PNNL

Abstract

Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment are lacking. In this study, biological small angle neutron scattering (BioSANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, namely PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from eucalyptus and spruce wood samples. Whereas the BioSANS analysis of spruce holocellulose revealed an increase in interfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of eucalyptus holocellulose revealed a reduction in packing number following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi ?-barrel (DPBB) domain surrounded by three ?-helices - the largest nestled against the DPBB core and the other two part of loops extending from the core.