Dataset Information

3D-Printed Labware for High-Throughput Immobilization of Enzymes.

ABSTRACT: In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme's immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring different support chemistries, buffer compositions, and temperatures. Thus, we report 3D-printed labware designed to measure and handle solids in multichannel format and expedite screening of enzyme immobilization conditions. To demonstrate the generality of these advances, alkaline phosphatase, glucose dehydrogenase, and laccase were screened for immobilization efficiency on seven resins. The results illustrate the requirements for optimization of each enzyme's loading and resin choice for optimal catalytic performance. Here, 3D-printed labware can decrease the requirements for an experimentalist's time by >95%. The approach to rapid optimization of enzyme immobilization is applicable to any enzyme and many solid support resins. Furthermore, the reported devices deliver precise and accurate aliquots of essentially any granular solid material.

SUBMITTER: Spano MB

PROVIDER: S-EPMC9096805 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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3D-Printed Labware for High-Throughput Immobilization of Enzymes.

Spano Michael B MB Tran Brandan H BH Majumdar Sudipta S Weiss Gregory A GA

The Journal of organic chemistry 20200617 13

In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme's immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring ...[more]

PMID: 32502347

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Project description:PurposeThermoplastic immobilizers are used routinely in radiation therapy to achieve positioning accuracy. These devices are variable in quality as they are dependent on the skill of the human fabricator. We examine the potential multi jet fusion (MJF) 3D printing for the production immobilizers with a focus on the surface dosimetry of several MJF-printed PA12-based material candidates. Materials are compared with the goal of minimizing surface dose with comparison to standard thermoplastic. We introduce a novel metamaterial design for the shell of the immobilizer, with the aims of mechanical robustness and low-dose buildup. We demonstrate first examples of adult and pediatric cranial and head-and-neck immobilizers.MethodsThree different PA12 materials were examined and compared to fused deposition modeling-printed polylactic acid (PLA), PLA with density lowered by adding hollow glass microspheres, and to perforated or perforated/stretched and solid status quo thermoplastic samples. Build-up dose measurements were made using a parallel plate chamber. A metamaterial design was established based on a packed hexagonal geometry. Radiochromic film dosimetry was performed to determine the dependence of surface dose on the metamaterial design. Full cranial and head-and-neck prototype immobilizers were designed, printed, and assessed with regard to dimensional accuracy.ResultsBuild-up dose measurements demonstrated the superiority of the PA12 material with a light fusing agent, which yielded a ∼15% dose reduction compared to other MJF materials. Metamaterial samples provided dose reductions ranging from 11% to 40% compared to stretched thermoplastic. MJF-printed immobilizers were produced reliably, demonstrated the versatility of digital design, and showed dimensional accuracy with 97% of sampled points within ±2 mm.ConclusionsMJF is a promising technology for an automated fabrication of patient immobilizers. Material selection and metamaterial design can be leveraged to yield surface dose reduction of up to 40%. Immobilizer design is highly customizable, and the first examples of MJF-printed immobilizers demonstrate excellent dimensional accuracy.

Project description:Biocatalysis is increasingly becoming an alternative method for the synthesis of industrially relevant complex molecules. This can be realized by using enzyme immobilized polysaccharide-based 3D scaffolds as compatible carriers, with defined properties. Especially, immobilization of either single or multiple enzymes on a 3D printed polysaccharide scaffold, exhibiting well-organized interconnected porous structure and morphology, is a versatile approach to access the performance of industrially important enzymes. Here, we demonstrated the use of nanocellulose-based 3D porous scaffolds for the immobilization of glycosyltransferases, responsible for glycosylation in natural biosynthesis. The scaffolds were produced using an ink containing nanofibrillated cellulose (NFC), carboxymethyl cellulose (CMC), and citric acid. Direct-ink-writing 3D printing followed by freeze-drying and dehydrothermal treatment at elevated temperature resulted in chemically cross-linked scaffolds, featuring tunable negative charges (2.2-5.0 mmol/g), pore sizes (10-800 μm), fluid uptake capacity, and exceptional dimensional and mechanical stability in the wet state. The negatively charged scaffolds were applied to immobilize two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Zbasic2-CGT; sucrose synthase, Zbasic2-SuSy), each harboring a cationic binding module (Zbasic2) to promote charge-based enzyme adsorption. Both enzymes were immobilized at ∼30 mg of protein/g of dry carrier (∼20% yield), independent of the scaffold used. Their specific activities were 0.50 U/mg (Zbasic2-CGT) and 0.19 U/mg (Zbasic2-SuSy), corresponding to an efficacy of 37 and 18%, respectively, compared to the soluble enzymes. The glycosyltransferases were coimmobilized and shown to be active in a cascade reaction to give the natural C-glycoside nothofagin from phloretin (1.0 mM; ∼95% conversion). All enzyme bound scaffolds showed reusability of a maximum of 5 consecutive reactions. These results suggest that the 3D printed and cross-linked NFC/CMC-based scaffolds could present a class of solid carriers for enzyme (co)-immobilization, with promising applications in glycosyltransferase-catalyzed synthesis and other fields of biocatalysis.

Dataset Information

3D-Printed Labware for High-Throughput Immobilization of Enzymes.

Publications

3D-Printed Labware for High-Throughput Immobilization of Enzymes.

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