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

   https://doi.org/10.3389/FENRG.2024.1372916  

   Nemmaru, Bhargava; Douglass, Jenna; Yarbrough, John M; DeChellis, Antonio; Shankar, Srivatsan; Thokkadam, Alina; Wang, Allan; Chundawat, Shishir_P S (July 2024, Frontiers in Energy Research)

   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 (fromThermobifida fusca) fused with endocellulase Cel5A (fromT. 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 inE. 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. 

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2. Ammonia-salt solvent promotes cellulosic biomass deconstruction under ambient pretreatment conditions to enable rapid soluble sugar production at ultra-low enzyme loadings

   https://doi.org/10.1039/C9GC03524A  

   Chundawat, Shishir P.; Sousa, Leonardo da; Roy, Shyamal; Yang, Zhi; Gupta, Shashwat; Pal, Ramendra; Zhao, Chao; Liu, Shih-Hsien; Petridis, Loukas; O'Neill, Hugh; et al (January 2020, Green Chemistry)

   Here, we report a novel ammonia : ammonium salt solvent based pretreatment process that can rapidly dissolve crystalline cellulose into solution and eventually produce highly amorphous cellulose under near-ambient conditions. Pre-activating the cellulose I allomorph to its ammonia–cellulose swollen complex (or cellulose III allomorph) at ambient temperatures facilitated rapid dissolution of the pre-activated cellulose in the ammonia-salt solvent ( i.e. , ammonium thiocyanate salt dissolved in liquid ammonia) at ambient pressures. For the first time in reported literature, we used time-resolved in situ neutron scattering methods to characterize the cellulose polymorphs structural modification and understand the mechanism of crystalline cellulose dissolution into a ‘molecular’ solution in real-time using ammonia-salt solvents. We also used molecular dynamics simulations to provide insight into solvent interactions that non-covalently disrupted the cellulose hydrogen-bonding network and understand how such solvents are able to rapidly and fully dissolve pre-activated cellulose III. Importantly, the regenerated amorphous cellulose recovered after pretreatment was shown to require nearly ∼50-fold lesser cellulolytic enzyme usage compared to native crystalline cellulose I allomorph for achieving near-complete hydrolytic conversion into soluble sugars. Lastly, we provide proof-of-concept results to further showcase how such ammonia-salt solvents can pretreat and fractionate lignocellulosic biomass like corn stover under ambient processing conditions, while selectively co-extracting ∼80–85% of total lignin, to produce a highly digestible polysaccharide-enriched feedstock for biorefinery applications. Unlike conventional ammonia-based pretreatment processes ( e.g. , Ammonia Fiber Expansion or Extractive Ammonia pretreatments), the proposed ammonia-salt process can operate at near-ambient conditions to greatly reduce the pressure/temperature severity necessary for conducting effective ammonia-based pretreatments on lignocellulose. 

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3. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass

   https://doi.org/10.1021/acssuschemeng.3c06266  

   DeChellis, Antonio; Nemmaru, Bhargava; Sammond, Deanne; Douglass, Jenna; Patil, Nivedita; Reste, Olivia; Chundawat, Shishir_P S (March 2024, ACS Sustainable Chemistry & Engineering)

   Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates. 

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4. Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity

   https://doi.org/10.3390/microorganisms8060871  

   Govil, Tanvi; Saxena, Priya; Samanta, Dipayan; Singh, Sindhu Suresh; Kumar, Sudhir; Salem, David R.; Sani, Rajesh K. (June 2020, Microorganisms)

   null (Ed.)

   The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain. 

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5. Abstract 1749 Probing the Active Site of Guanylurea Hydrolase: Insights into Catalysis and Substrate Specificity

   https://doi.org/10.1016/j.jbc.2025.109145  

   Abdi, Hanan; Tutak, Kennedy; Martinez-Vaz, Betsy (May 2025, Journal of Biological Chemistry)

   Metformin is one of the most regularly prescribed Type II diabetes drugs in the world, and its use is likely to expand as diabetes diagnoses rise globally. This drug and its main degradation byproduct, guanylurea, are not fully metabolized by humans and cannot be removed through conventional water treatment processes. These compounds have been detected in coastal waters around the world and are currently considered emerging pollutants. The goal of this research was to examine the catalytic mechanism and substrate specificity of Guanylurea Hydrolase (GuuH), a recently discovered enzyme that converts guanylurea to ammonia and guanidine. Bioinformatic analyses were conducted to predict the active site and three-dimensional structure of GuuH. Site-directed mutagenesis was performed to construct mutants in amino acids predicted to be part of the enzyme's catalytic triad and substrate binding site. The mutants created were K138R, N141K, E211D, E211Q, and E211N. The wild-type and mutant enzymes were purified using His-tag affinity chromatography. Enzyme activity was assessed by measuring ammonia released using Berthelot assays. The results showed that the K138R mutant had similar specific activity compared to the wild-type GuuH when reacting with guanylurea, while E211N and E221D showed low specific activity under the same conditions. All of the enzymes had no detectable activity when reacting with biuret, which suggests they have low affinity for this substrate. Future work will focus on kinetic analyses of the wild-type and K138R enzymes and additional mutagenesis to identify the amino acids that determine the substrate specificity to the enzyme. Understanding GuuH's catalytic activity and substrate specificity is essential to using this enzyme in the development of biotechnological applications for water treatment. 

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