Non-laser treatment for tattoo removal.

Tattoos are increasingly gathering attention in the young population, especially in second to fourth decade of life. With such trends, rate of its removal also has been on the rise. Treatment options for tattoo removal besides lasers are surgery, radiofrequency, infrared light, cryotherapy, dermabrasion and salabrasion. Unfortunately, none of these procedures are associated with satisfactory cosmetic results due to adverse effects such as scarring and dyspigmentation. Although laser treatment has become the gold standard for tattoo removal, it is also associated with some limitations. Some tattoo inks are resistant to laser, and multiple sessions and multiple wavelengths may be required for its complete removal. Considering these limitations, other treatment modalities for tattoo removal must be explored. This article highlights the non-laser treatment options for tattoo removal. We reviewed all published literature identified from electronic databases (MEDLINE and PubMed) till August 2021 to highlight the non-laser treatment options for tattoo removal.

A Novel Actinobacterial Cutinase Containing a Noncatalytic Polymer-Binding Domain.

The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino-acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origins. A modeled structure of KrCUT, although displaying a typical α/ß hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures (Tm) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar, at 50 to 51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C16 and C18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as the substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibers. This synergistic effect is relevant to the enzyme retting process of natural fibers. IMPORTANCE To date, only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of the microbial cutinases, named KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, and in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tools.

Development of a rat capnoperitoneum phantom to study drug aerosol deposition in the context of anticancer research on peritoneal carcinomatosis.

Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC) is a promising approach with a high optimization potential for the treatment of peritoneal carcinomatosis. To study the efficacy of PIPAC and drugs, first rodent cancer models were developed. But inefficient drug aerosol supply and knowledge gaps concerning spatial drug distribution can limit the results based on such models. To study drug aerosol supply/deposition, computed tomography scans of a rat capnoperitoneum were used to deduce a virtual and a physical phantom of the rat capnoperitoneum (RCP). RCP qualification was performed for a specific PIPAC method, where the capnoperitoneum is continuously purged by the drug aerosol. In this context, also in-silico analyses by computational fluid dynamic modelling were conducted on the virtual RCP. The physical RCP was used for ex-vivo granulometric analyses concerning drug deposition. Results of RCP qualification show that aerosol deposition in a continuous purged rat capnoperitoneum depends strongly on the position of the inlet and outlet port. Moreover, it could be shown that the droplet size and charge condition of the drug aerosol define the deposition efficiency. In summary, the developed virtual and physical RCP enables detailed in-silico and ex-vivo analyses on drug supply/deposition in rodents.

Light-Based Devices for the Treatment of Facial Erythema and Telangiectasia.

Facial erythema is one of the most common outpatient complaints in dermatology. There are various causes of facial erythema and several devices are available for its treatment. Pulsed dye laser (PDL) and intense pulsed light (IPL) are the two common light devices used for these conditions. In this review, we evaluated the literature to assess efficacy of IPL versus PDL in facial erythema and telangiectasia. We searched published articles including clinical trials or reviews articles, case series, and case reports. Electronic databases (MEDLINE and PubMed) were searched to retrieve the articles. Reference lists of selected articles were also considered for the review. Articles published in English language until June 2021 were considered for this review.

The effects of interventional mitral valve repair using the MitraClip System on the results of pulmonary function testing, pulmonary pressure and diffusing capacity of the lung.

BACKGROUND: The study analyzes changes in lung function, pulmonary pressure and diffusing capacity of the lung in patients with mitral valve regurgitation (MR) treated by MitraClip implantation. METHODS: A total of 43 patients (19 women and 24 men with an average age of 78.0 ± 6.6 years) who were able to perform pulmonary function testing including diffusing capacity of the lung for carbon monoxide (DLCO), vital capacity (VC), total lung capacity (TLC), residual volume (RV) and forced expiratory volume in 1 s (FEV1) before and 6 weeks after MitraClip implantation participated in this study. Furthermore, clinical and echocardiographic parameters including systolic pulmonary artery pressure (sPAP), left ventricular ejection fraction (LVEF) and left atrial diameter (LAD) measurements were recorded in all patients. RESULTS: The procedure was performed successfully in all 43 patients leading to a reduction of MR in 97.7% of cases. One patient died on day 4 after the intervention most likely due to pulmonary artery embolism. Six weeks after the implantation 79.1% of patients showed a MR of at most mild to moderate. Furthermore, we could demonstrate a significant reduction of systolic pulmonary artery pressure during follow-up (from 48.8 ± 11.4 mmHg to 42.9 ± 9.0 mmHg (t(41) = - 2.6, p = 0.01). However, no changes in LVEF were detected. Comparing pre and post implant lung function tests, no significant alterations were seen for VC, TLC, DLCO and FEV1. Though, in a subgroup of patients with moderate to severe preexisting deterioration of DLCO at the baseline (max. 50%) the MitraClip procedure resulted in a significant improvement in DLCO (37.8% ± 9.0 to 41.6% ± 10.0, p < 0.001). CONCLUSIONS: Treatment of MR with the MitraClip system successfully reduces MR severity in the vast majority of patients. Consecutively, a reduction in pulmonary pressure could be observed, however no changes in LVEF were obvious. Lung function tests remained unaltered during follow-up. However, in a subgroup of patients with severe preexisting deterioration of DLCO the MitraClip procedure resulted in a significant improvement in DLCO. TRIAL REGISTRATION: Name of the registry: Die Auswirkung der interventionellen Mitralklappenreparatur mit MitraClip-System auf die Ergebnisse der Lungenfunktionsmessung. TRIAL REGISTRATION NUMBER: DRKS00022435; Date of registration: 09/07/2020 'Retrospectively registered'; URL of trial registry record: https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00022435 .

