Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (17): 3587-3593.doi: 10.3864/j.issn.0578-1752.2013.17.007

• PLANT PROTECTION • Previous Articles     Next Articles

Prokaryotic Expression, Purification and Functional Activity Assay in vitro of Soluble Trehalse from Apolygus lucorum

 TAN  Yong-An, XIAO  Liu-Bin, SUN  Yang, BAI  Li-Xin   

  1. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2013-02-18 Online:2013-09-01 Published:2013-05-16

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Abstract: 【Objective】 The aims of this study are to obtain recombinant protein which has a trehalase enzyme activity and identify the best pH and the optimum temperature on the basis of cloned the soluble trehalase from Apolygus lucorum previous. This research expected to reveal the molecular mechanism of trehalase synthesis and offer the basic research of the development of trehalase inhibitor pesticide. 【Method】 The vector containing ALTre-1 gene was double enzyme restricted by Nde I and Not I, then the cDNA identified by sequencing was constructed to pET28a vector and transformed into BL21 of E. coli. The target recombinant protein was over expressed and been purified by using Ni-NTA agarose, and then its activity assay and enzymatic characteristics were studied. 【Result】 The ALTre-1 gene could over express in E. coli and the target recombinant protein which purified through Ni-NTA agarose had higher trehalase activity ((184.83±13.39)nmol•μg-1•min-1) by using trehalose as substrate, the suitable reaction temperature was 55℃ and the best pH was 7.0. 【Conclusion】 Recombinant protein with trehalase activity was obtained, and the suitable pH and temperature of the highest enzyme activity were 7.0 and 55℃, respectively.

Key words: Apolygus lucorum , soluble trehalase , prokaryotic expression , enzymatic characteristic

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