Identification of proteins involved in lignocellulose degradation using in gel zymogram analysis combined with mass spectroscopy-based peptide analysis of gut proteins from larval Asian longhorned beetles, Anoplophora glabripennis

Enzyme activities toward lignocellulose substrates were analyzed in the gut of larval Asian longhorned beetle (Anoplophora glabripennis). Total protein was extracted from gut contents of wild collected larvae from an invasive population in Worchester, MA, USA. From these protein extracts, lignocellulolytic activities were measured (β-1,4- endoglucanase, β-1,4-glucosidase and birch wood xylanase). β-1,4-glucosidase activity was 0.075 μmol glucose/mg protein per min, endoglucanase activity was measured at 0.41 μmol glucose/mg protein per min and xylanase activity was 0.058 μmol xylose/mg protein per min. To identify specific enzymes that may provide these activities, zymo- gram analysis was performed to detect enzymes active toward carboxymethyl cellulose (CMC), 4-methylumbelliferyl-β-D-glucopyranoside and birch wood xylan. Three protein bands were found to be active toward CMC, three displayed β-1,4-glucosidase, and one displayed xylanase activity. Proteins from active bands from these zymograms were then identifiedbyin-geltrypsindigestionsfollowedbypeptideidentificationbymatrix-assisted laser desorption ionization - time of flight - time of flight mass spectrometry (MS). A custom A. glabripennis transcriptome database was used for peptide identification, giv- ing highly significant matches in all MS analyses. These matches were then searched against the National Center for Biotechnology Information database to provide annotation to the transcripts and provide possible classification. From these analyses, we were able to detect enzymes active toward cellulose and xylan, and proteins putatively involved in lignocellulose degradation in the gut of this wood-feeding insect. Future research will be focused on characterizing these enzymes through cloning and expression experiments and understandinghowthelignocellulosedegradationsystemfunctionsinthegutofthisinsect.