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Continuous low-level supply or in situ generation of hydrogen peroxide (H 2 O 2 ) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C–H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO 2 or carbon have sufficient activity at ambient temperature and pressure to generate H 2 O 2 from H 2 and O 2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled. Continuous low-level supply or in situ generation of hydrogen peroxide (H 2 O 2 ) is essential for the stability of unspecific peroxygenases. Here, the authors demonstrate that AuPd / TiO 2 can generate sufficient H 2 O 2 for the engineered unspecific heme-thiolate peroxygenase PaDa-I to oxidise a range of C-H bonds.

Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c , ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called “uncoupling”, whereby NAD(P)H derived electrons are lost due to the reduced intermediates’ (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from C andida tropicalis ( Ct CPR) lacking the hydrophobic N -terminal part (Δ2–22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c . The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both Ct CPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of Ct CPR.

Summary Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large‐scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self‐sufficient P450s for their potential as whole‐cell biocatalysts. Most P450s are, however, not self‐sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450‐reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450‐CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad‐range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole‐cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450‐based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole‐cell biocatalyst was emphasized, supporting earlier findings. Significant improvements in P450 activity by the co‐expression of an unrelated partner were demonstrated. Impressively high substrate bioconversion was observed using Arxula adeninivorans whole‐cell catalytic system, compared to other reported studies. Reinforced prior observations of the potential of A. adeninvorans as a P450‐expressing whole cell biocatalyst.

Preventing plastic shrinkage cracks improves the durability of concrete. This is because plastic shrinkage cracks serve as pathways by which corroding agents can penetrate concrete. Freshly cast concrete is a saturated mixture of reactive and non-reactive materials. As water moves out of the concrete mass and as water is used in the hydration process, the free water in the mixture reduces. Eventually, the mixture can be considered to be unsaturated. In this research project, the viability of using soil water content sensors to measure the change in water availability in concrete from fresh state to early-age was explored. The soil water content sensors measured dielectric permittivity. The dielectric permittivity, cumulative evaporation and setting time of mortars with varying water/cement ratios were tested. It was found that the dielectric constant was influenced by changes in fresh mortar and that the sensors have the potential to qualitatively monitor cement content, bleeding, hydration and evaporation. Further work is required in this field.

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability.

Background The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. Results Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/g DCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. Conclusions This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.

Curing is one of the most crucial phases of concrete conditioning as it determines not only the long-term strength, but also influences the durability. Curing compounds are useful for reducing the evaporation rate of water from the concrete surface. They are typically sprayed on concrete as soon as possible after consolidation, especially for concrete members that have large areas exposed to the environment. These compounds have been proven to work well, however, how effective are different curing compounds in a variety of high wind and high temperature with low relative humidity conditions? The focus of this work is the effectiveness of different types of curing compounds at windy and dry conditions. The tests were done in a state-of-the-art Mobile Climate Chamber (MCC) where the weight of all samples was measured constantly to determine the water loss. It was concluded that the resin-emulsion based curing compound performed best at all environmentally tested conditions. The acrylic-based curing compound performed worse than the control, where nothing was applied to the top surface.

The feasibility of using a single vector to clone a cytochrome P450 monooxygenase (P450) in different yeasts and then compare whole‐cell hydroxylase activity was investigated. A broad‐range yeast expression vector using the ylTEFp to drive expression of the cloned gene and the scTEFp to drive the hygromycin resistance marker gene was used to clone the genes encoding two self‐sufficient P450s, CYP102A1 and CYP505A1. Both genes were cloned into Saccharomyces cerevisiae, Kluyveromyces marxianus, Yarrowia lipolytica (two strains) and Arxula adeninivorans. 4‐Hexylbenzoic acid (HBA), which is subterminally hydroxylated by both CYP102A1 and CYP505A1, was used to compare whole‐cell hydroxylase activity of transformants. Kluyveromyces marxianus and A. adeninivorans exhibited activity with both CYP102A1 and CYP505A1, while S. cerevisiae only displayed CYP102A1 activity and Y. lipolytica only CYP505A1 activity. The highest CYP102A1 activity (0.8 mM HBA converted in 24 h) was observed with concentrated resting‐cell suspensions of S. cerevisiae. The CYP505A1 activity observed with growing cultures of A. adeninivorans was however at least 12 times higher than the CYP102A1 activity of S. cerevisiae with up to 2 mM HBA converted within 6 h. The use of K. marxianus and A. adeninivorans for P450 expression has not previously been reported.

CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85 μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6-0.8 μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5-1.0 μmol P450 g^sub DCW^^sup -1^, for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7 g octanol L^sub BRM^^sup -1^ was obtained within 24 h (0.34 g L^sub BRM^^sup -1^h^sup -1^) with IPTG-induced cells containing only 0.20 μmol P450 g^sub DCW^^sup -1^, when glucose (22 g L^sub BRM^^sup -1^ ) was added for cofactor regeneration.[PUBLICATION ABSTRACT]

Epoxide hydrolases are useful catalysts for the hydrolytic kinetic resolution of epoxides, which are sought after intermediates for the synthesis of enantiopure fine chemicals. The epoxide hydrolases from Aspergillus niger and from the basidiomycetous yeasts Rhodotorula glutinis and Rhodosporidium toruloides have demonstrated potential as versatile, user friendly biocatalysts for organic synthesis. A recombinant A. niger epoxide hydrolase, produced by an overproducing A. niger strain, is already commercially available and recombinant yeast epoxide hydrolases expressed in Escherichia coli have shown excellent results. Within the vast body of activity information on the one hand and gene sequence information on the other hand, the epoxide hydrolases from the Rhodotorula spp. and A. niger stand out because we have sequence information as well as activity information for both the wild-type and recombinant forms of these enzymes.