4.6 Immobilization of OxdB from Bacillus sp. OxB-1 by crosslinking with
4.6.1 Overview of different enzyme immobilization strategies
Biocatalytic synthesis of linear dinitriles
4.6 I MMOBILIZATION OF O XD B FROM B
ACILLUS SP. O X B-1 BY CROSSLINKING
In principal, one can divide the immobilization methods for enzyme immobilization into three categories:
1. Carrier binding, where the carrier mostly consists of a polymer bead that can optionally be chemically modified on its surface.
2. Entrapment of the enzyme either by an anorganic or organic matrix.
3. Crosslinking of the enzyme with itself by applying a crosslinking agent.
4.6.1.1 Enzyme immobilization by carrier binding
Regarding carrier binding, this method is nowadays broadly applied due to the great variety of different available resins for immobilization. Some companies (Purolite, Resindion) have completely specialized themselves to the production of immobilization resins.[127,128] Most resins are made out of Poly(methyl methacrylate) (PMMA), styrene or copolymers of the former with other building blocks like divinylbenzene (DVB). The beads have a spherical shape and may be chemically modified to allow immobilization by different types of interaction between enzyme and carrier (Figure 25).
Figure 25: Chemically modified polymer beads (carriers) for enzyme immobilization with different modes of interaction.[127]
Modification of PMMA beads with short carbon linkers that contain a terminal amino or epoxy moiety results in resins that can bind enzymes by covalent binding. The epoxy ring can bind by nucleophilic attack of amino acid side chains like the ones of lysine, serine etc., while the amino spacers are preactivated with glutaraldehyde. After activation, the glutaraldehyde reacts with free amino side chains to form imino bridges between enzymes and carrier. However, the concrete binding mode is highly complex and the above mentioned description only represents a simplification.[129] As one can imagine, covalent binding represents a big interference in the complex interactions that determine the tertiary structure of an enzyme. As a result, major losses in enzyme activity are observed.
However, covalent binding is the strongest possible interaction and leads to no or only negligible leaching of the enzyme off the carrier.
Biocatalytic synthesis of linear dinitriles
Other resins adsorb the enzymes by hydrophobic interaction. Most prominently, octadecyl groups are attached to the surface of the resins to increase the hydrophobicity of the carrier. Alternatively, enzymes can be adsorbed by highly porous styrene resins with big cavities. The enzyme wanders into the cavities, being surrounded by a hydrophobic environment. This method is milder than covalent binding, but may still lead to strong distortion of the enzyme structure because the hydrophobic areas of the enzyme will mainly try to interact with the carrier. Many enzymes however are mainly hydrophilic on the surface, turning the enzyme inside out and potentially deactivating it.
Lastly, one can attach substituted amino groups on the surface of a resin. These amino groups can either directly be positively charged (for quarternary amines) or be preactivated by acidic treatment (for tertiary amines). If the isoelectric point of an enzyme is known, adjustment of the pH above it turns the enzyme into a polyanion. As a result, it is strongly bound to the carrier by ionic interaction without distorting its tertiary structure.
4.6.1.2 Enzyme immobilization by entrapment
Regarding the entrapment of an enzyme in an anorganic or organic matrix a broad variety of reported matrices exists, however only a small selection will be presented here. One of the oldest methods is the immobilization in calcium alginate beads. Due to the rather big pore size of the alginate beads, preferably whole cell catalysts are immobilized with this method. By dropping a suspension of whole cells and sodium alginate in a solution of calcium chloride, insoluble calcium alginate is formed and builds a protective barrier around the whole cells.[130] However, these beads are not very mechanically robust.
Another entrapment method is the immobilization in hydrogels. Hydrogels based on polyvinyl alcohol are already well established and utilized for industrial processes (Lentikats).[8,131] However, since these hydrogels represent an open polymer matrix, leaching of the enzyme can occur. As a consequence, mainly whole-cell catalysts are utilized for immobilization. Alternatively, prior crosslinking of the enzyme increases the size of the biocatalyst and reduces leaching as well.[131] A rather new approach is the immobilization of enzymes in hydrogels that are based on polyacrylic acid or polyacrylamide, so-called superabsorbers.[132] The superabsorbers provide an aqueous, natural environment for the enzyme, which is completely immobilized in the superabsorber. By applying a liquid, organic phase to the reaction, one can easily separate the immobilized enzyme and superabsorber by filtration. Hence, recycling of the immobilized biocatalyst is rather easily conducted.In 2014, Gröger et al. utilized the immobilization of an ADH in a superabsorber matrix for the combination of an organocatalytic, enantioselective aldol reaction followed up by a biocatalytic reduction with the immobilized ADH (Scheme 35).[132]
Scheme 35: Co-immobilization of a (S)-selective ADH from Rhodococcus sp. in superabsorber, reported by Gröger et al..[132]
Instead of using an open-pored prepolymerized organic matrix, one can also consider completely enclosing an enzyme in an inpenetrable organic matriy by polymerizing the organic matrix in situ as a suspension with the enzyme. In this case, the enzyme is contained in an aqueous environment surrounded by a solid organic matrix. As an example, polydimethylsiloxane (PDMS) can be mixed with a pre-made enzyme solution and dropped into a solution of polyvinylalcohol in water. The formed droplets slowly polymerize, irreversibly trapping the enzyme inside in discrete aqueous droplets. Starting in 2005, Ansorge-Schumacher’s group immobilized several lipases in PDMS beads for esterifications and dynamic kinetic resolutions.[133] They could impressively show the equally distributed aqueous droplets in the organic matrix, proving the native environment for the enzyme inside the PDMS beads.
