From the Discovery of Extremozymes to an Enzymatic Product: Roadmap Based on Their Applications

With the advent of the industrial revolution, the use of toxic compounds has grown exponentially, leading to a considerable pollution of the environment. Consequently, the development of more environmentally conscious technologies is an urgent need. Industrial biocatalysis appears as one potential solution, where a higher demand for more robust enzymes aims to replace toxic chemical catalysts. To date, most of the commercially available enzymes are of mesophilic origin, displaying optimal activity in narrow ranges of temperature and pH (i.e., between 20°C and 45°C, neutral pH), limiting their actual application under industrial reaction settings, where they usually underperform, requiring larger quantities to compensate loss of activity. In order to obtain novel biocatalysts better suited for industrial conditions, an efficient solution is to take advantage of nature by searching and discovering enzymes from extremophiles. These microorganisms and their macromolecules have already adapted to thrive in environments that present extreme physicochemical conditions. Hence, extremophilic enzymes stand out for showing higher activity, stability, and robustness than their mesophilic counterparts, being able to carry out reactions at nonstandard conditions. In this brief research report we describe three examples to illustrate a stepwise strategy for the development and production of commercial extremozymes, including a catalase from an Antarctic psychrotolerant microorganism, a laccase from a thermoalkaliphilic bacterium isolated from a hot spring and an amine-transaminase from a thermophilic bacterium isolated from a geothermal site in Antarctica. We will also explore some of their interesting biotechnological applications and comparisons with commercial enzymes.

Liability for robots II: an economic analysis

This is the second of two companion papers that discuss accidents caused by robots. In the first paper (Guerra et al., 2021), we presented the novel problems posed by robot accidents, and assessed the related legal approaches and institutional opportunities. In this paper, we build on the previous analysis to consider a novel liability regime, which we refer to as ‘manufacturer residual liability’ rule. This makes operators and victims liable for accidents due to their negligence – hence, incentivizing them to act diligently; and makes manufacturers residually liable for non-negligent accidents – hence, incentivizing them to make optimal investments in R&D for robots' safety. In turn, this rule will bring down the price of safer robots, driving unsafe technology out of the market. Thanks to the percolation effect of residual liability, operators will also be incentivized to adopt optimal activity levels in robots' usage.

Sulfonamide-salicylaldehyde imines active against methicillin- and trimethoprim/sulfonamide-resistant Staphylococci

Background: Increasing resistance has resulted in an urgent need for new antimicrobial drugs. A systematic me-too approach was chosen to modify clinically used sulfonamides to obtain their imines. Methods & results: Twenty-five compounds were synthesized and evaluated for their antibacterial activity. The most active compounds were also investigated against methicillin- and trimethoprim/sulfamethoxazole (SMX)-resistant Gram-positive species. Staphylococci shared the highest susceptibility including resistant strains with minimum inhibitory concentrations from 3.91 μM (≥2.39 μg ml-1). Crucially, the compounds inhibit MRSA and trimethoprim/SMX-resistant Staphylococci without any cross-resistance. Modification of parent sulfonamides turned a bacteriostatic effect into a bactericidal effect. Toxicity for HepG2 and hemolytic properties were also determined. Conclusions: The presence of a dihalogenated salicylidene moiety is required for optimal activity. Based on toxicity, promising derivatives for further investigation were identified.

Kinetics of Crude Peroxidase from the Rind of Watermelon Fruit

Aims: To study the kinetics of crude peroxidase from the rind of watermelon fruit in various assay conditions. Study Design: In vitro enzyme assay. Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between October 2015 and January 2016. Methodology: The activity of the crude peroxidase extracted from the rind of watermelon was determined by measuring the rate of oxidation of KI at 25oC in a 3.0 ml reaction mixture which contained 2.3 ml of 25 mM - 400 mM sodium acetate buffer (pH 3.5-6.0), 0.2 ml of 2 mM KI, 0.1ml of the crude peroxidase, and 0.2 ml of varying concentrations of chlorpromazine (0.01 mM - 0.1 mM). In all cases, 0.2 ml of 0.01 mM – 1 mM H2O2 was added last to initiate the reaction. Only one parameter was varied per assay. Assays were done in five replicates. The initial velocity of the crude peroxidase for KI oxidation was determined using the absorbance at 353 nm. Results: The concentration of H2O2 that generated an optimal activity for the crude peroxidase extracted was 0.2 mM, while a pH of 5.5 was optimal for the crude enzyme. The activity of the crude enzyme increased proportionately within a buffer concentration range of 25 mM and 400 mM. Chlorpromazine (0.01 mM - 0.1 mM) proportionately increased the enzyme activity, while promethazine within a range of 0.01 mM and 0.06 mM proportionally increased the enzyme activity. Further increase in promethazine concentration beyond 0.6 mM resulted in a decreased activity of the enzyme. Conclusion: This study suggests that the Rind of watermelon is an alternative source of peroxidase. The activity of this peroxidase can be enhanced by high buffer concentrations in the presence of some redox mediators like promethazine and chlorpromazine at a pH of 5.5.

