title: Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production.
abstract: Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lip BA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645 bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250 ± 16 U/ml and 1750 ± 153 U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40 °C respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04 ± 0.06 mM and 119.05 ± 7.16 μmol/(ml min), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3 h at 30 °C, with 92 % enzyme recovery) and the immobilized enzyme was applied in biodiesel production. This is the first report of the lipase activity and lipase gene obtained from B. amyloliquefaciens (including wild strain and recombinant strain) and the recombinant LipBA with the detailed enzymatic properties. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Xianghai
authors.initials: X
authors.lastname: Cai
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.forename: Dong-Zhi
authors.initials: DZ
authors.lastname: Wei
authors.forename: Jin-Ping
authors.initials: JP
authors.lastname: Lin
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D056804:Biofuels; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012492:Salts; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D045730:Soy Foods / Q000382:microbiology*; D013696:Temperature
chemical_list: D056804:Biofuels; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004800:Enzymes, Immobilized; D012336:RNA, Ribosomal, 16S; D012492:Salts; C017082:brine; D008049:Lipase