Albin Zonta

Efficient immobilization of lipases by entrapment in hydrophobic sol‐gel materials

The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl‐substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi‐(OMe)3 with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol‐gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed.

Directed evolution of an enantioselective lipase

BACKGROUND The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity. RESULTS A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions. CONCLUSIONS A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening up a large area of novel applications in biotechnology.

Entrapment of lipases in hydrophobic magnetite-containing sol-gel materials: magnetic separation of heterogeneous biocatalysts

The simultaneous entrapment of a lipase such as Pseudomonas cepacia or Candida antarctica and nanostructured superparamagnetic magnetite (Fe 3 O 4 ) in hydrophobic sol-gel materials derived from CH 3 Si(OCH 3 ) 3 (MTMOS) or other hydrophobic precursors leads to catalytically active, mechanically stable and magnetically separable heterogeneous biocatalysts. The relative enzyme activity in the test reaction involving the esterification of lauric acid by n-octanol in isooctane is typically 200-300%, with respect to the same reaction using a conventional suspension of the non-immobilized enzyme. Separation of the catalyst by applying a simple magnet poses no problems. In the kinetic resolution of 2-pentylamine, enantioselectivity is essentially complete (ee = 97-99%).

Bacterial lipases for biotechnological applications

Abstract Lipase genes originating from the Gram-negative bacteria Serratia marcescens and Pseudomonas aeruginosa were cloned. S. marcescens lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and finally refolded to give enzymatically active lipase. The lipase operon of P. aeruginosa consisting of genes lipA and lipH was cloned behind the T7 φ10 promoter and overexpressed in a lipase-negative P. aeruginosa strain carrying a chromosomal insertion of the gene encoding T7 RNA polymerase. A 3D structural model was built for P. aeruginosa lipase using the coordinates of the Burkholderia cepacia lipase structure which has recently been solved in its open conformation by X-ray crystallography. Both lipases have been purified to homogeneity and were tested for their potential to catalyze biotechnologically important reactions. S. marcescens lipase stereoselectively hydrolyzed racemic isopropylideneglycerol acetate which is a basic building block in a variety of organic synthesis reactions. P. aeruginosa lipase was successfully used for kinetic resolution of chiral alcohols and amines giving enantiomeric excess values of ≥ 95% at reaction rates of 40–50%. Our results demonstrate that both lipases can be produced at levels of 100 mg/l for S. marcescens and 150 mg/l for P. aeruginosa . The recombinant lipase proteins are promising candidates for biotechnological applications.

Biotechnological application of Pseudomonas aeruginosa lipase: efficient kinetic resolution of amines and alcohols

Abstract Pseudomonas aeruginosa secretes an extracellular lipase (EC 3.1.1.3), which has been isolated from culture media of either industrial fermentation of wild-type P. aeruginosa PAC1R or an overexpressing P. aeruginosa strain carrying a plasmid with the cloned lipase gene. Both culture supernatants contained enzymatically active lipase protein, as demonstrated by determination of hydrolytic activity using p-nitrophenylpalmitate and 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester as substrates and analysis by sodium dodacyl sulphate/polyacrylamide electrophoresis and Western blotting. Immobilization by entrapment into chemically inert hydrophobic silica gels was tested with crude enzyme preparations. A matrix consisting of tetramethoxysilane and propyltrimethoxysilane at a molar ratio of 1 : 5 yielded the highest enzyme activity as determined by esterification of lauric acid with 1-octanol in isooctane. The biotechnological potential of P. aeruginosa lipase to catalyse the kinetic resolution of chiral compounds was tested by enantioselective acylation of two different model compounds, racemic 1-phenylethanol and 2-pentylamine. Both compounds were acylated with high efficiency giving enantiomeric excess rates of more than 99% for the alcohol and 96% for the amine with an average conversion rate of 50%. These results demonstrated that P. aeruginosa lipase is an extremely useful enzyme for application in synthetic organic chemistry.

Characterization of Hydrophobic Sol-Gel Materials Containing Entrapped Lipases

The entrapment of lipases in hydrophobic sol-gel materials of RSi(OCH3)3 or mixtures of RSi(OCH3)3 and Si(OCH3)4 results in heterogeneous biocatalysts having dramatically enhanced enzyme activities as measured by the esterification of lauric acid by n-octanol in isooctane. These materials have been characterized by solid state NMR studies, revealing the degree of cross-linking. It is shown that this parameter generally does not correlate with relative enzyme activity. Likewise, the specific surface area or the pore size does not seem to be the decisive factor in determining the relative enzyme activities of the lipase-containing hybrid gels and the corresponding SiO2-gels obtained from Si(OCH3)4. Scanning electron microscopic studies (SEM) show that the hybrid gels all have a similar morphology. On the basis of these studies a model is proposed according to which most of the lipase enzyme is entrapped near the surface of the gel particles, where it is readily accessible by substrate molecules.

In situ fixation of lipase-containing hydrophobic sol–gel materials on sintered glass—highly efficient heterogeneous biocatalysts

The previously reported process of entrapping lipases in hydrophobic organic/inorganic hybride silica gels is extended in that the entrapment by the sol–gel process is performed in the presence of porous glass beads, e.g.(SIRAN®), resulting in the in situ fixation of the sol–gel material on the surface of the support and formation of highly active and mechanically stable heterogeneous biocatalysts.