The Depth-Dependent Mechanical Behavior of Anisotropic Native and Cross-Linked HheG Enzyme Crystals

Abstract

Enzymes are able to catalyze various specific reactions under mild conditions and can, therefore, be applied in industrial processes. To ensure process profitability, the enzymes must be reusable while ensuring their enzymatic activity. To improve the processability and immobilization of the biocatalyst, the enzymes can be, e.g., crystallized, and the resulting crystals can be cross-linked. These mechanically stable and catalytically active particles are called CLECs (cross-linked enzyme crystals). In this study, the influence of cross-linking on the mechanical and catalytic properties of the halohydrin dehalogenase (HheG) crystals was investigated using the nanoindentation technique. Considering the viscoelastic behavior of protein crystals, a mechanical investigation was performed at different indentation rates. In addition to the hardness, for the first time, depth-dependent fractions of elastic and plastic deformation energies were determined for enzyme crystals. The results showed that the hardness of HheG enzyme crystals are indentation-rate-insensitive and decrease with increases in penetration depth. Our investigation of the fraction of plastic deformation energy indicated anisotropic crystal behavior and higher irreversible deformation for prismatic crystal faces. Due to cross-linking, the fraction of elastic energy of anisotropic crystal faces increased from 8% for basal faces to 68% for prismatic crystal faces. This study demonstrates that mechanically enhanced CLECs have good catalytic activity and are, therefore, suitable for industrial use.

Biocatalytically Active and Stable Cross‐Linked Enzyme Crystals of Halohydrin Dehalogenase HheG by Protein Engineering

“…Crystallization of HheG wild type had previously been demonstrated using 10 % (w/v) PEG4000 as precipitant, HEPES buffer with a pH of 7.3 to 7.5 and protein concentrations between 6 and 32 mg/ml [26,30] . Hence, similar conditions were used to investigate the crystallizability of variants D114C and A221C (see supplementary for more details).…”

“…In contrast, HheG D114C CLECs cross‐linked with BMOE via soaking displayed high conversion in the azidolysis of 1 , albeit at 4 h reaction time conversion is slightly lower compared to the soluble enzyme (Figure 2). The latter, however, might be due to diffusion limitations within the crystal, [26,33] reducing substrate accessibility and product release. On the other hand, the limited enzyme motion within the CLEC could also explain the observed activity decrease.…”

“…In contrast, cross‐linking of HheG crystals with glutaraldehyde via vapor diffusion yielded CLECs that still displayed activity in the ring opening of cyclohexene oxide ( 1 ) with azide (Scheme 1). [26] This approach, however, is impractical in larger scales where protein crystallization is generally performed in stirred vessels [27] . Hence, we herein attempted to use protein engineering of HheG to insert desired residues in crystal contacts that would allow us to use chemoselective cross‐linkers for stable CLEC generation without compromising biocatalyst activity.…”

Unique Mechanical Properties of Gel-Incorporating Protein Crystals

“…By combining the gel media and protein crystals, they obtain the potential to toughen without sacrificing other mechanical properties. In previous studies, the mechanical properties such as the indentation hardness and elastic constants depends on the crystal characters such as protein types, crystal morphology, and solvent content in protein crystals. − As a next step, the effect of difference of these crystal characters for the macroscopic mechanical properties combined with the gel should be clarified. The combining methods will be more effective and powerful tool for mechanically unstable protein crystals, and the additive function by the combination with protein and polymer functions.…”

“…Studies on the macroscopic mechanical properties of protein crystals are limited because of the difficulty in growing large crystals of good quality. In early work, the Young’s modulus of the cross-linked tetragonal hen egg-white lysozyme crystals was estimated as 210 MPa via the periodic bending method. − Research on the deformation mechanism of protein crystals has been investigated primarily using the indentation method. − The slip systems and the dependence of the hardness on the crystal planes and crystal structures were characterized by the observation of the indentation marks and force–displacement curves. − In pioneer research on the elastic properties, dynamic elastic constants in the ranges of megahertz and gigahertz have been determined by the laser-generated ultrasound method, the ultrasonic pulse-echo method, − and the Brillouin scattering method. , …”