Production of Cross-Linked Lipase Crystals at a Preparative Scale

Abstract

The autoimmobilization of enzymes via cross-linked enzyme crystals (CLECs) has regained interest in recent years, boosted by the extensive knowledge gained in protein crystallization, the decrease of cost and laboriousness of the process, and the development of potential applications. In this work, we present the crystallization and preparative-scale production of reinforced cross-linked lipase crystals (RCLLCs) using a commercial detergent additive as a raw material. Bulk crystallization was carried out in 500 mL of agarose media using the batch technique. Agarose facilitates the homogeneous production of crystals, their cross-linking treatment, and their extraction. RCLLCs were active in an aqueous solution and in hexane, as shown by the hydrolysis of p-nitrophenol butyrate and α-methylbenzyl acetate, respectively. RCLLCs presented both high thermal and robust operational stability, allowing the preparation of a packed-bed chromatographic column to work in a continuous flow. Finally, we determined the three-dimensional (3D) models of this commercial lipase crystallized with and without phosphate at 2.0 and 1.7 Å resolutions, respectively.

Figure 1

Crystallization protocol from initial screening of BioL to the sequential scale up to 0.5 L. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel of BioL along the purification steps: lane 1, the protein marker; lane 2, after dialysis; lane 3, concentrated; lanes 4 and 5, the dissolved pellet and effluent of the precipitated sample; and lane 6, the commercial product. Crystallization hits found with conditions CSK-23 (b) and sodium formate pH 7.0 (SF7) (c). (d) Crystallization in the presence of 2.5% v/v TMOS (left capillary) and 0.1% w/v agarose (right capillary) using CSK-23 as a precipitating agent. (e) Batch crystallization experiment using agarose 0.1% w/v. (f) Scale-up process is illustrated going from 2 to 50 mL and (g) the last step growing-up to half liter in which the top-right inset corresponds to a closer look to show the homogeneity of lipase crystal size. The agarose concentration from 2 to 500 mL was set at 0.2% w/v.

Figure 2

(a) Adaptation of a crystallization instrument to extract the RCLLCs from the agarose matrix. (b) Lyophilized RCLLC final product collected in a 2 mL vial (c). (d) Magnification of the collected RCLLCs and (e) size distribution grouped in three categories to sum up the 100% of crystal population.

Figure 3

Operational setup of the RCLLC packed-bed column connected to a spectrophotometer and a pump. The system is schematically represented in (A), whereas (B) shows a picture of the column connected to the pump.

Figure 4

(A) Superposition of the lipase structural models showing the catalytic triad, Ser–His–Asp, of BioL and the NAG moiety at residue Asn33. BioL models in cyan and orange correspond to this work, PDB IDs 7APN and 7APP, respectively. The closed and open forms of TLL corresponding to the 1DT3 and 1EIN PDB models are shown in blue and green colors, respectively. (B, C) Crystal packing.

Figure 5

(A) Comparison of the activity of BioL in a solution (red, 50 μg·mL^–1) and as RCLLCs (blue, 50 μg RCLLCs·mL^–1), using pNPB as a substrate (25–600 μM). (B) Activity of RCLLCs (1.0 mg) vs the number of reaction cycles. (C) Hydrolysis of αMA using BioL in a solution (0.8 mg·mL^–1) and in a crystalline state (8.3 mg·mL^–1) showed in a thin-layer chromatography (TLC) plate: lane 1, α-methylbenzyl acetate (αMA); lanes 2 and 3, 1-phenylethanol (1-pOH) at two concentrations (pure and 1/10 dilution); lanes 4, 6, and 8, hydrolysis of αMA using soluble BioL in heptane at 40, 50, and 60 °C, respectively; and lanes 5, 7, and 9, hydrolysis of αMA using RCLLCs in heptane at 40, 50, and 60 °C, respectively. (D) Continuous production of pNP produced by the RCLLC packed column at different initial substrate concentrations (50–200 μM) under a continuous flow of 1.0 mL·min^–1.