X Y Sheng

Fudan University, Shanghai, Shanghai Shi, China

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Publications (6)14.1 Total impact

  • C N Ji, T Jiang, M Q Chen, X Y Sheng, Y M Mao
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    ABSTRACT: Thermostable alkaline phosphatase from Thermus sp. 3041 has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1)22(1), with unit-cell parameters a = 57.7, b = 69.9, c = 111.5 A. Diffraction data were collected to 2.54 A with a completeness of 91.1% (87.8% for the last shell), an R(merge) value of 0.105 (0.312) and an I/sigma(I) value of 9.5 (3.6).
    Acta Crystallographica Section D Biological Crystallography 05/2001; 57(Pt 4):614-5. · 14.10 Impact Factor
  • T Jiang, C N Ji, X Y Sheng, Y M Mao
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    ABSTRACT: In order to investigate the effects of amino acid replacement on the characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis. The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E. coli TG1 and purified. The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed. The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60 degrees C) and the Kcat/Km value of Pro229Ser and Glu243Gly (4.89 +/- 0.01 x 10(6) mol-1 s-1 and 5.88 +/- 0.01 x 10(6) mol-1 s-1, respectively) were reduced compared with the wild type enzyme (6.97 +/- 0.01 x 10(6) mol-1 s-1). However, the thermostability of Pro229Ser extremely decreased 10.2 degrees C and the thermostability of Glu243Gly slightly increased 1.5 degrees C. It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.
    Acta Genetica Sinica 02/2001; 28(3):278-84.
  • T. Jiang, C. N. Ji, X. Y. Sheng, y. Xie, Y. M. Mao
    Protein and Peptide Letters - PROTEIN PEPTIDE LETT. 01/2001; 8(4):327-332.
  • C N Ji, B Zhang, T Jiang, X Y Sheng, Y M Mao
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    ABSTRACT: Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained. Their enzymatic characteristics was determined. It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20 degrees C and its Tm descended 3 degrees C; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5 degrees C and its Tm descended 2 degrees C; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5 degrees C and its Tm descended 19 degrees C. These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity. The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.
    Acta Genetica Sinica 02/2000; 27(12):1100-7.
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    ABSTRACT: To reveal the mechanism of protein thermostability, we used in vivo random mutagenesis to generate variants of pTAP503F which contained thermostable alkaline phosphatase (FD-TAP). After screening about 5,000 clones, we obtained 4 temperature-sensitive mutants. The study of enzymatic properties of one mutant (TAPM3) showed that the thermostability of the mutant enzyme descended a lot, compared to the wild type, while the thermoactivity remained stable. DNA sequencing showed that the G-A transition in position 1,239 resulted in the substitution from glysine to serine in position 427. This mutation conspicuously affected thermostability, Michaelis constant and energy of activation. This suggests that only one substitution of amino acid will make great changes in thermostability and other properties, meanwhile, side-chain size, charge of residues and so on, which loosen the structure of protein, will result in the descent of thermostability.
    Acta Genetica Sinica 02/2000; 27(4):361-8.
  • Y Z Yuan, X Y Sheng, H Y Lu, S Tong, Y M Mao
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    ABSTRACT: A genomic library of Thermus sp. FD3041 which produces thermostable alkaline phosphatase (FD-TAP) was constructed with the vector pUC118 and the host E. coli TG1. 3-10kb inserted fragments of foreign DNA were identified in 90 percent of the 12,000 clones thus obtained. Five positive clones were detected after screening the plated library by the method of colony coloration for TAP in situ. Preliminary analysis of the enzyme expressed from one recombinant plasmid pTAP362 showed that the properties of the recombinant enzyme, such as the thermal stability and optimal temperature of reaction, were identical to those of the native enzyme. The gene of FD-TAP was located on the 2.0kb BamHI-HindIII fragment of the pTAP362, determined by its physical map and the change of enzyme activity in different partially deleted plasmids. Results of thermostability experiment in PCR thermal cycle showed that the FD-TAP would be suitable for labelling of primers and detection of PCR amplified products.
    Acta Genetica Sinica 02/1998; 25(4):375-80.