Review
Multi-enzyme co-immobilized nano-assemblies: Bringing enzymes together for expanding bio-catalysis scope to meet biotechnological challenges

https://doi.org/10.1016/j.ijbiomac.2021.07.064Get rights and content

Abstract

Co-immobilization of multi-enzymes has emerged as a promising concept to design and signify bio-catalysis engineering. Undoubtedly, the existence and importance of basic immobilization methods such as encapsulation, covalent binding, cross-linking, or even simple adsorption cannot be ignored as they are the core of advanced co-immobilization strategies. Different strategies have been developed and deployed to green the twenty-first century bio-catalysis. Moreover, co-immobilization of multi-enzymes has successfully resolved the limitations of individual enzyme loaded constructs. With an added value of this advanced bio-catalysis engineering platform, designing, and fabricating co-immobilized enzymes loaded nanostructure carriers to perform a particular set of reactions with high catalytic turnover is of supreme interest. Herein, we spotlight the emergence of co-immobilization strategies by bringing multi-enzymes together with various types of nanocarriers to expand the bio-catalysis scope. Following a brief introduction, the first part of the review focuses on multienzyme co-immobilization strategies, i.e., random co-immobilization, compartmentalization, and positional co-immobilization. The second part comprehensively covers four major categories of nanocarriers, i.e., carbon based nanocarriers, polymer based nanocarriers, silica-based nanocarriers, and metal-based nanocarriers along with their particular examples. In each section, several critical factors that can affect the performance and successful deployment of co-immobilization of enzymes are given in this work.

Introduction

Recent advancements in biotechnology and bioengineering have witnessed a rising trend in developing enzyme-mediated greener, safe, and sustainable bioprocesses. Exquisite selectivity, specificity, and catalytic performance have constituted enzymes as robust biocatalysts with wide-spectrum applications in biomedicine, biosensors, and bio-catalysis [1], [2], [3], [4]. Owing to low chemicals utilization and lack of hazardous metabolites/by-products, the use of biocatalysts is likely to facilitate green processes. However, low stability elevated operating costs, and lack of reuse capability of the native enzymes are major hurdles associated with the bio-based catalytic processes [5], [6], [7]. Enzyme immobilization on suitable carriers has been commonly recognized as a hopeful approach that gained a prominent place. This technology is capable of stabilizing or sheltering enzyme molecules against environmental and chemical attacks. In addition, the carrier-supported enzymes could be retrieved from the reaction system and re-processed in continuous processes [8], [9]. A variety of immobilization strategies and support materials (natural/synthetic polymers or inorganic materials) have been designed and employed for the immobilization of various classes of enzymes. Even though, the enzyme stability and activity can be preserved by optimized immobilization environments, applying carrier-incorporated biocatalysts in a bioreactor may result in reduced enzymatic activity because of alteration in the native enzyme structures after immobilization [10], [11]. Thus, identifying enzyme support matrices with robust, biocompatible, and separable properties, and most pertinent immobilization procedures are of utmost importance [12].

Contemporary progress in nanoscience and nanotechnology has furnished a plethora of multifunctional nanocarriers with unique attributes for effective enzyme immobilization. Enzymes immobilization onto nanoscale materials has gained considerable interest to improve the biocatalytic features of enzymes. Nanobiocatalysis is a growing field that synergistically combines the breakthroughs of nanotechnology and biotechnology. More specifically, the integration of biotechnology and nano-size materials, as support matrices, has arisen as a noteworthy platform to develop state-of-the-art multienzyme bionanostructures as nanobiocatalysts [13]. Nanobiocatalysts development implicates the attachment of enzyme molecules onto nanocarriers in an ordered state to achieve desired reaction kinetics and substrate selectivity. Highly effective and rigorous nanobiobiocatalytic mechanisms of multi-enzyme cascade reactions led to the development of strategies to co-localizing many enzymes on nano-size materials as support matrices. These multienzyme nanoassemblies and nanoarchitectures may provide better control over the spatial organization and molecular ratio of the contributing biocatalysts [14], [15]. Reports have evidenced that nanostructures exhibit the requirements to fabricate nanobiocatalytic system by providing greater surface areas that facilitate elevated enzyme binding and reduced mass transferring resistance [16], [17], [18], [19], [20]. Nanobiocatalysis represents an explicit assembly of functionalized nanocarrier and enzyme that holds incredible promise to augment enzyme stability, resistivity against harsh chemicals, and catalytic performance. It also produces a microenvironment around enzyme molecules for utmost reaction efficacy [21]. Immobilizing enzymes on nanocarriers can substantially expand the biocatalyst life cycles, thereby diminishing the process costs. Furthermore, based on literature evidence, immobilizing two or more enzymes induces their close in proximity, resulting in cascade kinetic properties and microenvironment influences that lead to an overall increased enzymatic performance as compared to single immobilized biocatalysts [17].

Multi-enzyme bio-catalysis is a new technological approach that assimilates different catalytic conversions to produce numerous chemicals of industrial importance [22], [23], [24], [25]. Considering in vivo multi-enzymatic reactions, researchers are striving to design multi-enzyme-based functional biocatalytic systems in vitro using various strategies, including co-immobilization, enzyme fusion, and enzyme-scaffold conjugates [17], [26], [27]. In multi-enzyme technology two or multiple enzymes are co-localized onto appropriate carriers or integrated enzyme molecules without supports in the presence of a linking agent [28], [29]. Co-immobilization process results in a reduction in mass transfer resistance, leading to improved enzyme activity through efficient substrate channeling and aggrandized reusability and stability [17], [23], [30]. To date, a vast number of functionalized nanostructured materials has been utilized to develop multi-enzyme immobilized nanobiocatalytic systems, such as carbon nanotubes, nanoparticles, graphene, metal-organic frameworks, silica, nanocomposites, and nanofiber scaffolds that are capable of protecting enzymes from harsh temperatures, heavy metals, and other challenging environments [29], [31], [32], [33], [34], [35]. A rising trend in the development of nanobiocatalysts is a driving force to fabricate new nanoscale carriers with structural diversity and unique attributes significantly enhance the engineering performances of enzymes. Herein, we spotlight the emergence of these new multienzyme co-immobilization strategies by bringing multi-enzymes together with various types of nanocarriers to expand the bio-catalysis scope (Fig. 1).

Section snippets

Random co-immobilization

A random co-immobilization approach is a simple but advanced version of basic immobilization strategies, where a multi-enzyme-based robust catalytic system can be engineered for multi-purpose applications. Using a random co-immobilization technique, multi-enzymes are randomly linked/conjugated into/onto the carrier matrices. Subject to the available functional entities and their involvement in the immobilization process, any or many of the basic immobilization methods, such as direct

Support materials for multi-enzyme immobilization

Many materials with various sizes/shapes, porous/non-porous architectures, and binding properties have been developed and employed as carrier supports for immobilizing multi-enzyme.

Conclusion and future directions

Nanobiocatalysis, incorporating enzymes into nanomaterials, is a rapidly emerging field that synergistically combines the breakthroughs of nanotechnology and biotechnology. Current progress in nanotechnology has contributed to a plethora of novel nanoscale nanostructures materials that exhibit the requirements to fabricate nanobiocatalytic systems by providing greater surface areas that facilitate elevated enzyme binding and reduced mass transferring resistance. The explicitly functionalized

Declaration of competing interest

The author(s) declare no conflicting interests.

Acknowledgments

Consejo Nacional de Ciencia y Tecnología (CONACYT) is thankfully acknowledged for partially supporting this work under Sistema Nacional de Investigadores (SNI) program awarded to Hafiz M.N. Iqbal (CVU: 735340).

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