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The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARSCoV- 2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment. [ABSTRACT FROM AUTHOR]

A type D ferulic acid esterase (FAE) was identified in the culture supernatant of Streptomyces werraensis, purified, sequenced, and heterologously produced in E. coli BL21(DE3)Star by co-expressing chaperones groES-groEL (69 U L). The unique enzyme with a mass of about 48 kDa showed no similarity to other FAEs, and only moderate homology (78.5%) to a Streptomycete β-xylosidase. The purified reSwFAED exhibited a temperature optimum of 40 °C, a pH optimum in the range from pH seven to eight and a clear preference for bulky natural substrates, such as 5-O- trans-feruloyl- l-arabinofuranose (FA) and β- d-xylopyranosyl-(1→2)-5-O- trans-feruloyl- l-arabinofuranose (FAX), compared to the synthetic standard substrate methyl ferulate. Treatment of wheat dough with as little as 0.03 U or 0.3 U kg reSwFAED activity resulted in a significant increase of the bun volume (8.0 or 9.7%, resp.) after baking when combined with polysaccharide-degrading enzymes from Aspergillus. For the first time, the long-standing, but rarely proven positive effect of a FAE in baking was confirmed. [ABSTRACT FROM AUTHOR]

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides. [ABSTRACT FROM AUTHOR]

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of~71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83mM-1 s-1, 7.63mM-1 s-1, 3.83mM-1 s-1 and 3.75mM-1 s-1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. [ABSTRACT FROM AUTHOR]

Abstract: An extracellular feruloyl esterase (PeFaeA) from the culture supernatant of Pleurotus eryngii was purified to homogeneity using cation exchange, hydrophobic interaction, and size exclusion chromatography. The length of the complete coding sequence of PeFaeA was determined to 1668 bp corresponding to a protein of 555 amino acids. The catalytic triad of Ser-Glu-His demonstrated the uniqueness of the enzyme compared to previously published FAEs. The purified PeFaeA was a monomer with an estimated molecular mass of 67 kDa. Maximum feruloyl esterase (FAE) activity was observed at pH 5.0 and 50 °C, respectively. Metal ions (5 mM), except Hg2+, had no significant influence on the enzyme activity. Substrate specificity profiling characterized the enzyme as a type A FAE preferring bulky natural substrates, such as feruloylated saccharides, rather than small synthetic ones. K m and k cat of the purified enzyme for methyl ferulate were 0.15 mM and 0.85 s−1. In the presence of 3 M NaCl activity of the enzyme increased by 28 %. PeFaeA alone released only little ferulic acid from destarched wheat bran (DSWB), whereas after addition of Trichoderma viride xylanase the concentration increased more than 20 fold. [Copyright &y& Elsevier]

The two Arxula adeninivorans -based bioassays (A-YGS and A-YGFS) described here provide sensitive and reliable screening for glucocorticoids in aquatic environments. The biocomponents of A-YGS and A-YGFS were constructed to carry the human glucocorticoid receptor ( hGRα ) and the phytase gene ( phyK , derived from Klebsiella sp. ASR1) or a fluorescence dsRED gene (derived from the Discosoma sp.), as reporter genes. The responses of A-YGS and A-YGFS were measured photometrically and spectrofluorometrically respectively. The half effective concentration (EC 50 ) and limit of detection (LoD) values for dexamethasone obtained with A-YGS and A-YGFS were 0.81 and 0.29 μM, and 9.42 and 0.47 μM, respectively. Furthermore, both bioassays exhibited different binding specificities for several natural and synthetic glucocorticoids: A-YGS – corticosterone > cyproterone acetate > mifepristone > dexamethasone > cortisol > methylprednisolone > prednisolone and A-YGFS – cortisol >corticosterone >dexamethasone >prednisolone >betamethasone >methylprednisolone. As proof of principle, A-YGS was used to assay total glucocorticoids in river water, wastewater influent and effluent taken from a wastewater treatment plant in Germany. Glucocorticoids were not detected in both A-YGS and LC–MS/MS in seven of the eight samples, however a dexamethasone equivalent (DEQ) was detected in an influent wastewater sample using A-YGS (0.055 μM), while LC–MS/MS analysis showed that hydrocortisone and cortisone were present at 0.063 and 0.083 nM, respectively. [ABSTRACT FROM AUTHOR]

A decarboxylase (IfPAD) from the ascomycete Isaria farinosa converted ferulic acid to 4-vinylguaiacol (4-VG), a volatile which imparts the distinct “smoke flavor” of pyrolized wood. The activity was enhanced by adding (E) -ferulic acid to the culture medium and peaked with 3.6 U g −1 mycelium (1 μmol 4-VG min −1 ). The coding sequence of 543 bp was translated into a 25 kDa protein with a homology of 91 % to putative phenolic acid decarboxylases of its teleomorph, Cordyceps militaris, and Beauveria bassiana, the anamorph of Cordyceps bassiana . Cold shock expression in Escherichia coli yielded 411 U g −1 wet mass. Substrate conversion required a hydroxyl substituent para to a trans -unsaturated C3-side chain of the aromatic ring. K m and k cat / K m values were determined to 0.3 mM and 78.4 mM −1 s −1 for p -coumaric acid and 1.9 mM and 45.1 mM −1 s −1 for (E) -ferulic acid, respectively. The native enzyme and its recombinant counterpart showed pH-optima at pH 6.0 and pH 5.5, and low temperature optima of 19 °C and 14 °C, respectively. IfPAD produced 4-VG from destarched wheat bran and sugar beet fiber, confirming activity on complex plant biomass. This is the first report on the biochemical characterization of a phenolic acid decarboxylase from a filamentous ascomycete. [ABSTRACT FROM AUTHOR]