We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags at the C-and N-termini,respectively.High-level expression of the lipase in E.coli BL21(DE3)was obtained upon induction with IPTG at 20°C.The recombinant lipase activity was approximately 1631-fold higher than the wild type.His-tagged recombinant lipase was purified rapidly and efficiently by using Ni-charged affinity chromatography with 63.5%recovery and a purification factor of 10.78.The purified lipase was stable in a broad range of temperatures and pH values,with the optimal temperature and pH being 50°C and 7.0,respectively.Its activity was stimulated to different degrees in the presence of metal ions such as Ca2+,Mg2+,and some non-ionic surfactants.In addition,the purified lipase was activated by a series of water-miscible organic solvents such as some short carbon chain alcohols and was highly tolerant to some water-immiscible organic solvents.
Recent progress in nanotechnology has provided high-performance nanomaterials for enzyme immobilization.Nanobiocatalysts combining enzymes and nanocarriers are drawing increasing attention because of their high catalytic performance,enhanced stabilities,improved enzyme-substrate affinities,and reusabilities.Many studies have been performed to investigate the efficient use of cellulose nanocrystals,polydopamine-based nanomaterials,and synthetic polymer nanogels for enzyme immobilization.Various nanobiocatalysts are highlighted in this review,with the emphasis on the design,preparation,properties,and potential applications of nanoscale enzyme carriers and nanobiocatalysts.
An extracellular chitinase produced by Paenibacillus pasadenensis CS0611was purified by ammoniumsulfate precipitation,HiTrap DEAE FF and HiLoad26/600Superdex200pg column chromatography.The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis was69kDa.The optimum pH and optimum temperature of the chitinase were5.0and50°C,respectively.The enzyme showed high stability at alkaline pH values and temperaturesbelow40°C.Additionally,the metal ions Mn2+,Mg2+,and Co2+inhibited activity of the chitinase.Thechitinase was active on colloidal chitin with an apparent Km of4.41mg/mL and Vmax of1.08mg/min.Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidicbond between GlcNAc‐GlcNAc.The enzymatic hydrolysate was analyzed by high‐performance liquidchromatography and thin layer chromatography,and clearly showed that a subunit of(GlcNAc)2was the main hydrolysis product.
A support made of mussel-inspired polydopamine-coated magnetic iron oxide nanoparticles （PD-MNPs） was prepared and characterized. The widely used Aspetyillus niger lipase （ANL） was immobilized on the PD-MNPs （ANL@PD-MNPs） with a protein loading of 138 mg/g and an activity recovery of 83.6% under optimized conditions. For the immobilization, the pH and immobilization time were investigated. The pH and thermal and storage stability of the ANL@PD-MNPs significant- ly surpassed those of free ANL. The ANL@PD-MNPs had better solvent tolerance than free ANL. The secondary structure of free ANL and ANL@PD-MNPs was analyzed by infrared spectroscopy, A kinetic study demonstrated that the ANL@PD-MNPs had enhanced enzyme-substrate affinity and high catalytic efficiency. The ANL@PD-MNPs was applied as a biocatalyst for the regioselective acylation of dihydromyricetin （DMY） in DMSO and gave a conversion of 79.3%, which was higher than that of previous reports. The ANL@PD-MNPs retained over 55% of its initial activity after 10 cycles of reuse. The ANL@PD-MNPs were readily separated from the reaction system by a magnet. The PD-MNPs is an excellent support for ANL and the resulting ANL@PD-MNPs displayed good potential for the efficient synthesis of dihydromyricetin-3-acetate by enzymatic regioselective acylation.
Preparation of biodiesel from waste oils containing 72% of free fatty acids catalyzed by a novel Br?nsted acidic ionic liquid 1-sulfobutyl-3-methylimidazolium hydrosulfate([BHSO_3MIM][HSO_4]) was systematically investigated.The optimum molar ratio of methanol to waste oils,catalyst amount,reaction temperature and reaction time were 8/1,10%(based on the mass of waste oils),140°C and 6 h,respectively,under which the obtained yield of biodiesel reached 94.9%.Also,[BHSO_3MIM][HSO_4] as a catalyst still retained around 97% of its original catalytic activity after successive re-use of 5 batches(6 h per batch),showing the excellent operational stability.Moreover,the acidic IL [BHSO_3MIM][HSO_4] was able to ef ficiently catalyze conversions of waste oils with different amounts of FFAs(free fatty acids) into biodiesel,and showed tremendous application potential.Therefore,an ef ficient and environmentally friendly catalyst is provided for the synthesis of biodiesel from waste oils with high acid value.
