[1S03a] 06. Enzyme Engineering/Enzymology - Part 2

3:30 PM - 3:45 PM JST(6:30 AM - 6:45 AM UTC)

[1ENZ-12] Engineering latex clearing protein for organic solvent tolerance to improve its potential in degrading rubber polymers

○Amulyasai Bakshi¹, Simone Morra¹, Anca Pordea¹ (1. University of Nottingham (UK))

Keywords:

Biocatalysis,Directed evolution,Waste re-valorisation,Mutant library screening

Rubber is a commonly used material in everyday objects and generates a large amount of waste globally. Current waste management methods are energy consuming and cause secondary pollution. Enzymes like latex clearing proteins (Lcps) provide a sustainable solution. Lcps degrade polyisoprene rubbers by oxidising the double bonds within the rubber chains, producing isoprene oligomers with terminal aldehyde and ketone functionalities. This provides a route for a circular rubber economy. Natural rubber latex is an emulsion of hydrophobic rubber chains suspended in a hydrophilic environment. Wild-type LcpK30 from Streptomyces sp. strain K30 is proven to degrade natural rubber and loses its efficiency with hydrophobic synthetic rubbers making it unfit for industrial usage.[1] This research hypothesises that decreased activity of the enzyme with synthetic rubber is because of the limited interaction between them due to the difference in hydrophobicity and can be improved by using organic solvents for a better dispersion of the substrate. Literature provides evidence for the formation of stable polymer emulsions in solvents which improved the efficiency of Lcps in synthetic rubber degradation.[2] However, the detrimental impact of the solvents on the enzyme activity is a limitation. This research aims to engineer LcpK30 to tolerate organic solvents in water-solvent biphasic mixtures, to improve its potential to degrade hydrophobic synthetic rubbers.Literature predicted three amino acids to play a role in the interaction between LcpK30 and a model decaisoprene substrate. In this project, rationally designed mutants were created through site-directed mutagenesis at the three sites to study the activity in 10 % tetradecane. Initial results from analysing 31 mutants using HPLC showed up to 40 % increase in oligomer formation compared to the WT.To increase the throughput of screening Lcps, a fluorescence-based method was developed, and the results showed that the screening process could be greatly accelerated, by avoiding protein purification and HPLC analysis of the oligomers. This methodology can accelerate the research on engineering Lcps and other rubber degrading enzymes.
1. Rose, K., K.B. Tenberge, and A. Steinbu¨chel, Identification and Characterization of Genes from Streptomyces sp. Strain K30 Responsible for Clear Zone Formation on Natural Rubber Latex and Poly(cis-1,4-isoprene) Rubber Degradation.
2. Biomacromolecules, 2005. 6. Adjedje, V.K.B., et al., Enzymatic degradation of synthetic polyisoprenes via surfactant-free polymer emulsification. Green Chemistry, 2021. 23(23): p. 9433-9438.