Designing and evolving ultra-stable enzymes for improved nutrition and reduced environmental damage

Lead Research Organisation: University of Oxford
Department Name: Interdisciplinary Bioscience DTP

Abstract

Enzymes are macromolecular proteins that serve as biological catalysts of chemical reaction and are critical in maintaining life. Humans have acknowledged and exploited the utility of enzymes for food and agriculture, giving rise to the biotechnology sector of today. The current industrial enzyme market is valued at over $5 billion, and reflects the desirability of enzymes as biocatalysts. Enzymes are highly specific in the reactions they catalyse, highly efficient, biodegradable, and found in abundance in nature. Despite such potential, many enzymes are unfit in their naturally occurring state for industrial applications due to their inherent instability, which limits their ability to withstand industrial processes. Over the years, many different approaches have been tested to stabilise enzymes to better exploit their catalytic potential with varying success, but all approaches have not been without pitfalls. One such approach has been SpyRing cyclisation, developed by the Howarth laboratory. Working on its success, I hope to develop a generic platform for enzyme stabilisation effective for a diverse range of enzymes in this DPhil project in conjunction with AB Vista. Priority area: Industrial Biotechnology and Bioenergy


BfH, ENWW

Publications

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Buldun CM (2018) SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation. in Journal of the American Chemical Society

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011224/1 01/10/2015 30/09/2023
1801400 Studentship BB/M011224/1 01/10/2016 30/09/2020 Jisoo Jean
 
Description Enzymes touch every aspect of our lives, from the detergent we use to the food we eat. The use of enzymes is critical in many sectors, including the pharmaceutical, food, drink and agricultural industries, and are featured prominently in many commercial products. Enzymes are ideal for industrial application due to their high degree of specificity and efficiency in the reactions they catalyse, but most enzymes found in nature require extensive manipulation and engineering since they are naturally fragile and cannot withstand the rigours of processing required for industrial use. Specifically, many industrial processes involve the application of heat, which denatures unstable enzymes to render them ineffective. Much research effort has been directed to stabilising enzymes to engineer resilience, enabling their industrial use. However, current methods are heavily laborious and involve engineering every possible site in each enzyme, then screening all possible combinations of such variants to produce a final, stabilised enzyme variant. However, this entails stabilisation on an enzyme-by-enzyme basis, which is ineffective and highly time consuming; it is not uncommon for an enzyme to take more than 5 years from discovery to commercialisation.

We have developed and implemented a novel method of stabilising enzymes, the SnoopLigase cyclisation system, which is not only genetically encodable and modular, but also functions as a general method of stabilisation. The SnoopLigase cyclisation system circumvents the need for enzyme-by-enzyme engineering, and enables quick, one-step thermal stabilisation of many enzymes. The system functions by placing two small peptide (protein) tags (SnoopTagJr and DogTag) at each end of the enzyme of interest, and adding SnoopLigase. SnoopLigase irreversibly locks the two peptide tags together to form a cyclised enzyme, whose two ends are fused together. We have demonstrated that this confers a great deal of thermal resilience to a variety of enzymes, including model enzyme beta-lactamase and phytase, an agriculturally and environmentally pertinent enzyme. In one step, SnoopLigase cyclisation was able to confer an increase in thermal resilience of over 60 C, and enabled the tested enzymes to retain their function even after boiling. This demonstrates that SnoopLigase cyclisation is a modular, efficient and effective way to capacitate enzymes for industrial use on a shorter timescale without compromises in thermal resilience.
Exploitation Route The SnoopLigase system is modular, and the enzyme flanked by the peptide tags (for peptide-peptide locking by SnoopLigase) can easily be swapped out. We have already demonstrated its applicability and efficacy on multiple enzymes, and the genetic components are available on Addgene (linked through the JACS publication). If others were to use the SnoopLigase cyclisation system, they would hopefully observe a similar degree of thermal resilience conferred without the need for laborious enzyme mutagenesis.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://pubs.acs.org/doi/10.1021/jacs.7b13237
 
Description The studentship is in conjunction with AB Vista, a company specialising in enzymes for use in animal feed in the agricultural sector. I have applied the SnoopLigase cyclisation system to phytase, an enzyme of special interest in the field, and also to Quantum Blue, the currently commercially available stabilised phytase variant patented by AB Vista. I am currently working with AB Vista, my industrial partner, and we are in the preliminary stages of testing SnoopLigase cyclisation on Quantum Blue. Should all the tests be successful and show improvements in thermal resilience, the system will be taken forward for implementation and commercialisation by AB Vista.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Economic