Theoretical study on the glycosidic C-C bond cleavage of 3''-oxo-puerarin.

Puerarin, daidzein C-glucoside, was known to be biotransformed to daidzein by human intestinal bacteria, which is eventually converted to (S)-equol. The metabolic pathway of puerarin to daidzein by DgpABC of Dorea sp. PUE strain was reported as puerarin (1) → 3''-oxo-puerarin (2) → daidzein (3) + hexose enediolone (C). The second reaction is the cleavage of the glycosidic C-C bond, supposedly through the quinoid intermediate (4). In this work, the glycosidic C-C bond cleavage reaction of 3''-oxo-puerarin (2) was theoretically studied by means of DFT calculation to elucidate chemical reaction mechanism, along with biochemical energetics of puerarin metabolism. It was found that bioenergetics of puerarin metabolism is slightly endergonic by 4.99 kcal/mol, mainly due to the reaction step of hexose enediolone (C) to 3''-oxo-glucose (A). The result implied that there could be additional biochemical reactions for the metabolism of hexose enediolone (C) to overcome the thermodynamic energy barrier of 4.59 kcal/mol. The computational study focused on the C-C bond cleavage of 3''-oxo-puerarin (2) found that formation of the quinoid intermediate (4) was not accessible thermodynamically, rather the reaction was initiated by the deprotonation of 2''C-H proton of 3''-oxo-puerarin (2). The 2''C-dehydro-3''-oxo-puerarin (2a2C) anionic species produced hexose enediolone (C) and 8-dehydro-daidzein anion (3a8), and the latter quickly converted to daidzein through the daidzein anion (3a7). Our study also explains why the reverse reaction of C-glycoside formation from daidzein (3) and hexose enediolone (C) is not feasible.

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens.

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.

Computer-aided discovery of a novel thermophilic laccase for low-density polyethylene degradation.

Polyethylene (PE) and industrial dyes are recalcitrant pollutants calling for the development of sustainable solutions for their degradation. Laccases have been explored for removal of contaminants and pollutants, including dye decolorization and plastic degradation. Here, a novel thermophilic laccase from PE-degrading Lysinibaccillus fusiformis (LfLAC3) was identified through a computer-aided and activity-based screening. Biochemical studies of LfLAC3 indicated its high robustness and catalytic promiscuity. Dye decolorization experiments showed that LfLAC3 was able to degrade all the tested dyes with decolorization percentage from 39% to 70% without the use of a mediator. LfLAC3 was also demonstrated to degrade low-density polyethylene (LDPE) films after eight weeks of incubation with either crude cell lysate or purified enzyme. The formation of a variety of functional groups was detected using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Damage on the surfaces of PE films was observed via scanning electron microscopy (SEM). The potential catalytic mechanism of LfLAC3 was disclosed by structure and substrate-binding modes analysis. These findings demonstrated that LfLAC3 is a promiscuous enzyme that has promising potential for dye decolorization and PE degradation.

Immobilization and Application of Fatty Acid Photodecarboxylase in Deep Eutectic Solvents.

Since its discovery in 2017, the fatty acid decarboxylase (FAP) photoenzyme has been the focus of extensive research, given its ability to convert fatty acids into alka(e)nes using merely visible blue light. Unfortunately, there are still some drawbacks that limit the applicability of this biocatalyst, such as poor solubility of the substrates in aqueous media, poor photostability, and the impossibility of reusing the catalyst for several cycles. In this work, we demonstrate the use of FAP in non-conventional media as a free enzyme and an immobilized preparation. Namely, its applicability in deep eutectic solvents (DESs) and a proof-of-concept immobilization using a commercial His-tag selective carrier, a thorough study of reaction and immobilization conditions in each case, as well as reusability studies are shown. We observed an almost complete selectivity of the enzyme towards C18 decarboxylation over C16 when used in a DES, with a product analytical yield up to 81 % when using whole cells. Furthermore, when applying the immobilized enzyme in DES, we obtained yields >10-fold higher than the ones obtained in aqueous media.

