Lipase B from C. antarctica

Anthonsen, T. and Hoff, B. (1998) Resolution of derivatives of 1,2-propanediol with lipase B from C antarctica. Effect of substrate structure, medium, water activity and acyl donor on enantiomeric ratio. Chem. Phys. Lipids, 93, 199-207. 

Enzymatic DKRs have also been applied in domino one-pot processes [97]. The combination of a lipase-catalyzed resolution with an intramolecular Diels-Alder reaction led to interesting building blocks for the synthesis of natural products such as compactin [98,99] or forskolin [100-102], A ruthenium catalyst is employed for the racemization of the slow reacting enantiomer of the starting material. The DKR with lipase B from C. antarctica delivered high enantiomeric excesses which could mainly be contained through the Diels-Alder reaction (Fig. 12). 

In the present work, green coconut fiber was successfiilly used to immobilize lipase B from C. antarctica by adsorption. During adsorption studies, it was observed that adsorption equilibrium was achieved afler a contact time of 2 h (in case of o=30 U/ml and Eo=(0 U/ml) or 6 h ( o=90 U/ml). Moreover, an improvement of hydrolytic activity of immobilized CALB is also observed with increasing concentrations of lipase offered to immobilization. This increase in activity is due to formation of multilayers, confirmed by thermal stability essays. Two plateaus of enzyme activity were observed when the pH of lipase solution during adsorption was varied in the range studied. This behavior is typical of an ionic support At 50 and 60 °C, the adsorbed enzyme was, respectively, 2- and 92-fold more stable than the soluble enzyme. At 60 °C, however, Novozyme 435 s stability was higher than that of CALB-7A. TUIer 10 h of incubation at 60 °C, Novozyme 435 retained more than 70% of its initial activity, whereas CALB-7A retained only 50%. Last but not least operational stabilities studies of butyl butyrate synthesis, compared to a commercial derivative, showed that C7U..B-7A is a suitable biocatalyst to be used in the synthesis of flavors. 

The Novozym 435 (Lipase B from C. antarctica, EC 3.1.1.3, a nonspecific lipase immobilized on macroporous acrylic resin, 1-2% water content, 10,000 propyl laurate... 

Arroyo M, Sanchez-Montero JM, Sinistetra JV (1999) Thermal satabiUzation of immobilized lipase B from C. antarctica on different supports effect of water activity on enzymatic activity in organic media. Enzyme Microb Technol 24 3—12 Balcao VM, Paiva AL, Malcata FX (1996) Bioreactors with immobilized Upases state of the art. Enzyme Microb Technol 18 392-398... 

In addition to homopolymers a number of copolymers of cyclic carbonates were synthesized by enzymatic ROP. Among them are amphiphilic block copolymers of PBTMC and PEG which were synthesized through enzymatic polymerization using immobilized porcine pancreas lipase (IPPL). The copolymerization of co-pentadecalactone (PDL) and TMC was also studied by using lipase catalysts. Of the six lipases evaluated for PDL/TMC copolymerizations in toluene at 70 ° C, an immobilized form of lipase-B from C. antarctica (Novozym-435) was preferred. Changing the PDL/TMC comonomers feed ratio from 1 10 to 10 1 (moLmol) provided copolymers that ranged... 

The most commonly used hydrolase for biocatalysis is lipase B from C. antarctica (CAL-B). The commercial material is usually Novozym 435, which is protein immobilized noncovalently on an acrylic resin. This immobilization is suitable for use in organic solvents, but in water, the lipase desorbs from the support. CAL-B is a recombinant protein from the yeast C. antarctica produced in a strain of the fungus Aspergillus oryzae [5]. CAL-B shows little or no interfacial activation and hydrolyzes long chain triglycerides only slowly. For this reason, it may be better classified as an esterase It shows high activity and enantioselertivity toward a... 

DESYMMETRIZATION OF MESO ClS-3,5-DIACETOXYCYCLOPENTENE TO (1R,4S)-(+)-4-HYDR0XY-2-CYCL0PENTENYL ACETATE CATALYZED BY LIPASE B FROM C. ANTARCTICA... 

