Novel enzyme diversity for improving cleaning and hygiene

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

The human body produces up to gram quantities each day of sebum that is deposited on our clothes. The common
enzymes added to many washing powders are protease and lipase enzymes to digest the proteins and lipids on the wash
fabric. The sebum contains a complex mixture of free fatty acids, triglycerides and cholesterol esters, wax esters, and
squalene. much of which remains on the clothes after the wash. In addition there is a need to remove resistant stains such
as tea stains which are difficult to remove without some bleaching of the fabric.
The are other enzyme activities which exist in nature that can breakdown these other products and also enzymes which
have a bleaching effect.
The project will aim to identify and characterise these enzymes from a large DNA resource that has been isolated from hot
environments around the globe. This resource can be mined using bioinformatic techniques to identify enzyme classes that
will tackle these cleaning problems. This will allow the production of small amounts of the new robust enzymes to be
produced for testing at the partner company, Unilever. In addition libraries of small pieces of DNA from different
thermophilic genomes have be made as libraries that can be screening for activity of the enzymes again specific fatty acids
etc. The overall project will allow access to novel stable new enzymes that could be used to improve the overall cleaning
process.
The use of enzymes in these processes is environmentally friendly and avoids the use of harsh bleaching agents.

Technical Summary

The technical approach is to use both bioinformatics mining and the screening of existing genomic libraries from the large DNA resource from thermophilic organisms and metagenomes accumulated from the recently completed EU Hotzyme project and other 'in house' resources available. The human sebum which is secreted daily from our bodies in not completely removed with the existing detergent enzymes. With knowledge of the human sebum composition the project will focus on the identification of the following 4 enzyme activities: (1) Novel carboxyl esterases and lipases since the conversion of triglycerides (TGs) to fatty acids found in sebum is only 70 % complete since the remaining 30% of TGs are non-saturated fatty acids that have poor affinity for the active site of the currently characterised TG lipases that are currently used in detergents. This is due to conformational restrictions of the cis-double bonds hindering the TG removal since the fatty alcohols do not carry a charge, which reduces the enzymes affinity for carboxylic acid products, which are likely to stay in the enzyme active site. (2) Cholesterol esterases - the Exeter group has previously solved a 3D structure of a novel potentially related esterase (Bourne et al.,2000). New novel cholesterol esterases will be mined from the genomic and metagenomics database. (3) Perhydrolases converting the available free fatty acids or carboxylic ester into an organic per-acid in the presence of hydrogen peroxide. This alpha/betahydrolase family member will contribute to the removal of superior sebum remnants and an improved hygiene by generating per-acetic acid for stain bleaching and will support the oxidation of the unsaturated fatty acids and triglycerides. (4) Squalene-hopene cyclase - converting squalene that is widely present in the human sebum into the sterol-like compound hopene.

Planned Impact

The enzymes discovered as part of this project will have an impact on the application biocatalysis in the industrial setting.
The development of new enzymes for fabric cleaning will benefit the general public as a whole. It will improve health and
overall quality of life. It will contribute to a sustainable economy.
As the world population steadily increases - from 7 billion in 2011 to forecasted 9 billion in 2030 (www.un.org) with people
migrating to the cities, the demand for water and other resources will multiply. By 2020, it is assumed that 1.4 billion people
will be living in urban areas, that is, 40% more than today. This strongly increases the risk for human hygiene and health
and severely impacts on the ecological and environmental balance. In this context, it is expected that more cleaning
products will be needed, for either hand or automated cleaning systems, which require more energy and more water while
producing more waste using the currently established cleaning techniques/methods. Also, higher living standards will
increase the demand for related products, notably laundry/cleaning products, per household and year, yielding more wash
cycles done per household and year. As a result, more frequent usage of warm wash cycles can be anticipated.
Considering these scenarios, energy consumption would increase by 43% in 2020 and additional 35% in 2030. Also, water
consumption would grow by 15% in 2020 and additional 18% by 2030, not to mention the produced waste in form of, e.g.
greenhouse gas. The application of new classes of robust enzymes in the production of consumer products for relevant
industries, such as Unilever will drive a change of personal habits. In this context, Unilever has initiated a sustainable living
plan, which sets out to decouple the company's growth from the environmental impact, while at the same time increasing
Unilever's social impact. There are 3 big goals to achieve here: (i) to improve health and well-being; (ii) reduce the
environmental impact and (iii) source 100% of raw materials sustainably. The introduction of sustainable processes using
new biocatalysts produced in this project for household (FMCG) and health/cosmetic products will provide a new
awareness of the general public for the advantages to the development of an 'environmentally friendly' economy.
As described in the proposal submitted to innovate UK
The academic beneficiaries of this research would be to provide general information in enzymology. It will help to identify
new classes of enzymes that can break down components of human sebum and perhydrolases that can act as bleaching
agents. It will allow a greater understanding of enzyme mechanism and features that are responsible for protein stabllity.
The enzymes discovered will be interesting in the fact that many will originate from metagenomes so this will represent
enzymes from organisms that currently cannot be cultured. It will provide information into the so called 'dark matter' of life.
It is estimated that we can only cultivate 1-2 percent of the microorganisms in the environment to date. The new enzymes
can contribute to our general knowledge of evolution.
The enzymes discovered will have potential industrial applications in other areas of biotechnology.
As described

