https://www.massey.ac.nz/%7Eychisti/Lipases.pdf

Production, purification, characterization, and applications of lipases.

Lipases for biotechnology.

Lipases as practical biocatalysts

Lipase promiscuity and its biochemical applications

Lipase was covalently bound onto Fe(3)O(4) magnetic nanoparticles (12.7 nm) via carbodiimide activation. The Fe(3)O(4) magnetic nanoparticles were prepared by coprecipitating Fe(2+) and Fe(3+) ions in an ammonia solution and treating under hydrothermal conditions. The analyses of transmission electron microscopy (TEM) and X-ray diffraction (XRD) showed that the size and structure of magnetic nanoparticles had no significant changes after enzyme binding. Magnetic measurement revealed the resultant lipase-bound magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu/g (only slightly lower than that of the naked ones (64 emu/g)), a remanent magnetization of 1.0 emu/g, and a coercivity of 7.5 Oe. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of lipase onto magnetic nanoparticles. The binding efficiency of lipase was 100% when the weight ratio of lipase bound to Fe(3)O(4) nanoparticles was below 0.033. Compared to the free enzyme, the bound lipase exhibited a 1.41-fold enhanced activity, a 31-fold improved stability, and better tolerance to the variation of solution pH. For the hydrolysis of pNPP by bound lipase at pH 8, the activation energy within 20-35 degrees C was 6.4 kJ/mol, and the maximum specific activity and Michaelis constant at 25 degrees C were 1.07 micromol/min mg and 0.4 mM, respectively. It revealed that the available active sites of lipase and their affinity to substrate increased after being bound onto magnetic nanoparticles.

Direct binding and characterization of lipase onto magnetic nanoparticles.

Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of </= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.

A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase

The rise of human populations and the growth of cities contribute to the depletion of natural resources, increase their cost, and create potential climatic changes. To overcome difficulties in supplying populations and reducing the resource cost, a search for alternative pharmaceutical, nanotechnology, and energy sources has begun. Among the alternative sources, microalgae are the most promising because they use carbon dioxide (CO2) to produce biomass and/or valuable compounds. Once produced, the biomass is ordinarily harvested and processed (downstream program). Drying, grinding, and extraction steps are destructive to the microalgal biomass that then needs to be renewed. The extraction and purification processes generate organic wastes and require substantial energy inputs. Altogether, it is urgent to develop alternative downstream processes. Among the possibilities, milking invokes the concept that the extraction should not kill the algal cells. Therefore, it does not require growing the algae anew. In this review, we discuss research on milking of diatoms. The main themes are (a) development of alternative methods to extract and harvest high added value compounds; (b) design of photobioreactors; (c) biodiversity and (d) stress physiology, illustrated with original results dealing with oleaginous diatoms.

https://www.mdpi.com/1660-3397/13/5/2629/pdf

Diatom milking: a review and new approaches.

The purified lipase from Pseudomonas cepacia (PS, Amano) was immobilized on a commercially available microporous polypropylene support. The enzyme was rapidly and completely adsorbed on the support. Special attention was devoted to the demonstration of the lack of diffusional limitations, either internal or external, when a soluble substrate (p-nitrophenylacetate, pNPA) was used. The activity yield was high (100%) with pNPA and very low (0.4%) with p-nitrophenylpalmitate (pNPP). These values clearly showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one. With the latter one, the low activity was due mainly to a slow rate of substrate diffusion inside the porous support. The same diffusional phenomenon could explain the complete change of fatty acid specificity of the immobilized lipase. After immobilization, the lipase was mainly specific for short chain fatty acid esters, whereas the free enzyme was mainly specific for long chain esters. The activity-versus-temperature profiles were not greatly affected by immobilization with maximal reaction rates in the range 45 degrees to 50 degrees C for both enzyme preparations. However, immobilization increased enzyme stability mainly by decreasing the sensitivity to temperature of the inactivation reaction. Half-lives at 80 degrees C were 11 and 4 min for the immobilized and free enzymes, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 181-189, 1997.

Gaëlle Pencreac'h

Papers

Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase

Diatom milking: a review and new approaches.

Properties of free and immobilized lipase from Pseudomonas cepacia.

Comparison of hydrolytic activity in water and heptane for thirty-two commercial lipase preparations.