In 2014, Langermann et al. expanded this method towards the biocatalytic, enantioselective cyanation of benzaldehyde with oxynitrilases utilizing commercially available Sylgard 184, the monomer of polydimethylsiloxane.[134] They stressed the point that the used organic phase for the reactions has to be saturated with water to prevent a slow extraction of the aqueous phase from the PDMS beads.
Drawbacks of this intriguing method are the rather long preparation time that can last several days because of the slow curing of PDMS at low temperature like room temperature. To decrease the curing time, one can increase the temperature. However, this may lead in conjunction with the curing time to strong inactivation of the biocatalyst.
On top of that, a highly reproducible protocol for highly monodisperse PDMS is very difficult to establish, as Rivadeneira could show in his bachelor thesis under supervision of the author of this thesis.[135] In his work, the aldoxime dehydratase from Bacillus sp. OxB-1 (OxdB) was immobilized as crude extract and as whole cell-catalyst (in E.coli) in PDMS beads.
To circumvent the drawbacks of the long curing time and reproduction issues, von Langermann et al. changed the polymer matrix in 2017 towards polyurethanes.[136] By premixing of the enzyme with the polymer precursor, the same highly dispersed aqueous dropelets are obtained as with the PDMS method. However, the polyurethane precursors rapidly and controllable polymerizes at ambient temperature once ultraviolet light is radiated upon him. Completely cured polyurethane is obtained in only five minutes, which drastically decreases the stress on the immobilized enzyme. The polyurethane is obtained as a solid plate that can be grinded to obtain it as small chips with a big surface area. As with the PDMS method, no or only negligible leaching can be observed.[136]
Biocatalytic synthesis of linear dinitriles
4.6.1.3 Enzyme immobilization by cross-linking
The last category to immobilize an enzyme is by crosslinking it with a crosslinking agent like glutaraldehyde. While several crosslinking agents exist, the most prominent is glutaraldehyde (1,5-pentanedial). The crosslinking can then be applied to the corresponding enzyme formulation to crosslink it. One of the first methods was to crosslink crystallized enzymes to obtain crosslinked enzyme crystals (CLECs).[8] While these represent a good formulation, the biggest drawback is the often not achieveable crystallization of the enzyme and the prior, expensive and time-intensive purification of the enzyme. Another approaches lies in the easily achieveable precipitation of an enzyme, followed by a crosslinking protocol. If one uses this approach, he obtains crosslinked enzyme aggregates (CLEAs). Instead of purified enzyme, one can simply utilize crude extract for the immobilization protocol. The precipitation acts as a purification step on itself, combining purification and immobilization in one step.
The benefit of CLEAs in comparison to free enzymes lies in the often improved operational stability in terms of tolerance against heat, organic solvents and autolysis. Additionally, the CLEAs show a low tendency of leaching and do not require an often rather expensive carrier to conduct the immobilization.[8] The crosslinking agent can also be mixed with other components that will help to finetune and optimize the immobilization, like amino containing sugars (chitosan) or siloxanes.[131] As with the immobilization on preactivated amino resins, the crosslinking of the enzymes in CLEA formation are rather complex and are not limited to imine formation.[129] As a consequence, further reduction with reagents as sodium hydroboride (NaBH4) may in principle conducted but to rarely show any benefit.
Especially the group of Sheldon has developed many contributions on the field of CLEA research, allowing a broad range of biotransformations with several enzyme classes to be conducted with CLEAs.[8,137,138,139]
Regarding Oxds, no investigations on immobilization have been conducted until today.
Because of that, the author decided to pursue this challenging endeavor by choosing CLEAs as the first method for the immobilization of Oxds because of the well documented and rather straight-forward protocols in literature. Additionally, as mentioned earlier, the encapsulation of Oxds in PDMS beads was conducted by Rivadeneira under the supervision of the author.[135] However, the encapsulation attempt was not investigated further.