Title: Rational redesign of Candida antarctica lipase B for the ring opening polymerization of D,D-lactide Takwa M, Larsen MW, Hult K, Martinelle M Ref: Chem Commun (Camb), 47:7392, 2011 : PubMed
Based on molecular modelling, the enzyme Candida antarctica lipase B was redesigned as a catalyst for the ring opening polymerization of D,D-lactide. Two mutants with 90-fold increased activity as compared to the wild-type enzyme were created. In a preparative synthesis of poly(D,D-lactide) the mutants greatly improved the rate and the degree of polymerization.
A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mM butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.
Two initiators containing a cleavable ester bond were compared in the lipase-catalyzed ROP of CL and PDL. The results show that transesterification reactions are present at high rates throughout the enzymatic ROP and start at low conversion. HEA and HEMA displayed similar reaction efficiencies as initiators (acyl acceptors) in the enzymatic ROP. However, transacylation reactions on the HEA-initiated polyesters were found to be 15 times faster. While in both cases the amount of HEA- and HEMA-initiated polymers could be maximized by short reaction times, a well-defined (meth)acrylation by this approach was not possible. Our results show that transesterification reactions have to be considered when performing an enzyme-catalyzed ROP.
2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.
A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M(r) xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.
Title: Polyester coating of cellulose fiber surfaces catalyzed by a cellulose-binding module-Candida antarctica lipase B fusion protein Gustavsson MT, Persson PV, Iversen T, Hult K, Martinelle M Ref: Biomacromolecules, 5:106, 2004 : PubMed
A new approach to introduce polymers to cellulosic materials was developed by using the ability of a cellulose-binding module-Candida antarctica lipase B conjugate to catalyze ring-opening polymerization of epsilon-caprolactone in close proximity to cellulose fiber surfaces. The epsilon-caprolactone was introduced to the cellulose surfaces either by simple addition of liquid monomer or through gas phase. The effects of water activity and temperature on the lipase-catalyzed polymerization process were investigated. Analysis showed that the water content in the system primarily regulated the obtained polymer molecular weight, whereas the temperature influenced the reaction rate. The hydrophobicity of the obtained surfaces did not arise from covalent attachment of the poly(epsilon-caprolactone) to the surface hydroxyl groups but rather from surface-deposited polymers which could be readily extracted. The degree of lipase-catalyzed hydrolysis through introduction of water to the polymer-coated cellulose fiber surfaces was also investigated and shown to be significant.
A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.
The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent. The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin. Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data. It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate. The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate. Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used. In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase. A reduction of both the acylation and deacylation reaction was consistent with the data.
Title: On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase Martinelle M, Holmquist M, Hult K Ref: Biochimica & Biophysica Acta, 1258:272, 1995 : PubMed
The interfacial activation of Candida antarctica lipase A (CALA) and B (CALB) has been investigated and compared with that of Humicola lanuginosa lipase (HLL). CALB displayed no interfacial activation towards p-nitrophenyl butyrate (PNPB) when exceeding the solubility limit of the substrate. No activation was observed towards p-nitrophenyl acetate (PNPA) at the addition of sodium dodecyl sulfate (SDS) nor in the presence of a solid polystyrene surface. The catalytic action of CALB was very different from that of Humicola lanuginosa lipase, which showed a pronounced interfacial activation with the same substrates. The basis for the anomalous behaviour of CALB is proposed to be due to the absence of a lid that regulates the access to the active site. In contrast to CALB, CALA expressed interfacial activation, but the activation was not as prominent as for Humicola lanuginosa lipase (HLL). The structural basis for the activation of CALA is unknown.
To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) from Humicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax = 6000 +/- 300 mmol.min-1.g-1 and an apparent Km = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: ES = 8.5; H. lanuginosa: ES = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (ER = 2.9). Chemical arginine specific modification of the R. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (ER = 2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (ES = 2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (ES = 1.9) and increased the enantioselectivity with the aromatic ester (ER = 4.4) as substrates. The mutation, Glu 87 Ala, in the lid of the H. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (ES = 17.4) and a decreased enantioselectivity with the phenyl ester (ER = 2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.
