Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

Nemmaru, Bhargava; Douglass, Jenna; Yarbrough, John M.; DeChellis, Antonio; Shankar, Srivatsan; Thokkadam, Alina; Wang, Allan; Chundawat, Shishir S.

doi:10.3389/fenrg.2024.1372916

Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

Journal Article · Mon Jul 01 00:00:00 EDT 2024 · Frontiers in Energy Research

DOI:https://doi.org/10.3389/fenrg.2024.1372916· OSTI ID:2434299

Nemmaru, Bhargava ^[1]; Douglass, Jenna ^[1]; Yarbrough, John M. ^[2]; DeChellis, Antonio ^[1]; Shankar, Srivatsan ^[1]; Thokkadam, Alina ^[1]; Wang, Allan ^[1]; ^[1]

Rutgers Univ., Piscataway, NJ (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca) fused with endocellulase Cel5A (from T. fusca) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning -14 to +6. These enzymes were recombinantly expressed in E. coli, purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (~3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.

View Accepted Manuscript (DOE)

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF); USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Grant/Contract Number:: AC36-08GO28308; 1604421; 1846797; FC02-07ER64494

OSTI ID:: 2434299

Report Number(s):: NREL/JA-2700-90999; MainId:92777; UUID:471107de-106a-4971-b28c-4d148caffc3e; MainAdminId:73434

Journal Information:: Frontiers in Energy Research, Vol. 12; ISSN 2296-598X

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

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09 BIOMASS FUELS
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
cellulase
carbohydrate-binding module
computational protein design
thermal stability
enzyme supercharging
lignin inhibition
non-productive binding
cellulosic biomass hydrolysis

Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

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