Part:BBa_K805011

Cel5A, endoglucanases

The endoglucanase genes cel5A is cloned from Penicillium decumbens 114-2. The expression levels of cel5A is repressed by glucose and induced by cellulose.It can hydrolyze β-1,4glucosan randomly and act on long cellulose chains. The main product is oligomeric glucose. It can digest glucosan to produce glucose monomer in high efficiency with synergy with β-1,4glucosidase.It shows high ability to hydrolyze the crystal cellulose but relative lower enzyme activity towards carboxymethyl cellulose, barley β-glucan, and PASC.

Usage and Biology

The expression levels of cel5A is repressed by glucose and induced by cellulose.It can hydrolyze β-1,4glucosan randomly and act on long cellulose chains. The main product is oligomeric glucose. It can digest glucosan to produce glucose monomer in high efficiency with synergy with β-1,4glucosidase.It shows high ability to hydrolyze the crystal cellulose but relative lower enzyme activity towards carboxymethyl cellulose, barley β-glucan, and PASC.

We construct the gene on the carrier displaying on the surface of Pichia pastoris and express it it in Pichia pastoris with electroporation. Then we detect the expression on the surface of Pichia pastoris by observing with fluorescent microscope and by flow cytometry analysis.

We fused FLAG-tag with N-terminal of cel5A gene on cel5A-pPIC9k vector. Label cells with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody. Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPIC9k empty vector is 1.14, with cel5A-pPIC9k is 5.55.

Left: pPIC9k negative control (x-mean: 1.14/13.4); Right: cel5A (x-mean: 5.55/16.5)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 874
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 307
  Illegal NgoMIV site found at 763
  Illegal AgeI site found at 1027
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1024