Cloning and Expression of beta-1,3-Glucanase Gene from Cellulosimicrobium cellulans in Escherichia coli DH5a

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Ozcan B. D., Özcan N., Baylan M., Güzel A. I.

KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI, vol.19, no.3, pp.523-528, 2013 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 19 Issue: 3
  • Publication Date: 2013
  • Doi Number: 10.9775/kvfd.2012.8225
  • Journal Name: KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, TR DİZİN (ULAKBİM)
  • Page Numbers: pp.523-528
  • Keywords: beta-1,3-Glucanase, Cellulosimicrobium cellulans, Cloning, Escherichia coli, OERSKOVIA-XANTHINEOLYTICA, MOLECULAR-CLONING, RECOMBINANT PROTEINS, ALPHA-AMYLASE, SECRETION, PURIFICATION, ENZYME, SEQUENCE, PATHWAY, LYSIS
  • Çukurova University Affiliated: Yes

Abstract

In this study, beta-1,3-glucanase gene of Cellulosimicrobium cellulans was amplified by PCR and cloned in pUC18 cloning vector to construct the recombinant plasmids pTEG5 and pTEG11. The recombinant plasmids pTEG5 and pTEG11 were transformed into competent Escherichia coli cells. Digestion of recombinant plasmids with SacI produced 1.9 kbp beta-1,3-glucanase gene band on agarose gel which indicated the gene integration. a-1,3-Glucanase gene amplification on the recombinant vectors also indicated 1.9 kbp gene insert. Recombinant enzyme was produced by E. coli intracellularly. Intracellular components of recombinant E. coli strains with pTEG5 or pTEG11 dropped on LB-laminarin-agar plate, showed clear positive zones by Congo-red staining revealing the activity of secreted protein. Based on the zymogram analysis, the intracellular produced recombinant beta-1,3-glucanase enzymes exhibited the same activity bands with C. cellulans enzyme with respect to molecular weight.