title: Isolation and characterization of a thermostable lipase from Bacillus thermoamylovorans NB501.
abstract: Two thermophilic bacterial strains, Bacillus thermoamylovorans NB501 and NB502, were isolated from a high-temperature aerobic fermentation reactor system that processes tofu refuse (okara) in the presence of used soybean oil. We cloned a lipase gene from strain NB501, which secretes a thermophilic lipase. The biochemical characteristics of the recombinant enzyme (Lip501r) were elucidated. Lip501r is monomeric in solution with an apparent molecular mass of 38 kDa on SDS-PAGE. The optimal pH and apparent optimal temperature of Lip501r were 8 and 60°C, respectively. Supplementation of 5 mM Ca2+enhanced the thermostability, and the enzyme retained 56% of its activity for 30 min at 50°C. Lip501r was active on a wide range of substrates with different lengths of p-nitrophenyl (pNP) esters, and showed a remarkably higher activity with pNP-myristate. The Kmand Vmaxvalues for pNP-butyrate in the presence of 5 mM CaCl2were 1.8 mM and 220 units/mg, respectively. The possible industrial use of the thermophilic lipase in modifying edible oil was explored by examining the degradation of soybean oil. A TLC analysis of the degraded products indicated that Lip501r is an 1,3-position specific lipase. A homology modeling study revealed that helix α6 in the lid domain of NB501 lipase was shorter than that of lipases from the Geobacillus group.
authors.affiliation: Department of Biotechnology, The University of Tokyo.
authors.forename: Chihaya
authors.initials: C
authors.lastname: Yamada
authors.forename: Kota
authors.initials: K
authors.lastname: Sawano
authors.forename: Noriyasu
authors.initials: N
authors.lastname: Iwase
authors.forename: Masaki
authors.initials: M
authors.lastname: Matsuoka
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Arakawa
authors.forename: Shigeo
authors.initials: S
authors.lastname: Nishida
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013024:Soybean Oil / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013024:Soybean Oil; D008049:Lipase; C428386:thermostable lipase