Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization.

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.

Hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media: implication for rate-limiting formation or breakdown of tetrahedral intermediates in acylation step.

A thermally stable esterase (SNSM-87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure-enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)-2-chloromandelates as the model substrates, an expanded Michaelis-Menten mechanism for the rate-limiting acylation step is adopted for the kinetic analysis. The Brønsted slope of 25.7 for the fast-reacting (S)-2-chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow-reacting (R)-2-chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is rate-limiting. However, the rate-limiting step shifts to the formation of tetrahedral intermediates for the (S)-2-chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate.

Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents.

In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.

Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: Implication for rate-limiting proton transfer in acylation step.

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators.

Recovery of Acinetobacter radioresistens lipase by hydrophobic adsorption to n-hexadecane coated on nonwoven fabric.

A simple and clean adsorption/desorption process was proposed for recovering Acinetobacter radioresistens lipase from fermentation broth. The adsorbent used was n-hexadecane coated on a hydrophobic nonwoven fabric (NWF). n-Hexadecane has a melting point of 16-18 degrees C, and its affinity for lipase decreases markedly from liquid to solid state. Accordingly, performing the adsorption and desorption above and below, respectively, the melting point would need no extraneous materials for separation. The adsorption isotherms at various temperatures were found to follow the Langmuir model. Simulation of the batch adsorption/desorption process showed that there exists an optimal amount of adsorbent for both concentration factor and enzyme recovery; the process is restrained by equilibrium. The performance of column adsorption/desorption could also be simulated using the adsorption isotherm, and it was shown that the concentration factor was proportional to the amount of adsorbent used. The benefits of this process include easy preparation of adsorbent, low operational cost, no extraneous materials needed, negligible enzyme denaturation, high efficiency, and simple process simulation.