Enantioselective hydrolysis catalyzed by lipase B from C. antarctica desymmetrized a meso diacetate to the chiral monoacetate. 

S)-2-Pentanol (Figure 11.15) is an intermediate in the synthesis of several potential anti-alzheimer s drugs which inhibit (3-amyloid peptide release and/or its synthesis [108,109]. The enzymatic resolution of racemic 2-pentanol and 2-heptanol by lipase B from C. antarctica has been demonstrated [110]. 

Classic resolntion has been performed by formation of diastereomeiic salts which could be separated. In a series of synthetic steps and when resolution is one step, it is of utmost importance that the correct chirality is introduced at an early stage. When a racemate is subject to enzyme catalysis, one enantiomer reacts faster than the other and this leads to kinetic resolution (Figure 2.2c). Results of hydrolysis using lipase B from Candida antarctica (CALB) and a range of C-3 secondary butanoates are shown in Table 2.1. 

Hansen, T.V., Waagen, V., Partali, V., Anthonsen, H.W. and Anthonsen, T. (1995) Cosolvent enhancement of enantioselectivity in lipase-catalysed hydrolysis of racemic esters. A process for production of homochiral C-3 building blocks using lipase B from Candida antarctica. Tetrahedron Asymmetry, 6, 499-504. 

Studies of the ability of the lipase B from Candida antarctica (CAL-B) to catalyse the enantioselective aminolysis of esters by cis- and firms-2-phenylcycloalkanamines (54 n = 1, 3, 4) have been followed up by molecular modelling approaches in order to probe the lipase-catalysed aminolysis mechanism. CAL-B possesses a typical serine-dependent triad, so it was possible, with access to an X-ray crystal structure of CAL-B, to model a series of phosphonamidates (55 n = 1, 3, 4) as analogues of the tetrahedral intermediate (TI) resulting from attack of the amine on the carbonyl of the acyl-enzyme. The results suggested as the most plausible intermediate for the CAL-B-catalysed aminolysis a zwitterionic TI resulting from the direct His-assisted attack of the amine on to a C=0 group of the acyl-enzyme.80... 

A more direct method for the enantioselective ring opening of unactivated bicyclic /3-lactams has been reported. When lipolase (modified lipase B from Candida antarctica adsorbed on a macroporous resin) was allowed to interact with racemates 126 (n 1 or 2) and 127 (n= 1 or 2) in aqueous diisopropyl ether at 70 °C for 4.5-7 h, the products were the enantiomerically pure ring-opened unsaturated alicyclic /3-amino acids 128 (n = 1 or 2) and 129 (n 1 or 2), and the bicyclic /3-lactams 130 ( = 1 or 2) and 131 (n = 1 or 2) in 45 48% yield with ee 95-99% (Equations 13 and 14) <2004TA2875>. 

The remaining activity of Lipase A was approximately 50% whereas the activity of Lipase B was almost inactivated. It is therefore assumed that Lipase A is responsible for the high thermostability observed in crude lipase preparations from C. antarctica. 

A similar resolution has also been achieved on large scale <20040PD22>. The KR of racemic isoxazoline 312 catalyzed by enzymes was studied. The best result was obtained with lipase B from Candida antarctica (CALB), which hydrolyzed the ethyl ester of (—)-312 to the corresponding monoacid (—)-313. The reaction, which was run in 0.1 M phosphate buffer/acetone at room temperature, spontaneously stopped at 50% conversion to yield monoacid (—)-313 and the residual ester (- -)-312 with ees higher than 99% <2004TA3079>. The C-5 epimer of 312 underwent enantioselective hydrolysis (>99% ee) of the methyl ester linked to C-5 in the presence of the protease proleather (subtilisin Carlsberg), whereas CALB and other lipases were not able to resolve it (Equation 53). 

An ethyl carbonate, prepared and cleaved by conditions similar to those described for a methyl carbonate, was used to protect a hydroxyl group in glucose. Ethyl chlo-roformate in pyridine or CH2CI2/TEA is the most common method of preparation for this carbonate. The carbonate may be prepared by exchange with diethyl carbonate in the presence of a MgLa mixed oxide catalyst. The carbonates of 2-hydroxycar-boxylic acids may also be prepared by the reaction of 2-ethoxy-l-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ). These carbonates can also be cleaved enzymatically with Lipase B from Candida antarctica (phosphate buffer, pH 7, 30-60°C)." ... 

