Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems.
Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.
Yamabhai
Montarop
M
School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima 30000, Thailand. montarop@sut.ac.th
Emrat
Suphap
S
Sukasem
Sirima
S
Pesatcha
Puntarika
P
Jaruseranee
Nanthnit
N
Buranabanyat
Bancha
B
D001407:Bacillus / Q000502:physiology*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000502:physiology*; D006868:Hydrolysis; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D000516:alpha-Amylases; D002688:Chitinases; D044902:beta-Mannosidase