Part:BBa_K4349000

PCCHI44 endochitinase from Pochonia chlamydosporia

PCCHI44 is an endochitinase derived from the fungus Pochonia chlamydosporia.

Chitinases are enzymes that are able to cleave chitin, a linear polysaccharide of N-acetyl-D-glucosamine units present in insect exoskeletons, fungal cell walls, cuticles and eggshells of nematodes. Chitinases are found in a vast variety of organisms, including fungi, viruses, bacteria, insects, plant, and animals (Wang et al., 2007). These enzymes are classified as endochitinases, which cleave the polysaccharide randomly, and exochitinases, which cleave the ends (Seidl, 2008).

This part does not have a stop codon because we used it to assemble Composite Parts.

Usage and Biology

PCCHI44 hydrolyzes the β-1,4-linkages in chitin. In a characterization study, PCCHI44 was purified as a protein of 44 kDa and presented an optimal activity at pH 6.0 and 50 °C. However, it also exhibited activity under a broad range of temperatures from 30°C to 60°C and under a broad pH range from 4.0 to 8.0. The same study showed that PCCHI44 can act as a biocontrol because it degraded the eggs of the nematode Meloidogyne incognita and the eggs of the insect Bombyx more (Mi et al., 2010).

The eggs of M. incognita showed scars on the surface and peeling of eggshell after 24 h of enzyme treatment. After 3d large vacuoles were observed. The eggs of B. mori eggshell had low density after 36 h of enzyme treatment. After 3d, they deformed and large vacuoles were also observed (Mi et al., 2010).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 619
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Construction:

To express the endochitinase PCCHI44, UFMG_UFV_Brazil Team synthesized the DNA sequence using the IDT grant. We received our parts, added an adenine overhang to the 3' extremities and successfully inserted our construction in TOPO vector via TA cloning.