Solid-binding peptides for immobilization of thermostable enzymes to hydrolyze biomass polysaccharides

Significance Statement

Immobilization of enzymes onto solid supports can be achieved by a number of physical and chemical methods including, covalent attachment, adsorption, crosslinking and encapsulation. Immobilized enzymes, as opposed to soluble enzymes, offer better stability and easier removal from reaction mixtures, enabling repetitive use in batch and continuous bioprocesses and rapid termination of reactions.

Unfortunately, typical enzyme immobilization approaches usually result in a non-uniform orientation of the enzyme as well as unwanted conformational changes that alter their active sites and may curtail the catalytic activity of the enzyme. Solid-binding peptides have binding affinity as well as selectivity to the surfaces of solid materials such as glass, polymers, silica, metals and zeolite, all support materials employed with biocatalysts.

Solid-binding peptides typically are used as molecular linkers for functional protein immobilization onto solid surfaces without the need for any chemical reactions or even physical treatments.

Researchers led by Professor Anwar Sunna at Macquarie University in Australia have presented the implementation of the solid-binding peptide-mediated immobilization of industrially-based enzymes onto a low cost solid zeolite matrix. The introduction of crosslinking of the immobilized enzymes to create single as well as multiple enzyme biocatalytic modules enabled the authors to highlight the feasibility of this technology for its integration in industrial-scale processes. Their work is published in Biotechnology for Biofuels.

The research team genetically fused the silica-binding linker peptide to three thermostable polysaccharide-degrading enzymes for potential application in industrial-scale biocatalysis. The linker had significant affinity for silica-containing supports allowing for directional immobilization of these enzymes onto the zeolite matrix. The enzymes were observed to retain their binding affinity for zeolite and their biological activity. The integration of the linker did not have adverse effects on the pH and temperature optima of the polysaccharide-degrading enzymes and the assembled single and multiple enzyme biocatalytic modules retained their specific hydrolytic activities upon several rounds of recycling at high temperatures.

Professor Sunna summarized the importance of this platform technology saying; “Despite the promising characteristics of solid-binding peptides, their practical application has been mostly in nanobiotechnology, where immobilization of biomolecules generally relies on exotic and expensive laboratory-based matrices that may not be realistic economically for large-scale processes. Inorganic bulk materials like zeolite and silica are excellent carriers due to their structural and operational stability and their lack of susceptibility to microbial degradation. The combination of solid-binding ability and low-cost bulk materials represents an ideal technology for production of industrial-scale biocatalysts.”

The linker system developed in their study minimizes the time wasted in choosing precipitants as well as crosslinking reagents. Its compositional and structural characteristics allows it to impart orientation and directionality to enzymes after crosslinking. This combination results in improved enzyme reusability. Therefore, this linker technology presents an inexpensive immobilization approach for industrially based enzymes.

Solid-binding peptides for immobilization of thermostable enzymes to hydrolyze biomass polysaccharides-Renewable Energy Global Innovations

About The Author

Andrew Care is a Research Fellow in the ARC Centre of Excellence for Nanoscale BioPhotonics, a transdisciplinary research centre that aims to develop innovative nanotechnologies to investigate complex living systems. He obtained his PhD from Macquarie University in Sydney, Australia.

His current research is focused on the use of solid-binding peptides to control the immobilization of proteins and enzymes onto solid matrices in a range of biotechnological applications, including biocatalysis.

About The Author

Kerstin Petroll obtained her Diploma degree in Food Chemistry at the Karlsruhe Institute of Technology (KIT) in Germany. She was awarded a Macquarie University Research Excellence Scholarship to join Macquarie University as a postgraduate student in 2015. Initially, she focused on analytical sciences of plant metabolites and proteins for medical applications before changing to the field of synthetic biology with a focus on environmental applications. Her PhD project aims at the assembly of a cell-free synthetic pathway to produce a platform chemical from renewable low-value compounds.

About The Author

Peter Bergquist is Emeritus Professor in the Biomolecular Discovery and Design Research Centre at Macquarie University. He has a PhD and DSc from the University of Auckland in New Zealand and has been a Postdoctoral Fellow and Research Fellow at Harvard Medical School, Yale and Oxford Universities and a Visiting Fellow at New York University School of Medicine.

He is one of the pioneers of cloning and expressing genes from extremely thermophilic bacteria. He has an interest in biofuels that stems from early studies of cellulolytic microorganisms and their enzymes for biomass breakdown. He has been on the editorial boards of several significant journals such as Applied and Environmental Microbiology and Journal of Bacteriology.

About The Author

Anwar Sunna is an Associate Professor in Synthetic Biology in the Department of Chemistry and Biomolecular Sciences at Macquarie University (MQ), Sydney, Australia. He obtained a PhD from the Hamburg University of Technology in Germany. He was manager of the Environmental Biotechnology Co-operative Research Centre at MQ and later was the recipient of the prestigious Vice-Chancellor’s Innovation Fellowship.

His recent research has been on the interaction between biomolecules and inorganic compounds including new synthetic peptide linkers with applications in enzyme immobilisation and functionalisation of nanomaterials. Anwar is a member of the MQ Biomolecular Discovery and Design Research Centre, MQ Biosecurity Futures Research Centre, Australian Research Council (ARC) Training Centre for Molecular Technology in the Food Industry and the ARC Centre of Excellence for Nanoscale BioPhotonics. He is also one of the directors of Synthetic Biology Australasia.

Reference

Andrew Care, Kerstin Petroll, Emily S. Y. Gibson, Peter L. Bergquist, and Anwar Sunna. Solid-binding peptides for immobilization of thermostable enzymes to hydrolyze biomass polysaccharides. Biotechnol Biofuels (2017) 10:29.

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