Emerging 3D Printing Strategies for Enzyme Immobilization: Materials, Methods, and Applications

Shao, Yun; Liao, Zhijun; Gao, Bing; He, Bingfang

doi:10.1021/acsomega.2c00357

Cited by 46 publications

(32 citation statements)

References 98 publications

(15 reference statements)

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“…Depending on the 3D printing technique and materials used, resulting structures can be rigid or flexible, hydrophobic or hydrophilic, or any combination of these, along with other desirable properties, such as water-permeable gel environments. Immobilizing enzymes on or within 3D printed materials enables high-resolution placement of enzymes in complex structures, simple scale-up with minimal material waste, and continuous-flow well-mixed reaction geometries without laborious separation steps while retaining all the benefits provided by conventional immobilized enzymes [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Advances in 3D Gel Printing for Enzyme Immobilization

Shen

Zhang

Fang

et al. 2022

Gels

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Incorporating enzymes with three-dimensional (3D) printing is an exciting new field of convergence research that holds infinite potential for creating highly customizable components with diverse and efficient biocatalytic properties. Enzymes, nature’s nanoscale protein-based catalysts, perform crucial functions in biological systems and play increasingly important roles in modern chemical processing methods, cascade reactions, and sensor technologies. Immobilizing enzymes on solid carriers facilitates their recovery and reuse, improves stability and longevity, broadens applicability, and reduces overall processing and chemical conversion costs. Three-dimensional printing offers extraordinary flexibility for creating high-resolution complex structures that enable completely new reactor designs with versatile sub-micron functional features in macroscale objects. Immobilizing enzymes on or in 3D printed structures makes it possible to precisely control their spatial location for the optimal catalytic reaction. Combining the rapid advances in these two technologies is leading to completely new levels of control and precision in fabricating immobilized enzyme catalysts. The goal of this review is to promote further research by providing a critical discussion of 3D printed enzyme immobilization methods encompassing both post-printing immobilization and immobilization by physical entrapment during 3D printing. Especially, 3D printed gel matrix techniques offer mild single-step entrapment mechanisms that produce ideal environments for enzymes with high retention of catalytic function and unparalleled fabrication control. Examples from the literature, comparisons of the benefits and challenges of different combinations of the two technologies, novel approaches employed to enhance printed hydrogel physical properties, and an outlook on future directions are included to provide inspiration and insights for pursuing work in this promising field.

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Section: Introductionmentioning

confidence: 99%

Advances in 3D Gel Printing for Enzyme Immobilization

Shen

Zhang

Fang

et al. 2022

Gels

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“…Enzyme biocatalysis plays a key role in various applications, including pharmaceuticals, food, biomedicine, biochemistry, etc. − For such applications, the enzymes immobilized on a suitable carrier (e.g., scaffold) are preferred over free enzymes (in solution), because they have the advantage of improving operational stability, cost efficiency, product separation, and enzyme reusability. − Both physical (enzyme entrapment in the matrix) or covalent (intermolecular cross-linking between enzyme and carrier, and conjugation with the carrier) immobilization methods have been used for the attachment of enzymes. , Among other methods, physical immobilization via electrostatic interactions is applicable to a large set of enzymes without the need for expensive case-to-case modifications. − The method is simple and rapid and allows the immobilization of enzymes at varying pH values and on different supports …”

Section: Introductionmentioning

confidence: 99%

3D Printed Porous Nanocellulose-Based Scaffolds As Carriers for Immobilization of Glycosyltransferases

Lackner

Liu

Štiglic

et al. 2022

ACS Appl. Bio Mater.

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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, Z basic2 -CGT; sucrose synthase, Z basic2 -SuSy), each harboring a cationic binding module (Z basic2 ) 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 (Z basic2 -CGT) and 0.19 U/mg (Z basic2 -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.

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“…Immobilization of enzymes exhibits several advantages, such as the feasibility of enzyme recovery and reuse, rapid termination of the enzymatic assay, enhanced storage, and thermal and operational stability [11,12]. Three-dimensional (3D) printing or additive manufacturing produces immobilization carriers that are easily isolated from the reaction media and have large specific surface areas, thereby improving the mass transfer effect [13]. Polylactic acid (PLA) is a naturally-derived polymer.…”

Section: Introductionmentioning

confidence: 99%

“…Polylactic acid (PLA) is a naturally-derived polymer. It is biodegradable, biocompatible, non-toxic, non-carcinogenic, and has good optical, mechanical, and rheological properties [13][14][15]. Moreover, its printing process is simple and consumes less energy than that required for other polymers.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, its printing process is simple and consumes less energy than that required for other polymers. Hence, PLA is one of the most popular choices for biotechnological applications [13,15]. So far, 3D printed scaffolds using PLA, carbon fiber-reinforced PLA (C-PLA), and nylon have been investigated extensively for optimizing enzyme immobilization [16][17][18].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

3D Printed Polylactic Acid (PLA) Well Plates for Enzyme Inhibition Studies: The Case of Pancreatic Lipase

STAMATIS¹,

Gournis²,

POLYDERA³

et al. 2022

Catal Res

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3D printed PLA has already been demonstrated for several biotechnological applications, including enzymes immobilization. The prerequisites for an efficient screening assay include using small volumes of reagents, low cost, and rapid screening of large numbers of compounds and extracts. Hence, assays based on microtiter plates are predominant. Thus, designing and fabricating scaffolds on a similar scale, which could serve as immobilization carriers, and their recruitment in inhibitors screening studies is of great significance, adding both enzyme stability and reuse potentiality of the biocatalytic system in assay merits. In this work, pancreatic lipase was immobilized on 3D-printed PLA microwells for enzyme inhibitor screening. XPS analysis demonstrated the successful modification of the PLA scaffolds. The immobilized enzyme displayed high levels of operational, thermal, and storage stability under the tested conditions. The IC<sub>50</sub> values for PPL inhibition were calculated for Orlistat, a model lipase inhibitor, and olive leaf extract, a promising natural compound. This is the first study reporting the use of 3D-printed PLA wells with an immobilized enzyme for inhibitor screening assay.

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Emerging 3D Printing Strategies for Enzyme Immobilization: Materials, Methods, and Applications

Cited by 46 publications

References 98 publications

Advances in 3D Gel Printing for Enzyme Immobilization

Advances in 3D Gel Printing for Enzyme Immobilization

3D Printed Porous Nanocellulose-Based Scaffolds As Carriers for Immobilization of Glycosyltransferases

3D Printed Polylactic Acid (PLA) Well Plates for Enzyme Inhibition Studies: The Case of Pancreatic Lipase

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