Isolation and Study of Biodegradation Capability of Hydrocarbonoclastic Bacteria from Industrial Waste Lubrication Oil Contaminated Sites

A research was carried out on Isolation and study of biodegradation capability of hydrocarbonoclastic bacteria from industrial waste lubricant oil contaminated sites. 12 different bacterial cultures are isolated from the soil samples collected from liquid effluent dump site of black galaxy granite, chimakurthy, prakasam Dist, and auto motive lubrication oil replacement garages, Auto Nagar, Vijayawada. Among the 12isolates 10 isolates were found to have capability of degrading used industrial lubrication oil VG 320 collected from NSL textiles LTD, Guntur. Among the isolates tested the isolate no BG4 obtained from liquid effluent dump site gave the maximum biodegradation potential of 18.78mm2followed by BG7 of 15.36, BG1 of 14.07and BG8 of 10.56 mm2 and the isolate no AL1 from automotive lubrication oil replacement garage soil sample found to be 9.03mm2 degradation potential. These isolates were identified based on physical and biochemical characters as Pseudomonas, Bacillus, Proteus, Flavobacterium and Enterococcussps respectively. For the future, our study will be focused on several data like the species of isolates, the optimal activity of isolates to degrade several Industrial lubricant oils.

Beauveria bassiana Xylanase: Characterization and Wastepaper Deinking Potential of a Novel Glycosyl Hydrolase from an Endophytic Fungal Entomopathogen

Beauveria bassiana is an entomopathogenic fungus widely used as a biopesticide for insect control; it has also been shown to exist as an endophyte, promoting plant growth in many instances. This study highlights an alternative potential of the fungus; in the production of an industrially important biocatalyst, xylanase. In this regard, Beauveria bassiana SAN01 xylanase was purified to homogeneity and subsequently characterized. The purified xylanase was found to have a specific activity of 324.2 Umg−1 and an estimated molecular mass of ~37 kDa. In addition, it demonstrated optimal activity at pH 6.0 and 45 °C while obeying Michaelis–Menton kinetics towards beechwood xylan with apparent Km, Vmax and kcat of 1.98 mgmL−1, 6.65 μM min−1 and 0.62 s−1 respectively. The enzyme activity was strongly inhibited by Ag2+ and Fe3+ while it was significantly enhanced by Co2+ and Mg2+. Furthermore, the xylanase was shown to effectively deink wastepaper at an optimal rate of 106.72% through its enzymatic disassociation of the fiber-ink bonds as demonstrated by scanning electron microscopy and infrared spectroscopy. This is the first study to demonstrate the biotechnological application of a homogeneously purified glycosyl hydrolase from B. bassiana.

A Thermophilic GH5 Endoglucanase from Aspergillus fumigatus and Its Synergistic Hydrolysis of Mannan-Containing Polysaccharides

In this study, we isolated and identified a thermophilic strain of Aspergillus fumigatus from the “Daqu” samples. Transcriptomic analysis of A. fumigatus identified 239 carbohydrate-active enzymes (CAZy)-encoding genes, including 167 glycoside hydrolase (GH)-encoding genes, 58 glycosyltransferase (GT)-encoding genes, 2 polysaccharide lyase (PLs)-encoding genes and 12 carbohydrate esterase (CEs)-encoding genes, which indicates that the strain has a strong potential for application for enzyme production. Furthermore, we also identified a novel endoglucanase gene (AfCel5A), which was expressed in Pichia pastoris and characterized. The novel endoglucanase AfCel5A exhibited the highest hydrolytic activity against CMC-Na and the optimal activity at 80 °C and pH 4.0 and also showed good stability at pH 3.0–11.0 and below 70 °C. The Km and Vmax values of AfCel5 were 0.16 ± 0.05 mg·mL−1 and 7.23 ± 0.33 mol mg−1·min−1, respectively, using CMC-Na as a substrate. Further, the endoglucanase exhibited a high tolerance toward NaCl as well as glucose. In addition, the finding that the endoglucanase AfCel5A in combination with β-mannanse (ManBK) clearly increased the release of total reducing sugars of glucomannan by up to 74% is significant.

Effect of the single mutation N9Y on the catalytical properties of xylanase Xyn11A from Cellulomonas uda: a biochemical and molecular dynamic simulation analysis

Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 ºC in the optimum temperature, and an upward shift of one unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 ºC, compared to that displayed by Xyn11A-WT. Molecular dynamics (MD) simulations of Xyn11A-WT and Xyn11A-N9Y suggest the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.