CaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels

The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity.


Effect of the ε-CL Concentration on the Esterification Ability of Non-Immobilized CaLB
The conversion of ε-CL to oligomers (Coligo) or polymers (Cpolym) respectively is determined from the 1 H-NMR spectra ( Figure S4) by using the discrete signals of the protons in γ−position for ε-CL, and the respective protons of 6-hydroxyhexanoic acid and the oligomer/polymer. While the signal at δ = 4.15 ppm (1a) is assigned to the ε-CL, the signal for the polymer/oligomer is found at δ = 3.97 ppm (1c) if the spectrum is measured in CDCl3. The signal of the end group (1b) of both the oligomer and the 6-hydroxyhexanoic acid is found at a shift of δ = 3.54 ppm. Therefore the conversion Coligo/Cpolym is calculated by (1) Figure S4. 1 H-NMR spectrum in CDCl3 of the polymerization product of experiment No. 2.6 in Table  S2 with the signals for the ε-CL monomer (a), the hydrolysis product 6-hydroxyhexanoic acid (b) and the poly/oligo(ε-CL) (c).

Cloning of Candida Antarctica Lipase B into the pGAPz Expression Vector
The gene of CaLB was ordered as a synthetic gene and transformed into E. coli DH5α [30]. Plasmid extraction of synthetic gene and pGAPzαA was done with the plasmid DNA purification kit. The CaLB gene was amplified from the vector via PCR with forward Primer (GCTGAAGCTGAATT CTTGCCATCTGGTTCTG) and reverse Primer (CACACTGGGTACCCGTTACTAGTGGATCCG). The PCR product and pGAPzαA were digested using EcoRI (100U) and KpnI (100 U) restriction enzymes. After 20-min heat inactivation at 80 °C and purification of the specific DNA fragments with the PCR purification gel extraction kit, the digested CaLB gene and vector pGAPzαA were ligated using T4 DNA ligase (5 U) resulting in pGAP_CaLB. Plasmid construct was subsequently transformed into E. coli DH5α (purchased from Agilent Technologies; Santa Clara, CA, USA). The plasmid pGAP_CaLB was extracted from E. coli DH5α by using a plasmid extraction kit. About 200 ng plasmid DNA linearized by AVRII was mixed with 80 μL of competent cells, and then it was transformed into Pichia cells (purchased Invitrogen GmbH, Karlsruhe, Germany) by electroporation conducted on Eppendorf Eporator (Eppendorf, Hamburg, Germany) according to the manufacturers instruction P. pastoris transformants via homologous recombination at the GAP promoter region between the transforming DNA and regions of homology within the Pichia genome. Positive clones were initially selected on YPDS plates containing 100 μg/mL Zeocin™ plates.

p-Nitrophenyl Butyrate (pNPB) Assay in MTP Format for CaLB Activity Measurement
Upon hydrolysis, para-nitrophenolate is released and its absorption is detected at 410 nm. The activity of the assay was determined by addition of TEA buffer (90 μL, 100 mm, pH 7.5) to supernatant (10 μL) and freshly prepared substrate solution (TEA buffer (100 μL) containing pNPB (0.5 mM) and acetonitrile (10%, v/v)) in each well. The release of para-nitrophenolate was recorded by measuring the absorption at 410 nm at room temperature over 8 min on the microtiter plate reader.

Production of CaLB in Shake Flask and Purification
Yeast cells pre-grown on YPD agar plate solid medium were inoculated in 10 mL YPD medium and incubated at 30 °C at 200 rpm for 16 h as a pre-culture. The main-culture was inoculated at OD600 of 0.2 and incubated at 20 °C at 220 rpm for 72 h. The supernatant containing the secreted enzyme was separated from the cells by centrifugation (Sorval RC 6; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 4 °C, at 4000 rpm.
Tris-acetate buffer (pH 7.2; 250 mM) was added to recovered supernatant in relation 1:10 and filtered with a glass fibre filter (pore size 0.45 μm; GE Healthcare). For enzyme purification first