Award details

Molecular breeding for the improvement of biocatalytic activity - directed evolution of a chimeric glucanase with enhanced ability to disrupt oral biofilms

Reference	BB/D522538/1
Principal Investigator / Supervisor	Professor Michael Wilson
Co-Investigators / Co-Supervisors	Dr Sean Nair, Dr Jonathan Pratten
Institution	University College London
Department	Eastman Dental Institute
Funding type	Research
Value (£)	202,985
Status	Completed
Type	Research Grant
Start date	14/11/2005
End date	13/11/2008
Duration	36 months

Abstract

Biofilms on the tooth surface (dental plaques) are responsible for two of the most prevalent microbial diseases of humans: caries and gingivitis. Frequent mechanical removal of these biofilms (ie. By brushing and flossing) is the main means by which the diseases are prevented but few individuals practice these procedures effectively and hence the high prevalence of these two diseases. There has, therefore, always been tremendous interest in developing ways of preventing or disrupting these biofilms to supplement mechanical plaque-removing procedures. One possible approach that has received considerable attention is the use of enzymes to degrade the polysaccharide matrix of the biofilm. However, use of such enzymes has had little success in vivo, mainly because of the variety of polysaccharides constituting the matrix and also because of the difficult circumstances under which the enzyme(s) have to operate. Hence, the pH may be very low and also the contact time may be extremely short. Screening for suitable naturally-occurring enzymes has not met with any success and we propose to create a new enzyme with the desired properties. In the proposed study we will use the technique of molecular breeding to produce a glucanase capable of degrading the main polysaccharides that constitute the matrix of dental plaques (water-insoluble glucans) thereby enabling the disruption of these biofilms. Molecular breeding will be used to produce large numbers of glucanases with different catalytic activities which will be screened for activity against glucans with different chemical structures and at different pHs. The most active enzymes will then be tested, in a biofilm model, for their ability to disrupt biofilms of Streptococcus mutans (the main aetiological agent of caries) and microcosm dental plaques under conditions similar to those that exist in the human mouth. The outcome of the research will be a plaque-degrading enzyme that could be incorporated into oral hygiene products(toothpastes, mouthwashes etc) that would help to increase the efficacy of mechanical plaque removal and hence reduce the incidence of caries and gingivitis.

Summary

The coating that forms on the teeth every day (which is known as dental plaque) consists of bacteria inside a jelly-like material. If this plaque isn¿t removed by toothbrushing every day if can produce acids which dissolve the teeth resulting in cavities which have to be filled by a dentist. The plaque can also irritate the gums and cause them to bleed: a condition known as gingivitis. Many people do not clean their teeth properly or else don¿t do it frequently enough, so the manufacturers of toothpastes and mouthwashes put into their products chemicals that are able to kill any bacteria left behind on the tooth after toothbrushing. Unfortunately, the jelly-like material in dental plaque protects the bacteria that are present by preventing the antibacterial chemicals from penetrating into the plaque and so many bacteria survive. In this project we are going to try to use enzymes that can break up the jelly-like material so that the biofilms can¿t be produced in the first place or can be broken down once they have formed. The problem is where can we get such an enzyme from? It would have to be able to work very quickly, do the job at low concentrations and work under the conditions found in the mouth. There may be enzyme somewhere on the planet that would be ideal for the job, but it would take a long time to find it. What we intend to do is to produce a super-enzyme that can very rapidly and efficiently break up the biofilms or prevent the biofilms from forming in the first place. To do this we will need to start with genes that carry the information for enzymes that could break up the biofilm: but that are not good enough to use in humans because they work too slowly, or else because we¿d need very high concentrations or because they wouldn¿t really work under the conditions present in the mouth. From these we will `breed¿ the super-enzyme we¿re looking for. To do this we will chop up each of these genes into small sections, mix them all up and then join the bits together again in different ways. This will give us thousands of different enzymes, some of which are certain to be very good at destroying biofilms or preventing them from being formed. Once we¿ve got these enzymes we can test them to find out which of these has the properties we¿re looking for. We can then incorporate the enzyme into toothpastes or mouthwashes so that they can help to prevent the production of dental plaque or else break it down and this will help to prevent people getting cavities in their teeth and bleeding gums.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Industrial Biotechnology, Microbiology, Synthetic Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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