Allyl Aryl Ether Cleavage by Blautia sp. MRG-PMF1 Cocorrinoid O-Demethylase.

Coabalamin-dependent O-demethylase in Blautia sp. strain MRG-PMF1 was found to catalyze the unprecedented allyl aryl ether cleavage reaction. To expand the potential biotechnological applications, the reaction mechanism of the allyl aryl ether C-O bond cleavage, proposed to utilize the reactive Co(I) supernucleophile species, was studied further from the anaerobic whole-cell biotransformation. Various allyl naphthyl ether derivatives were reacted with Blautia sp. MRG-PMF1 O-demethylase, and stereoisomers of allyl naphthyl ethers, including prenyl and but-2-enyl naphthyl ethers, were converted to the corresponding naphthol in a stereoselective manner. The allyl aryl ether cleavage reaction was regioselective, and 2-naphthyl ethers were converted faster than the corresponding 1-naphthyl ethers. However, MRG-PMF1 cocorrinoid O-demethylase was not able to convert (2-methylallyl) naphthyl ether substrates, and the conversion of propargyl naphthyl ether was extremely slow. From the results, it was proposed that the allyl ether cleavage reaction follows the nucleophilic conjugate substitution (SN2') mechanism. The reactivity and mechanism of the new allyl ether cleavage reaction by cobalamin-dependent O-demethylase would facilitate the application of Blautia sp. MRG-PMF1 O-demethylase in the area of green biotechnology. IMPORTANCE Biodegradation of environmental pollutants and valorization of biomaterials in a greener way is of great interest. Cobalamin-dependent O-demethylase in Blautia sp. MRG-PMF1 exclusively involves anaerobic C1 metabolism by cleaving the C-O bond of aromatic methoxy group and also produces various aryl alcohols by metabolizing allyl aryl ether compounds. Whereas methyl ether cleavage reaction is known to follow the SN2' mechanism, the reaction pattern and mechanism of the new allyl ether cleavage reaction by cobalamin-dependent O-demethylase have never been studied. For the first time, stereoselectivity and the SN2' mechanism of allyl aryl ether cleavage reaction by Blautia sp. MRG-PMF1 O-demethylase is reported, and the results would facilitate the application of Blautia sp. MRG-PMF1 O-demethylase in the area of green biotechnology.

Biodegradation of polyethylene and polystyrene: From microbial deterioration to enzyme discovery.

The global production of plastics has continuously been soaring over the last decades due to their extensive use in our daily life and in industries. Although synthetic plastics offer great advantages from packaging to construction and electronics, their low biodegradability induce serious plastic pollution that damage the environment, human health and make irreversible changes in the ecological cycle. In particular, plastics containing only carbon-carbon (C-C) backbone are less susceptible to degradation due to the lack of hydrolysable groups. The representative polyethylene (PE) and polystyrene (PS) account for about 40% of the total plastic production. Various chemical and biological processes with great potential have been developed for plastic recycle and reuse, but biodegradation seems to be the most attractive and eco-friendly method to combat this growing environmental problem. In this review, we first summarize the current advances in PE and PS biodegradation, including isolation of microbes and potential degrading enzymes from different sources. Next, the state-of-the-art techniques used for evaluating and monitoring PE and PS degradation, the scientific toolboxes for enzyme discovery as well as the challenges and strategies for plastic biodegradation are intensively discussed. In return, it inspires a further technological exploration in expanding the diversity of species and enzymes, disclosing the essential pathways and developing new approaches to utilize plastic waste as feedstock for recycling and upcycling.

Semi-rational Engineering of a Promiscuous Fatty Acid Hydratase for Alteration of Regioselectivity.

