Overexpression and characterization in Bacillus subtilis of a positionally nonspecific lipase from Proteus vulgaris.
A Proteus vulgaris strain named T6 which produced lipase (PVL) with nonpositional specificity had been isolated in our laboratory. To produce the lipase in large quantities, we cloned its gene, which had an opening reading frame of 864 base pairs and encoded a deduced 287-amino-acid protein. The PVL gene was inserted into the Escherichia coli expression vector pET-DsbA, and active lipase was expressed in E. coli BL21 cells. The secretive expression of PVL gene in Bacillus subtilis was examined. Three vectors, i.e., pMM1525 (xylose-inducible), pMMP43 (constitutive vector, derivative of pMM1525), and pHPQ (sucrose-inducible, constructed based on pHB201), were used to produce lipase in B. subtilis. Recombinant B. subtilis WB800 cells harboring the pHPQ-PVL plasmid could synthesize and secrete the PVL protein in high yield. The lipase activity reached 356.8 U/mL after induction with sucrose for 72 h in shake-flask culture, representing a 12-fold increase over the native lipase activity in P. vulgaris. The characteristics of the heterologously expressed lipase were identical to those of the native one.
Lu
Yaping
Y
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
Lin
Qian
Q
Wang
Jin
J
Wu
Yufan
Y
Bao
Wuyundalai
W
Lv
Fengxia
F
Lu
Zhaoxin
Z
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D007218:Industrial Microbiology / Q000379:methods*; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D011514:Proteus vulgaris / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D008049:Lipase