Head orthosis therapy in positional plagiocephaly: longitudinal 3D-investigation of long-term outcomes, compared with untreated infants and with a control group.

Background: As there are very few long-term studies on the effects of head orthosis on deformational plagiocephaly (DP), we investigated the outcomes of patients, including facial symmetry and dental occlusion. Methods: Forty-five infants with DP [cranial vault asymmetry index (CVAI) > 3.5 per cent] were divided into two groups: one treated with head orthosis (32 infants) and another without (13 infants). Another group without head asymmetry (CVAI ≤ 3.5 per cent) served as control. Using 3D-stereophotogrammetry, cranial asymmetry was analysed using symmetry-related variables [CVAI, posterior cranial asymmetry index (PCAI), and ear offset]. Data acquisition was performed before (T1) and at the end of treatment (T2), and at the age of 4 years (T3) for the treated group and at T1 and T3 for the remaining groups. Parameters of facial symmetry and dental occlusion were assessed at T3 for infants with DP. Results: Symmetry-related variables (∆T1-T3) improved significantly more in the treated than the control group, whereas these parameters did not differ significantly between the untreated and control group. Comparing the treated and untreated groups between T1 and T3, the reduction in the asymmetry at the treated group was significantly higher for the CVAI and PCAI. In follow-up, the untreated group showed higher incidences of facial asymmetries than the treated group. Seventy-five per cent of all lateral crossbites found in patients with DP were contralateral to the posterior flattening. Limitations: Due to ethical reasons, the investigation is a non-randomized study. Parameters of facial symmetry were only assed for the treated and untreated groups. Conclusion: Head orthosis therapy in patients with DP leads to significantly better long-term outcomes. Facial asymmetries are more frequent in patients with DP who do not receive this treatment.

Hyperthermic intracavitary nanoaerosol therapy (HINAT) as an improved approach for pressurised intraperitoneal aerosol chemotherapy (PIPAC): Technical description, experimental validation and first proof of concept.

Background: The delivery of aerosolised chemotherapeutic substances into pressurised capnoperitonea has been reported to be more effective than conventional liquid chemotherapy for the treatment of peritoneal carcinomatosis. However, recent reports reveal limitations of the currently available technology. Material and Methods: A novel approach for pressurised intraperitoneal aerosol chemotherapy (PIPAC), called hyperthermic intracavitary nanoaerosol therapy (HINAT), based on extracavitary generation of hyperthermic and unipolar charged aerosols, was developed. The aerosol size distribution, the spatial drug distribution and in-tissue depth penetration of HINAT were studied by laser diffraction spectrometry, differential electrical mobility analysis, time of flight spectrometry, scintigraphic peritoneography and fluorescence microscopy. All experiments were performed contemporaneous with conventional PIPAC for the purpose of comparison. Furthermore, a first proof of concept was simulated in anesthetised German Landrace pigs. Results: HINAT provides a nanometre-sized (63 nm) unipolar-charged hyperthermic (41 °C) drug aerosol for quasi uniform drug deposition over the whole peritoneum with significantly deeper drug penetration than that offered by conventional PIPAC.

Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae.

The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any ß-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (K m=3.2 ± 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (T m) of the two proteins, estimated to be in the range of 49.5-53.7 °C indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings.

Treatment of major depression with bilateral theta burst stimulation: a randomized controlled pilot trial.

