tomohiko Ishii, Ken Izumori, Shigehiro Kamitori
Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined...
December 2016: Applied Microbiology and Biotechnology
Weiwei He, Wanmeng Mu, Bo Jiang, Xin Yan, Tao Zhang
An integrative food-grade expression system with tandem repeat target genes was constructed for the expression of d-psicose 3-epimerase (DPEase; EC The DPEase gene fused with the P43 promoter constituted an independent monomeric expression cassette. Multimers of the expression cassette were constructed in vitro using the isocaudamer strategy. The recombinant integration plasmids pDG-nDPE (n = 1, 2, 3), which contained one, two, or three consecutive P43-DPEase tandem repeats, were integrated into the genome of B...
July 20, 2016: Journal of Agricultural and Food Chemistry
Robert-Jan van Putten, Jan C van der Waal, Martin Harmse, Henk H van de Bovenkamp, Ed de Jong, Hero J Heeres
The acid-catalysed dehydration of the four 2-ketohexoses (fructose, sorbose, tagatose and psicose) to furanics was studied in methanol (65 g L(-1) substrate concentration, 17 and 34 mm sulfuric acid, 100 °C) with Avantium high-throughput technology. Significant differences in the reactivities of the hexoses and yields of 5-hydroxymethylfurfural (HMF) and its methyl ether (MMF) were observed. Psicose and tagatose were the most reactive, and psicose also afforded the highest combined yield of MMF and HMF of approximately 55 % at 96 % sugar conversion...
July 21, 2016: ChemSusChem
Syed Muniruzzaman, Megan McIntosh, Ahamed Hossain, Ken Izumori, Partha S Bhattacharjee
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...
June 2016: Journal of Pharmacological Sciences
Satya Narayan Patel, Manisha Sharma, Kusum Lata, Umesh Singh, Vinod Kumar, Rajender S Sangwan, Sudhir P Singh
The aim of the present work was to improve stability of d-psicose 3-epimerase and biotransformation of fruit and vegetable residues for d-psicose production. The study established that N-terminal fusion of a yeast homolog of SUMO protein - Smt3 - can confer elevated optimal temperature and improved operational stability to d-psicose 3-epimerase. The Smt3-d-psicose 3-epimerase conjugate system exhibited relatively better catalytic efficiency, and improved productivity in terms of space-time yields of about 8...
September 2016: Bioresource Technology
Kiran S Dhar, Volker F Wendisch, Kesavan Madhavan Nampoothiri
Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively...
July 20, 2016: Journal of Biotechnology
Jingqi Chen, Liuqun Zhao, Gang Fu, Wenjuan Zhou, Yuanxia Sun, Ping Zheng, Jibin Sun, Dawei Zhang
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...
April 28, 2016: Microbial Cell Factories
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