Publications

10 25 50
 
Description New enzyme classes that are not currently used in commercial detergents have been identified in know genomes and metagenomes. These have been are cloned and over-expressed. purified and biochemically characterised regarding their ability to degrade components of human sebum which are not currently targeted in detergent products. Several of the novel enzymes have been shown to be more effective than the commercial enzymes currently used by Unilever. Patents are currently being filed.
The enzymes have interesting properties in their own right and can be applied to other industrial applications. Several of the enzymes do not have any related structures deposited in the Protein Data Bank so more academic studies have commenced to determine their crystallographic structure to understand their enzymatic mechanisms and allow rational design for commercial applications.
One enzyme class SHC has been selected to be taken forward in another grant with Innovate UK. This enzyme has now been crystallised for structural solution. The crystals diffract but need further improvement to change crystal form before full data collection.
Exploitation Route The use of these new stable enzymes can be used in other industrial biotechnology applications. At least 5 of the new enzymes are of sufficient interest to be patented by Unilever to address the breakdown of human sebum in new detergent products.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The newly identified enzymes have been cloned and over-expressed in a soluble form and have been purified and characterised biochemically. Samples of the enzymes have been supplied to Unilever for testing in assays developed at Exeter and Unilever for digestion against components of human sebum. They have also been assessed in small scale wash studies and have been shown to be more effective than the commercial enzymes currently used by Unilever. These results will result in another 5 patents between Exeter and Unilever which are currently in preparation for filing in March 2018. One enzyme is being taken forward for larger scale production and further assessment for industrial applications within an application between Exeter and Unilever for a Innovate UK/BBSRC grant to be submitted at the end of March 2018.
First Year Of Impact 2018
Sector Chemicals,Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic

 
Description Industrial Contribution from Unilever
Amount £10,000 (GBP)
Organisation Unilever 
Department Unilever UK R&D Centre Port Sunlight
Sector Private
Country United Kingdom
Start 08/2016 
End 08/2018
 
Description Industrial contribution from Unilever
Amount £60,000 (GBP)
Organisation Unilever 
Department Unilever UK R&D Centre Port Sunlight
Sector Private
Country United Kingdom
Start 11/2013 
End 12/2017
 
Description Synergy of novel chemistry and Biology to Combat unmet cleaning needs
Amount £690,556 (GBP)
Funding ID Innovate UK 104457 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 11/2018 
End 10/2020
 
