TY - JOUR
T1 - A screening approach for assessing lytic polysaccharide monooxygenase activity in fungal strains
AU - Dixit, Pooja
AU - Basu, Biswajit
AU - Puri, Munish
AU - Tuli, Deepak Kumar
AU - Mathur, Anshu Shankar
AU - Barrow, Colin James
PY - 2019/7/22
Y1 - 2019/7/22
N2 - Background: Efforts to develop efficient lignocellulose-degrading enzymatic preparations have led to the relatively recent discovery of a new class of novel cellulase boosters, termed lytic polysaccharide monoxygenases (LPMOs). These enzymes are copper-dependent metalloenzymes that initiate the biomass deconstruction process and subsequently work together with cellulases, hemicellulases, and other accessory enzymes to enhance their hydrolytic action. Given their wide distribution and diversity, screening and isolation of potent LPMOs from natural fungal diversity may provide an important avenue for increasing the efficiency of cellulases and thereby decreasing cellulosic ethanol production costs. However, methods for quick screening and detection are still not widely available. In this article, a simple and sensitive method is described by combining nonhydrolytic activity enhancement followed by LC-MS-based quantitation of LPMOs. Results: In this study, a screening approach has been developed for the detection of nonhydrolytic cellulase-enhancing enzymes in crude fungal supernatants. With the application of a saturating benchmark cocktail of Celluclast 1.5L, fungal isolates were selected which had the capability of hydrolyzing pretreated rice straw by their synergistic enzyme fractions. Subsequently, these fungal extracts along with an LPMO-enriched commercial enzyme were investigated for their ability to produce Type I LPMO activity. An LC-MS-based methodology was applied to quantitate gluconic acid in enzymatic hydrolysates as an indirect measurement of Type I LPMO activity. Conclusion: The present study describes an LC-MS-based separation method to detect and quantitate LPMO activity in a commercial enzyme. This method was also applied to screen fungal extracts. The developed screening strategy has enabled detection of LPMO activity in two industrially important Penicillium strains.
AB - Background: Efforts to develop efficient lignocellulose-degrading enzymatic preparations have led to the relatively recent discovery of a new class of novel cellulase boosters, termed lytic polysaccharide monoxygenases (LPMOs). These enzymes are copper-dependent metalloenzymes that initiate the biomass deconstruction process and subsequently work together with cellulases, hemicellulases, and other accessory enzymes to enhance their hydrolytic action. Given their wide distribution and diversity, screening and isolation of potent LPMOs from natural fungal diversity may provide an important avenue for increasing the efficiency of cellulases and thereby decreasing cellulosic ethanol production costs. However, methods for quick screening and detection are still not widely available. In this article, a simple and sensitive method is described by combining nonhydrolytic activity enhancement followed by LC-MS-based quantitation of LPMOs. Results: In this study, a screening approach has been developed for the detection of nonhydrolytic cellulase-enhancing enzymes in crude fungal supernatants. With the application of a saturating benchmark cocktail of Celluclast 1.5L, fungal isolates were selected which had the capability of hydrolyzing pretreated rice straw by their synergistic enzyme fractions. Subsequently, these fungal extracts along with an LPMO-enriched commercial enzyme were investigated for their ability to produce Type I LPMO activity. An LC-MS-based methodology was applied to quantitate gluconic acid in enzymatic hydrolysates as an indirect measurement of Type I LPMO activity. Conclusion: The present study describes an LC-MS-based separation method to detect and quantitate LPMO activity in a commercial enzyme. This method was also applied to screen fungal extracts. The developed screening strategy has enabled detection of LPMO activity in two industrially important Penicillium strains.
KW - AA9 LPMOs
KW - Acid-treated rice straw
KW - Biorefinery
KW - Cellobionic acid
KW - Cellulose
KW - Gluconic acid
KW - Hemicellulose
KW - Penicillium sp. LPMOs
KW - Synergy in cellulases
KW - UPLC-ESI-MS
UR - http://www.scopus.com/inward/record.url?scp=85069891483&partnerID=8YFLogxK
U2 - 10.1186/s13068-019-1526-4
DO - 10.1186/s13068-019-1526-4
M3 - Article
AN - SCOPUS:85069891483
SN - 1754-6834
VL - 12
JO - Biotechnology for Biofuels
JF - Biotechnology for Biofuels
IS - 1
M1 - 185
ER -