Doctoral research at Macquarie University tested functional inclusion bodies immobilized on polypropylene fibers in SpinChem’s Rotating Bed Reactor to convert fructose to tagatose. The work shows that engineered protein particles can be handled, contained, and evaluated in an RBR, a practical step toward scale-up.
The challenge: turning active protein particles into usable biocatalysts
Functional inclusion bodies are often discussed as protein particles produced during expression. The work of Santhosh Vijayakumar, PhD researcher at Macquarie University, under the supervision of Anwar Sunna, explored a more applied question: could these particles be used as functional materials for immobilized biocatalysis?
His PhD research used a P40 fusion strategy to produce enzymes as active inclusion bodies. The P40 domain made it possible to influence yield, shape, and size, turning the inclusion bodies into a more controlled enzyme format.
The next challenge was practical. To become useful in process development, the active particles needed to be attached to a support material that could be handled in a reaction system.
Santhosh’s work identified a reversible solubilization and re-aggregation behavior. When the inclusion bodies were solubilized using guanidinium hydrochloride and mixed with a porous matrix, they could re-aggregate during dilution and bind tightly to the material.
This created a route to functionalize beads, membranes, and fibers with active enzyme particles. The broader immobilization work was described as achieving close to 100% functionalization efficiency with retained enzymatic activity.
The approach: immobilizing T4E on polypropylene fibers
In trials performed in SpinChem’s Rotating Bed Reactor (RBR), Santhosh used tagatose 4-epimerase, or T4E. This enzyme converts fructose directly into tagatose.
The reaction was suitable for a proof-of-concept trial because it was clean and easy to interpret:
- fructose was the single substrate
- magnesium was the only required cofactor
- no side products were observed in the specific reaction discussed
- product formation was detected using HPAEC-PAD, anion exchange chromatography
- very low residual fructose was observed
Santhosh expressed T4E as P40-fused inclusion bodies, solubilized them, and re-aggregated them onto polypropylene non-woven fibers. These fibers, originally sourced from an Australian manufacturer of insulation materials, became the support matrix for the immobilized enzyme particles.
The result was a new type of immobilized enzyme format: active inclusion bodies bound onto a thin, porous fiber matrix.
The solution: testing the functionalized fibers in the Rotating Bed Reactor
Once the polypropylene fibers were functionalized, Santhosh packed them into the Rotating Bed Reactor compartment and performed the reaction.
This step moved the work beyond simple tube-based trials. The RBR provided a contained reactor format where the functionalized fibers could remain in place while the liquid reaction medium moved through and around the solid phase.
The practical learning: handling functionalized fibers matters
During Santhosh’s process development work and trials, one clear practical difference emerged between simple magnetic stirring and the Rotating Bed Reactor.
When similar functionalized fiber matrices were placed in a beaker with a magnetic stirrer, significant leaching of protein particles was observed. The mechanical environment caused material to come out from the fibers.
Inside the Rotating Bed Reactor, the behavior was different. Even when some leaching occurred, the reactor geometry appeared to help particles re-settle back into the fiber matrix. This made leaching less problematic than under open stirring conditions.
“Because of the SpinChem geometry, the leach out particles are again settling back into the fiber.”
Santhosh Vijayakumar, PhD, Macquarie University
This observation is important because a reactor for novel immobilized materials must do more than drive conversion. It must also support practical handling:
- keeping the solid phase contained
- maintaining contact between substrate and catalyst
- reducing mechanical stress on the support
- enabling recovery and repeated-cycle testing
- providing a route toward scale-up
Santhosh described SpinChem as a “good tool for scale-up” and noted that results from the system were reliable and transferable toward larger-scale experiments.
The outcome: a proof of concept, not a finished process
The system was not fully optimized. Santhosh noted that several parameters would need further development, including:
- protein loading ratio
- protein loading on the matrix
- rotational speed
- temperature
- long-term recyclability
- leaching control
The strongest outcome is that Santhosh successfully brought a novel inclusion body-based immobilization platform into the Rotating Bed Reactor and showed that the concept could work.
Why this matters for future biocatalysis development
Santhosh’s work shows how engineered protein assemblies can become functional materials for biocatalytic processes. The polypropylene fibers provided a support format where active material was concentrated near the surface, rather than buried inside a larger carrier.
This has potential relevance for process scientists working with immobilized enzymes, fiber-supported catalysts, and multi-enzyme systems.
The work also opened several possible next steps. Santhosh discussed future directions including:
- multi-enzyme cascades
- inclusion body agglomerates
- Mini RBR experiments
- glutaraldehyde-crosslinked particles
- magnetic particles and magnetosomes
- diagnostic kit materials
- broader biocatalysis applications
For SpinChem,this work demonstrates a new use case for the RBR platform: testing functional inclusion bodies immobilized on fiber matrices in a contained reactor format.