BACKGROUND: Current efforts to improve clinical effectiveness and utility of repetitive transcranial magnetic stimulation (rTMS) in the treatment of major depression (MD) include theta burst stimulation (TBS), a patterned form of rTMS. Here, we investigated the efficacy of bilateral TBS to the dorsolateral prefrontal cortex (dlPFC) in patients with MD in additon to ongoing medication and psychotherapy. METHODS: In this randomized-controlled trial, thirty-two patients with MD were treated for six weeks (thirty sessions) with either successively intermittent, activity enhancing TBS (iTBS) to the left and continuous, inhibiting TBS (cTBS) to the right dlPFC or with bilateral sham stimulation. Primary outcome measure was the proportion of treatment response defined as a Montgomery-Åsberg Depression Rating Scale (MADRS)≤50% compared to baseline. Secondary outcomes comprised response and remission rates of the Hamilton Depression Rating Scale (HAMD) and the Beck Depression Inventory (BDI). RESULTS: A larger number of responders were found in the cTBS (n=9) compared to the sham-stimulation (n=4) group (odds ratio: 3.86; Wald χ(2)=3.9, p=0.048). On secondary endpoint analysis, patient-reported outcome as assessed by the BDI, pointed towards a higher rate of remitters in the cTBS (n=6) than in the sham (n=1) group (odds ratio: 9; Wald χ(2)=3.5, p=0.061). LIMITATIONS: With regard to the pilot character of the study and the small sample size, the results have to be considered as preliminary. CONCLUSIONS: These findings provide first evidence that six weeks treatment of MDD with iTBS to the left and cTBS to the right dlPFC for six weeks is safe, feasible and superior to sham stimulation applied add-on to pharmacological and psychotherapeutic treatment.

Genome mining for new α-amylase and glucoamylase encoding sequences and high level expression of a glucoamylase from Talaromyces stipitatus for potential raw starch hydrolysis.

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ∼8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71-87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.

Characterization of CpdC, a large-ring lactone-hydrolyzing enzyme from Pseudomonas sp. strain HI-70, and its use as a fusion tag facilitating overproduction of proteins in Escherichia coli.

There are few entries of carbon-carbon bond hydrolases (EC 3.7.1.-) in the ExPASy database. In microbes, these enzymes play an essential role in the metabolism of alicyclic or aromatic compounds as part of the global carbon cycle. CpdC is a ω-pentadecalactone hydrolase derived from the degradation pathway of cyclopentadecanol or cyclopentadecanone by Pseudomonas sp. strain HI-70. CpdC was purified to homogeneity and characterized. It is active as a dimer of 56,000 Da with a subunit molecular mass of 33,349. Although CpdC has the highest activity and reaction rate (kcat) toward ω-pentadecalactone, its catalytic efficiency favors lauryl lactone as a substrate. The melting temperature (Tm) of CpdC was estimated to be 50.9 ± 0.1°C. The half-life of CpdC at 35°C is several days. By virtue of its high level of expression in Escherichia coli, the intact CpdC-encoding gene and progressive 3'-end deletions were employed in the construction of a series of fusion plasmid system. Although we found them in inclusion bodies, proof-of-concept of overproduction of three microbial cutinases of which the genes were otherwise expressed poorly or not at all in E. coli was demonstrated. On the other hand, two antigenic proteins, azurin and MPT63, were readily produced in soluble form.

The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: a proposal for an evolutionary driving force.

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

Structural analysis of a novel cyclohexylamine oxidase from Brevibacterium oxydans IH-35A.

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.

Camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions.

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 µM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 µM; kcat = 283 s(-1)) in preference to flavin adenine dinucleotide (FAD) (Km = 19 µM; kcat = 128 s(-1)). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.

Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453.

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140-152, 1983). Here we cloned and overexpressed the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP(+) at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP(+). A comparison of several crystal forms of OTEMO bound to FAD and NADP(+) revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k(cat)/K(m)) favors 2-n-hexyl cyclopentanone (4.3 × 10(5) M(-1) s(-1)) as a substrate, although its affinity (K(m) = 32 µM) was lower than that of the CoA-activated substrate (K(m) = 18 µM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

New feruloyl esterases to access phenolic acids from grass biomass.

In the Sorangium cellulosum strain So ce56 genome, two putative esterase-encoding genes (loci sce1896 and sce8927) were cloned, expressed in Escherichia coli, and the resulting enzymes (designated ScFAE1 and ScFAE2) were used to assess the possible release of ferulic acid (FA) from triticale and wheat brans, and an aqueous fraction of steam-exploded wheat straw. The two polypeptides, sharing only 30% sequence identity, exhibit a typical catalytic Ser-Asp-His triad, a characteristic of α/ß-hydrolase fold proteins. Both ScFAE1 (35 kDa) and ScFAE2 (34 kDa) were purified to apparent homogeneity and comparison of their kinetic parameters indicated an apparent higher affinity of ScFAE2 than ScFAE1 towards the various feruloyl substrates. This property was reflected by the observation that ScFAE2 was capable of yielding up to 85% of FA from destarched triticale bran. In the steam-exploded wheat sample, more than 85% yield of FA or p-coumaric acid was also effected by ScFAE2 without the decomposition of valuable chemical such as furfural. The two cloned FAEs represent the first of myxobacterial origin to be characterized and they are classified as new members of the type D family of FAEs.

Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme.

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a ß-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the ß-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.

Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran.

A putative alpha/beta hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75 degrees C, the enzyme retained at least 95% of its original activity for over 80 min; at 80 degrees C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3-5 °C increase in the apparent melting temperature (T(m)) of the mutants over the native BcEST that has a T(m) of 50 °C was outperformed by TtEST, a naturally occurring homologue with a T(m) of 65 °C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/ß-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.