Description Collaboration with Unilever as part of Catalyst Grant 
Organisation Unilever
Department Unilever UK R&D Centre Port Sunlight
Country United Kingdom 
Sector Private 
PI Contribution Unilever Industrial collaboration funded 50% by Innovate UK A newly appointed scientist in Unilever will be establishing an authority recognised standard body soiling model, preparing formulations and conducting wash studies with the newly identified enzymes from Exeter (up to medium scale) at Unilever, either in their high-throughput facility or in semi-automated single pot devices. The Exeter Biocatalysis Centre will be involved with bioinformatic identification of new novel enzymes and their cloning and over-expression allowing their biochemical characterisation and potential substrate specificity with regards to industrial applications. This will be complemented by structural studies, molecular modelling and site-directed mutagenesis which will provide complementary academic knowledge on enzyme mechanism and stability issues.
Collaborator Contribution Unilever will collaborate with industrial aspects of the project regarding applications of newly identified enzymes towards improved cleaning properties. Newly identified enzymes will be analysed for their ability to use substrate components of human sebum and their stability in surfactants and other components present in the commercial detergent product.
Impact The project kick-off meeting was in September 2016, followed by a meeting with the Innovate UK monitoring officer in Dec 2016 at Unilever, Port Sunlight. The next meeting will be in Exeter on the 22nd March 2017. Enzymes have been identified by bioinformatic analysis, cloning and overexpression is continuing and assays and Mass Spec analysis of activity to analyse substrate specificity is being developed.
Start Year 2016
 
Title Detergent compositions with lipase and biosurfactant 
Description Composition comprising lipases and biosurfactants, especially psychrophilic lipases and biosurfactants. The invention relates to certain lipases and to composition comprising said lipases. Preferably the composition also includes a biosurfactant. Biosurfactants preferred includes mannosylerythritol lipid (MEL). 
IP Reference WO2017/036902 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact Not at the moment but being incorporated into new detergent formulation at Unilever. Using cold sensitive enzyme for washing at reduced temperature for energy conservation. Sustainable use of enzymes rather than chemicals.
 
Title Improved wash compositions 
Description Improvement of the enzyme content in washing powders. Laundry composition comprising lipases from Psychromonas ingrahamii. Uses of such composition in methods of laundry, especially at low temperature 
IP Reference WO2017/036901 A1 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact None to date but used by Unilever for new washing detergents Long term impact on enabling washing at lower temperatures thereby saving energy. Also more sustainable by using enzymes rather than chemical components in wash powders.
 
Title Liquid Detergency Composition Comprising Protease and Non-Protease Enzyme. 
Description Improvements of composition of liquid detergents containing protease enzymes. A liquid detergency composition comprising: protease and non-protease enzyme wherein at least part of the protease or non-protease enzyme is cross-linked enzyme aggregate. 
IP Reference WO2017/036915 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact Not at the moment but being incorporated in new liquid detergent design by Unilever
 
Title Liquid detergency composition comprising lipase and protease 
Description A liquid detergency composition comprising: protease and a lipase, wherein the lipase comprises a polypeptide having an amino acid sequence which has at least 90 percent sequence identity with the wild-type lipase derived from Thermomyces lanuginosa strain DSM 4109 and compare to said wild-type lipase, comprise a substitution of an electrically neutral or negatively charged amino acid within 15 Å of E1 or Q249 with a positively charged amino acid. 
IP Reference WO2017/036917 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact None to date. Will be used for new detergent formulations by Unilever
 
Title Process to manufacture cross-linked enzyme aggregates. 
Description Development for stabilising and immobilisation of lipase enzymes for improved activity in laundry detergents.A process for the manufacture of cross-linked enzyme aggregates comprising of step: 1) providing a liquid mixture of the enzyme; 2) precipitating the enzyme comprised in the liquid mixture; 3) cross-linking the enzyme by addition of a cross-linking agent, having at least two reactive group suitable for cross-linking amino acids; 4) stopping the crosslinking by the addition of a suitable buffer; wherein step 2) is performed in the presence of a compound selected from a crown ether or a surfactant: and wherein step 4) is preferably performed when the mean diameter of the particle is at most 4.0 µm. 
IP Reference WO2017/036916 A8 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact Not at present but being developed by Unilever for new detergent formulation
 
Description Poster at 5th International Conference on Novel Enzymes, Groningen, the Netherlands 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation: Stabilisation of a lipolytic enzyme for Commercial Applications, Simone Antonio De Rose and Jennifer Littlechild
Year(s) Of Engagement Activity 2016