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1. Modifying Surface Charges of a Thermophilic Laccase Toward Improving Activity and Stability in Ionic Liquid

   https://doi.org/10.3389/fbioe.2022.880795  

   Stevens, Joseph C.; Shi, Jian (June 2022, Frontiers in Bioengineering and Biotechnology)

   The multicopper oxidase enzyme laccase holds great potential to be used for biological lignin valorization alongside a biocompatible ionic liquid (IL). However, the IL concentrations required for biomass pretreatment severely inhibit laccase activity. Due to their ability to function in extreme conditions, many thermophilic enzymes have found use in industrial applications. The thermophilic fungal laccase from Myceliophthora thermophila was found to retain high levels of activity in the IL [C 2 C 1 Im][EtSO 4 ], making it a desirable biocatalyst to be used for lignin valorization. In contrast to [C 2 C 1 Im][EtSO 4 ], the biocompatibility of [C 2 C 1 Im][OAC] with the laccase was markedly lower. Severe inhibition of laccase activity was observed in 15% [C 2 C 1 Im][OAc]. In this study, the enzyme surface charges were modified via acetylation, succinylation, cationization, or neutralization. However, these modifications did not show significant improvement in laccase activity or stability in [C 2 C 1 Im][OAc]. Docking simulations show that the IL docks close to the T1 catalytic copper, likely interfering with substrate binding. Although additional docking locations for [OAc] - are observed after making enzyme modifications, it does not appear that these locations play a role in the inhibition of enzyme activity. The results of this study could guide future enzyme engineering efforts by showing that the inhibition mechanism of [C 2 C 1 Im][OAc] toward M. thermophila laccase is likely not dependent upon the IL interacting with the enzyme surface. 

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2. 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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3. Single amino acid bionanozyme for environmental remediation

   https://doi.org/10.1038/s41467-022-28942-0  

   Makam, Pandeeswar; Yamijala, Sharma S. R. K. C.; Bhadram, Venkata S.; Shimon, Linda J. W.; Wong, Bryan M.; Gazit, Ehud (March 2022, Nature Communications)

   Abstract Enzymes are extremely complex catalytic structures with immense biological and technological importance. Nevertheless, their widespread environmental implementation faces several challenges, including high production costs, low operational stability, and intricate recovery and reusability. Therefore, the de novo design of minimalistic biomolecular nanomaterials that can efficiently mimic the biocatalytic function (bionanozymes) and overcome the limitations of natural enzymes is a critical goal in biomolecular engineering. Here, we report an exceptionally simple yet highly active and robust single amino acid bionanozyme that can catalyze the rapid oxidation of environmentally toxic phenolic contaminates and serves as an ultrasensitive tool to detect biologically important neurotransmitters similar to the laccase enzyme. While inspired by the laccase catalytic site, the substantially simpler copper-coordinated bionanozyme is ∼5400 times more cost-effective, four orders more efficient, and 36 times more sensitive compared to the natural protein. Furthermore, the designed mimic is stable under extreme conditions (pH, ionic strength, temperature, storage time), markedly reusable for several cycles, and displays broad substrate specificity. These findings hold great promise in developing efficient bionanozymes for analytical chemistry, environmental protection, and biotechnology. 

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4. Evaluation of anammox biocathode in microbial desalination and wastewater treatment

   https://doi.org/https://doi.org/10.1016/j.cej.2018.02.088  

   Bahareh Kokabian, Veera Gnaneswar (June 2018, Chemical engineering journal)

   This study presents the use of an autotrophic microorganism, Anammox bacteria, as a sustainable biocatalyst/biocathode in microbial desalination cells (MDCs) for energy-positive wastewater treatment. We report the first proof of concept study to prove that anammox mechanism can be beneficial in MDCs to provide simultaneous removal of carbon and nitrogen compounds from wastewater while producing bioelectricity. A series of experiments were conducted to enrich and evaluate the anammox mechanism and the process performance in continuous, fed-batch mode conditions. Coulombic efficiency of MDCs and nitrite and ammonium removal of wastewater increased in successive batch studies. A maximum power density of 0.092 Wm−3 (or a maximum current density of 0.814 A m−3) with more than 90% of ammonium removal was achieved in this system. We calculated the Nernst potential for the nitrite reduction in the anammox biocathode chamber and compared with experimental values. Sequential removal of carbon and nitrogen compounds in anode and cathode chambers respectively, was also evaluated. Further, the inhibition effect of high nitrogen concentrations and the variations in microbial community profiles, especially, anammox presence was studied at different carbon and ammonia concentrations. Experimental studies and microbial community analysis are presented in detail. 

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5. The challenge of non-reactive phosphorus: Mechanisms of treatment and improved recoverability using electrooxidation

   https://doi.org/10.1016/j.jece.2023.110295  

   Mallick, Synthia Parveen; Hossain, Mohammad Shakhawat; Takshi, Arash; Call, Douglas F; Mayer, Brooke K (October 2023, Journal of Environmental Chemical Engineering)

   Recalcitrant phosphorus (P) species, i.e., soluble non-reactive phosphorus (sNRP), are generally not effectively removed or recovered in conventional wastewater treatment processes. This was substantiated in our meta-analysis, which showed that nearly one-third of wastewater facilities’ effluent P was primarily in the non-reactive form. Transformation of sNRP to more readily removable/recoverable soluble reactive phosphorus (sRP) may offer a viable pathway to enhance P removal and recovery. Electrooxidation (EO) may offer one route for sNRP to sRP transformation. During EO, different sNRP transformation pathways may occur, influencing the extent and efficiency of sNRP transformations as a function of water quality. To explore these mechanisms, we conducted oxidant quenching tests as well as cyclic voltammetry and chronoamperometry experiments using a synthetic water matrix spiked with the sNRP compound beta-glycerol phosphate (BGP). We found that direct electron transfer was responsible for BGP transformation. To assess the applicability of EO for wastewater sNRP to sRP transformation and improved recoverability, EO was used to treat municipal wastewater centrate, followed by tests of sNRP recoverability using the P-selective LayneRT™ ion exchanger. Complete transformation of centrate sNRP to sRP was not achieved with EO, but subsequent removal of sNRP using ion exchange increased after 2 hr of EO treatment. Longer periods of EO treatment did not improve sNRP removal. Improved sNRP adsorption after EO was likely due to decreased competing organics in the centrate after EO treatment. Overall, this study showed that EO can improve sNRP removal using subsequent ion exchange and facilitate enhanced P recovery. 

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