Fatty acid hydratases (FAHs) catalyze regio- and stereo-selective hydration of unsaturated fatty acids to produce hydroxy fatty acids. Fatty acid hydratase-1 (FA-HY1) from Lactobacillus Acidophilus is the most promiscuous and regiodiverse FAH identified so far. Here, we engineered binding site residues of FA-HY1 (S393, S395, S218 and P380) by semi-rational protein engineering to alter regioselectivity. Although it was not possible to obtain a completely new type of regioselectivity with our mutant libraries, a significant shift of regioselectivity was observed towards cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA). We identified mutants (S393/S395 mutants) with excellent regioselectivity, generating a single hydroxy fatty acid product from EPA (15-OH product), which is advantageous from application perspective. This result is impressive given that wild-type FA-HY1 produces a mixture of 12-OH and 15-OH products at 63 : 37 ratio (12-OH : 15-OH). Moreover, our results indicate that native FA-HY1 is at its limit in terms of promiscuity and regiospecificity, thus it may not be possible to diversify its product portfolio with active site engineering. This behavior of FA-HY1 is unlike its orthologue, fatty acid hydratase-2 (FA-HY2; 58 % sequence identity to FA-HY1), which has been shown earlier to exhibit significant promiscuity and regioselectivity changes by a few active site mutations. Our reverse engineering from FA-HY1 to FA-HY2 further demonstrates this conclusion.

A new thermophilic extradiol dioxygenase promises biodegradation of catecholic pollutants.

Extradiol dioxygenases (EDOs) catalyze the meta cleavage of catechol into 2-hydroxymuconaldehyde, a critical step in the degradation of aromatic compounds in the environment. In the present work, a novel thermophilic extradiol dioxygenase from Thermomonospora curvata DSM43183 was cloned, expressed, and characterized by phylogenetic and biochemical analyses. This enzyme exhibited excellent thermo-tolerance, displaying optimal activity at 50 °C, remaining >40% activity at 70 °C. Structural modeling and molecular docking demonstrated that both active center and pocket-construction loops locate at the C-terminal domain. Site-specific mutants D285A, H205V, F301V based on a rational design were obtained to widen the entrance of substrates; resulting in significantly improved catalytic performance for all the 3 mutants. Compared to the wild-type, the mutant D285A showed remarkably improved activities with respect to the 3,4-dihydroxyphenylacetic acid, catechol, and 3-chlorocatechol, by 17.7, 6.9, and 3.7-fold, respectively. The results thus verified the effectiveness of modeling guided design; and confirmed that the C-terminal loop structure indeed plays a decisive role in determining catalytic ring-opening efficiency and substrate specificity of the enzyme. This study provided a novel thermostable dioxygenase with a broad substrate promiscuity for detoxifying environmental pollutants and provided a new thinking for further enzyme engineering of EDOs.

A Bi-Enzymatic Cascade Pathway towards Optically Pure FAHFAs*.

Recently discovered endogenous mammalian lipids, fatty acid esters of hydroxy fatty acids (FAHFAs), have been proved to have anti-inflammatory and anti-diabetic effects. Due to their extremely low abundancies in vivo, forging a feasible scenario for FAHFA synthesis is critical for their use in uncovering biological mechanisms or in clinical trials. Here, we showcase a fully enzymatic approach, a novel in vitro bi-enzymatic cascade system, enabling an effective conversion of nature-abundant fatty acids into FAHFAs. Two hydratases from Lactobacillus acidophilus were used for converting unsaturated fatty acids to various enantiomeric hydroxy fatty acids, followed by esterification with another fatty acid catalyzed by Candida antarctica lipase A (CALA). Various FAHFAs were synthesized in a semi-preparative scale using this bi-enzymatic approach in a one-pot two-step operation mode. In all, we demonstrate that the hydratase-CALA system offers a promising route for the synthesis of optically pure structure-diverse FAHFAs.

Thai Curcuma Species: Antioxidant and Bioactive Compounds.

