Enzyme technology for next generation biofuels

The project's overall goal was to develop expertise in enzymatic degradation of lignocellulose-rich biomass with special focus on development of enzyme mixtures and processes for biomass of the type produced by Borregaard from Norwegian spruce. This means that we worked with pretreated raw materials where some non-cellulose components (lignin, hemicellulose) are removed. This means that one probably can use enzyme mixtures containing only a few components, rather than mixtures of very many components found in commercial enzyme mixtures. If it becomes possible to use tailored few-component compositions it will be easier to make such compositions more thermostable, which has technical process advantages. It was important in this project to test enzymes with "real" substrates, ie industrially relevant substrates. The project had a special focus on so-called "lytic polysaccharides monooxygenases" (LPMOs; also called "GH61" or "CBM33") which were discovered by NMBU in 2010 and which have proven to be of great importance for the efficiency of current commercial enzyme mixtures for cellulose degradation. In this project we have studied these enzymes and we have examined the extent to which these enzymes will be important in future few-component mixtures. It is relatively difficult to produce these (and other relevant) enzymes, and much time has been used on this. Fortunately, we have had a collaboration with a group in Austria, which has complementary interests, giving us access torelevant enzymes, including 5 different GH61-type LPMOs. We have worked with cloning, production and purification of these exciting enzymes, both in Austria and in our lab. The enzymes were tested for activity, first in the laboratory, with model substrates, and then under realistic (ie industrially relevant) conditions. We have generated several groundbreaking results. We have discovered new LPMOs with new features and possibly new applications, we have found that not all LPMOs suitable for all saccharification purposes and we have identified LPMOs which may be of particular interest to Borregaard processes. We have also developed knowledge of how process conditions affect LPMO activity and thus the effectiveness of all the enzymatic process. The project has been aligned with a project called New Norwegian Biorefinery (NFR 219 633) which was led by Borregaard. The publication record of the project is good and there has been considerable knowledge transfer to business partner and industry in general.

Effective biochemical conversion of lignocellulosic biomass to biofuels depends on a combination of optimal thermochemical pretreatments followed by efficient enzymatic depolymerisation of biomass polysaccharides. Despite recent progress, the enzymatic co nversion of many lignocellulosic biomasses is still insufficiently efficient and there is an urgent need to find better processes, enzymes and enzyme blends. In this project, we will develop innovative new processes for saccharification of biomass by comb ining leading research on biomass enzymology with proprietary pretreatment technologies developed by Borregaard. Borregaard?s processing strategies, aimed at fully exploiting all fractions of the biomass, lead to production of cellulose-enriched lignin-fr ee pulps that are an excellent test case for developing novel enzyme blends that are simpler than current commercial blends. We will optimize the enzymatic conversion of Borregaard?s substrates and of a steam-exploded biomass for reference, using commerci al enzyme preparations as well as blends of monocomponent enzymes. We will have special focus on enzymes classified as CBM33 and GH61, whose function and huge potential have recently been discoverd by the applicant. We will identify speed- and/or yield-li miting factors (enzymes) in the commercial and home made blends and use this to optimize blends and identify targets for further research (optimizing specific enzyme activities, stability engineering). Eventually, this approach will lead to identification of "minimal enzyme blends" (i.e. containing only few enzyme activities) for cellulose-rich substrates, such as the Borregaard substrates. In addition, novel fundamental insights into enzymatic cellulose conversion will be obtained. Interestingly, improvi ng the thermostability of such "minimal blends" is easier than improving the thermostability of multi-component blends and this may open up possibilities to run processes at higher temperatures.