Dramatic Increase in Catalytic Performance of Immobilized Lipases by Their Stabilization on Polymer Brush Supports

Abstract

Despite their widespread use in biocatalysis, the marginal stability of lipases can significantly limit their catalytic performance in industrial biotransformations. Here, we demonstrate that this limitation can be overcome by immobilization on poly­(sulfobetaine methacrylate) (PSBMA) polymer brushes. Specifically, the immobilization of Bacillus subtilis lipase A (lipA) on PSBMA brushes resulted in a 100-fold enhancement in turnover frequency relative to ambient conditions at the temperature optimum of the immobilized enzyme, which was also improved by immobilization. This significant enhancement in catalytic performance was due to the structural stabilization of lipA as well as changes in lipA conformational dynamics as measured using single-molecule Förster resonance energy transfer. Interestingly, the enhancement in catalytic performance of lipases depended strongly on the chemistry of the brush. These findings demonstrate that tuning the brush chemistry can lead to marked improvements in the catalytic efficiency of immobilized lipases, which may have major ramifications in industrial biocatalysis.

Engineering the Composition of Heterogeneous Lipid Bilayers to Stabilize Tethered Enzymes

“…The site‐specific labeling and FRET activity of the enzyme constructs used for SM‐FRET experiments were reported previously. [ 19,33–35 ] As shown in Figure , these constructs exhibited a range of net surface charge, including both negative and positive charges. Specifically, the zeta potentials of soluble wild‐type NfsB, OPH, LipA, and T4L at pH 8.0 (the same pH used for all experiments reported here) were −11.6, −16.3, +3.6, and +7.0 mV, respectively.…”

“…For SM‐FRET imaging, enzyme variants containing a single cysteine and a p‐azidophenylalanine unnatural amino acid were expressed, purified, and labeled as described previously. [ 19,33–35 ] Dual‐labeled enzymes were immobilized to vesicles through the interaction between hexa‐histidine tag on the enzymes and DGS‐NTA‐Ni lipids (Avanti Polar Lipids) that were incorporated into the vesicles at a concentration of 0.3 × 10 –9 m , as described previously. [ 33 ] To form SLBs, vesicles with tethered enzymes were incubated in a custom‐built flow cell with fused silica wafers (Mark Optic) that had been previously cleaned in piranha solution and treated with UV‐ozone, for 30 min.…”

“…[ 15–18 ] Importantly, because each enzyme exhibits unique properties, a material that stabilizes one enzyme may destabilize another. [ 19,20 ] A grand challenge in this field involves the discovery of rules that permit rational design of materials that stabilize a given enzyme under a wide range of extreme conditions. [ 21–25 ]…”

Tunable Polymeric Scaffolds for Enzyme Immobilization

“…Furthermore, polymeric brushes act as an extension of the surface into a third dimension, granting a high and tunable density of functional groups (epoxide, carboxylic acid, hydroxyl, aldehyde, and amine groups) that can be eventually used for the conjugation of enzymes (Jiang and Xu, 2013). Several publications have pointed out that hydrophilic or zwitterionic polymer brushes create favorable microenvironments for catalysis (Weltz et al, 2019). However, there is still some work to do in the fabrication of efficient enzyme-polymer brush systems.…”

Mapping protein–polymer conformations in bioconjugates with atomic precision

“… 11 Recent work on surface-immobilization of enzymes has shown that lipase catalytic performance can be dramatically improved by attachment to hydrophilic surface-grafted polymers. 12 In a subsequent publication, by the same group, it was found that there is a trade-off in this system – more protein–polymer attachments resulted in higher stability but lower activity due to decreased motion in the folded state of the protein. 13 This experimental result conflicts with recent work on soluble chymotrypsin conjugates, where MD models showed more motion in the conjugate with the lowest enzymatic activity, a manifestation of a measured decrease in the substrate binding constant, K m .…”