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Publisher Summary This chapter describes the engineering of the thermotolerance and pH optimum of family 11 xylanases by site-directed mutagenesis. The potential of using xylanases in industrial applications generated an intensive academic and industrial activity to hunt xylanases with desired properties from all kinds of natural sources. The thermostability of mesophilic xylanases has been increased considerably by designed mutations. T. reesei xylanase II (XYNII) is stable at 40–45°C, whereas temperatures above 50°C cause conformational changes and the enzyme activity is lost quite quickly. A combination of various mutations has often a cumulative effect on the thermostability of family 11 xylanases. It is observed that when mutations with only a small stabilizing effect were combined with the disulfide bridge engineered into α helix, a considerable increase in thermostability was achieved. It is found that xylanase activity is determined by using the 3,5-dinitrosalicylic acid assay to measure the amount of reducing sugars liberated from 1% birchwood xylan.

Synthesis of bis-4,5-diazafluoren-9-one silver(I) nitrate I (dafone = 4,5-diazafluoren-9-one) and the low temperature X-ray single crystal structure of [Ag(4,5-diazafluoren-9-one)2NO3], crystal form 1, and a re-determination of [Ag(4,5-diazafluoren-9-one)2]NO3·H2O, crystal form 2 are presented. Crystal form 1 has a distorted trigonal planar coordination geometry around Ag(I) with an N–Ag–N bond angle of 123.45(7)°. Crystal form 2 has a perfect linear coordination around Ag, with N–Ag–N 180.0°. Compound I was characterized by 1H-NMR, biological activity and ESI-MS in DMSO at room temperature. The biological activity was determined against 6 different resistant clinical isolates; two Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and four Gram-negative (Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella sp.) in comparison with 15 known antibiotics used in the treatment of diabetic foot infections. Compound I showed broad spectrum activity against all the test organisms. P. mirabilis and S. aureus and K. pneumoniae were the most sensitive clinical isolates (MIC = 4, 6 and 4 μg ml−1, respectively). Three different hydrogels containing I or Ag2SO4 were prepared and the antimicrobial activity against Ps. aeruginosa (ATCC 15442) compared, showing more or less equal activity on a weight basis, but I seems to have a significant better performance per silver ion. The Ag(I) complex also binds more effectively to calf thymus DNA than the dafone ligand itself.

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4-->3) that lacks the loop between beta-strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4-->3 even further, we mutated asparagine 118 on the bottom of the ligand-binding pocket to methionine, which simultaneously caused a significant drop in biotin-binding affinity. The X-ray structure of cpAvd4--> 3(N118M) allows an understanding of the effect of mutation to biotin-binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion-fold between wild-type avidin and cpAvd4-->3(N118M). To demonstrate the versatility of the cpAvd4-->3 construct, we have shown that it is possible to link cpAvd4-->3 and cpAvd5-->4 to form the dual-chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self-organising nanoscale avidin building blocks.

The pyrolysis of scrap tires is a very attractive strategy to valorize chemically these end-of-life wastes. The products of this step and any additional one, such as hydrotreating, are relatively complex in nature entangling the understanding and limiting the viability. In this work, we have investigated in detail the composition of a tire pyrolysis oil blended with light cycle oil (from a refinery) and its hydrotreated products using a bifunctional NiW/HY catalyst at 320-400 °C. We have applied a set of analytical techniques to assess the composition, namely simulated distillation, ICP, GC/FID-PFPD, GC × GC/MS, and APPI FT-ICR/MS. Our results show the strength of our analytical workflow to highlight the compositional similarities of this pyrolysis oil with the standard refinery streams. The main differences arise from the higher boiling point species (originated during the pyrolysis of tires) and relatively high concentration of oxygenates. These effects can be minimized by hydrotreating the feed which effectively removes heteroatomic compounds from the feed while boosting the quantity and quality of gasoline and diesel fractions.

Extractives are an important class of compounds in plants because they contribute to many of their physicochemical properties such as color, odor, density, strength, permeability, and hygroscopicity. Moreover, they also possess significant biological activity and are thus an important part of the plants’ defense mechanisms against biotic and abiotic stresses. Tree needles are a rich source of extractives, counting for as much as 40% of their dry weight. In this study, chemical fingerprinting of essential oils and solvent extracts, obtained from the needles of four conifer tree species (i.e., pine, spruce, larch, and juniper), was performed by using ultrahigh-resolution Fourier transform ion cyclotron mass spectrometry. A wide variety of compounds were detected in the oil samples, including mono-, sesqui-, and diterpenes, terpenoids, fatty and resin acids, esters, and different phenolic compounds. Although the main compounds were present in all the four essential oil samples, large variations in their relative abundances were observed. In contrast, pine needle hexane and toluene extracts showed a high content of resin acids, including pinifolic acid, a rare labdane-type diterpene diacid, and its mono- and dimethyl esters. Thus, by selecting a suitable solvent, specific types of compounds may be isolated from tree needles for further biotechnological or medicinal applications.

Predicting the hydrotreating performance of industrial catalysts used for upgrading heavy oils is hampered by the unknown chemistry behind it. In this work, we have used a set of chromatographic and mass spectrometric techniques (APPI/ESI FT-ICR MS, FID-MS GC × GC and PFPD GC) for acquiring a more precise composition of the feed and products of the hydrotreatment of a blend of light cycle oil and scrap tire oil (20 vol%) using three benchmark catalysts: CoMo/Al2O3, NiMo/SiO2-Al2O3 and NiW/USY zeolite. Despite the different nature of the catalysts, the composition of the products was relatively similar, indicating the slower and controlled transformation of the heaviest molecules of the feed, particularly in tire oil. A faithful analysis of these molecules by combining the results of the analysis clarifies the multiple mechanisms affecting hydrotreating simultaneously: hydrodearomatization, hydrocracking, hydrodesulfurization, hydrodeoxygenation and hydrodenitrification. An effort has been made to use these results in a quantitative manner for catalyst screening.