Novel enzyme diversity for improving cleaning and hygiene

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.

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.

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