title: Cellulose binding domain assisted immobilization of lipase (GSlip-CBD) onto cellulosic nanogel: characterization and application in organic medium.
abstract: A cbd gene was cloned into the C-terminal region of a lip gene from Geobacillus stearothermophilus. The native lipase (43.5 kDa) and CBD-Lip fusion protein (60.2 kDa) were purified to homogeneity by SDS-PAGE. A highly stable cellulosic nanogel was prepared by controlled hydrolysis of microcrystalline cellulose onto which the CBD-lip fusion protein was immobilized through bio-affinity based binding. The nanogel-bound lipase showed optimum activity at 55 °C, and it remains stable and active at pH 10-10.5. Furthermore, the immobilized lipase showed an over two-fold increase of relative activity in the presence of DMSO, isopropanol, isoamyl alcohol and n-butanol, but a mild activity decrease at a low concentration of methanol and ethanol. The immobilized biocatalyst retained ~50% activity after eight repetitive hydrolytic cycles. Enzyme kinetic studies of the immobilized lipase showed a 1.24 fold increase in Vmax and 5.25 fold increase in kcat towards p-NPP hydrolysis. Additionally, the nanogel bound lipase was tested to synthesize a biodiesel ester, ethyl oleate in DMSO. Kinetic analysis showed the km 100.5 ± 4.3 mmol and Vmax 0.19 ± 0.015 mmolmin(-1) at varied oleic acid concentration. Also, the values of km and Vmax at varying concentration of ethanol were observed to be 95.9 ± 13.9 mmol and 0.22 ± 0.013 mmolmin(-1) respectively. The maximum yield of ethyl oleate 111.2 ± 1.24 mM was obtained under optimized reaction conditions in organic medium. These results suggest that this immobilized biocatalyst can be used as an efficient tool for the biotransformation reactions on an industrial scale.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Shaowei
authors.initials: S
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Cheng Chao
authors.initials: CC
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: JunPeng
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: R
authors.initials: R
authors.lastname: Baskaran
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: lzd@mail.hzau.edu.cn.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005782:Gels; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D049329:Nanostructures; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D005782:Gels; D002482:Cellulose; D008049:Lipase
keywords: Ethyl oleate; Immobilization; Lipase; Nanogel; Reusability