Sybrin Schröder & Casper de Boer
Long sugar chains from plant cell walls are an attractive feedstock for industry, but they are recalcitrant to break down. Many fungal species, however, easily degrade polysaccharides with hydrolytic enzymes. These enzymes are of high interest for industrial use. Sybrin Schröder and Casper de Boer developed a new tool to find, identify and characterise an important class of such enzymes.
Tools to easily find novel biomass-degrading enzymes
Plant cell walls have to be strong, as their function is to protect cell contents. Cell walls mostly consist of polymers: long and stable chains of sugar molecules. These sugar polymers (polysaccharides) are of interest to industry as a versatile and renewable feedstock that is available in huge quantities. But their strength is also a disadvantage: they are not easily broken down in smaller, usable pieces. ‘To chemically degrade them, you need extreme conditions,’ Sybrin Schröder says. But what is difficult in industry, is common practice in many fungal species that grow on dead or living wood. They excrete enzymes that accelerate breakdown of sugar chains. Schröder: ‘Such enzymes would enable the industry to degrade polymers efficiently under prevailing conditions.’
How do you get hold of these enzymes? ‘Genomic research enables scientists to quickly sequence fungal genomes and identify genes encoding enzymes that help to cut down sugar chains. But you cannot tell under which conditions these enzymes are stable and functional. So you don’t know which enzymes are useful for industry,’ Casper de Boer states.
A better approach would be to catch enzymes while they are doing their job under whatever conditions desired. That can be done: a wood-living fungus species is grown on a substrate of cell wall polymers of interest. The fungus secretes a mixture of compounds that includes enzymes needed to digest the substrate. To this ‘secretome’ a probe is added: a molecule from which a part is designed to mimic a part of the sugar chain. To this part, targeted enzymes are attracted and irreversibly bound. The probe also bears a fluorescent dye. The mixture is then moved through an electrophoretic gel, and probe-enzyme addition products appear as fluorescent bands. With conventional chemical techniques, such as crystallography, the enzymes can be further characterised.
‘Our probe to search for biomass-degrading enzymes is now available to scientists, and several groups are already using it. The tool even marked the start of a new research field in our group.’
‘Similar methods to quickly discover and identify other enzymes are already being used,’ De Boer explains. ‘But until now, only probes with one single sugar unit were constructed. They attract enzymes that are active at the ends of sugar chains. We aimed to find enzymes that attack the middle of a chain. Such enzymes recognise a sequence of a few sugar units, and so we tried to design a probe with multiple sugar units instead of one.’ Schröder adds: ‘It was a challenge to construct such a new probe that is stable under desirable conditions. Many chemists even thought it would be impossible. But thanks to many colleagues from different disciplines who collaborated, we succeeded.’ A paper in ACS Central Science, which appeared in May 2019, is the result.
The team grew the well-known fungus Aspergillus niger on xylan, a polysaccharide that forms an important component of plant cell walls and is present in for instance wood and flour. With their newly designed probe, Schröder and De Boer indeed detected enzymes that help degrade xylan. ‘These enzymes were already known, but it is a proof of concept,’ De Boer says. ‘Our probe to search for this class of degrading enzymes from xylan is applicable to unknown enzymes as well. It is now available to scientists, and several groups are already using it now.’ Schröder adds: ‘In our group, this was the start of a new field of research for which a number of PhD students is already appointed.’
Sybrin Schröder (1989) earned his master’s degree in Chemistry at Radboud University Nijmegen; Casper de Boer (1991) studied Molecular Science and Technology at Leiden University and Delft University of Technology, followed by a master’s degree in Chemistry at Leiden University. The two of them met when doing their PhD research in Leiden, and it soon turned out that they shared an interest in understanding the structure and function of molecules in view of biological and industrial applications. And so they started to collaborate.