Effect of the chitin binding domain deletion from Bacillus thuringiensis subsp. kurstaki chitinase Chi255 on its stability in Escherichia coli.
Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Delta5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Delta5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50 degrees C and 40 degrees C, respectively for Chi255 and Chi255Delta5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.
Driss
F
F
Laboratory of Biopesticides, Centre of Biotechnology of Sfax, Sfax, Tunisia.
Baanannou
A
A
Rouis
S
S
Masmoudi
I
I
Zouari
N
N
Jaoua
S
S
D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006596:Hexosaminidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion
D011994:Recombinant Proteins; D002686:Chitin; C079639:1,4-beta-chitobiosidase; D006596:Hexosaminidases; D002688:Chitinases