Waste oil was collected from the restaurant in South China University of Technology. The saponification value was 200.3 mg KOH/g, from which, the average molecular weight of the waste oil was known to be 840.2. Novozym 435 (lipase B from Candida antarctica, 164 U/g, limit corresponds to the amount of enzyme that produces 1 pmol methyl oleate from triolein per minute at 35 °C) was kindly donated by Novozymes Co. (Denmark). Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate and methyl heptadecanoate (as an internal standard) were purchased from Sigma (USA). All other chemicals were also obtained commercially and of analytical grade. Ultrasonic irradiation experiments were carried out using an ultrasonic bath (Type NP-B-400-15 Newpower Co. Ltd., China). 

Fig. 5 Thermal stability of derivatives of lipase B from Candida antarctica obtained by adsorption, incubated in 0.1 M sodium phosphate buffer, pH 7, at 50 or 60 °C...

Recently Gross et. al. reported that polymerization of e-CL catalyzed by Novozyme-43S (immobilized lipase B from Candida Antarctica) was improved by reaction in toluene because of higher log P (76). In situ NMR measurement in toluene-dg was employed to study die effect of reaction temperature on propagation kinetics and average molecular weight. The polymerization conducted at 90 °C gave the most rapid polymerization rate. 

This interfacial activation does not appear in all hydrolytic enzymes. For example, cutinase from fusarium solani, which hydrolyzes cutin, usually has the fatty acids -Ci6 and -C,8 and does not have a lid and does not exhibit these characteristics (Carvalho et al 1998 Schrag et al 1997). In addition, the presence of a lid does not always cause the enzyme to be active at the interface. The exception is lipase B from Candida antarctica that does not show interfacial activation despite having a lid (Kuo and Gardner, 2002 Uppenberg et al., 1994). Additionally, Staphylococcus hyicus and P. aeruginosa show an interfacial activation with some substrates but not with others (Verger, 1997). 

Another demonstration of a continuous flow operation is the psi-shaped microreactor that was used for lipase-catalyzed synthesis of isoamyl acetate in the 1-butyl-3-methylpyridinium dicyanamide/n-heptane two-phase system [144]. The chosen solvent system with dissolved Candida antarctica lipase B, which was attached to the ionic liquid/n-heptane interfacial area because of its amphiphilic properties, was shown to be highly efficient and enabled simultaneous esterification and product removal. The system allowed for simultaneous esterification and product recovery showed a threefold reaction rate increase when compared to the conventional batch. This was mainly a consequence of efficient reaction-diffusion dynamics in the microchannel system, where the developed flow pattern comprising intense emulsification provided a large interfacial area for the reaction and simultaneous product extraction. Another lipase-catalyzed isoamyl acetate synthesis in a continuously operated pressure-driven microreactor was reported by the same authors [145]. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. Controlling flow rates of both phases reestablished a parallel laminar flow with liquid-liquid boundary in the middle of the microchannel and a separation of phases was achieved at the y-shaped exit of the microreactor (Figure 10.25). The microreactor approach demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations and has proven more effective and outperformed the batch operation, which could be attributed to the favorable mass and heat transfer characteristics. 

The thermostability of the lipase B from Candia antarctica was also improved by direrted evolution. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70°C compared with the wild-type lipase B [66]. 

Experiments were typically performed at 70°C in toluene (125 mL) in a glass reactor at atmospheric pressure under H2 flow (AGA 99.999, 295 mL/min). The initial reactant concentration was 0.02 mol/L. Ethyl acetate with the concentration of 0.06 mol/L was used as an acyl donor. The catalytic hydrogenation of acetophenone (Acros, 99%) was carried out over 2% (w/w) Pd/N-CNT (312.5 mg) and the formed / -l-phenylethanol was acylated in the same pot to / -l-phenylethyl acetate with an immobilized lipase (Novozym 435, lipase B from Candida antarctica) (62.5 mg). The supported Pd catalysts were pre-reduced at 200 °C prior to the experiment. 

---