1.Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase.
Yuxia WEI ; Xian ZHANG ; Mengkai HU ; Yu SHAO ; Shan PAN ; Morihisa FUJITA ; Zhiming RAO
Chinese Journal of Biotechnology 2021;37(12):4303-4313
D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.
Bacillus subtilis/metabolism*
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Carbohydrate Epimerases/genetics*
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Enzyme Stability
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Enzymes, Immobilized/metabolism*
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Fructose
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Hydrogen-Ion Concentration
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Racemases and Epimerases
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Temperature
2.Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis.
Lan WU ; Zhong QIAN ; Jun FU ; Shi-ying MIAO ; Lin-fang WANG
Acta Academiae Medicinae Sinicae 2009;31(6):696-701
OBJECTIVETo purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.
METHODSThe tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200).
RESULTThe purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained.
CONCLUSIONSWe successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.
Bacterial Proteins ; biosynthesis ; chemistry ; isolation & purification ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; isolation & purification ; Cloning, Molecular ; Crystallization ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Thermoanaerobacter ; enzymology ; genetics ; Transformation, Bacterial
3.Expression optimization and molecular modification of heparin C5 epimerase.
Bingbing WANG ; Zhengxiong ZHOU ; Xuerong JIN ; Jianghua LI ; Zhongping SHI ; Zhen KANG
Chinese Journal of Biotechnology 2020;36(7):1450-1458
Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Animals
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Carbohydrate Epimerases
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biosynthesis
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chemistry
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genetics
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Escherichia coli
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Gene Expression
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Heparin
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metabolism
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Heparitin Sulfate
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metabolism
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Iduronic Acid
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metabolism
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Zebrafish Proteins
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biosynthesis
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chemistry
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genetics
4.TSTA3 gene promotes esophageal cancer invasion through MAPK-ERK pathway and downstream MMP2/9.
En Wei XU ; Jie YANG ; Ling ZHANG
Chinese Journal of Pathology 2022;51(1):50-52
Carbohydrate Epimerases/metabolism*
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Cell Line, Tumor
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Cell Movement
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Cell Proliferation
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Esophageal Neoplasms/genetics*
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Gene Expression Regulation, Neoplastic
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Humans
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Ketone Oxidoreductases/metabolism*
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MAP Kinase Signaling System
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Matrix Metalloproteinase 2/genetics*
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Matrix Metalloproteinase 9
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Neoplasm Invasiveness/genetics*