Construction and characteristics of recombinant cellulose-bindingdomain-glutamate decarboxylase of Enterococcus faecium

  • YANG Shengyuan ,
  • LIN Qian ,
  • LAI Liping ,
  • HUANG Tingting
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  • 1 (School of Chemistry and Chemical Engineering, Lingnan Normal University, Zhanjiang 524048, China)
    2 (College of Biology and Pharmacy, Yulin Normal University, Yulin 537000, China)

Received date: 2019-02-25

  Online published: 2019-11-15

Abstract

In order to develop high-quality glutamate decarboxylase (GAD, EC4.1.1.15) for efficient immobilization, the construction of cellulose-binding domain (CBD) GAD fusion enzyme (CBD-GAD) and its enzymatic properties were investigated using Enterococcus faecium GDMCC60203 as gadB donor. The results showed that the recombinant Escherichia coli GDMCC60445 carrying pRPOCB-EfagadB could efficiently express CBD-GAD, and the activity of the cellulose immobilized CBD-GAD prepared from 120 mL fermentation broth was 347.93±27.63 U/g. When continuously transferred five batches in LB medium without ampicillin, the capability of CBD-GAD expression in the recombinant E. coli GDMCC60445 was still stable. The CBD-GAD purified by one-step purification showed a single band on SDS-PAGE electrophoresis with a molecular weight of 74.02 kDa. The purified CBD-GAD showed maximum activities at pH 4.6 and 60 ℃, respectively, and was stable in the range of pH 4.8 to 5.6 and temperature from 4 to 40 ℃. CBD-GAD reacted only with L-glutamic acid with apparent Km and Vmax of 10.58 mmol/L and 3.83 μmol/(mL·min), respectively, and the reaction activities were not inhibited by substrate and product. Hence, this strain has the potential to be applied in industrial production of gamma-aminobutyric acid and D-glutamic acid due to its high stability and excellent enzymatic properties.

Cite this article

YANG Shengyuan , LIN Qian , LAI Liping , HUANG Tingting . Construction and characteristics of recombinant cellulose-bindingdomain-glutamate decarboxylase of Enterococcus faecium[J]. Food and Fermentation Industries, 2019 , 45(21) : 22 -30 . DOI: 10.13995/j.cnki.11-1802/ts.020312

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