For the functional food applications, antioxidant properties and the bioactive compounds of the 23 Curcuma species commercially cultivated in Thailand were studied. Total phenolic content and DPPH radical scavenging activity were determined. The concentrations of eight bioactive compounds, including curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (4), germacrone (5), furanodienone (6), zederone (7), and ar-turmerone (8), were determined from the Curcuma by HPLC. While the total phenolic content of C. longa was highest (22.3 ± 2.4 mg GAE/g, mg of gallic acid equivalents), C. Wan Na-Natong exhibited the highest DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging activity. Twenty-three Curcuma species showed characteristic distributions of the bioactive compounds, which can be utilized for the identification and authentication of the cultivated Curcuma species. C. longa contained the highest content of curcumin (1) (304.9 ± 0.1 mg/g) and C. angustifolia contained the highest content of germacrone (5) (373.9 ± 1.1 mg/g). It was noteworthy that 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (4) was found only from C. comosa at a very high concentration (300.7 ± 1.4 mg/g). It was concluded that Thai Curcuma species have a great potential for the application of functional foods and ingredients.

Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity.

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C18 polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.

[Effect of cigarette smoke extract on phagocytosis of Staphylococcus aureus by macrophages]. / Staphylococcus aureus'un makrofajlar tarafindan fagositozu üzerine sigara dumani ekstraktinin etkisi.

Staphylococcus aureus is one of the most important pathogen that causes community acquired and nosocomial infections worldwide. Phagocytosis by macrophages plays an important role in the first line defense against infections caused by S.aureus. On the other hand, the conducted studies have indicated that cigarette smoke has negative effects on both innate and acquired immune responses. The aim of this study was to investigate the effects of cigarette smoke on macrophage viability and their capacity of S.aureus phagocytosis. For this purpose THP-1 cell lines (human leukemic monocyte cell culture) were used and after the differentiation of the cells with PMA (phorbol myristate acetate) treatment, the macrophages were exposed to cigarette smoke extract (CSE) for 2- and 4-hours at concentrations of 1%, 5%, 10%, 25%, and 50%. Afterwards, the cells were stained with propidium iodide and the viability of the cells was analyzed by a flow cytometer. Two different methods were used to investigate the effect of CSE on the phagocytosis of S.aureus. The first one was the classical bacteriological method, in which macrophages were exposed to CSE for 2 hours in five different concentrations and were infected with 100 MOI (multiplicity of infection) S.aureus. After 1 hour of incubation, macrophages were lysed with PBS-0.1% Triton X-100 and plated on Luria-Bertani (LB) agar following serial dilutions. Newly formed colonies were counted and the number of bacteria phagocytosed were evaluated as colony forming units (CFU). The second method for the detection of phagocytosis was flow cytometric analysis in which SYBR(®) Green-labeled bacteria were used. To confirm that the macrophages were infected, bacteria were stained with SYBR(®) Green and macrophages were analyzed following infection via flow cytometry. Macrophages were exposed to 10% and 50% CSE and infected with bacteria stained with SYBR(®) Green. The level of phagocytosis was analyzed by flow cytometry in terms of median fluorescence intensity. Macrophage death rate was 24% and 30% following 2-hour exposure to 25% and 50% CSE, respectively, while 4-hour exposure increased death rate to 38% and 51%, respectively (p< 0.001). At 10% and higher concentrations of CSE, cell death increased with an average of 1.5-fold between 2- and 4-hour exposures (p< 0.05). Macrophages were successfully infected (99.8%) with SYBR(®) Green-stained S.aureus. Phagocytosis of S.aureus by macrophages decreased significantly upon exposure to 10% or more CSE concentrations (p< 0.0001). Median fluorescence intensity, which indicates phagocytosed bacterial cell number, decreased with no statistical significance when macrophages exposed to 10% and 50% CSE were infected with SYBR(®) Green-stained S.aureus. The results of this study revealed that, macrophage viability and phagocytosis of S.aureus were reduced depending on CSE concentration and time of exposure. In addition, it was shown that, SYBR(®) Green dye is a proper stain for the flow cytometric analysis of bacteria that were phagocytosed by macrophages.