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.

Cloning, Expression, and Characterization of a Novel Thermostable and Alkaline-stable Esterase from Stenotrophomonas maltophilia OUC_Est10 Catalytically Active in Organic Solvents

“…After incubation in 100% n-hexane, Est7 retained 83.8% of the activity in the absence of organic solvent, which indicated that Est7 had good stability in both hydrophilic and hydrophobic organic solvents. In particular, compared with other reports, Est7 showed higher stability than rEstSL3 [30] and LipBA45 [31] in organic solvents. These features are good prerequisites for the application of this enzyme in biocatalytic reactions.…”

The Impact of Microbial Diversity on Biogenic Amines Formation in Grasshopper Sub Shrimp Paste During the Fermentation

“…produce various fungicidal agents, amylases, hyaluronic acid, and plasmin, which not only play an important role in fermentation, but also confer a health effect on humans (Zhao et al, 2009;He et al, 2013). Several studies have suggested that the growth of Bacillus cereus is inhibited by the presence of LAB because of the acidic environment created as a result of the increased growth of LAB (Cai et al, 2014). Bacilli did not constitute a major microbiota in grasshopper sub shrimp paste, but their effect on the flavor and nutrition of grasshopper sub shrimp paste and the interaction between Bacilli and LAB require further study (Gu et al, 2018).…”

Sugary Kefir: Microbial Identification and Biotechnological Properties

“…Due to the rigid regulation of lipase synthesis, the cloning of lipase genes is often preferred. The first documentation of lipase gene from B. amyloliquefaciens cloning and application in biodiesel production was reported by Cai et al [61]. B. amyloliquefaciens Nsic-8 lipase gene, lipBA, was isolated from a stinky tofu brine and successfully cloned in E. coli.…”