1.Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system.
Xin HUANG ; Yimin LI ; Cong DU ; Wenjie YUAN
Chinese Journal of Biotechnology 2022;38(12):4669-4680
Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.
Sphingobacterium/metabolism*
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Phosphotransferases (Phosphate Group Acceptor)/metabolism*
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Amino Acids
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Adenosine Triphosphate
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Regeneration
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Polyphosphates/metabolism*
2.Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production.
Sijia WU ; Jie LI ; Chenlong HU ; Junyu TIAN ; Tong ZHANG ; Ning CHEN ; Xiaoguang FAN
Chinese Journal of Biotechnology 2020;36(5):1002-1011
Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.
Cytidine Monophosphate
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metabolism
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Escherichia coli
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genetics
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Industrial Microbiology
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methods
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Phosphotransferases (Phosphate Group Acceptor)
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metabolism
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Uridine Kinase
3.Biosynthesis of poly (3-mercaptopropionate) and poly (3-mercaptopropionate-co-3-hydroxybutyrate) with recombinant Escherichia coli.
Shuang-Jiang LIU ; Tina LÜTKE-EVERSLOH ; Alexander STEINBÜCHEL
Chinese Journal of Biotechnology 2003;19(2):195-199
Polythioesters newly emerged as a type of novel polymer and they have showed great potential for application in industries. In this study, genes of butyrate kinase (buk) and phosphotransbutyrylase (ptb) from Clostridium acetobutylicum, and poly (3-hydroxybutyrate) (PHB) synthase gene from Thiocapsa pfennigii were used for construction of a metabolic pathway to synthesize the polythioesters. When 3-mercaptopropionate and 3-hydroxybutyrate were fed, poly (3-mercaptopropoinate) [poly (3MP)] and poly(3-mercaptopropionate-co-3-hydroxybutyrate) [poly(3MP-co-3HB)] were synthesized by recombinant Escherichia coli JM109 (pBPP1) harboring the constructed metabolic pathway. Results indicated clearly that all these genes are necessary for the synthesis of poly(3MP) and poly(3MP-co-3HB).
3-Hydroxybutyric Acid
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chemistry
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3-Mercaptopropionic Acid
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chemistry
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Acyltransferases
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genetics
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metabolism
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Escherichia coli
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genetics
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metabolism
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Gas Chromatography-Mass Spectrometry
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Models, Biological
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Molecular Weight
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Phosphate Acetyltransferase
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genetics
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metabolism
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Phosphotransferases (Carboxyl Group Acceptor)
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genetics
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metabolism
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Plasmids
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Polymers
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chemistry
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metabolism
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Spectrophotometry, Infrared
4.Low dietary inorganic phosphate affects the lung growth of developing mice.
Cheng Xiong XU ; Hua JIN ; Youn Sun CHUNG ; Ji Young SHIN ; Soon Kyung HWANG ; Jung Taek KWON ; Sung Jin PARK ; Eun Sun LEE ; Arash MINAI-TEHRANI ; Seung Hee CHANG ; Min Ah WOO ; Mi Suk NOH ; Gil Hwan AN ; Kee Ho LEE ; Myung Haing CHO
Journal of Veterinary Science 2009;10(2):105-113
Inorganic phosphate (Pi) plays a critical role in diverse cellular functions, and regulating the Pi balance is accomplished by sodium-dependent Pi co-transporter (NPT). Pulmonary NPT has recently been identified in mammalian lungs. However, to date, many of the studies that have involved Pi have mainly focused on its effect on bone and kidney. Therefore, current study was performed to discover the potential effects of low Pi on the lung of developing transgenic mice expressing the renilla/firefly luciferase dual reporter gene. Two-weeks old male mice divided into 2 groups and these groups were fed either a low PI diet or a normal control diet (normal: 0.5% Pi, low: 0.1% Pi) for 4 weeks. After 4 weeks of the diet, all the mice were sacrificed. Their lungs were harvested and analyzed by performing luciferase assay, Western blotting, kinase assay and immunohistochemistry. Our results demonstrate that low Pi affects the lungs of developing mice by disturbing protein translation, the cell cycle and the expression of fibroblast growth factor-2. These results suggest that optimally regulating Pi consumption may be important to maintain health.
Animals
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Blotting, Western
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Carrier Proteins/metabolism
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Immunohistochemistry
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Lung/drug effects/enzymology/*growth & development/metabolism
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Male
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Mice
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Mice, Transgenic
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Phosphoproteins/metabolism
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Phosphorus, Dietary/*administration & dosage
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Phosphorylation
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Phosphotransferases (Alcohol Group Acceptor)/metabolism
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Proto-Oncogene Proteins c-akt/metabolism
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Sodium-Phosphate Cotransporter Proteins, Type IIa/*metabolism